IMPROVING THE DIAGNOSIS AND PREVENTION OF MENTAL RETARDATION DUE TO GENETIC FACTORS

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1 Investeşte în oameni! Proiect cofinanţat din Fondul Social European prin Programul Operaţional Sectorial Dezvoltarea Resurselor Umane Axa prioritară Educatia si formarea profesională în sprijinul cresterii economice si dezvoltării societătii bazate pe cunoastere Domeniul major de intervenţie 1.5 Programe doctorale si post-doctorale în sprijinul cercetării Titlul proiectului: Burse doctorale pentru cresterea competitivitatii in domeniul medical si farmaceutic Numărul de identificare al contractului: POSDRU/88/1.5/S/58965 Beneficiar : Universitatea de Mdicina si Farmacie Gr. T. Popa Iasi Partener : Universitatea de Medicina si Farmacie Iuliu Hatieganu Cluj Napoca UNIVERSITY OF MEDICINE AND PHARMACY GRIGORE T.POPA IAŞI FACULTY OF MEDICINE IMPROVING THE DIAGNOSIS AND PREVENTION OF MENTAL RETARDATION DUE TO GENETIC FACTORS SUMMARY OF THESIS SCIENTIFIC COORDINATOR, PROF. UNIV. DR. MIRCEA COVIC DOCTORAL STUDENT, ADRIANA SIRETEANU 2013

2 TABLE OF CONTENTS I. INTRODUCTION... 1 II. CURRENT STATE OF KNOWLEDGE GENERAL INFORMATION ABOUT MENTAL RETARDATION Definition Classