A global optimized diagnostic approach of dysmorphology and intellectual disability

Transcription

1 Grigore T. Popa University of Medicine and Pharmacy A global optimized diagnostic approach of dysmorphology and intellectual disability HABILITATION THESIS Prof. Cristina Rusu MD PhD 1

2 Table of Contents THESIS SUMMARY... 3 REZUMATUL TEZEI... 4 SECTION I - SCIENTIFIC ACHIEVEMENTS INTELLECTUAL DISABILITY AND BEHAVIORAL DISTURBANCES Introduction and conceptual background Subtelomeric rearrangements and microdeletions/duplications as an important cause of nonspecific intellectual disability X-linked intellectual disability Autism spectrum disorders Conclusions and further enhancements References OPTIMIZATION OF DYSMORPHOLOGIC DIAGNOSIS AND MANAGEMENT OF PATIENTS WITH GENETIC DISORDERS Introduction and conceptual background Prader Willi syndrome RAS-opathies Skeletal dysplasia Conclusions and further enhancements References INTRODUCTION OF INNOVATIVE TECHNIQUES IN MEDICAL GENETICS PRACTICE AND OPTIMIZATION OF THEIR USE Introduction and conceptual background MLPA use for patients with intellectual disability Role of MLPA test in the identification of complex chromosomal rearrangements Use of arraycgh in the diagnosis of complex cases Diagnosis of Duchenne/ Becker muscular dystrophy using MLPA test Conclusions and further enhancements References SECTION II PAST, PRESENT AND FUTURE PROFESSIONAL, SCIENTIFIC AND ACADEMIC CONTRIBUTIONS Career overview PhD thesis Academic activity Research projects International recognition FUTURE DIRECTIONS SECTION III LIST OF PUBLICATIONS SECTION IV - CURRICULUM VITAE

3 Thesis summary The Habilitation Thesis is structured in 4 major sections: Section I (and the most important) is dedicated to my scientific achievements during the postdoctoral work ( ). I have concentrated to the following research areas: 1. Intellectual disability and behavioural disturbances: I have introduced new tests in our daily practice (e.g. antifmrp test for fragile X screening and MLPA for subtelomeric rearrangements detection). Later, MLPA was extended to many other applications (e.g. microdeletions, Fragile X syndrome, autism etc); 2. Optimization of dysmorphologic diagnosis and management of patients with genetic disorders: Some of the data referring to Prader Willi syndrome, RAS-opathies and bone dysplasia have been included. Basicly, the work tried to identify new clinical features and to facilitate genetic testing for the benefit of the patient and his family; 3. Introduction of innovative techniques in Medical Genetics practice and optimization of their use: for this direction, I have tried to introduce simple, robust, inexpensive lab techniques that will provide us with results comparable with more complex and expensive techniques. After optimization, we have extended the method for many other aplications (some of them new in the field, enabling us to publish the results). Section II is dedicated to my professional, scientific and academic contributions, as well as to future research directions. I have included a career overview, a synthesis of my PhD thesis, succinct information on my academic activity, a comprehensive presentation of my research projects, as well as some data on my international visibility. Future research directions include: Use of new molecular techniques for the diagnosis of intellectual disability; Comprehensive study of autism spectrum disorders; Definition of clinical picture in adults and infants with certain genetic disorders and maybe also for other populations; Panel testing for spectra like RAS-opathies, ciliopathies etc; Identification of Romanian cases for new microdeletion syndromes; Extension of pharmacogenetic tests; Panel testing in cancer and leukemia. Section III illustrates my list of publications, whereas Section IV presents my Curriculum vitae. 3

4 Rezumatul tezei Teza de abilitare este structurată în 4 secţiuni majore: Secţiunea I (şi cea mai importantă) este dedicată realizărilor ştiinţifice din perioada postdoctorală ( ). M-am concentrat pe următoarele domenii de cercetare: 4. Dizabilitatea intelectuală şi tulburările de comportament: am introdus teste noi în practica noastră zilnică (ex. test antifmrp pentru screening-ul Sindromului X fragil şi MLPA pentru detecţia rearanjamentelor subtelomerice). Ulterior, testul MLPA a fost extins pentru multe alte aplicaţii (ex. microdeleţii, Sindrom X fragil, autism etc); 5. Optimizarea diagnosticului dismorfologic şi a managementului pacienţilor cu boli genetice: Am inclus unele date privind Sindromul Prader Willi, RAS-opatii şi displazii osoase. În principiu, activitatea a încercat să identifice semne clinice noi şi să faciliteze testarea în beneficiul pacienţilor şi familiilor lor; 6. Introducerea de tehnici innovative în practica Geneticii Medicale şi optimizarea folosirii lor: pentru această direcţie, am încercat să introduc tehnici de laborator simple, robuste, necostisitoare care să ne furnizeze rezultate comparabile cu alte tehnici mai complexe şi mai scumpe. După optimizare, am extins metoda la multe alte aplicaţii (unele în domenii noi, permiţându-ne realizarea de publicaţii). Secţiunea II este dedicată contribuţiilor mele profesionale, ştiinţifice şi academice, precum şi viitoarelor direcţii de cercetare. Am inclus o trecere în revistă a carierei, o sinteză a tezei de doctorat, o informare succintă privind activitatea mea academică, o prezentare detaliată a proiectelor mele de cercetare, precum şi unele date privind vizibilitatea mea internaţională. Direcţiile viitoare de cercetare includ: vitae. Folosirea de tehnici moleculare noi pentru diagnosticul dizabilităţii intelectuale; Studiu multidisciplinary al anomaliilor din spectrul autist; Definirea tabloului clinic la adulţi şi nou născuţi cu anumite boli genetice şi poate şi la alte etnii; Testare de tip panel pentru spectre ca RAS-opatii, ciliopatii etc; Identificarea de cazuri în România pentru sindroame noi de microdeleţie; Extensia testelor farmacogenetice; Testare în panel pentru cancer şi leucemii. Secţiunea III ilustrează lista mea de publicaţii, iar Secţiunea IV prezintă Curriculum 4

5 Section I - Scientific achievements Intellectual disability and behavioral disturbances 1.1. Introduction and conceptual background Intellectual disability (formerly known as mental retardation) is a generalized neurodevelopmental disorder characterized by significantly impaired intellectual and adaptive functioning. It is defined by an IQ score below 70 in addition to deficits in two or more adaptive behaviors that affect everyday, general living. Intellectual disability affects approximately 3% of the individuals in the general population and represents a very heterogeneous category of disorders. It is usually classified upon IQ value into mild, moderate and severe, but must also take into consideration a person's adaptive functioning. Major causes include genetic and environmental factors, but in approximately half of the cases the cause is still unknown. Causes are different according to the severity of the disorder in moderate/ severe intellectual disability genetic causes are much more frequently involved, compared to mild intellectual disability that is mostly determined by social causes. Genetic causes that could lead to intellectual disability range from visible chromosomal abnormalities (numerical and structural unbalanced) to microdeletions/ microduplications (visible using FISH, MLPA or arraycgh testing), subtelomeric rearrangements (identified by MLPA) and even monogenic defects (identified by Sanger sequencing or panel exome sequencing). For practical reasons, intellectual disability could be classified as: Syndromic or specific situation in which intellectual disability is associated with certain birth defects or behavioural disturbances that could be grouped in a recognizable pattern; Nonsyndromic or nonspecific situation in which intellectual disability is solely present or associated with very few, nonspecific anomalies (that cannot be grouped in a certain pattern); A particular category of intellectual disability is represented by X-linked intellectual disability, meaning that the mental problem is determined by mutations on chromosome X. Usually the distribution of the affected persons within the family is highly suggestive (at least 2 affected males related on the maternal side e.g. affected son and brother of an apparently normal woman). Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders defined by core deficits in social interaction and communication, restrictive interests and repetitive behaviors appearing before age 3 (American Psychiatric Association, 2013). The spectrum encompasses autistic disorder, Asperger syndrome, pervasive developmental disorder not otherwise specified, childhood disintegrative disorder and Rett syndrome. Frequently autistic children associate comorbidities like intellectual disability, seizures, schizophrenia, sleep disorders or gastrointestinal symptoms (e.g. diarrhea, 5

6 constipation, bloating and gastro-esophageal reflux) (Rossignol and Frye, 2012). The mechanism that leads to ASD is very complex, involving genetic, epigenetic, immune and environmental factors that could act in different proportions, at different developmental stages (prenatal, perinatal or postnatal) and on different pathways. The general prototype consists in an initial systemic dysfunction, such as immune dysregulation, inflammation, impaired detoxification or oxidative stress (Rossignol and Frye, 2012). In this context, ASD may arise due to the harmful action of environmental factors. Autism is more frequent in males (4 male/ 1 female ratio) (Baron-Cohen et al., 2011) and different genetic, epigenetic, metabolic and social hypotheses tried to explain this finding. In USA, ASD prevalence has increased in time, from 1 in 3,000 individuals in 1966 (Lotter, 1966), to 1 in 150 in 2007 (Kuehn, 2007) and 1 in 88 in 2012, with specific prevalence in boys (1 in 54) comparative to girls (1 in 252) (CDC, 2012). This recent increase pointed to environmental factors as a key issue for ASD determinism and stimulated research in the field. Surprisingly, research showed that ASD could be triggered in a genetically predisposed individual not only by classical external environmental factors (e.g. toxicants, pollutants, pesticides), but also by maternal imbalances or disorders (e.g. hormonal or inflammatory), as well as by disturbances of gut microbiota in the affected child (considered as internal environment) Subtelomeric rearrangements and microdeletions/duplications as an important cause of nonspecific intellectual disability Intellectual disability (ID) is a major public health issue, with a prevalence that varies from less than 1% to 3% in the general population, depending on defining criteria used by different studies (Roeleveld et al 1997). ID etiology is complex, including exogenous and genetic factors, yet in approximately 50% of cases it remains unknown, either because patients were not genetically evaluated or because a genetic defect was not identified. Recent studies have shown that 15-20% of ID cases are caused by submicroscopic copy number variations (CNVs) (Miller et al 2010, Cooper et al 2011). The introduction of FISH (Fluorescence In Situ Hybridization) and MLPA (Multiplex Ligation-dependent Probe Amplification) techniques (Schouten et al 2002) enabled the detection of microdeletions / microduplications in syndromic ID patients (ID associated with dysmorphic features and/or multiple congenital anomalies). In the past decade, the introduction of the microarray technology has allowed the detection of submicroscopic CNVs with unprecedented resolution leading thus to the identification of numerous syndromes with microdeletions and microduplications (Slavotinek et al 2008), now chromosome microarray being recommended as a first-tier diagnostic test in patients with ID and/or multiple congenital anomalies (Miller et al 2010). However, this technology requires expensive equipment and consumables that are hardly accessible to all diagnostic centres. Some of the chromosomal abnormalities identified by microarray tests can also be detected using different MLPA kits. Taking into consideration the cost difference between whole genome microarray platforms and MLPA, screening ID patients using the MLPA technique represents a reasonable option in the diagnostic evaluation of ID, especially in the developing countries. The aim of this study was to evaluate the ability of a combination of MLPA kits to establish the etiologic diagnosis in a group of patients with syndromic ID. Material and method The study group consisted of 369 patients with syndromic ID of unknown etiology, older than three months and with normal (358 patients) or uncertain karyotype results at band level (11 patients). Patients that were clinically suggestive for aneuploidies (21, 13 and 18 trisomies) and Fragile X syndrome were not included in the study. All patients have been clinically evaluated by a geneticist. The standard evaluation included family and medical history (pre-, peri- and postnatal data), anthropometric measurements, detailed 6

7 physical examination and psychological examination. A written informed consent was obtained prior to evaluation from either patients parents or legal representatives and the study was approved by the Ethics Committee of the University of Medicine and Pharmacy, Iasi. All patients were assessed for chromosomal imbalances using commercially available SALSA MLPA kits (MRC-Holland, Amsterdam, The Netherlands). Patients with a clinical suspicion of a microdeletion syndrome (186 patients, subgroup A) were tested with P064 kit (156 patients) or P096 kit (30 patients), which contain probes for common microdeletion/microduplication syndromes. For patients without clinical suspicion of a specific syndrome and for those with uncertain karyotype results (183 patients, subgroup B) the P036 and P070 kits were used. These kits have been developed to screen for subtelomeric CNVs and contain one MLPA probe for each subtelomeric region, except for the short arms of acrocentric chromosomes (13p, 14p, 15p, 21p and 22p), for which a probe on the q arm, close to the centromere is included instead. Abnormal results detected by these MLPA kits were further characterized using appropriate follow-up MLPA kits (SALSA MLPA Telomere Follow-up set, P029-A1 Williams-Beuren Syndrome probemix, P250-B2 DiGeorge probemix and ME028-B1 Prader Willi/Angelman probemix). The details of regions detected by each kit are available at The gene content of abberations was analyzed using the UCSC genome browser (NCBI36/hg18, Parental DNA samples were not available. The DNA extraction from peripheral blood was performed using Wizard Genomic DNA Purification Kit (Promega Corp., Madison, WI, USA). The standard MLPA analysis was performed according to the manufacturer s instructions. Briefly, 200 nanograms of genomic DNA was denatured and hybridized with SALSA probes at 60 C for hours. After 15 minute ligation at 54 C, PCR was performed in a Gradient Palm-Cycler (Corbett Research, Mortlake, NSW, Australia) available in a 96-well format, using Cy5 universally labeled primers. Fluorescent amplification products were subsequently separated through capillary electrophoresis, in a CEQ 8000 GeXP Genetic Analysis System (Beckman Coulter), and were analyzed using the default software. The number of DNA copies was estimated using the Coffalyser.Net software, which calculates the ratio of peak areas in test samples over those of normal controls for each target sequence. Results In this study a combination of MLPA kits was used to detect chromosomal aberrations in a group of 369 patients with unexplained syndromic ID. For subgroup A, P096 kit showed reduced ratios for all of 16 the probes targeting 4p telomeric region (Wolf-Hirschhorn syndrome) in 1 of the 30 individuals examined. P064 kit detected abnormalities in 24/156 (15.3%) patients: 14 22q11.21 deletions (Velocardiofacial/ DiGeorge syndrome), seven 7q11.23 deletions (Williams syndrome) and three 15q11.2 deletions (Prader Willi syndrome). The imbalances found using P064 microdeletion kit and their confirmation by the appropriate folllow-up kits are presented in Table For subgroup B, subtelomeric rearrangements were detected in 24 patients by both P036 and P070 kits, while in 6 patients the rearrangements were detected by a single MLPA kit (three 4q deletions, one 21q deletion which was confirmed by P365 kit and included only the probe that targets PRMT2 gene, one 5q duplication and one 16p deletion not confirmed by P277 and P365 kit, respectively). Clinical and molecular description of subtelomeric rearrangements detected by both screening and follow-up kits is presented in Table

8 Table 1.2.1: Details of the imbalances found using microdeletion kits P064 P250-B2 P029-A1 ME028-B1 14 patients with velocardiofacial syndrome 14 patients with deleted probes a 7 patients with Williams syndrome - 5 patients with 12 deleted probes 2 patients with 9 deleted probes b - 3 patients with Prader Willi syndrome patients with 29 deleted probes c a Breakpoint between low-copy repeats A and D, commonly deleted DiGeorge region b Two sisters without the typical Williams phenotype. The deletion includes probes related to the following genes: FKBP6, FZD9, TBL2, STX1A, ELN and LIMK1. c Proximal breakpoint located between NIPA1 and MKRN3 genes, distal breakpoint located between GABRB3 and APBA2. Discussion Subgroup A. The incidence of microdeletion/microduplication syndromes was 13.4% (25/186), most of them (24 out of 25) being identified using the P064 kit. The low detection rate of P096 kit observed in this study might be explained by the fact that the frequency of the syndromes covered is lower compared to those detected by P064 kit (except for Down syndrome, but the patients with phenotype suggestive of trisomy 21 were not included in this study). Other studies that have used MLPA P064 kit had detection rates of % if patients were selected based on the presence of ID and/or multiple congenital anomalies (Kirchhoff et al 2007, Jehee et al 2011), and of 14,1%, if patients were selected based on phenotype suggestive of a microdeletion syndrome ( Kirchhoff et al 2007). All these data emphasize the fact that submicroscopic anomalies are involved in a large number of cases with ID/multiple congenital anomalies, and consequently the selection of patients based on clinical suspicion of microdeletion syndrome may increase the detection rate. The 22q11.21 deletion was the most frequent abnormality in our study, being detected in 7.5% (14/186) of patients and representing more than half of the abnormalities identified in this subgroup of patients. The outcome is concordant with the results reported in other studies, in which the 22q11.21 deletion was also the most frequent abnormality, detected in 4.6-7% of patients (Kirchhoff et al 2007, Jehee et al 2011). Using the P250 kit we were able to establish that all 14 patients have the common ~3.0 Mb deletion with breakpoints between low copy repeats A and D. P029-A1 follow-up kit showed the deletion of the entire single copy region of Williams syndrome in five patients and a smaller deletion that does not extend telomerically further than the LIMK1 gene in two sisters, which do not display the classical phenotype of Williams syndrome. A few patients with atypical deletions in Williams syndrome region have been reported (Fusco et al 2014), and they are of particular interest for genotype phenotype studies. Methylation-specific MLPA kit for Prader Willi/Angelman syndrome allowed us to approximate the size of deletions in all patients, from breakpoint 2 to breakpoint 3 (typical type 2 deletions, ~5.3 Mb in size) (Butler et al 2008). 8

9 Subgroup B. Both the manufacturer and the outcomes of previous studies (Northrop et al 2005, Kirchhoff et al 2005, Rooms et al 2006) regarding the use of MLPA for subtelomeric rearrangements detection recommend the use of two different probes for the identification of abnormal regions, as an independent confirmation measure. However, establishing the clinical significance of confirmed subtelomeric rearrangements is quite complicated, especially in cases where the DNA samples of the parents are not available. Out of a total number of 183 investigated patients, in 24 patients subtelomeric rearrangements were identified by both kits and most of them were concordant with the phenotype. Regarding only the patients with normal karyotype (172 cases), cryptic subtelomeric rearrangements were detected in 13 cases (7.5%). Previously reported studies that performed subtelomere analysis showed an overall abnormality rate of 6%, varying between different studies from 2 to 29% (Northrop et al 2005, Kirchhoff et al 2005, Rooms et al 2006, Biesecker et al 2002, Ravnan et al 2006, Koolen et al 2004, Ahn et al 2007, Ahn et al 2008, Stegmann et al 2008, Wu et al 2010, Medina et al 2014). The reasons for these differences are the inclusion criteria and the assay used in the study, the size of the cohort and the complete exclusion (or not) of the polymorphisms. A general overview upon the main previous studies using MLPA to identify subtelomere imbalance is presented in Table The detection rate of 7.5% found in these patients is above the average 6% reported in a review by Biesecker et al. (13), which illustrates the efficiency of using two subtelomeric screening kits in all patients along with the assessment of abnormal results using follow-up kits. Table A general overview upon the main studies using MLPA to identify subtelomere imbalance Reference Number of cases Selection criteria Number of patients with clinically significant abnormalities Koolen et al., ID 9 (4.3%) Kirchhoff et al., ID, dysmorphic features 13 (5%) Rooms et al., ID 8 (2.9%) Ahn et al., Developmental 27 (5.9%) a delay +/- dysmorphism Ahn et al., Developmental 22 (5.5%) a,b delay +/- dysmorphism Stegmann et al., ID +/- congenital abnormalities 18 (3.9%) Wu et al., Moderate to severe ID 23 (5.1%) Medina et al., ID 5 (4.2) a Polymorphisms are included. b Abnormalities detected by karyotype are included. Out of 30 patients displaying abnormal MLPA results, 6 (20%) were detected by one kit, but remained undetected by the other. Such rearrangements detected by a single kit were previously reported in the literature (Kirchhoff et al 2005, Koolen et al 2004, Stegmann et al 9

10 2008) and could be explained by the fact that the probes of P036 and P070 kits hybridize to sequences in different positions of the same subtelomeric region. The imbalances detected by only one probe can be either a false-positive result (due to a mutation or a polymorphism in the sequence detected by a probe or due to different sensitivity of the probes to the DNA sample purity and the conditions in which the experiment is performed) or a true positive result (e.g. if the genomic imbalance is small or the breakpoint is situated between the regions covered by the two probes). Half of the rearrangements identified by a single kit in our study were 4q deletions detected by older versions of the P036 kit, and they were due to the presence of a SNP in the probe site, an aspect that the manufacturer confirmed as well. As shown by other studies, most abnormal results detected by a single kit are clinically irrelevant inherited polymorphisms that can also be detected in a healthy parent (Kirchhoff et al 2005, Koolen et al 2004, Stegmann et al 2008). In our study the parental DNA samples were not available, but considering the size and gene content of the 21q duplication and the fact that 5q duplication and 16p deletion could not be confirmed by follow-up kits, we considered them as non-causative variants. The use of a follow-up kit allows both the confirmation of the abnormality and the estimation of its size. By using different follow-up kits in 24 patients we have managed to identify the material of unknown origin noticed in the standard karyotype in 10 out of 11 patients and we have established the approximate size of 14 of the 31 (45%) singular anomalies characterized by follow-up (Table 1.2.3). Table Clinical and molecular characterization of subtelomeric rearrangements detected in patients in subgroup B Cas Main features Followup CNV size eno. F P036 + Karyotype H P070 Age kit 1. 14y 2. 5y FD, brachydactyly, unstable gait, severe ID - 46,XY del1p P147 ~2.9 Mb Short stature, FD, CHD, severe ID - 46,XX del1p P147 ~4.6 Mb 3. 23y Microcephaly, FD, tapered fingers, moderate ID - 46,XX dup3p P208 ~24 kb* del14q P291 ~3.9 Mb 4. 14y FD, short hands and feet, thin skin, mild ID, epilepsy + 46,XY del2q P264 >4.9 Mb dup9q P286 ~3,4 Mb 5. 8y Microcephaly, FD, short hands and feet, thin skin, mild ID + 46,XX del2q P264 >4.9 Mb dup9q P286 ~3.4 Mb 6. 9y Intrauterine growth retardation, microcephaly, FD, severe ID - 46,XX del15q P291 ~3 Mb dup16q P291 >5.4 Mb 10

11 7. 9y Short stature, microcephaly, FD, brachydactyly, moderate ID - 46,XY del11q P286 >6.2 Mb 8. 2y Tall stature, FD, GDD - 46,XX dup15q P291 >5.6 Mb 9. 1y Short stature, FD, cleft palate, GDD - 46,XY dup22q P356 ~1.1 Mb y Microcephaly, FD, scoliosis, short fourth metatarsals, severe ID - 46,XX del10q P286 ~3.9 Mb dup12q P286 ~2.9 Mb 11. 5y 12. 9y y Short stature, microcephaly, FD, CHD, inguinolabial hernia, hypoplastic labia majora, sacral dimple, severe ID Microcephaly, congenital scoliosis, congenital hip dysplasia, myelomeningocele, sacrococcyx agenesis, mild ID Short stature, obesity, FD, CHD, short hands and feet, short fourth metatarsals, mild ID - 46,XX del7q P277 >5.5 Mb - 46,XX del7q P277 >5.5 Mb - 46,XY del2q P264 >4.9 Mb 14. 3y Short stature, microcephaly, FD, CHD, hirsutism, cryptorchidism, GDD + 46,XY,add(5)( p15) del5p P358 ~487 kb* dup1q P264 >9.1 Mb y Short stature, microcephaly, FD, CHD, hirsutism, severe ID + 46,XY,add(5)( p15) del5p P358 ~487 kb* dup1q P264 >9.1 Mb 16. 7mo FD, CHD, labia hypoplasia, GDD + 46,XY,add(5)( p15) del5p P358 ~487 kb* dup1q P264 >9.1 Mb 17. Short stature, FD, - 46,XY,add(14) dup7q P277 >5.5 Mb 11

12 3mo hydrocephaly, GDD (q32) 18. 3mo FD, short neck, CHD, GDD - 46,XX,add(7)( q36) dup6q P277 >4.9 Mb del7q P277 ~1.2 Mb 19. 3mo FD, Dandy Walker anomaly, uterine agenesis, coloboma of the optic nerve, GDD 46,XX,add(12) (p13.1) dup7q P277 >5.5 Mb del12p P230 ~368 kb* 20. 3y Short stature, microcephaly, FD, umbilical hernia, GDD - 46,XY,add(18) (q22) del18q P320 >5.2 Mb dup5p P358 >13.9 Mb y FD, short neck, CHD, rocker-bottom feet, hypoplastic toenails, arthrogryposis, hypotonia, GDD - 46,XX,add(9)( p24) dup9p P230 >5.1 MB 22. 8y Obesity, FD, hypotonia, mild ID, speech delay - 46,XX,add(5)( p15.1) del5p P358 P358 del ~1,9 Mb dup >7,1 Mb 23. 6mo Microcephaly, FD, short neck with excess nuchal skin, CHD, clenched hands, hypotonia, GDD - 46,XY,add(2)( q37) del2q P264 P264 del ~1,7 Mb dup >2,6 Mb 24. 4y FD, short neck, pectus excavatum, tapering fingers, joint contractures, severe developmental and speech delay - 46,XX,add(8)( p21)** del8p P208 >6.4 MB FH - family history, FD - facial dysmorphism, CHD congenital heart defects, GDD - global developmental delay * These aberrations were considered not related to phenotype due to their small size and limited gene content. **To elucidate the origin of additional material a SNP array was performed revealing an 8p terminal deletion of ~6.8 Mb, and an 8p duplication of ~31.3 Mb. More than that, in the case of two patients (22 and 23), for which the screening kits indicated the presence of deletions (5pter, and 2qter respectively), the use of follow-up kits (P358, and P264 respectively) has also indicated the presence of duplications of the same chromosome arms. In these two cases and in case 26 (for which the origin of additional material was identified using SNP array) we hypothesize the presence of inverted duplications contiguous to terminal deletions, but further studies are needed for confirmation. In four patients (case 4, 5, 6 and 10) with normal karyotype results (4/13, 30.7%) we detected both a deletion and a duplication, which suggests the presence of a cryptic unbalanced 12

13 translocation. This prevalence is within the range of % reported in previous studies (Jehee et al 2011, Wu et al 2010, Mundhofir et al 2013). Until now, only one study that mentions the use of follow-up kits for the confirmation and measurement of subtelomeric imbalances has been reported (Pohovski et al 2013). Its authors established the size of subtelomeric imbalances in two thirds of the identified anomalies. Follow-up kits offer better results when compared to FISH technique that can confirm the MLPA result and/or offer information regarding the position and breakpoints, but the physical size of a FISH probe can prevent the detection of smaller abnormalities that can be otherwise detected by MLPA and the technique has a limited resolution in detection of microduplications. Moreover, the price of the follow-up MLPA kit is lower compared to the corresponding FISH test. By summarizing the data provided by the two groups, the combined use of the MLPA kits led to the diagnosis in 38 out of 358 cases with normal karyotype results (10.6%) and helped to establish the origin of the additional material and the type of rearrangement in 10 out of 11 cases with extra segments of unknown origin. Other studies that used a combination of MLPA kits (subtelomeric screening and P064 kit) in all patients had detection rates of 14% (22), respectively 20.7% (7), but the inclusion criteria in the study were different: patients with ID with or without dysmorphic features or additional congenital abnormalities in the first study and patients with multiple congenital malformations with or without ID in the second study. A study performed on patients with ID and/or dysmorphic features where the same three kits were used separately, on groups of patients, had a detection rate of 7.2% (17). All these studies show that the combined use of MLPA kits has a relatively high detection rate in ID patients, close to the ~19% reported by a review of 29 microarray-based studies of unselected multiple congenital anomalies/id patients (23). A recent study that compared different investigation approaches regarding ID patients (24) has suggested that the replacement of chromosomal analysis with MLPA as first diagnostic test, followed by microarray can prove to be efficient as far as the detection rate and the cost-efficiency balance are concerned. Conclusions In our study, the detection rate of subtelomeric abnormalities was 7.5%, higher than the average value reported in the literature, which illustrates the efficiency of using two subtelomeric screening kits for all patients and of assessing the abnormal results by means of follow-up kits. The use of a follow-up kit allows both the confirmation of the abnormality and estimation of its size, which facilitates the establishment of the clinical significance of subtelomeric anomalies, especially in cases where parental DNA samples are not available. Further characterization of additional material of unknown origin noticed in the standard karyotype is also possible due to combined use of screening and follow-up subtelomeric kits. The combined use of karyotype and MLPA kits for the screening of the most frequent submicroscopic anomalies represents an efficient strategy for establishing the etiologic diagnosis in ID patients, particularly when microarrays are unavailable as a first line approach. To increase detection rate, the best MLPA kit should be selected according to patients phenotype X-linked intellectual disability The term X-linked intellectual disability is used for the forms of intellectual disability determined by mutated genes located on X chromosome. There are approximately 100 genes involved, half of them producing syndromic forms and half nonsyndromic forms. My research in the field focused more on Fragile X syndrome (see section 1.4) and Aarskog syndrome (see below). 13

14 Aarskog syndrome (Facio- digito- genital syndrome) is a form of X-linked intellectual disability characterized by short stature, hypertelorism, downslanting palpebral fissures, joint hyperextensibility and shawl scrotum. The affected gene is called FGD1, it is located on Xp11 and encodes a Rho/Rac guanine nucleotide exchange factor, being involved in development. We have studied 23 cases (14 males and 9 females coming from 5 unrelated families) diagnosed with Aarskog syndrome in Iaşi Medical Genetics Center and followed for a long time. The major aims of the study were to evaluate: Frequency of clinical features associations suggestive for the diagnosis; Evolution of the clinical features; Genotype phenotype correlation; Particularities of our patients. The follow-up protocol for Aarskog patients included: Measure height, weight, head circumference every year; Ophtalmologic assessment at 1 year of age and surgical correction of ptosis; reevaluation once a year; glasses before starting school if needed; Orthodontic examination in older children and specific correction; reevaluation once a year; Surgical examination for cryptorchid testes, followed by surgical correction; Orthopedic assessment for metatarsus adductus, followed by adapted treatment (cast or surgery); reevaluation once a year. Psychologic evaluation at the moment of diagnosis; reevaluation once a year; infant stimulation program, special education; behavioural therapy if needed. The frequencies of the main characteristics for males are presented in Fig and those for females in Fig below. The long-term follow-up of the patients (for up to 20 years) enabled us to record the following changings in time: The shape of the face becomes more elongated with age; Prominent forehead becomes less evident; In the adult the nose becomes coarse; Orthodontic problems appear mainly in the secondary dentition; During childhood the ears are relatively small, lowset, posteriorily rotated; with age ear size increases and thay become vertical; Onset of puberty is mildly delayed; 14

15 Fig 1.3.1: Frequency of clinical features in males with Aarskog syndrome studied Mental retardation Cryptorchid testes Overriding scrotum Metatarsus adductus Interdig. Web Hyper-ext. fingers Brachydactyly Short/ broad hands/ feet Promin. umbilicus Pectus excavatum Short stature Orthodontic probl. Thin upper lip Long/ broad/ prom philtrum Flat nose Broad nasal bridge Prom. nose root Short nose Ptosis Palp. fis. slant down Hypertelorism Widow's peak Prominent forehead Yes No? 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 15

16 Fig 1.3.2: Frequency of clinical features in females with Aarskog syndrome studied Interdigital web Brachydactyly Short/ broad hands/ feet Short stature Narrow ears Small ears Broad central upper incis. Yes No Broad nasal bridge Promin. nose root Hypertelorism Widow's peak 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 16

17 In some patients short stature accentuates with age (further endocrinologic investigations should be performed to clarify this aspect); In the adult (male) the dysproportionate aspect (long trunk, short limbs) is more prominent; many adult patients have an athletic appearance. Genotype phenotype correlation for the patients studied is presented in Table 1.3.1: Family Mutation Phenotype 1 Substitution (G1829A) that changes arginine with glutamine at amino-acid position 610 Males classical Females no expression 2 Substitution (G1138A) that changes lysine with glutamate at amino-acid position 380 Male mild expression Female no expression 3 Deletion of 10 bp starting at nucleotide 297 frameshift from amino-acid position 100, with premature termination at amino-acid 124 Male classical/ moderate expression Female no expression 4 Exon 3 (deleted C342) stop codon at position 114 Males severe expression; Females no/mild expression 5 In due course Male - classical Table 1.3.1: Genotype phenotype correlation in Aarskog syndrome The particularities recorded included: Intellectual disability is more common (78.6%) compared to the literature (30%); in most of the cases it is mild, but in family 3 affected males have moderate intellectual disability; Family 3 associates 3-5 camptodactyly (both hands); The nose becomes coarse in many affected male adults; In many patients the face becomes coarse with age (check thyroid function?); Orthodontic problems, cryptorchid testes and metatarsus adductus are very common features; In some patients short stature accentuates with age; 17

18 Ptosis is partially treatable using surgical methods, whereas orthodontic problems, cryptorchidism and metatarsus adductus respond well to specific treatments. The evolution in time of one of the cases followed is illustrated in Fig Fig 1.3.3: Clinical features evolution in time for an Aarskog patient 1.4. Autism spectrum disorders As mentioned before, autism spectrum disorders represent a very complex field of pathology because there are so many different factors involved in the pathogenic mechanism genetic, epigenetic, metabolic, immunologic, endocrinologic, as well as a multitude of environmental factors (air pollutants and pesticides, factors related to the mother, that represents the environment of the child during the pregnancy, as well as microbiota unbalances in the affected person) just to mention a few of them. Most of the studies in the literature concentrate on a single causal agent, in an attempt to evaluate if that category of factors is really involved and then to appreciate which specific factor from that category is more important for the determinism of the disorder. We have produced a review on autism spectrum disorder (number 285 in the list of publications), aiming to combine all the different factors in an integrative model. This model is presented in Fig and illustrates the fact that autism spectrum disorders are produced in individuals with genetic predisposition (determined by specific CNVs), in which additional factors like epigenetic dysregulation, 18

19 oxidative stress, maternal disorders, infections, electromagnetic fields or environmental chemicals trigger an immune dysregulation that finally leads to neuroinflammation. The major consequences consist of abnormal Ca ++ signaling, abnormal neuronal development and networking that express clinically as disorders of social interaction and communication, restrictive interests and repetitive behaviors appearing before age 3, enabling specialists to establish the clinical diagnosis of autism spectrum disorders. This theoretical study will be continued with a practical one a multidisciplinary approach of our autistic patients (see chapter Future directions ) that will establish the contribution of the different factors involved in the pathogenic mechanism of the disorder. Fig : Integrative model for the complex mechanism of autism spectrum disorders Regarding genetic causes of autism spectrum disorders, there are two major categories of defects involved: specific chromosomal abnormalities or monogenic defects that lead to genetic disorders that include autism in their clinical picture e.g. Fragile X syndrome due to 19

20 a full mutation of the FMR1 gene that leads to a fragile site on Xq27.3. These disorders usually associate other clinical features (like dysmorphic face and macroorchydism for Fragile X syndrome) that group in a characteristic pattern that enable the specialist to establish the diagnosis; these syndromes account for 20% of the autistic cases; single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) that determine the genetic predisposition on which other factors act to build ou the clinical features of autism. Fragile X syndrome is a common cause of autism according to literature data. During my doctoral study dedicated to X-linked intellectual disability (named X-linked mental retardation at that time) I have selected families with intellectual disability (with affected individuals distribution compatible with X-linked inheritance) as well as individuals with suggestive phenotype for Fragile X syndrome, as it was mentioned in the literature that half of the cases of X-linked intellectual disability are represented by this condition. Only 10% of the cases (5/50) have been confirmed by molecular testing, leading to the hypothesis that either Fragile X syndrome frequency is lower in our region, or we should look for other clinical features that are highly suggestive for the diagnosis. So, I continued my study in the field by introducing anti-fmrp testing an immunohistochemical staining performed on hair root, using specific antibodies against FMRP protein that is typically missing in Fragile X patients (see Section II, Chapter 1.4 Research projects). This test was very convenient for us at that time, as it was unexpensive and was validated in the literature to be used as a screening test and because molecular tests (PCR and Southern blot used to confirm the suspicion of diagnosis) were not easily accessible. We have tested 150 individuals, 12 proved to be positive for Fragile X syndrome and only 7 have been confirmed by molecular tests. The conclusions of the study were that indeed, the frequency of Fragile X syndrome seems to be lower in our region (and to elucidate this aspect we need a more extensive and a more in-depth study) and that there are some features that are highly suggestive for the diagnosis and they should be checked with priority when considering the diagnosis of Fragile X syndrome. These features include: positive family history compatible with X-linked inheritance, normal motor development and delayed speech development in young children and a deep plantar crease in the first interdigital space. Typical aspect (illustrated in Fig 1.4.2) is not always present and in most of the cases it is noticed only after puberty. 20

21 Fig : Typical clinical features in a patient diagnosed with Fragile X syndrome Fragile X study will be continued with a more detailed study using modern technology (See chapter Future directions ). To continue the study of genetic causes of autism, I have designed an algorithm (to be used for children with autism associated with other clinical features) that aims to facilitate the recognition of different syndromes and direct genetic testing in an optimized way. The algorithm includes the following steps: Detailed history and physical examination: o Depigmented spots on the skin Tuberous sclerosis; o X-linked type family history, normal motor development and delayed speech development, plantar crease Fragile X syndrome; o Girl with normal initial development, loss of skills Rett syndrome; o No speech, ataxic movements, depigmented Angelman syndrome; o Ptosis, genital hypoplasia, syndactyly Smith Lemli - Opitz syndrome; o Cafe-au-lait spots on the skin Neurofibromatosis; o Long QT, immune deficiency, intellectual disability, seizures, dysmorphic face Timothy syndrome; Perform karyotype: o Abnormal result diagnosis; o Normal result continue the investigations; MLPA test using dedicated kit for autism; More in-depth molecular tests. By using this protocol for autistic cases we have identified special cases that have been communicated (items 55, 190, 249, 273, 275 in the list of publications). 21

22 1.5. Conclusions and further enhancements The major conclusions of the MLPA study for microdeletions and subtelomeric rearrangements show that: The detection rate increases (7.5% in our case) if two subtelomeric screening kits are used and the result may be confirmed by follow-up kits; The use of a follow-up kit allows both the confirmation of the abnormality and estimation of its size, which facilitates the establishment of the clinical significance of subtelomeric anomalies, especially in cases where parental DNA samples are not available; Further characterization of additional material of unknown origin noticed in the standard karyotype is also possible due to combined use of screening and follow-up subtelomeric kits; The combined use of karyotype and MLPA kits for the screening of the most frequent submicroscopic anomalies represents an efficient strategy for establishing the etiologic diagnosis in ID patients, particularly when microarrays are unavailable as a first line approach. To increase detection rate, the best MLPA kit should be selected according to patients phenotype. Referring to the Aarskog syndrome study, the most important conclusions include: We have analysed the frequency of definitory clinical features in 23 cases confirmed with Aarskog syndrome and found that widow s peak, short nose and broad nasal bridge, as well as short/ broad hands and feet, brachydactyly and interdigital webbing are very common in males; in carrier females, relatively frequent features are widow s peak, hypertelorism, small ears and brachydactyly; The follow-up showed that dysmorphic features change with age (the face elongates and becomes coarse), the onset of puberty is a little delayed, the degree of mental retardation is relatively constant and orthodontic problems, cryptorchidism and metatarsus adductus can be treated using specific methods; We have identified some particularities in our patients: common mental retardation (moderate in some cases), severe camptodactyly, coarse face in some adults, short stature accentuated with age in some cases. For these particular cases more in depth investigations should be performed. Autism is a very complex developmental disorder and genetic, epigenetic, immune and environmental factors should be considered in a comprehensive approach when investigating a case. In most of the situations these factors act together in a sequence of events that leads to neuroinflammation and abnormal brain development. The knowledge of the factors involved provides ways for medical intervention and complication prevention. The recent increase in ASD prevalence underlines the growing importance of environmental factors in autism determinism. Environmental factors involved in ASD do not refer only to classic extrinsic agents (like environmental pollutants, electromagnetic fields etc.), but also involve maternal disorders or lifestyle factors, as well as intrinsic factors (hormones, 22

23 inflammatory mediators and gut bacteria), that may influence the developing fetal or neonatal brain. Early diagnosis is a key issue because it allows a multimodal intervention plan, including education, medical intervention aiming to prevent complications and genetic counseling References 1. American Psychiatric Association, (2013), Diagnostic and Statistical Manual of Mental Disorders, 5th ed., American Psychiatric Association, Arlington, VA. 2. Rossignol D.A., Frye R.E., (2012), A review of research trends in physiological abnormalities in autism spectrum disorders: immune dysregulation, inflammation, oxidative stress, mitochondrial dysfunction and environmental toxicant exposures, Molecular Psychiatry, 17, Baron-Cohen S., Lombardo M.V., Auyeung B., Ashwin E., Chakrabarti B., Knickmeyer R., (2011), Why are autism spectrum conditions more prevalent in males?, PLOS Biology, 9, e Lotter V., (1966), Epidemiology of autistic conditions in young children: Some characteristics of the parents and children, Social Psychiatry, Kuehn B.M., (2007), CDC: autism spectrum disorders common, Journal of the American Medical Association, 297, CDC, (2012), Prevalence of autism spectrum disorders - Autism and Developmental Disabilities Monitoring Network, 14 sites, United States, 2008, MMWR: Morbidity and Mortality Weekly Report, Surveillance Summaries, 61, Roeleveld N, Zielhuis GA, Gabreels F. The prevalence of mental retardation: a critical review of recent literature. Dev Med Child Neurol Feb;39(2): Miller DT, Adam MP, Aradhya S, Biesecker LG, Brothman AR, Carter NP, et al. Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies. Am J Hum Genet May 14;86(5): Cooper GM, Coe BP, Girirajan S, Rosenfeld JA, Vu TH, Baker C, et al. A copy number variation morbidity map of developmental delay. Nat Genet Sep;43(9): Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res Jun 15;30(12):e Slavotinek AM. Novel microdeletion syndromes detected by chromosome microarrays. Hum Genet Aug;124(1): Kirchhoff M, Bisgaard AM, Bryndorf T, Gerdes T. MLPA analysis for a panel of syndromes with mental retardation reveals imbalances in 5.8% of patients with mental retardation and dysmorphic features, including duplications of the Sotos syndrome and Williams-Beuren syndrome regions. Eur J Med Genet Jan-Feb;50(1): Jehee FS, Takamori JT, Medeiros PF, Pordeus AC, Latini FR, Bertola DR, et al. Using a combination of MLPA kits to detect chromosomal imbalances in patients with multiple congenital anomalies and mental retardation is a valuable choice for developing countries. Eur J Med Genet Jul-Aug;54(4):e

24 14. Fusco C, Micale L, Augello B, Teresa Pellico M, Menghini D, Alfieri P, et al. Smaller and larger deletions of the Williams Beuren syndrome region implicate genes involved in mild facial phenotype, epilepsy and autistic traits. Eur J Hum Genet Jan;22(1): Butler MG, Fischer W, Kibiryeva N, Bittel DC. Array comparative genomic hybridization (acgh) analysis in Prader-Willi syndrome. Am J Med Genet A Apr 1;146(7): Northrop EL, Ren H, Bruno DL, McGhie JD, Coffa J, Schouten J, et al. Detection of cryptic subtelomeric chromosome abnormalities and identification of anonymous chromatin using a quantitative multiplex ligation-dependent probe amplification (MLPA) assay. Hum Mutat Nov;26(5): Kirchhoff M, Gerdes T, Brunebjerg S, Bryndorf T. Investigation of patients with mental retardation and dysmorphic features using comparative genomic hybridization and subtelomeric multiplex ligation dependent probe amplification. Am J Med Genet A Dec 15;139(3): Rooms L, Reyniers E, Wuyts W, Storm K, van Luijk R, Scheers S, et al. Multiplex ligation-dependent probe amplification to detect subtelomeric rearrangements in routine diagnostics. Clin Genet Jan;69(1): Biesecker LG. The end of the beginning of chromosome ends. Am J Med Genet Feb 1;107(4): Ravnan JB, Tepperberg JH, Papenhausen P, Lamb AN, Hedrick J, Eash D, et al. Subtelomere FISH analysis of cases: an evaluation of the frequency and pattern of subtelomere rearrangements in individuals with developmental disabilities. J Med Genet Jun;43(6): Koolen DA, Nillesen WM, Versteeg MH, Merkx GF, Knoers NV, Kets M, et al. Screening for subtelomeric rearrangements in 210 patients with unexplained mental retardation using multiplex ligation dependent probe amplification (MLPA). J Med Genet Dec;41(12): Ahn JW, Ogilvie CM, Welch A, Thomas H, Madula R, Hills A, et al. Detection of subtelomere imbalance using MLPA: validation, development of an analysis protocol, and application in a diagnostic centre. BMC Med Genet. 2007;8: Ahn JW, Mann K, Docherty Z, Mackie Ogilvie C. Submicroscopic chromosome imbalance in patients with developmental delay and/or dysmorphism referred specifically for Fragile X testing and karyotype analysis. Mol Cytogenet. 2008;1: Stegmann AP, Jonker LM, Engelen JJ. Prospective screening of patients with unexplained mental retardation using subtelomeric MLPA strongly increases the detection rate of cryptic unbalanced chromosomal rearrangements. Eur J Med Genet Mar-Apr;51(2): Wu Y, Ji T, Wang J, Xiao J, Wang H, Li J, et al. Submicroscopic subtelomeric aberrations in Chinese patients with unexplained developmental delay/mental retardation. BMC Med Genet. 2010;11: Medina A, Pineros L, Arteaga C, Velasco H, Izquierdo A, Giraldo A, et al. Multiplex ligation-dependent probe amplification to subtelomeric rearrangements in idiopathic intellectual disability in Colombia. Pediatr Neurol Mar;50(3): Mundhofir FE, Nillesen WM, Van Bon BW, Smeets D, Pfundt R, van de Ven- Schobers G, et al. Subtelomeric chromosomal rearrangements in a large cohort of 24

25 unexplained intellectually disabled individuals in Indonesia: A clinical and molecular study. Indian J Hum Genet Apr;19(2): Pohovski LM, Dumic KK, Odak L, Barisic I. Multiplex ligation-dependent probe amplification workflow for the detection of submicroscopic chromosomal abnormalities in patients with developmental delay/intellectual disability. Mol Cytogenet. 2013;6(1): Hochstenbach R, van Binsbergen E, Engelen J, Nieuwint A, Polstra A, Poddighe P, et al. Array analysis and karyotyping: workflow consequences based on a retrospective study of 36,325 patients with idiopathic developmental delay in the Netherlands. Eur J Med Genet Jul-Aug;52(4): Kriek M, Knijnenburg J, White SJ, Rosenberg C, den Dunnen JT, van Ommen GJ, et al. Diagnosis of genetic abnormalities in developmentally delayed patients: a new strategy combining MLPA and array-cgh. Am J Med Genet A Mar 15;143(6): Stevenson R., Schwartz C., Curtis Rogers R. Atlas of X-linked Intellectual Disability Syndromes. Oxford University Press 2012,

26 2. Optimization of dysmorphologic diagnosis and management of patients with genetic disorders 2.1. Introduction and conceptual background Dysmorphologic diagnosis is a highly specialized medical activity with specific particularities that include: Difficult needs a trained eye ; Knowledge on normal variants! Patience complete evaluation of all the defects; Ability to differentiate essential by nonessential features; Ability to differentiate abnormal cases by minor familial variants; Capacity to establish the diagnosis ( build the puzzle ). Most of the genetic disorders are rare and the clinical diagnosis could be really difficult if you have never seen such a patient or if you don t know how to interprete a trait. Moreover, Dysmorphology field is a very dinamic one due to the use of more and more detailed laboratory testing new defects and new disorders are described permanently, whereas old disorders are regrouped based on new criteria (usually pathogenic). And last but not the least, new molecular data enable the scientists to think of innovative therapies for disorders that seemed hopeless not so long ago. So, in this rapidly changing environment, to establish a clinical diagnosis of a certain genetic disorder could be challenging. To help overcome all these difficulties, computer assisted diagnosis and databases may be of real support Prader Willi syndrome I have studied more in detail Prader Willi syndrome because this is a clinical entity that totally changes in time. The newborn is hypotonic and has marked feeding difficulties that gradually improve. As feeding difficulties get better, the appetite increases, finally leading to monstrous obesity. Due to the dynamic aspect and the severe consequences in adults, nowadays specialists aim to an early diagnosis, estecially because growth hormone therapy proves to be so efficient. Moreover, the disorder could be produced by different types of genetic defects of chromosome 15 (deletion, uniparental disomy or imprinting defect) and I expect for some of them specific therapies will become available sooner or later. Prader Willi syndrome (PWS) is a complex disease caused by the lack of expression of paternally inherited imprinted genes on chromosome 15q11.2-q13. The main molecular mechanisms are paternal deletion in about 70% of subjects, maternal uniparental disomy (UPD) in about 25% and translocations or imprinting centre mutations in 1-3% (Grechi et al 2012). PWS has a characteristic phenotype with newborn hypotonia and feeding difficulties during infancy, hyperphagia with progressive obesity during childhood, distinctive dysmorphic features, intellectual disability (ID) and behavioural problems. Consensus criteria for clinical diagnosis of PWS were first established in 1993 by Holm (Holm et al 1993) and 26

27 are used as a screening test for identifying appropriate patients for genetic tests. Patients with UPD have an increased risk of psychosis (Vogels et al 2003) and autism spectrum disorders (Veltman et al 2004), but the facial dysmorphism is mild ( Gillessen-Kaesbach et al 1995, Cassidy et al 1997), the verbal IQ is higher (Roof et al 2000) and jigsaw puzzles skills are less developed compared with patients with deletion (Dykens 2002). Most cases of PWS occur sporadically (Cataletto et al 2011). The recurrence risk is depending on the molecular mechanism that leads to PWS: usually less than 1%, except for inherited mutation in imprinting centre (up to 50% risk) and parental translocation (up to 25% risk) (Grechi et al 2012). Associations between PWS and sex chromosome aneuploidies are very rare and the chance for their occurrence is very low. Four studies reported an association between PWS and 47, XXX syndrome (Ferrante et al 1986, Devriendt et al 1987, Butler et al 1997, Pascanu et al 2010). PWS patients have hypothalamic - pituitary insufficiency which can lead to the development of multiple endocrinological disorders such as central adrenal insufficiency, GH deficiency, LH/FSH abnormalities and central hypothyroidism. The main objectives of our study were to evaluate clinical, metabolic and endocrine aspects in five genetically confirmed patients with PWS. Patients and methods The study included five patients (four females and one male, aged years), admitted into our department with the suspicion of genetic obesity. Written informed consent from parents was obtained before detailed evaluation. The patients were selected according to Holm's criteria and genetically confirmed in our laboratory by Methylation-Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA using SALSA ME028-B1 Prader Willi/Angelman probe mix). Patients for which MS-MLPA showed a deletion were subsequently tested by FISH (Vysis Prader- Willi/Angelman Region Probe - LSI D15S10 Spectrum Orange/CEP 15 - D15Z1 Spectrum Aqua/PML Spectrum Green Probe). Anthropometric measurements, detailed physical examination and blood pressure measurement were performed for all patients. Medical and surgical history was recorded. Fasting blood samples were collected for determination of glucose, HbA1c, total cholesterol, HDL-cholesterol, triglycerides and hormones such as basal GH, IGF1, TSH, ft4, cortisol, FSH, LH and sex hormones using commercially available chemiluminescent immunoassays (IMMULITE 1000). Provocative GH stimulation and oral glucose tolerance tests were not performed because of the food seeking behaviour and the range of cognitive abilities in patients with PWS. Bone mineral density (BMD) of lumbar spine (L1-L4) and femoral neck was assessed using Dual Energy X-ray Absorptiometry (DXA), according to the standard procedure using a Hologic Delphi W instrument. The patients BMD results were compared with data matched for age and sex and reported as Z-scores. The World Health Organization definition of osteoporosis/osteopenia was applied (World Health Organization 1994). Bone X-rays were performed if skeletal abnormalities were present. Polysomnographic studies were performed in all patients. Results Features included in Holm's diagnostic criteria and present in all our patients were: marked hypotonia and feeding difficulties in infancy, obesity (weight between SD and +5.3 SD), dysmorphic face, viscous saliva, small hands and feet, ID and characteristic behaviour (hyperphagia, stubbornness, repetitive skin picking, repetitive questioning and 27

28 insistence on routine, high pain threshold) (Table 2.2.1). Patient 1 associated macrocephaly (+3 SD), genu varum and premature pubarche (at 7 years of age). Patient 5 presented primary amenorrhea. Table Consensus diagnostic criteria for PWS (Holm, Cassidy) Patient Patient Patient Patient Patient Age 10 y 11 y 8 mo 10 y 9 mo 5 y 6 mo 19 y Sex F M F F F Major criteria (1 point each) Neonatal/ infantile hypotonia/ poor suck Feeding problems and failure to thrive as infant Weight gain at 1-6 years/ obesity/ hyperphagia Characteristic dysmorphic facial features Genital hypoplasia/ cryptorchidism/ pubertal delay/ hypogonadism Developmental delay/ ID Sum of the major criteria points

29 Minor criteria (1/2 point each) Decreased foetal movement or??? - + infantile lethargy Characteristic behaviour problems Sleep apnoea?? Short stature for family by 15 na * Na na na + years of age Hypopigmentation for the family Small hands and feet for height Narrow hands with straight ulnar border Esotropia, myopia Thick, viscous saliva Speech articulation defects Skin picking Sum of the minor criteria points Total sum Clinical diagnosis: 5 points (at least 4 of them from major criteria) at age < 3 years; 8 points (at least 5 of them from major criteria) at age > 3 years. * na - not applicable due to the young age of the patients. MS-MLPA revealed presence of two methylated copies and normal dosage at 15q11.2-q13 in four patients (patients 1-4) (Fig ) and a type II heterozygous deletion (between breakpoint 2 and breakpoint 3) within 15q11.2-q13 in patient 5 (Fig ). The deletion was confirmed by FISH test. Blood pressure was normal for all cases. Biochemical evaluations revealed normal glucose and normal HbA1c. Lipid assessment revealed: hypertriglyceridemia (patients 1 and 2), hypercholesterolemia (patient 5), combined hyperlipidemia (patient 4) and normal values (patient 3). 29

30 Endocrine evaluation: Hypothalamic pituitary GH axis: in all patients basal GH and IGF1 levels were lower, but still within normal limits for age and sex, according to the reference values of the assay. Patients 1 and 2 received GH therapy at a dose of 0.05 mg/kg/day administered daily by subcutaneous injection. GH treatment was not initiated in patient 3, 4 and 5 due to sleep apnoea; Hypothalamic pituitary thyroid axis: TSH and ft4 levels and thyroid ultrasound were normal in patients 2-5; for patient 1 TSH was 4.4 μui/ml ( ) and ft4 was 1.3 ng/dl (0.8-2); Fig 2.2.1: Detection of two methylated copies (similar signals in digested and undigested reactions) and normal dosage at 15q11.2-q13 in patient 1 by MLPA. Fig : Detection of a type II heterozygous deletion within 15q11.2-q13 in patient 5 by MLPA (~50% reduction in peak area of the amplification product). 30

31 Hypothalamic pituitary gonadal axis: FSH and LH levels were in the normal range for sex and age. Sex hormones levels (testosterone, estradiol) were normal for age and sex in patients 1-4 and low, showing hypogonadotropic hypogonadism (prepubertal range) in patient 5, aged 19; Hypothalamic-pituitary-adrenal axis: basal cortisol levels were within normal range, but no dynamic assays were performed; BMD: mean Z-scores were reduced for the spine, but normal for the hip in three patients. Skeletal X-rays and orthopaedic examination revealed severe sinistro-convex scoliosis of the dorsal spine (patient 5), mild scoliosis (patient 2) and tibia vara - Blount disease (patient 1). Polysomnographic studies revealed moderate obstructive sleep apnoea for patient 4 and mild mixed sleep apnoea for patients 3 and 5. Psychological evaluation showed mild ID in patients 1-4 and severe ID in patient 5. Further imagistic investigations (MRI) showed that patient 5 associates frontal cortical atrophy with secondary seizures and patient 1 had hydrocephaly with EEG abnormalities in mesodiencephalic areas. Patient 5 associated also compound myopic astigmatism and multiple food and drug allergies. The multiple drug allergies included recurrent antihistamine-induced urticaria (after taking cyproheptadine and clorpheniramine), allergic reactions to topiramate and ibuprofen, anaphylactic reactions to penicillin, penicillin derivatives and metamizole. Furthermore, she had pollen, mold and detergent induced rhinoconjunctivitis and also allergies to insect stings (bees). Discussion GH deficiency PWS patients are characterized by low growth velocity, obesity, reduced lean body mass and decreased bone density, as well as delayed bone maturation, features indicative of deficient GH production (Jin 2012, Wollmann et al 1998). More than 85% of patients with PWS have low levels of basal GH and IGF-I and, depending on the stimulation test used, % of children fulfil the criteria for GH deficiency (Burman et al 2001). GH secretory pattern is different in patients with UPD (lower basal GH values, poor answer to GHRH or arginine test) compared to patients with deletion, suggesting a better response to GH therapy in UPD patients (Badiu 2012). GH treatment in PWS patients improves short stature, body composition, fat utilization and motor function ( Carrel et al 1999, Carrel et al 2010). Growth-promoting effects of GH therapy seem to be more prominent in hyperleptinemic GH deficient children (Popa et al 2007). Although all our patients had normal growth parameters (height and height velocity) and GH levels within lower normal range, in accordance with literature data (Goldstone et al 2008), we considered beginning GH therapy. For GH treatment we used as exclusion criteria severe obesity, uncontrolled diabetes mellitus, untreated sleep apnoea, active cancer and psychosis (Grechi et al 2012, Deal et al 2013). Scoliosis was not considered a contraindication to GH treatment because neither its incidence nor its rate of progression are influenced by this treatment (Deal et al 2013, de Lind van Wijngaarden et al 2009). Therefore, patient 3, 4 and 5 (who presented sleep apnoea) did not receive treatment. There is no consensus on age of starting GH treatment, although the benefits of treating before the onset of obesity, which often begins by 2 years of age is documented (Deal et al 2013). Patients 1 and 2 received GH therapy from 5.5 years of age and 8 years 8 months, respectively. We used a dose of 0.05 mg/kg/day, calculated using actual weight, according to 31

32 previous guidelines (Richmond et al 2010). New consensus guidelines for GH therapy in PWS recommend starting with a daily dose of 0.5 mg/m2/day subcutaneously with subsequent adjustments toward 1.0 mg/m2/day every 3 6 months according to clinical response (Deal et al 2013). GH therapy in conjunction with dietary, environmental and lifestyle interventions showed significant improvement of mental/motor development and no adverse effects, despite the late start of GH therapy (Goldstone 2008). Recent studies have shown that long-term GH treatment in adults with PWS has favourable effects on abnormal body composition without clinically significant side effects (Hoybye 2007, Sode-Carlsen et al 2012). If no exclusion criteria are present, when arrived at adult age our patients should receive a starting dose of mg/day based on age, presence of edema, prior GH exposure and sensitivity, and concomitant oral estrogen use (Deal et al 2013). From the best of our knowledge, there is only one case of PWS who associates Blount disease reported in the literature (Dulka 2013). One of our cases who received GH treatment (patient 1) had Blount disease, and showed no skeletal anomalies progression during one year therapy. The GH treatment was stopped to perform orthopedic surgery for Blount disease. Thyroid function Previous studies reported a frequency of hypothyroidism in PWS patients varying widely, from 2% (Butler et al 2007) to 25% (Miller 2012, Diene et al 2010). Only one of our cases had subclinical hypothyroidism (patient 1) and received low dose substitute therapy (Levothyroxine 25 μg/day) due to metabolic particularities. Hypothyroidism in PWS patients may be congenital or of late onset (Vaiani 2010), therefore the levels of TSH and ft4 need to be monitored at birth and thereafter yearly, or every six months during GH therapy (Grechi 2012). Gonadal function Previous studies reported isolated premature pubarche, without other signs of puberty, in approximately % of PWS patients, probably due to early maturation of zona reticularis of the adrenal glands (Schmidt et al 2001, Crino et al 2003). In our study only patient 1 (who presented hydrocephalus) had premature pubarche. Children with hydrocephalus may have frequently short stature and precocious puberty (Lopponen et al 1998), therefore the premature pubarche in our patient may be also due to the damage of the hypothalamus or pituitary gland caused by increased intracerebral pressure. Delayed or incomplete pubertal development, including primary amenorrhea, are more common in PWS patients (Jin 2012). Consistent with this data, patient 5 presents delayed puberty. Cryptorchidism is present in 80-90% of PWS boys and should be treated in the first year of life to prevent germ cells destruction (Miller 2012). The most efficient treatment is surgery, but some authors recommend human chorionic gonadotropin as first intention therapy in order to avoid general anaesthesia risk existing at PWS children (McCandless 2011). In our only male (patient 2) cryptorchidism was treated in the second year of life, by surgery, without anaesthethic incidents. At some moment in the future our patients will need sex steroid replacement in order to induce or maintain puberty. There is no agreement on the most appropriate therapy for puberty development in patients with PWS, options depending on the local team experience and pharmaceutical market possibilities. In adolescents and adults with PWS, the main objective of this therapy is prevention of osteoporosis (Goldstone 2008). 32

33 Adrenal insufficiency Our patients had normal basal cortisol and no signs of adrenal insufficiency. According to a recent study adrenal insufficiency in PWS patients has a low prevalence (7.5%), but clinicians are advised to test the patients for central adrenal insufficiency. The same study also showed that basal cortisol was closely correlated with adrenal response to stimulation, indicating that its measurement may be helpful in selecting patients for low-dose short Synacthen test (Grugni 2013). Bone mineral density (BMD) Three of our patients presented low Z-scores, suggestive of osteopenia/osteoporosis. After one year therapy with vitamin D and calcium supplements, Z-scores increased notably (from -3.6 to -2.2 and from -3.7 to -2.4 in two patients, and from -1.9 to -1.2 in one patient). Reduced BMD in PWS patients can be related to hypogonadism, low GH hormone level and low physical activity. Salles et al. showed that osteopenia is not present at early stage in PWS patients, but develops later, specially during puberty (Salles 2007). Therefore, we suggest that all PWS patients must be periodically investigated by using DXA. Additional medical issues Body composition studies in PWS patients showed increased body fat from infancy to adulthood (Brambilla et al 1997, Eiholzer et al 2000), which is considered a risk factor for cardiovascular disease and diabetes mellitus type 2. Our patients presented dyslipidemia, whereas blood pressure and glucose homeostasis were normal, which is consistent with data reported by previous studies (de Lind van Wijngaarden et al 2010). Considering that 25% of adults with PWS have been reported with type 2 diabetes mellitus (mean age of onset about 20 years) (Butler et al 2002) and hypertension may be present in up to 38% in adults (Vogels et al 2004), the patients must be periodically reevaluated. The management of type 2 diabetes mellitus in PWS patients is difficult due to uncontrolled food intake. New studies suggest single dose of glucagon-like peptide 1 receptor agonists as a novel therapy, considering its potential effects on glycemic control, delayed gastric emptying and increasing satiety (Uzuki et al 2012). Food allergy and atopy is uncommon in patients with PWS, the first case being reported in 2004 (Becker et al 2004). Patient 5 had multiple allergies which, associated with characteristic food seeking behaviour, increase the risk of anaphylaxis. Patient 5 had also recurrent antihistamine-induced urticaria, which was rarely reported. No precise mechanism had been established, but some authors consider this an immunoglobulin (Ig) E mediated reaction (Rodriguez et al 2009). Epileptic seizures are found in 18-26% of PWS patients (Vendrame et al 2010, Fan et al 2009). One of our cases (patient 5) associated epilepsy, challenging aspect due to the fact that this patient associated also multiple allergies, including to some anticonvulsivant drugs. Genotype-phenotype correlations performed in PWS patients revealed that hypopigmentation and epilepsy are seen primarily in patients with deletion (Goldstone 2008, Vendrame et al 2010). Four of our patients presented abnormal methylation profile and one presented a deletion, but we did not observe hypopigmentation in any of our patients. Other studies showed that patients with UPD have higher verbal intelligence scores and less maladaptive behaviors compared with patients with deletions (Veltman et al 2004, Whittington et al 2004). Considering that the vast majority of PWS patients with abnormal methylation profiles are due to UPD, the mild ID which was diagnosed in patients 1-4 can be explained by the involvement of this molecular mechanism. 33

34 Conclusions Our study aimed to evaluate clinical, metabolic and endocrine aspects in five patients with PWS. PWS is a complex disease, associated with endocrine disfunctions, that requires a multidisciplinary approach. Marked hypotonia and feeding difficulties in infancy (found in all our patients) are early evocative features for PWS. Early diagnosis of PWS is essential for starting GH therapy and for appropriate genetic counseling. Although we started relatively late the GH therapy and it was administered to patients with unevolutive skeletal anomalies, we observed significant improvement in mental development and in body habitus and no adverse effects. Our study also illustrated the challenges raised by some features very rarely described in PWS (Blount disease and multiple allergies) RAS-opathies RAS-opathies (Neuro cardio facio skeletal spectrum) represent another challenging field in Clinical Genetics because we deal with a group of similar disorders, but with different prognosis, reason why a precise diagnosis is a key issue. INTRODUCTION Syndrome definition Noonan syndrome (NS, OMIM ) is an autosomal dominant multisystem disorder with variable expressivity and an estimated prevalence of 1 in 1,000-2,500 individuals. It is characterized by distinctive facial features, short stature, congenital heart defects, unusual chest shape, broad or webbed neck, cryptorchidism and developmental delay (Roberts et al 2013, Allanson 2010, Allanson 2009). Most cases associate lymphatic malformations and bleeding diathesis. NS shares many features with Cardio-facio-cutaneous, Costello, LEOPARD, Neurofibromatosis - Noonan, Legius and Noonan - loose anagen hair syndromes, as part of neuro - cardio - facio - cutaneous spectrum (Sarkozy et al 2009, Tartaglia et al 2010). These disorders are produced by defects of the Ras - MAPK pathway, a signal transduction pathway through which extracellular ligands (growth factors, cytokines, and hormones) stimulate cell proliferation, differentiation, survival and metabolism (1). PTPN11 (50% of cases), SOS1 (10%), KRAS (3%), RAF1 (5%), BRAF, NRAS, MEK1, SHOC2 and CBL mutations account for 70% of NS individuals (Roberts et al 2013, Tartaglia et al 2010, Tartaglia et al 2009). Clinical features Facial appearance changes with age, but some features are relatively constant, being very suggestive for NS diagnosis (relative macrocephaly, diamond-shaped eyebrows, wide spaced and down-slanting palpebral fissures, ptosis and low set ears with thickened helices) (Allanson 2009, Sarkozy et al 2009). Typical heart defects are: pulmonary valve stenosis with dysplastic valves (50-60%), hypertrophic cardiomyopathy (20%) and ostium secundum atrial septal defect (6-10%). Electrocardiograms show wide QRS complexes with predominantly negative pattern in left precordial leads and left axis deviation with giant Q waves (Roberts et al 2013). Chest deformity (superior pectus carinatum and inferior pectus excavatum), with wide spaced and low set nipples is also very suggestive, especially if associated with short/ webbed neck (Allanson 2010). Cubitus valgus and scoliosis are frequently found (Roberts et al 2013). 34

35 Eighty percent of males associate cryptorchidism, but male gonadal dysfunction seems to be determined more by primary Sertoli cell dysfunction. Fertility is not impaired in females (Roberts et al 2013). Twenty percent of cases present learning difficulties, social cognition being the most affected field (Roberts et al 2013, Pierpont 2009). Birth size is normal (in the lower range), with important neonatal weight loss. Infants associate feeding difficulties that lead to failure to thrive (self-limited by 18 months of age). Later (up to 12 years in boys and 10 years in girls), NS growth follows the general population 3 rd centile growth curve with normal growth velocity, after which mean height decreases below normal range (more obvious for males) (Padidela et al 2008, Romano et al 2009). Puberty is delayed (mean age of onset years in males and years in females), with a diminished pubertal growth spurt and a rapid tempo (less than 2 years) (Romano et al 2010). However, delayed bone maturation (2 years) (Romano et al 2010) seems to ensure prolonged growth potential into the 20s (Allanson 2009). Mean adult height is approximately -2 SD ( cm in females and cm in males) (Noonan et al 2003, Binder 2009, Ranke et al 1988, Shaw et al 2007, Witt et al 1986). Specific NS growth curves are available in the literature (Witt et al 1986, Hall et al 2007). In adults, prevalent clinical findings are: pulmonary valve stenosis (71%), easy bruising (63%), gastro-esophageal reflux (60%), constipation (51%), scoliosis (54%), joint pain (54%), lymphedema (49%), depression/ anxiety (49%) and osteopenia/osteoporosis (14%) (Smpokou et al 2012). NS diagnosis is challenging in adults, because clinical features become less characteristic. Van der Burgt scoring system (Table 2.3.1) is the most used model patients are first classified as having typical/ suggestive facial dysmorphy; typical face + 1 major/ 2 minor signs establish the diagnosis of NS; suggestive face + 2 major/ 3 minor criteria confirm NS diagnosis (van der Burgt 2007, Jorge et al 2009). Table 2.3.1: van der Burgh scoring system for Noonan syndrome Feature Major Minor Facial A = Typical face dysmorphology B = Suggestive face dysmorphology 2 Cardiac Pulmonary valve stenosis, hypertrophic obstructive cardiomyopathy and/or ECG typical of NS Other defect 3 Height < p3 < p0 4 Chest wall Pectus carinatum/ excavatum Broad thorax 5 Family history 1 st degree relative - definite NS 1 st degree relative - suggestive NS 6 Other Mental retardation, cryptorchidism and lymphatic dysplasia One of mental retardation, cryptorchidism and lymphatic dysplasia Different genes of the Ras-MAPK pathway may be involved in NS, each of them producing common and specific features. See Tables and for genotype - phenotype correlation and differential diagnosis with other entities of the Neuro - cardio - facio - cutaneous spectrum. 35

36 Table 2.3.2: Genotype-phenotype correlation in Noonan sdr (modified after Roberts 2013) Gene PTPN 11 (50%) SOS 1 (10%) RAF 1 (10%) KRAS (<2%) NRAS, BRAF,CB LMEK1 Short stature Heart defect PVS, ASD - ++ PVS, ASD HCM + + PVS, HCM Macrocephaly Dysmorphic face Short/webbed neck Pectus Scoliosis Cryptorchidisn Ectodermal defects Bleeding dyathesis Intellectual disability Tumors +/- + +/ / * / /- - - ** / *** **** Few cases reported; phenotype seems typical for NS; NS + florid ectodermal manifestations (BRAF, MEK 1) PVS pulmonary valve stenosis; HCM hypertrophic cardiomyopathy; ASD atrial septal defect; * - juvenile myelo-monocytic leukemia, multiple giant cell lesions; ** - facial keratosis pilaris; *** - pigmented nevi, cafe au lait spots; **** - cerebral and ocular abnormalities. Other Table 2.3.3: Differential diagnosis in Noonan syndrome (entities included in neuro- cardiofacio- cutaneous spectrum) (modified after Jorge 2009) Disorder Causative gene Phenotype Noonan syndrome Costello syndrome Cardio - facio - cutaneous syndrome LEOPARD syndrome Neurofibromatosis type 1 Neurofibromatosis Noonan (Watson sdr) PTPN 11, SOS 1, RAF 1, KRAS, NRAS, BRAF, MEK 1 (MAP2K1), CBL HRAS, KRAS, BRAF, MEK 1 KRAS, BRAF, MEK 1, MEK 2 PTPN 11, RAF 1 NF1 NF 1 Dysmorphic face, congenital heart defect, short stature, broad/ webbed neck, pectus, cryptorchidism, developmental delay Intellectual disability, large birth weight, neonatal feeding difficulties, curly hair, coarse face, thick lips, nasal papillomata, diffuse skin hyperpigmentation, nail dystrophy Coarse face, congenital heart defect, ectodermal anomalies (follicular and palmar hyperkeratosis), short stature, intellectual disability (moderate/ severe), facial features reminiscent of Noonan and Costello Multiple lentigines (L), EKG conduction abnormality (E), ocular hypertelorism (O), pulmonic stenosis (P), abnormal genitalia (A), growth retardation (R), deafness (D) Familial cancer syndrome; hyper pigmented skin lesions and benign neurofibromas; common learning disability; Lower incidence of plexiform neurofibromas, skeletal anomalies, internal tumors + hypertelorism, ptosis, low set ears, heart defects Legius SPRED 1 Axillary freckling, cafe-au-lait spots, macrocephaly, NS-like face learning difficulties, ADHD, lipomas; No Lisch nodules, neurofibromas, central nervous system tumors; Noonan like loose anagen hair SHOC 2 Reduced growth, GH deficiency, cognitive deficit, hyperactive behavior, hair anomalies, dark skin with eczema, dystrophic nails, heart defect 36

37 Endocrine aspects in Noonan syndrome PTPN11 mutations down-regulate cellular response to GH in NS children, leading to short stature (Binder 2009). In NS due to SOS 1 mutations growth is less compromised, but mutations in other NS genes (KRAS, RAF1, BRAF) also lead to short stature, deregulation of Ras-MAPK pathway itself probably playing a role in growth impairment (Binder 2009). Studies in the literature (Binder 2009, Binder 2009, Noordam 2001) have identified the hormonal profile in NS: low IGF I and IGFBP 3 and mild increase of spontaneous GH secretion with high trough GH concentrations, values that suggest post receptor signaling GH resistance (more prominent in PTPN11 mutations). In spite of this profile, GH therapy proved to be effective (Binder 2009, Allanson 2013, Ferreira 2005, Zbranca 2008, Cooke et al 2011). Growth velocity is mostly enhanced in the first year of therapy. Mean gain in height SD of +1 SD appears to be sustained, even if bone maturation is also accelerated (Allanson 2009, Padidela et al 2008, Noordam et al 2008, Ferreira et al 2005). Predictors of a good response to GH treatment seem to be the following ones (Romano et al 2010, Romano et al 2009, Noordam et al 2008, Cooke et al 2011): earlier initiation and number of prepubertal years spent on GH therapy; age of pubertal onset (earlier onset of puberty determines a longer pubertal growth period, contributing to a greater height gain during puberty); duration of puberty from Tanner stage II to IV (the longer it lasts, more cm are gained) height SDS at puberty (the better height at puberty onset, the taller NS adult). Therapy monitoring includes periodic evaluation of cardiac status, growth velocity, bone age, insulin, glucose, HbA1c, lipids, IGF I, thyroid function, blood chemistry and scoliosis (Allanson 2010, Noordam et al 2008). In cases resistant to GH therapy and severely abnormal growth, rh IGF I therapy could be an option, but for the moment IGF I levels in NS do not fulfil the strict criteria postulated by Food and Drug Administration (FDA) and European Medicines Agency (-3SD) (Padidela et al 2008, Binder 2009). Sertoli cell dysfunction in NS is suggested by low inhibin B, impaired negative feedback and raised FSH, as well as normal levels of LH and testosterone. It may be the result of aberrant Ras function, Ras playing a role in normal germ cell proliferation and migration (Marcus et al 2008). We aimed to evaluate clinical, endocrine and genetic aspects in 3 patients with NS (2 of them under GH therapy) and to establish genotype phenotype correlations. 37

38 PATIENTS AND METHODS The study included three patients admitted to our department due to short stature associated with heart defect. Written informed consent from parents was obtained before detailed evaluation (personal and family history, anthropometric measurements, physical examination completed with photos, imagistic and lab investigations). The suspicion of NS diagnosis was confirmed by positive van der Burgt score (B + 3 major + 2 minor in case 1; A + 2 major + 2 minor in case 2; A + 1 major + 3 minor in case 3) and DNA sample analysis was performed. As PTPN11 is the most frequently affected gene in NS (50% of cases) (Roberts 2013, Tartaglia et al 2009), molecular testing started with the direct sequencing of the PTPN11 gene (exons 2, 3, 4, 7, 8, 12 and 13) in each case. Negative cases (2, 3) have been further tested by direct sequencing of KRAS gene (exon 2 case 2) and SOS 1 gene (exon 4 case 3). DNA testing was done according to international protocols (Romano et al 2010, Allanson et al 2013) and confirmed the diagnosis of NS for all three cases. Basal GH and IGF1 levels were measured after centrifugation of a serum sample by means of solid phase, two-site chemiluminescent immunometric assay (IMMULITE 2000 from Siemens). The sensitivity of the GH assay 0.01 ng/ml, the reportable range was ng/ml, and the normal range was up to 3ng/mL in men and up to 8 ng/ml in women. The analytical sensitivity of the IGF1 assay was 20 ng/ml. Normal range was detailed function of age and sex. In some cases, before having the confirmation of the diagnosis, due to the important growth delay, stimulation insulin test was performed (0.1 units/kg, 0.05 units/kg for children under 5 years of age) under careful monitorisation. Once blood glucose concetration has reached 47 mg/dl GH sample is taken and the child eats a high carbohidrate meal and, if needed, 10% glucose is administrated. A peak GH concentration below 7 ng/ml support the diagnosis of GH deficiency. GH therapy was started after molecular testing and after correction of the heart defect in cases 1 and 2, with periodic reevaluation every 6 months, according to literature data (Allanson 2010, Noordam et al 2008). GH therapy was contraindicated in case 3 due to severe scoliosis. 38

39 RESULTS Case 1: 7 years (y) old male, the only child of a young, unrelated couple. Mother: affected by NS (diagnosed after her son s diagnosis), probably inherited from her father. Father: normal. Pregnancy: polyhydramnios. Birth: cesarean section, at 36 weeks (wk), Apgar score 7, weight (Wt) 2,900 g, length (Ht) 46 cm, head circumference (HC) 35 cm. Postnatal staturo-ponderal and psycho-motor development: mild delay (see attached NS growth curve Fig ). Diagnosed with heart defect at 1 month (Mo) (valvular/ infundibular pulmonary artery stenosis, atrial septal defect, persistent ductus arteriosus surgical reconstruction at 3 y) and at 2 Mo with NS (PTPN11 mutation identified later). At 3 Mo - diagnosed with hypothyroidism and substitutive therapy (Levothyroxine 25 µg/day) was introduced. Surgical correction of bilateral cryptorchidism performed at 2 y 6 Mo. Easy bruising noted in time. hgh therapy (Omnitrope 0.04 mg/kg/day) started at 3 y of age with good evolution (Ht velocity > 1cm/Mo, 9.4 cm gain Ht compared to normal Ht growth chart; constant speed of Ht gain) and no adverse events recorded. Measurements on normal growth chart: Ht: -3.2 SD, improved after GH therapy to -1.3 SD; Wt: -2.8 SD (relatively constant till GH therapy), improved to -1.0 SD; HC: -1.2 SD (relatively constant). Physical examination: relative macrocephaly; dysmorphic face that became more triangular in time (tall/ broad forehead, hypertelorism, short/ prominent phyltrum, thick lips, low set large ears with thick helices, normal hearing see Fig 2.3.4), short neck with normal insertion of hair in the back, abnormal thorax (broad, mild upper pectus carinatum, lower pectus excavatum, low set nipples), normal spine (no scoliosis), excess of skin on hands/ feet with wrinkled palms/ soles (mainly in the first 3 years of life), mild talus valgus, systolic murmur III/VI (before correction), bilateral cryptorchidism (surgically corrected), hypertrophic scars after surgery and hyperactive behavior. Investigations: o Platelet count and blood sugar: normal; o Chest X-ray: ICT 0.48, mild enlargement of right heart; o EKG: sinusal rhythm, 90/min, QRS axis extreme right deviation, PQ 0.12 sec, right ventricle hypertrophy, right branch block; o Wrist X-ray: delayed bone age (1 year delay); o IGF I: initially low levels (33.4 ng/ml, normal ranges ), later on normalized (61.8 ng/ml); o Vitamin D: 14.2 ng/ml (normal >30 ng/ml); o Thyroid ultrasound and function: normal (under therapy); o Abdominal ultrasound: normal; 39

40 DNA test: PTPN 11 exon 8: c.923a G heterozygous (p.n308s). Fig 2.3.1: NS growth curve for case 1 (PTPN 11 mutation); green arrow marks start of GH therapy; blue arrow marks heart defect correction (graph adapted from Hall, 2007) Case 2: 12 y old female, first child of a young, normal, unrelated couple. Negative family history. Uneventful pregnancy. Birth: natural, at term, cranial presentation, Wt 3,500 g, neonatal jaundice (2 days). 40

41 Postnatal development: mild delay. Staturo-ponderal development (see NS growth curve attached Fig 2.3.2): delay with particular characteristics - normal Ht growth in the first year, later failure to thrive (-3SD) and mild recovery after GH therapy. Fig 2.3.2: NS growth curve for case 2 (KRAS mutation); green arrow marks start of GH therapy (graph adapted from Hall, 2007) Diagnosed with heart defect at 6 Mo (atrial septal defect later closed; anterior mitral valve prolapse, mild mitral insufficiency); diagnosed clinically with NS at 6 Mo (KRAS mutation identified later). hgh therapy (Omnitrope) started at 8 y of age (0.04 mg/kg/day, interrupted without medical advice for three months, subsequently reintroduced), with relatively good 41

42 evolution (Ht velocity > 1cm/Mo, 5 cm extra Ht compared to the standard Ht grow chart) and no adverse events recorded. Measurements on normal growth chart: Ht: -3.3 SD, improved after GH therapy to SD; Wt: -2.9 SD and HC: -1.1 SD (both relatively constant). Physical examination: dysmorphic face that has changed in time (tall and broad forehead became less prominent, marked hypertelorism, epicanthic fold, prominent phyltrum, dystrophy of temporary teeth, low set ears with thick helices and mild hearing loss see Fig 4), short/webbed neck with low/trident insertion of hair in the back, abnormal thorax (upper pectus carinatum, lower pectus excavatum, low set nipples), normal spine (no scoliosis), cubitus valgus, mild brachydactyly, systolic murmur II/VI, liminar intellectual function. No puberty onset. Easy bruising. Investigations: o Platelet count and blood sugar: normal; o Wrist X-ray: delayed bone age (2 years delay); o low basal GH (1.82 ng/ml) with no Insulin stimulation (8.02 ng/ml) and low IGF I (49.1 ng/ml; normal ranges ), later on normalized (226 ng/ml, normal ranges ); o Thyroid ultrasound and function: normal; o Abdominal ultrasound: normal; o Ophthalmologic exam: mild hypermetropia; o Barr test: 18% positive (normal); DNA test: PTPN 11 gene (exons 2, 3, 4, 7, 8, 12 and 13) normal; KRAS gene - exon 2: c.9178a G heterozygous (Gly60Ser). Case 3: 14 y old male, first child of a young, normal, unrelated couple. Negative family history. Uneventful pregnancy. Birth: natural, at term, cranial presentation, Wt 3,200 g. Intellectual development: initially normal, later on difficulties in nursery/ school with school drop-out. Staturo-ponderal development (see NS growth curve attached Fig 2.3.3): growth at -1 SD till age 10 y, then marked growth delay. Diagnosed with heart defect at 1 Mo (ventricular septal defect surgically corrected at 3 y). Diagnosed clinically with NS at 1 y 4 Mo (SOS 1 mutation identified later). Scoliosis detected at 3 y 7 Mo, severe evolution in time, surgical correction at 13 y. hgh therapy contraindicated due to severe scoliosis. Measurements on normal growth chart: Ht: -3 SD (constant till 10 y), then -3.9 SD; Wt: -2.7 SD till 10 y, -4 SD after; HC: -1.3 SD till 12 y, -2.5 SD later. Physical examination: disproportionate appearance (short trunk due to marked scoliosis), dysmorphic face that has changed in time (tall/ broad forehead became less prominent, hypertelorism, prominent phyltrum, low set ears with thick helices and normal hearing see Fig 2.3.4), webbed neck with low/trident insertion of hair in the 42

43 back, broad thorax with low set nipples, marked dorso-lumbar scoliosis and rib cage deformation, mild intellectual disability. No puberty onset. Fig 2.3.3: NS growth curve for case 3 (SOS 1 mutation); blue arrow marks heart defect correction (graph adapted from Hall, 2007) Investigations: o platelet count and blood sugar: normal. o Low basal GH (1.66 ng/ml) and low IGF I (45.1 ng/ml; normal ranges ); o normal thyroid function; 43

44 o Abdominal ultrasound: normal; o Orthopedic examination: initially - thoraco-lumbar scoliosis with double curve; complex thoracic surgical correction (at 13 y); o Wrist X-ray: delayed bone age (4 years delay); Knee X-ray: fertile growth plate; Skull X-ray: small, normal shaped sella turcica; Spine X-ray: thoracolumbar scoliosis with dextro-convex curvature of the thoracic region with maximum at T7-T8, dextro-concave in the lumbar region with important spin of the axis in the thoracic T2-T12 and lumbar L1-L5 spine; o Ophthalmologic exam: sinuous retinal blood vessels; DNA test: PTPN 11 gene (exons 2, 3, 4, 7, 8, 12 and 13) normal; SOS 1 gene - exon 4: c.233t G heterozygous (p.phe78cys). Echocardiographic results have been constant in time in all cases. None of them recorded constipation, joint pain or mood disorder (frequent manifestations found in adults with NS) or lymphatic or oncologic abnormalities. (1) (2) (3) Fig : Patients 1, 2 and 3 at different ages 44

45 DISCUSSION Genotype phenotype correlation Genotype phenotype correlation is a key issue in NS and related disorders, mainly because neuro-cardio-facio-cutaneous spectrum includes many entities that share some features in common, each of them being determined by different defects of genes involved in Ras-MAPK pathway (Sarkozy et al 2009, Tartaglia et al 2010). So, molecular investigation of patients selected with van der Burgt score should consider first PTPN11 gene (as it is involved in 50% of cases), but for PTPN11 mutation negative cases, genotype phenotype correlation should be the main criteria to select next gene to be tested (Roberts et al 2013, Tartaglia et al 2010, Tartaglia et al 2009). We have evaluated the clinical features in our cases (followed for a long time) in an attempt to identify which of the common NS features were present and which not, to identify particular features, as well as to evaluate how clinical picture changed with age. Out of the common features, short stature at a young age (1-3 years old), as well as relative macrocephaly, hypertelorism, prominent phyltrum, low set ears with thick helices, short/ webbed neck, pectus/ low set nipples, heart defect and mild intellectual disability have been present in all patients and did not change in time, suggesting that these features could orientate the diagnosis of a short child towards NS. Other common features cited in the literature are: postnatal feeding difficulties leading to failure to thrive, diamond eyebrows, down slanting palpebral fissures and ptosis (Allanson 2009, Tartaglia et al 2010), but none of them was present in our patients, suggesting that more patients should be studied to evaluate the real frequency of these features in the Romanian NS population. Particularities were noted in the second case: her KRAS mutation was not reported in the literature (Tartaglia et al 2010, Allanson et al 2013) and this may explain why her phenotype is not as severe as we would expect for the severe end of the NS spectrum. Moreover, she associates relative macrocephaly (compared to Ht and Wt) and not true macrocephaly, as stated in the literature (Roberts et al 2013, Sarkozy et al 2009). Striking differences were noted in the third case: according to literature data (Roberts et al 2013, Sarkozy et al 2009, Tartaglia et al 2010, Allanson et al 2013, Pierpont et al 2009), SOS 1 mutations (including the one present in our patient) are characterized by relatively normal height and intellectual development, ectodermal features (keratosis pilaris, curly hair, sparse eyebrows), pectus and pulmonary valve stenosis/ hypertrophic cardiomyopathy/ atrial septal defect; our case associates marked short stature (-3.9 SD), intellectual disability that led to school drop-out, neither ectodermal features, nor pectus and the heart defect is represented by ventricular septal defect; moreover, severe scoliosis is associated and this feature was not associated with SOS 1 mutations until now. Probably the clinical picture attributed to SOS 1 mutations is broader than described till now. Clinical picture changed with age in all patients (see Fig 2.3.4), data consistent with the literature: the forehead became less prominent and broad, the chin became pointed, whereas the hand aspect reminiscent of cardio-facio-cutaneous syndrome (wrinkled skin) was present only in the first years of life in the first child, underlining the importance of periodic follow-up for a correct diagnosis. Growth in Noonan syndrome According to literature data, most cases show feeding difficulties in the first months of life (frequently needing tube-feeding) that lead to failure to thrive in the first year. Later on, due to the dysfunction of GH action, Ht growth follows the normal curve in the lower range (3 rd percentile) up to puberty (Padidela et al 2008, Romano et al 2009). Puberty is delayed, as well as bone age (mean delay 2 years). We have evaluated growth (Ht, Wt, and 45

46 HC) on normal growth curves (because criteria used to appreciate GH therapy efficiency refer to normal growth curves) and Ht growth on NS growth curves (see Fig 2.3.1, 2, 3) available in the literature (Witt et al 1986, Hall et al 2007). None of our cases associated postnatal feeding difficulties. The first case was small for gestational age and even if he did not have overt feeding difficulties, his Ht growth was markedly delayed (-3.2 SD on normal growth curves, -1 SD on NS growth curve); he has recovered 1.9 SD after GH therapy. The second case was normal at birth and grew normally in the first year of life (nutrition dependent growth phase). Later on (GH dependent phase), her Ht growth was delayed (-3.3 SD on normal growth curves, mean on NS growth curve); she recovered 0.9 SD in Ht after GH therapy. In the third case Ht growth was in the lower range of NS growth curve till 9 y of age and then growth was delayed (totally different aspect compared to literature data for SOS 1 mutations). Wt and HC growth curves have been constant in the first two cases (Wt at -2.6 and -2.9 in both cases, HC at -2.6 in the first case and at -1.1 SD in the second case), whereas in the third case Wt and HC growth was more severely affected after age 10. We appreciate that initial growth deficit could be attributed also to the heart defect, after surgical correction child growth evolving better (see Fig and 3). The hormonal profile (GH, IGF I) was different compared to literature data (low IGF I and mild increase of spontaneous GH secretion suggesting post receptor signaling GH resistance). Initial somatotropic axis evaluation showed low basal GH with no stimulation in the insulin test and low IGF I, profile that normalized after GH therapy, indicating a lack of GH secretion instead of GH resistance. More patients should be studied to evaluate this theory. We also appreciate that NS growth charts should be used for any NS child, to be able to appreciate if the evolution is typical, or if there are other associated conditions that impede child growth. However, for children on GH therapy, normal growth charts should be used in parallel, because the aim of the therapy is to get them as close as possible to the normal staturo - ponderal development. GH therapy Studies in the literature show that PTPN11 mutation positive children respond less efficiently to GH compared to PTPN11 mutation negative NS children and the response to GH is more marked in the first two years, later on decreasing (Romano et al 2010, Ferreira et al 2005). We have recorded different results: In the first case (PTPN11 mutation positive, expected to react less) in the first year of GH therapy Ht growth went in parallel with NS growth chart and then he started to grow more (constantly), after four years of therapy the extra Ht gained being 1.9 SD. In the second case (KRAS mutation positive, expected to react better), the extra cm were gained only in the third year of GH therapy. We have no clear explanation for this and further studies should be done, as well as longer follow-up of these children. During GH therapy we did not record any classic adverse event (cardiomyopathy, scoliosis, oncologic events etc.). However, after heart surgery first case developed hypertrophic scars, that could be interpreted either as consequences of defective connective tissue recorded in NS, or as an adverse event of GH therapy not recorded for the moment. Both children reacted well to GH therapy and because the treatment was initiated early (3 y of age in case 1 and 8 y of age in case 2), and there are no signs of puberty onset in case 2, we expect that adult Ht will be close to normal range. 46

47 CONCLUSIONS Our study aimed to evaluate clinical, endocrine and genetic aspects in three patients with NS (due to PTPN 11, KRAS and SOS 1 mutations), first two under GH therapy. NS is a complex autosomal dominant disease, associated with endocrine dysfunctions, that requires a multidisciplinary approach. Early recognition of the syndrome based on classical association of dysmorphic face, heart defect, short/ webbed neck, pectus and short stature is essential for starting GH therapy and for appropriate genetic counseling. We have identified common features that are very suggestive for the diagnosis (short stature, relative macrocephaly, hypertelorism, low set ears with thick helices, short neck, pectus, low set nipples, heart defect and mild intellectual disability), as well as the changing of facial aspect in time. In case 2 the aspect was milder than expected for a KRAS mutation, whereas in case 3 (SOS1 mutation) the aspect was more severe. Severe scoliosis associated seems to be a new feature. We have detected particular patterns of growth in our patients before and after GH therapy. Unlike literature data, our PTPN11 mutation positive child reacted well to GH therapy, whereas our KRAS mutation positive case started to gain Ht only after 3 years of GH therapy. We have recorded hypertrophic scars either as a new feature of NS, or as a possible adverse event of GH therapy. GH therapy was successful in our patients, without classical adverse events recorded. Somatotropic axis dysfunction is discussed Skeletal dysplasia Skeletal dysplasias represent another very challenging field in Clinical Genetics because it is so heterogeneous and because some conditions are really rare. To overcome these difficulties and to be able to investigate some of our special cases, we have joint big research groups in the field. The work led to the description of new clinical features, new mechanisms and the discovery of new genes. Globally, the investigation provided very interesting results materialized in highly cited articles. The most suggestive results in the field of bone dysplasia include: Cranio- fronto- nasal dysplasia: soon after the identification of the causal gene our case was tested and confirmed as affected; because of the multidisciplinary approach of the investigation, the child followed intensive plastic surgery, with a marked improvement of the quality of life; a healthy sister was born later due to prenatal diagnosis available; the research has been published in the article listed at number 63 in the list of publications; Fuhrmann symphalangism: proximal symphalangism is a very rare condition expressed by the fusion of the proximal and middle phalanges, fusion of carpal and tarsal bones, elbow fusion and hearing loss. Typical inheritance is autosomal dominant. We have followed an affected family for a long time. When molecular tests have become available we have performed the test and the results were normal. Later on, because the clinical features were very suggestive for the diagnosis in our family and in a different one, more extensive work has been performed and a new gene has been identified, confirming the diagnosis. The pathogenicity has been confirmed with transgenic animals. The research has been published in the article listed at number 168 in the list of publications; 47

48 Schimke immune- osseous dysplasia: this is a rare condition that associates nephrotic syndrome and a bone dysplasia with a particular immunodeficiency. We have diagnosed a patient with this disorder and followed him in a multidisciplinary team. The clinical features were very suggestive, but molecular testing has been normal. However, because the clinical picture was so convincing, our patient was taken into consideration when top specialists described new clinical and radiological features of the disorder. The results of the study have been published in the articles listed at number 87 and 171 in my list of publications. Ten years later, due to the use of new molecular techniques, a new gene has been identified and the diagnosis of our patient was finally confirmed Conclusions and further enhancements The most important conclusions referring to Prader Willi syndrome are: PWS is a complex disease, associated with endocrine disfunctions, that requires a multidisciplinary approach. Marked hypotonia and feeding difficulties in infancy (found in all our patients) are early evocative features for PWS. Early diagnosis of PWS is essential for starting GH therapy and for appropriate genetic counseling. Although we started relatively late the GH therapy and it was administered to patients with unevolutive skeletal anomalies, we observed significant improvement in mental development and in body habitus and no adverse effects. Our study also illustrated the challenges raised by some features very rarely described in PWS (Blount disease and multiple allergies). Multidisciplinary approach is also required for Noonan syndrome, a complex autosomal dominant disease associated with endocrine dysfunctions. Early recognition of the syndrome based on classical association of dysmorphic face, heart defect, short/ webbed neck, pectus and short stature is essential for starting GH therapy and for appropriate genetic counseling References 1. Grechi E, Bruna C, Benedetta M, Stefania DC, Giuseppe C. Prader-Willi syndrome: clinical aspects. J Obes. 2012;2012: Holm VA, Cassidy SB, Butler MG, Hanchett JM, Greenswag LR, Whitman BY, et al. Prader-Willi syndrome: consensus diagnostic criteria. Pediatrics Feb;91(2): Vogels A, Matthijs G, Legius E, Devriendt K, Fryns JP. Chromosome 15 maternal uniparental disomy and psychosis in Prader-Willi syndrome. J Med Genet Jan;40(1):72-4. Veltman MW, Thompson RJ, Roberts SE, Thomas NS, Whittington J, Bolton PF. Prader-Willi syndrome--a study comparing deletion and uniparental disomy cases with reference to autism spectrum disorders. Eur Child Adolesc Psychiatry Feb;13(1):

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52 50. Roberts AE, Allanson JE, Tartaglia M, Gelb BD. Noonan syndrome. Lancet 2013; 381(9863): Allanson JE. Noonan Syndrome. In: Cassidy SB, Allanson JE, eds. Management of Genetic Syndromes, 3rd ed. Hoboken, New Jersey: John Wiley& Sons, Inc., 2010: Allanson JE. The Clinical Phenotype of Noonan Syndrome. In: Zenker M, eds. Monographs in Human Genetics: Noonan syndrome and related disorders: A matter of deregulated RAS signaling. Vol 17. Basel: Karger Press, 2009: Sarkozy A, Digilio MC, Marino B, Dallapiccola B. Genotype - Phenotype Correlations in Noonan Syndrome. In: Zenker M, eds. Monographs in Human Genetics: Noonan syndrome and related disorders: A matter of deregulated RAS signaling. Vol 17. Basel: Karger Press, 2009: Tartaglia M, Zampino G, Gelb BD. Noonan syndrome: clinical aspects and molecular pathogenesis. Mol Syndromol 2010; 1(1): van der Burgt I. Noonan syndrome. Orphanet J Rare Dis 2007; 2: Jorge AA, Malaquias AC, Arnhold IJ, Mendonca BB. Noonan syndrome and related disorders: a review of clinical features and mutations in genes of the RAS/MAPK pathway. Horm Res 2009; 71(4): Padidela R, Camacho-Hubner C, Attie KM, Savage MO. Abnormal growth in Noonan syndrome: genetic and endocrine features and optimal treatment. Horm Res 2008; 70(3): Binder G. Endocrine Regulation of Growth and Short Stature in Noonan Syndrome. In: Zenker M, eds. Monographs in Human Genetics: Noonan syndrome and related disorders: A matter of deregulated RAS signaling. Vol 17. Basel: Karger Press, 2009: Romano AA, Allanson JE, Dahlgren J, Gelb BD, Hall B, Pierpont ME, Roberts A, Robinson W, Takemoto C, Noonan J. Noonan syndrome: clinical features, diagnosis, and management guidelines. Pediatrics 2010; 126(4): Noonan JA, Raaijmakers R, Hall BD. Adult height in Noonan syndrome. Am J Med Genet A 2003; 123A(1): Binder G. Noonan syndrome, the Ras-MAPK signalling pathway and short stature. Horm Res 2009; 71 Suppl 2: Ranke MB, Heidemann P, Knupfer C, Enders H, Schmaltz AA, Bierich JR. Noonan syndrome: growth and clinical manifestations in 144 cases. Eur J Pediatr 1988; 148(3): Shaw AC, Kalidas K, Crosby AH, Jeffery S, Patton MA. The natural history of Noonan syndrome: a long-term follow-up study. Arch Dis Child 2007; 92(2): Witt DR, Keena BA, Hall JG, Allanson JE. Growth curves for height in Noonan syndrome. Clin Genet 1986; 30(3):

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54 3. Introduction of innovative techniques in Medical Genetics practice and optimization of their use 3.1. Introduction and conceptual background Globally, the evolution of the society is based on innovative ideas. This concept is a key issue when we discuss about lab techniques used in Medical Genetics, where any discovery triggers an entire network of subsequent research projects. Moreover, knowledge develops so quickly, that anybody could get outdated without using innovative techniques. I appreciate that this rapid pace of technological development might have two major advantages for us: Because the technology is changing so quickly and in the same time becoming cheaper and cheaper, this could represent an opportunity to skip some developmental stages and go directly to the last step, for the benefit of the entire society; Due to the relatively limited resources, techniques used in Romania tend to be optimized and innovative techniques are encouraged. Moreover, the use of a certain innovative technique could be extended to other applications, making it more successful. In this chapter I shall present innovative techniques we have introduced in our practice and how useful they proved to be MLPA use for patients with intellectual disability In 2005 in Romania the investigation of individuals with intellectual disability was simply performed by karyotype, long range PCR for Fragile X syndrome and rarely by FISH. In Europe, MLPA technique was introduced in 2002, but it was not extensively used because array techniques were introduced meanwhile and they produced more interesting results. Subtelomeric rearrangements were just described and that was the time for the description of many new microdeletion/ microduplication syndromes. So, due to the accessible price, simple and reliable technique of the MLPA method, as well as due to its ability to detect subtelomeric rearrangenments that represent a relatively frequent cause of nonspecific intellectual disability, we have introduced MLPA in our daily practice. The results of the study have been presented in Chapter 1.2. The protocol mentions that an abnormal result should be confirmed with a different test and in most of the countries FISH or arraycgh is used for this purpose. We have continued to use innovative methods and for confirmation we have introduced MLPA follow-up kits, that both confirm the presence of the defect and estimate its size at a more economic cost. After introducing MLPA technique for the detection of subtelomeric rearrangements, we have extended its use to other causes of intellectual disability (microdeletions, Fragile X syndrome and autism) enabling us to optimize the investigation protocol for intellectually disabled individuals. 54

55 Illustrations of our results are presented in Chapter 1.2 and in Section II in the part dedicated to research projects Role of MLPA test in the identification of complex chromosomal rearrangements After the optimization of the MLPA test for the detection of subtelomeric rearrangements, we have thought of other innovative uses of this simple method. A possible answer to the question was represented by complex chromosomal rearrangements or minute chromosomes, situations in which a chromosomal fragment is too small to be identified using only chromosomal banding. So, to check this innovative use of the MLPA test, we have taken 54 cases of complex chromosomal rearrangements and minute chromosomes and we have applied subtelomeric MLPA testing. In 6 cases the telomeres were involved and we were able to identify the abnormality at a low cost. An example is illustrated in Fig 3.3.1, showing a deletion of chromosome 7 not identified on the karyotype. Fig : Subtelomeric MLPA used for complex chromosomal rearrangements identifies a deletion of chromosome 7. A different innovative use of subtelomeric MLPA was for the detection of mosaics in the very specific situation of a suspicion of Pallister Killian syndrome mosaic 12p tetrasomy due to the presence of an extra isochromosome. Usually the abnormal cells are not present in blood and to confirm the diagnosis you need other cells. The gold standard method in this case is considered the FISH test / array CGH performed on fibroblast from a skin biopsy. In our case we looked for other innovative ways to confirm the presence of the mosaic in other tissues. So, we have extracted DNA from blood, hair root, saliva and urine 55

56 and by using subtelomeric MLPA we were able to confirm the tresence of the 12p tetrasomy in saliva and urine (paper in print). An illustration of the MLPA test performed on the DNA extracted from saliva is presented in Fig The result has been confirmed with the gold standard method (array CHG done on a DNA sample from saliva). Fibroblast culture from skin did not grow. Fig Mosaic tetrasomy 12p identified with subtelomeric MLPA performed on DNA extracted from saliva 3.4. Use of arraycgh in the diagnosis of complex cases To illustrate the importance of array techniques for solving complex cases, I shall present another research we performed in the field of intellectual disability. Introduction Monosomy 18p was first reported in 1963 by de Grouchy et al. [de Grouchy 1963] and has an incidence of 1:50,000 live-born infants [Turleau 2008]. About 2/3 of cases are de novo deletions, 1/6 are due to de novo translocations between the long arms of an acrocentric and 18q, and the rest come from familial translocations, inversions, complex translocations or direct transmission [Schinzel 2001]. Main clinical features of monosomy 18p are mild to moderate intellectual disability (ID), postnatal growth retardation, and dysmorphic features including ptosis, hypertelorism, strabismus, broad flat nose, micrognathia, and low-set large ears [Wester 2006]. We report on a girl with ID, mild dysmorphic face and Turner like features who had a de novo 14;18 translocation. Copy number analysis with SNP array detected a terminal deletion flanked by a duplication on 18p and a duplication of 16p

57 Material and methods Case report The patient is a 20 year old female, the first child of non-consanguineous, healthy Caucasian parents (mother - 24 year old, father - 27 year old at birth of the proband). There was no family history of ID, congenital anomalies or psychiatric disorders. Pregnancy was uneventful. She was born at term, by normal delivery, with a birth weight of 2,200 g (below 3 rd percentile), while length and head circumference were not recorded. All developmental milestones were delayed: she achieved head control at 6 months, walked without support at 2 years, spoke first clear words at 1.5 years. The girl was referred for genetic evaluation at the age of 5.5 year old, due to ID and single central maxillary incisor. Her growth parameters were: height 95.5 cm (-3.68 SD), weight 13 kg ( SD), and head circumference 47 cm ( SD). She had a triangular face, horizontal palpebral fissures, blue sclera, short, slightly protruding philtrum and upper lip, blunted Cupid's bow, slightly everted lower lip, mild microretrognathia, bilateral preauricular sinus (Fig ). Oral cavity examination showed absent maxillary and mandibular frenulum and single central maxillary incisor. Her language was limited to single words (she could not produce sentences). She had nocturnal and diurnal enuresis, for which she received therapy. No hearing impairment has been identified. Echocardiography showed an atrial septal defect. Abdominal ultrasound, routine biochemical and hematological tests, endocrine investigations (GH, free T4, TSH) were normal. No metabolic tests have been performed. Craniocerebral CT scan and MRI of the spine did not show any changes. Fig : Facial aspect of the patient studied When examined at the age of 20 years, her height was 147 cm (-2.89 SD), weight was 43 kg (-1.76 SD, body mass index 19.9 kg/m 2 ), and head circumference was 53 cm (-1.75 SD). She presented a particular posture (widespread legs and leaning slightly forward) and slowness in motion and action. The face became elongated, mature for age, with slightly coarse features (Fig ). She had mild webbed neck, broad chest and narrow hips, normal posterior hairline, kyphoscoliosis, pectus excavatum, short and wide hands and feet (below 3 rd percentile), with mild brachydactyly. Puberty was normal, but she developed asymmetric mammary glands and excessive hair growth in presternal, circumareolar and subumibilical regions (Ferriman-Gallwey score of 2). Psychological testing established a moderate ID (IQ 45), with impaired speech and language skills, difficulties with interpersonal relationships and oppositional behavior. She presented giggle incontinence. Endocrine investigations were 57

58 as follows: normal levels of FSH, LH, estradiol, prolactin, elevated plasmatic levels of testosterone, DHEA-sulfate, and 17-OH progesterone. Cytogenetic studies The chromosome analysis was performed for the patient and her parents using G banding technique on metaphase chromosomes from peripheral blood lymphocytes, according to standard protocol. Chromosome C banding was performed by the standard BSG (Barium hydroxide/saline solution/giemsa) method [Sumner 1972] with slight modification. SNP array Genomic DNA was purified from peripheral blood using Wizard Genomic DNA Purification Kit (Promega Corp., Madison, WI, USA). SNP array was performed using HumanCytoSNP-12 v2.1 BeadChip platform (Illumina Inc., San Diego, CA), containing approximately SNPs per sample, according to the manufacturer's instructions. The data were processed using Genome Studio V software (Illumina). Genomic positions were defined using GRCh37/hg19. Results The cytogenetic G-banding revealed an unbalanced translocation between chromosomes 14 and 18 (Fig ). C-banding showed two centromeres on the derivative chromosome; in all metaphases examined the 14 chromosome centromere was inactivated (Fig ). The karyotype was thus 45,XX,psu dic(14;18)(p11.1;p13.1). Parental karyotypes were normal. SNP array analysis detected a terminal deletion of approximately Mb, from 18p11.32 to 18p11.22 (274-10,242,742), flanked by a duplication of approximately 1.15 Mb, from 18p11.22 to 18p11.21 (10,249,343-11,401,062) (Fig ). In addition, the SNP array revealed a duplication of 516,590 bp in 16p11.2 (29,568,718-30,085,308). The relative small size of the duplications did not allow for FISH to determine orientation. Blood samples from the parents were not available for SNP array analysis. Fig : Karyotype of the patient studied 58

59 Fig 3.4.3: SNP array of the patient identifying the defect on chromosome 18 Discussion We describe a female patient with features evocative of Turner syndrome, mild dysmorphic face, minor features of holoprosencephaly, small hands and feet, excessive hair growth on anterior trunk and ID. The karyotype showed an unbalanced translocation between chromosomes 14 and 18 resulting in the formation of a dicentric derivative chromosome. SNP array analysis revealed three abnormalities: an 18p deletion flanked by a duplication, and a 16p11.2 duplication. The translocation is de novo as both parents had a normal karyotype. Non-Robertsonian dicentric autosomes are rare findings, in a review by Lemyre et al. [Lemyre 2001] being reported only 26 cases. The majority of cases involve the acrocentric chromosomes, with a short arm breakpoint, followed in frequency by chromosome 18. Most of the heterodicentric autosomes have only one primary constriction on metaphase chromosomes, and the constriction is noticed mostly at the site of the non-acrocentric centromere [ Lemyre 2001], as in our case. Deletions of p arms of acrocentrics containing NOR regions are not known to be associated with phenotypic anomalies and therefore probably have not contributed to the phenotype. Also, the 1.15 Mb 18p duplication is not likely to have contributed to the phenotype, since most patients with trisomy 18p have normal or mild phenotypes, and may or may not have ID [Marical 2007]. Our patient displays some of the features of 18p- syndrome, such as ID, features of the HPE spectrum (mild microcephaly, single central maxillary incisor), and features evocative of Turner syndrome (short stature, mild webbed neck, broad trunk, pectus excavatum) (Table 1). Facial dysmorphism (triangular face, blue sclera, bilateral preauricular sinus) is different from that described for 18p deletion, excepting the oromandibular region. Facial appearance has changed over time, becoming elongated (Fig. 2), as described also by Tsukahara et al. [Tsukahara 2001]. Congenital cardiac defects, present in our case, have been observed in 10% of cases of 18p- [Digilio 2000]. Although the phenotype described above is not characteristic for Monosomy 18p, the standing position with widespread legs and leaning slightly forward as well as marked slowness in motion and action are very suggestive for this chromosomal syndrome. Recurrent 16p11.2 microduplications have been initially associated with phenotypes ranging from normal to ID, autistic spectrum disorders and psychiatric problems [Fernandez et al 2010; Weiss et al 2008;Kumar et al 2009; McCarthy et al 2009]. Other studies showed that these duplications can manifest with dysmorphic features without a recognizable pattern, microcephaly, congenital anomalies (including torticollis, cleft lip and palate, pectus excavatum, pectus carinatum, mild scoliosis, hypospadias, phimosis, tethered cord, pes planus), and seizures [Shinawi et al 2010]. Jacquemont et al. showed that 16p11.2 duplication is associated with a body mass index 18.5 kg per m 2 in adults and -2 SD from the mean in 59

60 children [Jaquemont et al 2011]. Among the features mentioned above, our patient exhibits mild microcephaly, pectus excavatum, mild scoliosis and ID, but these features are also described in 18p deletion. She was underweight during childhood, but recovered later, her body mass index being within normal range as an adult. Considering that empiric estimate for penetrance of proximal 16p11.2 duplication established a penetrance of 27.2%, and the likelihood of a normal phenotype is ~73% [Rosenfeld et al 2012], we cannot clearly conclude how this CNV influences the phenotype. More recently, a patient with thoracolumbar syringomyelia and 16p11.2 duplication has been described [Schaaf et al 2011]. Although our patient presented kyphoscoliosis and nocturnal enuresis, MRI of the spine showed no changes. The short stature, excess hair growth on anterior trunk, regular menses and the hormonal profile of our patient may be associated with adult-onset CAH-21-hydroxylase deficiency and further genetic investigation would be useful to confirm it. In a study of three patients with 18p deletion, Portnoi et al. suggested that there might be a critical region for GH deficiency between 18p11.23 and 18pter [Portnoi et al 2007]. Our patient has a deletion which includes that region, but the level of GH is normal and the craniocerebral CT did not show any pituitary gland anomalies. Critical region for ID has been tentatively mapped between 18p11.1 and 18p11.21 [Wester et al 2006]. Our patient has a deletion distal to this point and moderate ID, but this feature may be due to the 16p11.2 microduplication. Brenk et al. [Brenk et al 2007] proposed round face to map to the distal 1.6 Mb of 18p, and post-natal growth retardation and seizures to the distal 8 Mb. Our patient has a terminal deletion larger than 10 Mb, but she had no history of seizures, and the face was triangular in childhood and elongated in adulthood. Considering that pointed chin can be noticed in 5 out of 13 patients with 16p11.2 duplication for which the facial features were presented [Fernandez et al 2010, Shinawi et al 2010], we appreciate that the triangular aspect of the face may be due to this rearrangement. Ptosis and short neck, features frequently associated with 18p- [Turleau 2008], were attributed by Brenk et al. to the proximal half of 18p. These features were absent in our patient, in whom the proximal 5,1 Mb of 18p was not deleted. Thus, haploinsufficiency of genes located in this region may be responsible for these features. Our patient has a microform of HPE, although only 10% of patients carrying a 18p deletion (including the TGIF gene) present HPE [Schinzel 2001]. HPE is a complex developmental disorder in which multiple genetic and environmental factors can affect the severity of the phenotype [Ming et al 2002]. A recent array CGH study of a large group of HPE patients demonstrated a high frequency of submicroscopic anomalies involving known but also novel HPE loci, including 16p11.2 [Bendavid et al 2009]. Therefore, the 16p11.2 microduplication present in our patient can be a second genetic event contributing to HPE manifestation. In conclusion, we report a female patient with a pseudodicentric 14;18 chromosome that carries two additional CNVs. These CNVs confer phenotypic variability to 18p- syndrome, leading to difficulties in establishing the contribution of each abnormality to the phenotype. Although the phenotype of 18p- syndrome is not as typical as for other syndromes, HPE microform and Turner-like phenotype association with characteristic posture and marked slowness in motion and action is very suggestive for this syndrome. Microarray analysis of our patient allowed us to define precise molecular characterization of the translocation breakpoints and to uncover two unsuspected cryptic abnormalities, improving genotypephenotype correlations and management. 60

61 3.5. Diagnosis of Duchenne/ Becker muscular dystrophy using MLPA test INTRODUCTION Dystrophinopathies are the most frequent spectrum of neuromuscular disorders, caused by mutations in the DMD gene that encodes for dystrophin, which is a key element for sarcolemma stability during muscle contraction (Juan-Mateu et al 2013). Duchenne muscular dystrophy (DMD; MIM# ) is the most severe form, being characterized by progressive symmetrical muscle weakness, calf hypertrophy (before age 5), wheelchair dependency by age 12 and death in the 2 nd -3 rd decade due to heart or respiratory failure. Becker muscular dystrophy (BMD; MIM# ) is a less severe allelic form of DMD, affected individuals surviving till the 7 th decade (Hedge et al 2008). As X-linked recessive disorders, DMD and BMD fully express in males and carrier females rarely present features, the clinical expression becoming evident in very specific situations (Juan-Mateu et al 2012) and being usually mild. Typical dystrophin isoform involved in DMD/BMD has 4 main domains actin-binding NH 2 - terminal, rod domain, cysteine-rich domain and COOHterminal. Most DMD mutations occur in the central rod domain and have different consequences depending if they affect or not the reading frame ( reading frame rule ) (Monaco et al 1988). In DMD cases the mutation alters the reading frame (out-of-frame mutation), the result being a severely truncated, nonfunctional or even absent dystrophin, while in BMD cases the mutation doesn t alter the reading frame (in-frame mutation), the result being a partly functional dystrophin (Aartsma-Rus et al 2006). Different methods have been used for DMD mutation detection ( Hedge et al 2008, Stockley et al 2006, Schwartz et al 2004, Janssen et al 2005, Borun et al 2014, Zeng et al 2008, Hamed et al 2006, Wang et al 2014). However, the molecular genetic workup can be performed in two steps: MLPA, followed by sequencing of the coding regions and splice sites (Grimm et al 2012). For the approximately 2% of the cases in which both MLPA and sequencing fail to identify a mutation, a muscle biopsy with specific dystrophin immunohistochemical staining or Western blotting is recommended (Laing et al 2011). The aim of this study is to evaluate the efficiency of MLPA as a DMD mutations screening tool (both for patients and carriers), as well as to appreciate the frequency of different types of mutations, and to check the validity of the reading frame rule. To our knowledge this is the first full-text study regarding the efficiency of MLPA for the screening of exonic copy number variations in dystrophin gene in Romania. MATERIAL AND METHODS This study included 53 individuals (30 affected males and 23 asymptomatic female relatives) referred to the Medical Genetics Unit of Sfanta Maria Children's Hospital in Iasi for evaluation and genetic counseling due to the clinical suspicion of DMD/BMD. The study was approved by the Ethics Committee of the Grigore T. Popa University of Medicine and Pharmacy from Iasi and informed consent was signed by the parents or by the patient. The diagnosis was based on physical examination, serum CK levels and family history. For asymptomatic carriers (mothers/sisters of affected males) we asked about symptoms related to exercise and checked serum CK level. Genomic DNA was extracted from peripheral blood samples using QIAamp DNA Blood Mini Kit (Qiagen). The standard MLPA analysis was performed according to the manufacturer s instructions using SALSA MLPA probemixes P034-A2 (dystrophin gene exons 1-10, 21-30, 41-50, and 61-70) and P035-A2 (dystrophin gene exons 11-20, 31-40, 51-60, and 71-79). Briefly, 200 nanograms of genomic DNA was denatured and then hybridized with SALSA probemixes. Following ligation, PCR was performed in a Gradient Palm-Cycler (Corbett Research, Mortlake, NSW, Australia), using 61

62 Cy5 universally labeled primers. Fluorescent amplification products were subsequently separated by capillary electrophoresis on a CEQ 8000 GeXP Genetic Analysis System (Beckman-Coulter) and the data obtained were analyzed with the Coffalyser.Net software, which uses block normalisation in order assess copy numbers of the target sequences. The deletions/duplications obtained by MLPA were subjected to the reading-frame checker from Leiden Database Muscular Dystrophy (White et al 2006), which generates a prediction of the effect of whole-exon changes upon the reading frame. RESULTS Out of the 30 affected males referred for evaluation, 21 had the clinical diagnosis of DMD, whereas 9 were diagnosed with BMD. MLPA analysis detected abnormalities in 63.5% (19/30) of male patients (DMD 71.5% - 15/21 and BMD 44.5% - 4/9). Deletions were detected in 53.5% of cases (16/30) and duplications in 10% (3/30). Deletions account for 84% of mutated cases (16/19) and duplications account for 16% (3/19). Out of the 16 males with deletion, 4 had deletion of a single exon. Deletions were grouped in the actin-binding (NH 2 - terminal) domain and rod-like domain, none of the mutations affecting the cysteinerich or COOH- terminal domain. The most frequently deleted exons were exons 45, 46, 47, 8 and 9, and the most frequently duplication involved exons 3-5 (see Figure 3.5.1). The most frequent breakpoints were recorded in introns 44 and 47, intron 2 breakpoint being involved both in deletions and duplications. Fig Mutations identified within DMD gene Out of the 23 females referred for evaluation and/or genetic counseling, none was symptomatic. The mutation rate in females was 43.5% (10/23), deletions affecting 35% of cases (8/23) and duplications 9% (2/23). Most of the mutated cases (80%, 8/10) were deletions. Unexpectedly, MLPA proved helpful in detecting numerical chromosomal abnormalities. The mother of a child with normal MLPA result had 35-50% amplification in relative peak area for all probes, suggesting the presence of triple X syndrome, situation confirmed by a karyotype performed afterwards. 62

63 Table 3.5.1: Abnormal MLPA results in the patients studied Nr. Clinical Serum CK MLPA FS Potential MLPA result in FH* diagnosis level result in carrier potential carrier (x normal) proband 1 DMD 27 dup 2 Yes mother N - 2 DMD 29 del 2-13 Yes mother del DMD 68 del 8-9 Yes 4 DMD 48 del 8-9 Yes mother del DMD 42 del 8-9 Yes mother del DMD 28 del 44 Yes mother del DMD 45 del 45 Yes mother del DMD 51 del 45 Yes mother NT + 9 DMD 22 del 48 No mother NT - 10 DMD 29 del Yes sister del DMD 72 del Yes mother N - 12 DMD 67 del Yes mother N - 13 DMD 18 del 3-30 No mother del DMD 24 del Yes mother NT - 15 DMD 18 dup 3-5 No mother dup BMD 17 del No 17 BMD 11 del No mother del BMD 9 del No mother NT - 19 BMD 7 dup 3-10 No mother dup FS frame-shift, FH* family history, more than one affected male; DMD Duchenne muscular dystrophy; BMD Becker muscular dystrophy; N- normal, NT not tested. DISCUSSION Efficiency of DMD gene mutation screening Various studies reported a detection rate of 60-70% for deletions and 10% for duplications (see Table 3.5.2). In our study, by selecting patients based on detailed physical examination and increased plasma CK levels, the detection rate has been 53.5% for deletions and 10% for duplications. We expect the difference to be due to a geographic variation, such differences being cited in the literature. After performing MLPA, 11/30 affected males received a normal result, in spite of the typical features of DMD/BMD. These cases are further candidates for gene sequencing in order to identify point mutations. 63

64 Table 3.5.2: DMD/BMD mutation frequency in the literature Type of mutation DMD BMD References Deletion (1/ more exons) 60-65% 65-70% Yan et al., 2004; Dent et al., 2005; Dolinsky et al., 2002; Takeshima et al., 2010 Duplication 5-10% 10-20% White et al., 2002; White et al., 2006; Flanigan et al., 2009; Takeshima et al., 2010 Point mutation (small deletion/ insertion, single base change or splicing mutation) 25-35% 10-20% Dolinsky et al., 2002; Hofstra et al., 2004; Takeshima et al., 2010 An important aspect is that MLPA is an inexpensive, rapid and reliable technique that proved repeatedly to be useful for DMD diagnosis (Table 3.5.2). Moreover, MLPA can be performed on DNA samples extracted from paraffin embedded muscle biopsies and this could be a valid solution for the specific situation when blood samples are not available and a person with positive family history is asking for genetic counseling. An alternative to the combined use of MLPA and sequencing analysis can be the use of a complex MLPA variant that checks in the same time for large exon deletions/duplications and for the most common point mutations in DMD gene (Bunyan et al 2007). Frequency of different types of mutations The precise identification of the mutation is essential in DMD, since clinical trials developed recently are personalized (therapy aimed to a specific mutation only) (Ferlini et al 2013). Most of the deletions and duplications cluster in two hotspot regions of the dystrophin gene (exons 2-20 and 44-53) (Marquis-Nicolson et al 2013), unlike small deletions and point mutations that seem to be evenly distributed throughout the gene (Hedge et al 2008). The mutations we have detected are indeed located in those areas, but slightly different the first region covered exons 2-36, whereas the second region covered exons (see Figure 3.5.1). If we consider the areas covered by multiple mutations, we can narrow these intervals to exons 3-30 for the first hotspot and for the second hotspot. To evaluate if this is a particularity of the local population, further studies should be performed. We have found that the major deletion breakpoint was in intron 44, similar to literature data (Prior et al 2005) (see Figure 3.5.1). Deletions of a single exon should be differentiated by small mutations or polymorphisms situated close to the probe ligation site, which can influence the peak area of the amplification product of that probe (Gatta et al 2005). We have identified 4 deletions of a single exon (cases 6-9), the most frequently involved (2/4) being exon 45, as reported also in Leiden database (White et al 2006). These deletions were associated with a DMD phenotype, so we don t expect this to be due to a polymorphism, but sequencing should be performed to confirm the diagnosis. The most frequent deletions in the literature are those involving exons (7% of cases), 45 (5.3%), (5.1%) and for duplications the most common one is that involving exon 2 (8.6% of cases) (White et al 2006). We have found all these types of defects in our patients. However, besides the common mutations, we have also found mutations that are rarely cited. Deletion (case 14) is the rarest mutation identified in our group, being reported only once in the literature (Schwartz et al 2004). Other rare deletions found (2-13 and 3-30) are probably related to the common breakpoint in exon 2 (involved mainly in 64

65 duplications). Deletion 8-9 was found in two different families, living in remote areas and to exclude their relatedness we shall perform a linkage study. Fig 3.5.2: Illustration of results from the study above: exon 44 deletion in an affected male; below: his carrier mother According to the literature data the duplications are frequently located near the 5 end of the gene, the most common one is exon 2 duplication and most of them have grandpaternal origin, with a high recurrence risk (White et al 2006). The mechanism for exon 2 duplication seems to be nonhomologous end joining and not unequal crossing over (White et al 2006). For duplication cases the genotype phenotype relationship is difficult to evaluate because the reading frame rule is not always valid. Tandem duplications are common, but non-tandem duplications may also occur and the orientation of the duplicated 65

66 fragment is very important (Flanigan et al 2009). All duplications identified in our study (exon 2, 3-5, 3-10) were close to the 5 end of the gene, and involved a breakpoint in intron 2. Validity of reading frame rule Most of the phenotypic differences between BMD and DMD cases can be explained by the reading frame rule, meaning that out-of-frame mutations are associated with a severe phenotype (DMD), whereas in-frame mutations are associated with a milder phenotype (BMD) (Aartsma-Rus et al 2006). The rule has been valid in 84% of our mutated cases (16/19), except cases 9, 13 and 15. We appreciate that in these cases, even if we have detected a defect, DMD gene sequencing should be performed to look for associated defects that could explain these particularities. An important issue when analyzing genotype phenotype relationship is to keep in mind that in advanced stages of disease the plasma CK level decreases, as a result of the progressive elimination of dystrophic muscle fibers (Hoffman et al 1988). We have identified this particularity in two adult patients (cases 10, 18). Carriers Carriers identification is an essential issue for genetic counseling in a DMD family, especially in cases with a single affected male in the family. In such a situation the mother could be carrier, gonadal mosaic or normal (the child being affected due to a new mutation). In our study MLPA identified heterozygous deletions in 10/23 women, enabling an appropriate genetic counseling, especially in case 13, where family history was negative. CONCLUSIONS In our study, the use of MLPA in patients with suspicion of DMD/BMD had a detection rate of 63.5%, similar with the average value reported in the literature. The mutations identified cover the two hotspots described in the literature, but with slightly different limits. The most frequently deleted exon was exon 45 and the most frequently duplicated exons were exons 3-5, confirming the presence of both hotspots. The reading frame rule was valid in 84% of our cases, allowing both for diagnostic confirmation and for differential diagnosis of DMD versus BMD. MLPA is an accurate, reliable method for the detection of deletions/duplications in all dystrophin gene exons in carriers and affected males, and also has the ability to determine the size of the abnormality, which is critical for personalized gene therapy strategies Conclusions and further enhancements The use of innovative techniques in the DNA lab is a key issue, mainly because we deal with expensive tests in most of the cases. In our experience, we found that when financial resources are limited, the best option is to use optimized protocols that include simple, robust, inexpensive methods that should diagnose an important proportion of the cases tested. For the rest, more complex diagnostic methods could be used, but due to the combined use of different techniques, the global cost of testing is markedly reduced. Moreover, we have combined molecular and cytogenetic tests to facilitate the diagnosis in challenging situations like complex chromosomal rearrangements. 66

67 3.7. References 1. J. de Grouchy, M. Lamy, S. Theffry, M. Arthuis, C. Salmon, Dysmorphic complexe avec oligophrenie: deletion des bras courts d un chromosome 18, CR Acad Sci III (1963) C. Turleau, Monosomy 18p, Orphanet J Rare Dis, 3 (2008) A. Schinzel, Catalogue of Unbalanced Chromosome Aberrations in Humans 2nd ed., Walter de Gruyter, Berlin, U. Wester, M.L. Bondeson, C. Edeby, G. Anneren, Clinical and molecular characterization of individuals with 18p deletion: a genotype-phenotype correlation, Am J Med Genet A, 140 (2006) A.T. Sumner, A simple technique for demonstrating centromeric heterochromatin, Exp Cell Res, 75 (1972) E. Lemyre, V.M. der Kaloustian, A.M. Duncan, Stable non-robertsonian dicentric chromosomes: four new cases and a review, J Med Genet, 38 (2001) H. Marical, M.J. Le Bris, N. Douet-Guilbert, P. Parent, J.P. Descourt, F. Morel, M. De Braekeleer, 18p trisomy: a case of direct 18p duplication characterized by molecular cytogenetic analysis, Am J Med Genet A, 143A (2007) M. Tsukahara, K. Imaizumi, K. Fujita, H. Tateishi, M. Uchida, Familial Del(18p) syndrome, Am J Med Genet, 99 (2001) M.C. Digilio, B. Marino, A. Giannotti, R. Di Donato, B. Dallapiccola, Heterotaxy with left atrial isomerism in a patient with deletion 18p, Am J Med Genet, 94 (2000) B.A. Fernandez, W. Roberts, B. Chung, R. Weksberg, S. Meyn, P. Szatmari, A.M. Joseph-George, S. Mackay, K. Whitten, B. Noble, C. Vardy, V. Crosbie, et al., Phenotypic spectrum associated with de novo and inherited deletions and duplications at 16p11.2 in individuals ascertained for diagnosis of autism spectrum disorder, J Med Genet, 47 (2010) L.A. Weiss, Y. Shen, J.M. Korn, D.E. Arking, D.T. Miller, R. Fossdal, E. Saemundsen, H. Stefansson, M.A. Ferreira, T. Green, O.S. Platt, D.M. Ruderfer, et al., Association between microdeletion and microduplication at 16p11.2 and autism, N Engl J Med, 358 (2008) R.A. Kumar, C.R. Marshall, J.A. Badner, T.D. Babatz, Z. Mukamel, K.A. Aldinger, J. Sudi, C.W. Brune, G. Goh, S. Karamohamed, J.S. Sutcliffe, E.H. Cook, et al., Association and mutation analyses of 16p11.2 autism candidate genes, PLoS One, 4 (2009) e S.E. McCarthy, V. Makarov, G. Kirov, A.M. Addington, J. McClellan, S. Yoon, D.O. Perkins, D.E. Dickel, M. Kusenda, O. Krastoshevsky, V. Krause, R.A. Kumar, et al., Microduplications of 16p11.2 are associated with schizophrenia, Nat Genet, 41 (2009) M. Shinawi, P. Liu, S.H. Kang, J. Shen, J.W. Belmont, D.A. Scott, F.J. Probst, W.J. Craigen, B.H. Graham, A. Pursley, G. Clark, J. Lee, et al., Recurrent reciprocal 16p

68 rearrangements associated with global developmental delay, behavioural problems, dysmorphism, epilepsy, and abnormal head size, J Med Genet, 47 (2010) S. Jacquemont, A. Reymond, F. Zufferey, L. Harewood, R.G. Walters, Z. Kutalik, D. Martinet, Y. Shen, A. Valsesia, N.D. Beckmann, G. Thorleifsson, M. Belfiore, et al., Mirror extreme BMI phenotypes associated with gene dosage at the chromosome 16p11.2 locus, Nature, 478 (2011) J.A. Rosenfeld, B.P. Coe, E.E. Eichler, H. Cuckle, L.G. Shaffer, Estimates of penetrance for recurrent pathogenic copy-number variations, Genet Med, (2012). 17. C.P. Schaaf, R.P. Goin-Kochel, K.P. Nowell, J.V. Hunter, K.A. Aleck, S. Cox, A. Patel, C.A. Bacino, M. Shinawi, Expanding the clinical spectrum of the 16p11.2 chromosomal rearrangements: three patients with syringomyelia, Eur J Hum Genet, 19 (2011) M.F. Portnoi, N. Gruchy, S. Marlin, L. Finkel, F. Denoyelle, C. Dubourg, S. Odent, J.P. Siffroi, Y. Le Bouc, M. Houang, Midline defects in deletion 18p syndrome: clinical and molecular characterization of three patients, Clin Dysmorphol, 16 (2007) C.H. Brenk, E.C. Prott, D. Trost, A. Hoischen, C. Walldorf, B. Radlwimmer, D. Wieczorek, P. Propping, G. Gillessen-Kaesbach, R.G. Weber, H. Engels, Towards mapping phenotypical traits in 18p- syndrome by array-based comparative genomic hybridisation and fluorescent in situ hybridisation, Eur J Hum Genet, 15 (2007) J.E. Ming, M. Muenke, Multiple hits during early embryonic development: digenic diseases and holoprosencephaly, Am J Hum Genet, 71 (2002) C. Bendavid, L. Rochard, C. Dubourg, J. Seguin, I. Gicquel, L. Pasquier, J. Vigneron, A. Laquerriere, P. Marcorelles, C. Jeanne-Pasquier, C. Rouleau, S. Jaillard, et al., Array-CGH analysis indicates a high prevalence of genomic rearrangements in holoprosencephaly: an updated map of candidate loci, Hum Mutat, 30 (2009) Juan-Mateu J, Gonzalez-Quereda L, Rodriguez MJ, Verdura E, Lazaro K, Jou C, et al. Interplay between DMD point mutations and splicing signals in Dystrophinopathy phenotypes. PLoS One. 2013;8(3):e Hegde MR, Chin EL, Mulle JG, Okou DT, Warren ST, Zwick ME. Microarray-based mutation detection in the dystrophin gene. Hum Mutat. 2008;29(9): Juan-Mateu J, Rodriguez MJ, Nascimento A, Jimenez-Mallebrera C, Gonzalez- Quereda L, Rivas E, et al. Prognostic value of X-chromosome inactivation in symptomatic female carriers of dystrophinopathy. Orphanet J Rare Dis. 2012;7: Monaco AP, Bertelson CJ, Liechti-Gallati S, Moser H, Kunkel LM. An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics. 1988;2(1): Aartsma-Rus A, Van Deutekom JC, Fokkema IF, Van Ommen GJ, Den Dunnen JT. Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve. 2006;34(2):

69 27. Stockley TL, Akber S, Bulgin N, Ray PN. Strategy for comprehensive molecular testing for Duchenne and Becker muscular dystrophies. Genet Test. 2006;10(4): Schwartz M, Duno M. Improved molecular diagnosis of dystrophin gene mutations using the multiplex ligation-dependent probe amplification method. Genet Test. 2004;8(4): Janssen B, Hartmann C, Scholz V, Jauch A, Zschocke J. MLPA analysis for the detection of deletions, duplications and complex rearrangements in the dystrophin gene: potential and pitfalls. Neurogenetics. 2005;6(1): Borun P, Kubaszewski L, Banasiewicz T, Walkowiak J, Skrzypczak-Zielinska M, Kaczmarek-Rys M, et al. Comparativehigh resolution melting: a novel method of simultaneous screening for small mutations and copy number variations. Hum Genet. 2014;133(5): Zeng F, Ren ZR, Huang SZ, Kalf M, Mommersteeg M, Smit M, et al. Array-MLPA: comprehensive detection of deletions and duplications and its application to DMD patients. Hum Mutat. 2008;29(1): Hamed SA, Hoffman EP. Automated sequence screening of the entire dystrophin cdna in Duchenne dystrophy: point mutation detection. Am J Med Genet B Neuropsychiatr Genet. 2006;141B(1): Wang Y, Yang Y, Liu J, Chen XC, Liu X, Wang CZ, et al. Whole dystrophin gene analysis by next-generation sequencing: a comprehensive genetic diagnosis of Duchenne and Becker muscular dystrophy. Mol Genet Genomics Grimm T, Kress W, Meng G, Muller CR. Risk assessment and genetic counseling in families with Duchenne muscular dystrophy. Acta Myol. 2012;31(3): Laing NG, Davis MR, Bayley K, Fletcher S, Wilton SD. Molecular diagnosis of duchenne muscular dystrophy: past, present and future in relation to implementing therapies. Clin Biochem Rev. 2011;32(3): White SJ, den Dunnen JT. Copy number variation in the genome; the human DMD gene as an example. Cytogenet Genome Res. 2006;115(3-4): Yan J, Feng J, Buzin CH, Scaringe W, Liu Q, Mendell JR, et al. Three-tiered noninvasive diagnosis in 96% of patients with Duchenne muscular dystrophy (DMD). Hum Mutat. 2004;23(2): Dent KM, Dunn DM, von Niederhausern AC, Aoyagi AT, Kerr L, Bromberg MB, et al. Improved molecular diagnosis of dystrophinopathies in an unselected clinical cohort. Am J Med Genet A. 2005;134(3): Dolinsky LC, de Moura-Neto RS, Falcao-Conceicao DN. DGGE analysis as a tool to identify point mutations, de novo mutations and carriers of the dystrophin gene. Neuromuscul Disord. 2002;12(9): Takeshima Y, Yagi M, Okizuka Y, Awano H, Zhang Z, Yamauchi Y, et al. Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center. J Hum Genet. 2010;55(6): White S, Kalf M, Liu Q, Villerius M, Engelsma D, Kriek M, et al. Comprehensive detection of genomic duplications and deletions in the DMD gene, by use of multiplex amplifiable probe hybridization. Am J Hum Genet. 2002;71(2):

70 42. White SJ, Aartsma-Rus A, Flanigan KM, Weiss RB, Kneppers AL, Lalic T, et al. Duplications in the DMD gene. Hum Mutat. 2006;27(9): Flanigan KM, Dunn DM, von Niederhausern A, Soltanzadeh P, Gappmaier E, Howard MT, et al. Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort. Hum Mutat. 2009;30(12): Hofstra RM, Mulder IM, Vossen R, de Koning-Gans PA, Kraak M, Ginjaar IB, et al. DGGE-based whole-gene mutation scanning of the dystrophin gene in Duchenne and Becker muscular dystrophy patients. Hum Mutat. 2004;23(1): Bunyan DJ, Skinner AC, Ashton EJ, Sillibourne J, Brown T, Collins AL, et al. Simultaneous MLPA-based multiplex point mutation and deletion analysis of the dystrophin gene. Mol Biotechnol. 2007;35(2): Ferlini A, Neri M, Gualandi F. The medical genetics of dystrophinopathies: molecular genetic diagnosis and its impact on clinical practice. Neuromuscul Disord. 2013;23(1): Marquis-Nicholson R, Lai D, Lan CC, Love JM, Love DR. A Streamlined Protocol for Molecular Testing of the DMD Gene within a Diagnostic Laboratory: A Combination of Array Comparative Genomic Hybridization and Bidirectional Sequence Analysis. ISRN Neurol. 2013;2013: Prior TW, Bridgeman SJ. Experience and strategy for the molecular testing of Duchenne muscular dystrophy. J Mol Diagn. 2005;7(3): Gatta V, Scarciolla O, Gaspari AR, Palka C, De Angelis MV, Di Muzio A, et al. Identification of deletions and duplications of the DMD gene in affected males and carrier females by multiple ligation probe amplification (MLPA). Hum Genet. 2005;117(1): Hoffman EP, Fischbeck KH, Brown RH, Johnson M, Medori R, Loike JD, et al. Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy. N Engl J Med. 1988;318(21):

71 Section II Past, present and future 1. Professional, scientific and academic contributions 1.1 Career overview My entire career is presented in the Curriculum vitae included in Section IV. However, I considered useful to insert here a very brief synthesis of my experience to facilitate the interpretation of the scientific achievements presented in detail in Section I and their correlation with future research directions mentioned below. Thus, the most representative landmarks of my activity include: Fully trained (consultant) in two medical specialties Pediatrics and Medical Genetics; Progressive development of the academic career up to the level of Professor in the Medical Genetics Department of Gr T Popa University of Medicine and Pharmacy in Iaşi; PhD thesis in the field of X-linked intellectual disability; 290 papers since PhD thesis presentation, 60 of them in extenso, 15 of them published in ISI journals; 16 books written in the field of Medical Genetics or Pediatrics between 2000 and 2015, 7 of them as first/ single author; 97 citations (h-index 6); 12 research grants, 5 of them won by international competition (national coordinator in all of them) and 6 won by national competition (in two of them project director and in one administrative manager). It should be noted that one of these project was classified on the first place (out of 200 projects) in the national competition; As a recognition of the academic activity, appointed member of the Profesoral Council and member in different commissions of the university; Coordinator of Iasi Regional Center of Medical Genetics and responsible with 3 national health programs (since 2010); Member of the National Council for Rare Disorders (extended specialty commission of the Health Ministry); Appointed member of the editorial board of European Journal of Medical Genetics (since 2005); Founder member and treasurer of the Romanian Society of Medical Genetics; later on elected vicepresident ( ); Special prizes: best reviewer of the European Journal of Medical Genetics in 2011; prize of the Romanian Medical Council in 2014 for Orphanet project; 1 st prize in the 71

72 dysmorphology quiz of the German Romanian Human Genetics Course (2 nd and 3 rd edition). 1.2 PhD thesis Title: X-linked mental retardation (XLMR); Supervisor: Prof Dr. Ioan Tansanu; Presentation: ; Abstract Objectives: 1. Optimization of clinical evaluation, selection and exclusion criteria for XLMR; 2. Optimization of clinical and differential diagnosis in different forms of XLMR; 3. Study importance of different XLMR categories in the studied group; 4. Molecular study of some XLMR families; 5. Discuss genetic counselling issues in XLMR; 6. Discuss care optimization in XLMR; 7. Present some particular XLMR cases; Group definition and research methodology: the cases were selected from 2 major sources Medical Genetics Center, Children s Hospital Iaşi, Romania (1,132 files examined, 328 children evaluated) and institutions in Iaşi county (1,561 filed examined, 1,027 children evaluated); I have selected initially the cases upon the presence of at least 2 affected males in the family and a suggestive phenotype for a certain XLMR syndrome; I have identified groups (syndromic XLMR 97 cases; nonsyndromic XLMR 27 cases; nonspecific XLMR 106 cases; suspicion XLMR 243 cases); the cases were completely evaluated (clinical, paraclinical); for syndromic XLMR cases I have analysed the frequency of clinical features, introduced algorithms and diagnostic scores, performed molecular tests for some disorders (Fragile X, DMD, Aarskog, dyskeratosis congenital, Lowe), analysed the efficiency of computerized diagnostic software, and I have introduced diagnostic and follow-up protocols and analysed genetic counselling aspects. General conclusions: Clinical evaluation of the MR child is difficult (I have provided solutions); I have established a scale of the clinical evaluation methods ( diagnostic utility); I have adapted selection criteria (more cases in the family, no social cases etc) I have used 2 categories of exclusion criteria ( nongenetic / not X-linked); I have adapted XLMR classification upon associated signs (new syndrome grouping, new features); I have proposed adding X chromosome abnormalities to monogenic forms; I have designed diagnostic algorithms for syndromic XLMR upon associated defects; In Fragile X and Aarskog syndrome I have analysed feature and feature association frequency and importance; Based on literature data I have designed diagnostic scores (upon sex and age); 72

73 I have compared computerized diagnostic software; found abnormal frequencies ( Aarskog, Fragile X); I have introduced in our lab practice DNA extraction and Fragile X test; molecular tests performed identified particular results; I have proposed evaluation protocols and a discussion plan to be used for genetic counselling in XLMR; the study identified particularities of Romanian institutions; ethical aspects were discussed. 1.3 Academic activity I shall refer below to the most important achievements in the field of teaching activities, dissertations coordinated, books published, as well as my attendance in different committees and commissions of the Gr T Popa University. A brief synthesis of the practical lessons and lectures taught during my career is presented in the table below. Please note that all these activities are practical, interactive and centred on the specific of the year of study (e.g. teeth anomalies discussed for Dental Medicine students). The content is permanently updated. Table 1.3.1: Teaching activities during my postdoctoral career ( ) Med Dental med Phar macy Nutri tion Opt Med Residents Ped, Gen Col nurses Opt Col nurs Lecture Practical lesson Romanian English Apart of the basic curricula, I have introduced optional and facultative lectures, in an attempt to reinforce knowledge referring to practical issues related to Medical Genetics (i.e. to recognize, be able to name and know how to prevent common plurimalformative syndromes; to be able to draw a pedigree and to recognize typical patterns; to know the major indications of genetic testing, genetic counselling and prenatal diagnosis etc). The list of newly introduced lectures includes: Introduction to Dysmorphology clinical recognition, elements of antropometry, molecular pathogeny of birth defects (Medicine Romanian series); Clinical Genetics (Medicine English series); Management complexity in genetic disorders (College of nurses). 73

74 Based on the theoretical and practical experience accumulated, between 2000 and 2015 I have supervised 37 dissertations (in Romanian and in English), all of them in the field of Clinical Genetics. The major aim for every thesis was to teach the students how to do a literature review (how to select good articles, how to extract useful information and then how to build a proper review) and a basic clinical study (which information should be extracted from every file, how to organize them, how to do simple statistical interpretations, how to correlate the results and how to formulate conclusions). Students also learned how to do a references list and how to design and to present a Power Point presentation. All the theses passed the final evaluation with maximum mark. My academic activity also includes writing books. In the interval mentioned above, I have written or contributed to 16 books, all of them in the field of Medical Genetics and Paediatrics. It should be noted the chapter Mental retardation written for the textbook Medical Genetics (1 st and 2 nd Edition, main authors M. Covic, D. Ştefănescu, I. Sandovici), because it represents the synthesis of my own experience gathered during my PhD study, as well as during my postdoctoral research. Another book of interest is Usual techniques for the screening and diagnosis of genetic disorders, book published as a coordinator. It reflects the experience of many specialists working in Medical Genetics, in an attempt to familiarize young doctors or specialists from other specialties with the basic principles of the most used techniques in Cytogenetic and Molecular laboratories, as well as the indications of these tests and the interpretation of test results. Manuals of interest are both editions of the English manuals (both for lectures and practical lessons), books that contain practical information presented in a concise, yet comprehensive way, with many applications and keep in mind sections that summarize essential information. Concerning my attendance in different commissions of the university, I think I should mention that I have attended the commission for the entrance exam for graduate students every year, as well as the residency exam in the last 2 years. Since 2012 I was appointed member of the commission that selects overseas candidates that apply to become students of Gr T Popa University of Iaşi. I have also been a member of many promotion and doctoral commissions in our own and in other universities, as well as member of commissions for specialist or consultant degree exam in Medical Genetics. Moreover, I have taught postgraduate lectures and supervised the practical training of residents in different specialties (Medical Genetics, Paediatrics, Endocrinology etc) and I have been a tutor of graduate students (Romanian and English series) in the last 5 years. And last, but not the least, I have been appointed member of the board of professors in our university for two years. For all these jobs I have accomplished my mission as good as I could, trying to get the maximum benefit for all the parts involved and to encourage people to develop their potential. 74

75 1.4 Research projects My research projects developed in parallel with my postdoctoral work. The topics concentrated in the beginning on intellectual disability (mental retardation) and introduced new lab techniques for Romania at that time. Later on, my research extended to other fields of Clinical Genetics and also to rare disorders. The entire list of my research grants is presented below, with detailed data for each of them, including results. 1) Competition: 2004 ; Title: Fragile X syndrome diagnosis optimization by introducing immunohistochemical tests in the investigation protocol of mentally retarded patients; Contract number: Grant CNCSIS 1242/ 2004; Period: ; Project director: Cristina Rusu; Value: RON ( ROL); This project was the first one to continue my doctoral work. In my PhD research (dedicated to X-linked mental retardation/ intellectual disability) - XLID - I have tried to identify as many cases as possible and out of these to check if the proportion between different XLID syndromes is the same like in the literature. The literature mentions that half of the XLID cases are represented by Fragile X syndrome, but in spite of the efforts, I found very few cases. So, the mission of this project was to introduce a screening test (inexpensive, reliable, technically simple and accessible) to be used in any boy with ID without a specific diagnosis and for cases with family history of ID suggestive of an X-linked inheritance. At the moment there were 2 methods for the immunohistochemical testing for Fragile X syndrome (anti-fmrp test) to be used as a screening test done on blood smear or on hair root. I have followed a short training in the Medical Genetics Department of Erasmus University, Rotterdam, Netherlands and learned both techniques. The technique performed on blood smear proved to be technically more difficult, more dangerous in our conditions (because we were working with blood) and unpleasant for the patient. Finally, we have applied the technique on hair root in our patients. 75

76 Fig 1.4.1: Anti-FMRP testing specific immunohistochemical staining done on blood smear (left) and on hair root (right). Upper images positive testing (pink staining confirms the presence of the FMRP protein). Lower images negative testing (no staining means FMRP protein is absent, thus confirming the diagnosis of Fragile X syndrome) The major conclusions of the study were: Anti-FMRP testing is a reliable technique to be used for the screening of Fragile X syndrome in males, especially for countries with reduced financial resources that cannot afford extensive molecular testing; however, the suspicion of diagnosis has to be confirmed by specific molecular tests (PCR + Southern blot/ Dichblotting); In females, the results of anti-fmrp testing are very difficult to evaluate and in families known with Fragile X syndrome for an accurate genetic counselling molecular tests should be applied; Indeed, the frequency of Fragile X syndrome seems to be lower in our region compared to literature data (and a more extensive study is needed to confirm this hypothesis); Suggestive family history is a key issue when selecting a case for further genetic testing. Positive and negative results from the study are presented in Fig The technique was introduced in the daily practice of our Medical Genetics Centre and used for 3 years, until molecular testing has become more affordable for us. 76

77 2) Competition : 2006 ; Title: Optimization of diagnosis and management of mentally retarded patients by introducing MLPA test in the evaluation protocol; Contract number: Grant CNCSIS 832/2006; Period: ; Project director: Cristina Rusu; Value: RON; This project continued my research in the field of ID. MLPA testing was a newly described molecular method at that time. Subtelomeric rearrangements were recently described as a major cause of nonspecific ID and MLPA kits were just introduced for the detection of subtelomeric deletions and duplications, so we organized a national partnership and submitted the application. The project was classified the first out of 200 competitive projects in the national competition and represented a major success. We have thoroughly designed the investigation protocol to enable us to exclude other major causes of ID. Only patients with multiple birth defects associated to ID have been selected, by using De Vries score proposed in the literature. The investigation of the patient was performed in a specific sequence: Detailed family and personal history: positive family history was a good point; events during the pregnancy or birth trauma could explain ID and exclude the patient from testing; Karyotype: detects relatively big chromosomal defects as a major cause of ID; if a defect was found, parents and eventually other family members were tested subsequently; if the karyotype was normal, the investigation proceeded to the nest step; If the phenotype was suggestive of a microdeletion syndrome (e.g. Williams or Prader Willi syndrome), specific FISH testing was performed; Fragile X testing (anti-fmrp test followed by long range PCR) for cases that associate relatively normal motor development and speech delay or cases with ID and autistic features or if family history was suggestive of XLID; if the test was normal, we went to the next step; MLPA testing using 2 different kits for subtelomeric rearrangements (P036 and P070) to enable us to exclude polymorphisms. MLPA testing was first done in Bucharest and in the second part of the project (since 2007, after a short training in the Medical Genetics Department of Radboud University, Nijmegen, Netherlands) we have introduced it in our medical practice. During the project we have tested almost 200 cases from all over the country (as we were a national partnership) and our detection rate has been 8% (compared to 5% in the literature). 77

78 The major conclusions of this study included: MLPA is an inexpensive, simple and reliable testing for the detection of subtelomeric rearrangements as a cause of nonsyndromic ID; the method is very useful if financial resources are limited; MLPA should be used as a screening test and positive results should be confirmed with another method (e.g. FISH testing); The evaluation in steps, as well as the application of a clinical diagnostic score are important points that increase detection rate; Two different kits (complementary) should be used to enable polymorphism exclusion; Particularities of each kit should be known when interpreting data (some probes do not work very well) to avoid misdiagnosis; the use of 2 kits helps with this difficulty. Some examples of results from the project are presented in Fig After the end of the project we have continued to use MLPA testing for the detection of subtelomeric rearrangements in our daily practice and we have extended the use of the method for other applications (e.g. microdeletions, Prader Willi/ Angelman syndrome using a kit that detects also methylation defects, Fragile X syndrome, autism, Duchenne muscular dystrophy and spinal muscle atrophy). After detecting subtelomeric rearrangements or microdeletions we use MLPA follow-up kits that are much better than FISH testing for confirmation, because they enable us to appreciate the size of the defect at a much lower price. We have also extended the use of subtelomeric MLPA to the detection of complex chromosomal rearrangements or minute chromosomes. 78

79 Fig 1.4.2: Above clinical and MLPA data for 1p del (1 st case identified in the subtelomeric MLPA study for nonspecific ID); Below 7q del correlated with sacral agenesis; 3) Competition: 2006; Title: Interdisciplinary platform of molecular medicine; Contract number: Grant platformă CNCSIS 29/2006; Contract number 32/ ; Period: ; Rusu; Project director: Prof. Eugen Carasevici; coordinator administrative activity Cristina Value: RON; The project has introduced a molecular platform to be used by many disciplines of Gr T Popa University of Medicine. It represented the base for many subsequent projects and articles. 79

80 4) Competition: 2006 ; Title: Dyscerne a network of centres of expertize for dysmorfology; Contract number: European Commission Public Health Executive Agency (DG Sanco) Project: ; Period: ; Project director: Prof. Jill Clayton Smith, United Kingdom; Cristina Rusu responsible with the case submission node in Romania (Iasi); This project represented an European network for Dysmorphology that joined 85 centres (32 centres of expertize, the rest of 53 centres being case submission nodes for a webbased electronic diagnostic systems). The major aim of this project was to help with the diagnosis and investigation in complex cases. Usually, individual cases were submitted to a national case submission node (Iaşi for Romania), from where data were introduced in the system. Depending on the type of case, appropriate specialists were selected and after data analysis they used to provide diagnostic suggestions and eventually investigations to be done. The site also contained an educational part, aiming to guide and educate clinicians on key aspects of clinical dysmorphology. The network of specialists in the field also developed best practice management guidelines for Angelman, Kabuki, Noonan and Williams syndrome Dyscerne project represented an excellent opportunity to diagnose some of our most special cases. An example of case submission solved is presented in Fig

81 Fig 1.4.3: Model of form for a case submitted to Dyscerne network 81

82 Ratio ACP CHL IRF BET1L NDN STCH IL17R X SHOX HABILITATION THESIS 5) Competition: 2008; Title: Correlation of clinical, genetic and epigenetic aspects involved in the etiology of Prader Willi/ Angelman syndrome: model of multidisciplinary approach of rare disorders in Romania; Contract number: Grant CNMP (Parteneriat) PN II / 2008; Period: ; Rusu; Project director: Assoc. Prof. Maria Puiu; responsible partner 5 (UMF Iaşi): C Value: RON (UMF Iaşi RON); This project represented a national partnership that joined major Genetics Centres, cytogenetic and DNA labs and Romanian Prader Willi Association (patient support group) in an attempt to introduce in Romania complete patient evaluation (clinical and molecular) and early diagnosis (to enable growth hormone therapy for the benefit of the patients). The most important conclusions for our centre have been: We have increased awareness concerning this disorder in our region and cases are sent for evaluation at a much younger age, for the benefit of the patient and the family; every floppy infant sent for evaluation in our Medical Genetics Centre is tested both for spinal muscular atrophy and Prader Willi syndrome (dedicated MLPA kits); We have introduced the diagnostic score in our practice, increasing thus the efficiency of genetic testing; A multidisciplinary team (geneticist, paediatrician, endocrinologist, nutritionist, psychiatrist, orthopaedist) familiarized with the particularities of this syndrome has been formed; 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0 P D08_ IO^CEQsCSV.csv Mapview Fig : Clinical features of an atypical Prader Willi patient that proved to have a subtelomeric 2q deletion 82

83 We have compared the diagnostic efficiency of the association karyotype + FISH with the Prader Willi MLPA kit (that checks in the same test both for microdeletions and for methylation defects) and proved that the last one is less time-consuming and less expensive, reason why we have introduced it in our daily practice; the kit can be also used for the diagnosis of Angelman syndrome patients; Special patients that were considered as atypical Prader Willi syndrome have been diagnosed with other disorders (e.g. subtelomeric deletion 2q) after complete genetic investigation; Some particularities have been identified (high rate of allergies, seizures etc) and published this to increase awareness of the practitioners taking care of Prader Willi patients. Fig : MLPA test results using ME028 Prader Willi/Angelman MRC Holland kit (above deletion; below imprinting defect of the sequence involved in PWS ) 83

84 Some of the results found are illustrated in Fig (an atypical Prader Willi case that proved later to be subtelomeric deletion 2q) and in Fig (typical results using the dedicated Prader Willi kit that investigates for both microdeletions and imprinting defects). 6) Competition: 2008; Title: Optimization and implementation of some molecular biology techniques for the detection of hereditary predisposition to breast and ovarian cancer; Contract number: Grant tip Idei; Period: ; Project director: Senior lecturer Anca Mihaela Negură; Cristina Rusu team member; Value: RON. This project aimed to familiarize specialists in other specialties than Medical Genetics with the family history/ pedigree suggestive for genetic breast and ovarian cancer (associated with a high recurrence risk in the family). Another major aim was to introduce BRCA testing for families with typical pedigree, aim accomplished successfully. 7) Competition: 2014; Title: Implementation of a diagnostic algorithm based on complex analysis of the genomic profile for patients with congenital and developmental anomalies; Contract number:?; Period: ; Project director: Carmen Diaconu; Cristina Rusu team member; Value: RON. The project represents a consortium of three Medical Genetics Centres, aiming to evaluate patients with birth defects using a complex protocol (PCR, MS-MLPA, sequencing, array CGH and Next Generation Sequencing). The workload just started, we cannot discuss results yet. 8) Competition: 2011 internal grants UMF Iaşi (registered number 17232); Title: Building of an integrated model for the evaluation of Clopidogrel resistance by genotyping Cyp2c19 cytochromes and ABCB1 gene; Contract number: 28214/ ; Period: ; Project director: Costache Irina Iuliana; Rusu Cristina team member; 84

85 Value: Euros. The major aim of this project was to introduce a quick and accessible test to evaluate Clopidogrel responsiveness of critically ill patients with heart attacks. The study reavealed that a high proportion of patients (95%), due to specific polymorphisms, do not respond appropriately to Clopidogrel therapy (see Table 1.4.1). This indicates the use of alternative drugs with subsequent improvement of patients survival and quality of life. Table 1.4.1: Polymorphisms related to Clopidogrel metabolism (allele/ genotype frequency) Polimorfism Genotip Numar probe Frecventa CC 12 15% ABCB1 c.c3435t CT 48 60% TT 20 25% Alela T 55% GG % CYP2C19*2,c.G681A GA % AA % Alela A 15.3% CC % CYP2C19*3,g.-806C>T CT 0 0% TT 0 0% Alela T 0% CC 4 5% CYP2C19*17, c.c806t CT % TT % Alela T 78.1% 9) Competition 2006; Title: Orphanet PC7 (Rare Disease Portal) (European Project - collaboration INSERM France European Comission); Contract number: ; Period: ; Project director: Segolene Ayme, France; national coordinator for Romania Prof. Mircea Covic , Cristina Rusu ; 10) Competition: 2009; Title: Development of the European portal of rare diseases and orphan drugs - Joint Action for Orphanet (Rare Disease Portal 2) (European Project - collaboration INSERM France European Comission); Contract number: ; Period: ; 85

86 Project director: Segolene Ayme, France; Cristina Rusu national coordinator for Romania; Value: Euro; 11) Competition: 2010; Title: Orphanet Europe (European Project - collaboration INSERM France European Comission); Contract number: ; Period: ; Project director: Segolene Ayme, later Odile Krempf and then Anna Rath, France; Cristina Rusu national coordinator for Romania; Value: Euro; 12) Competition: 2015 Title: Promoting Implementation of Recommendations on Policy, Information and Data for Rare Diseases (European Project - collaboration INSERM France European Comission); Contract number: RD-ACTION 3rd Health Programme Type of action: HP-PJ Proposal number: Call: HP-JA-2014 Topic: JA ; Period: ; Project director: Anna Rath, France; Cristina Rusu national coordinator for Romania; Orphanet projects became more and more complex in time. The major aim is to increase awareness on rare disorders, to facilitate specialists access to updated literature and legislation in the field, as well as patients access to specialists and diagnostic services. The project started like a free Internet encyclopedia with permanently updated information both for specialists and patients. In time, major changings have been added, including: Translation of the information into other major languages apart of English (French, Spanish, German, Italian, Portughese and Dutch); Leaflets with data of interest (e.g. prevalence data for the most important rare disorders); European documents in the field of rare disorders; National Orphanet entry point with the key information translated into the native language of the country; Orphacode and corresponding code in other widely used databases (e.g. ICD-10, OMIM, UMLS, MeSH, MedDRA); If the diagnostic service is accredited or not; 86

87 Research projects and clinical trials in the field; Age of onset and Emergency guidelines; Permanently updated information referring to the medicines used for the therapy of specific rare disorders; Patient support groups; Connection to other databases (e.g. OMIM, Gene reviews etc); Orphanews (new data in the field of rare disorders published every 2 weeks) and Orphanet Journal of Rare Disorders (highly rated ISI journal); Extension of the partnership to other non-european countries (Australia, Canada, Israel and Japan). Our attendance in these European projects represented a major advance as the information referring to Romanian specialists, services, research projects and patient support groups has become more accessible to the public (patients and specialists in other fields treating patients with rare disorders). It also contributed to the prestige of Gr.T. Popa University of Medicine and Pharmacy, because the university has been nominated coordinator center for Romania. It also facilitated the development of the National Plan for Rare Disorders in Romania. An illustration of the Romanian Orphanet entry point is presented in Fig Fig : Illustration of the Romanian Orphanet entry point. 87

88 13) Competition: 2010; Title: Parents, back to school! Contract number: 8386/ ; Period: ; Project director: Crăciun Izabela Bethany Social Services Foundation; Rusu Cristina representative of Sf Maria Iaşi Children s Hospital; Value: RON (Iasi local council contribution , cofinancing Bethany Social Services Foundation ). This project aimed to support parents with children with different disorders (e.g. Down syndrome, autism, seizures etc) by teaching them about the causes, clinical features, management plan and recurrence risk of the specific disorder. A multidisciplinary team has been involved (geneticist, paediatrician, psychologist, social assistant etc) and team discussions proved very useful for all the parts involved. The project has been really welcome and the parents were very content to find out more about the disorder of their child. 1.5 International recognition Some of the criteria that justify my international visibility include: Appointment in the editorial board of the European Journal of Medical Genetics (2005) and the prize for the best reviewer of the journal (2011); Two invitations of the Elsevier Publishing House to present conferences on how to get published ( How to Write a World Class Paper From Title to References, From Submission to Revision ) in three major university centres within Romania (Iaşi , Cluj Napoca , Bucureşti ); Invitation of the organizing committee of the European Conference of Rare Diseases to present the situation of the expertize centres for rare disorders in Romania (5th European Conference on Rare Diseases Krakow, Poland, ); Invitation of the European Society of Human Genetics to attend an ESHG workshop (ESHG Symposium, Clinical Genetics Society Meeting, Liverpool, UK, ) and present the situation of the Medical Genetics Specialty in Romania; Delegated by the Romanian Health Ministry for the European Comission session where the European recognition of the Medical Genetics specialty was debated (2009); also delegated for the EUCERD (European Comission of Experts in Rare Disorders) meeting on how to introduce Orphacodes in the national health codification system (2014); As a vicepresident of the national society, invited to represent Romanian Society of Medical Genetics in the Balkan Conference of Human Genetics (8th Balkan Meeting on Human Genetics, Cavtat-Dubrovnik, Croatia, ) and in the meetings of the International Federation of the National Societies of Human Genetics ( ); 88

89 97 international citations (h-index 6) See Fig Fig : Citations extracted from Scopus. 89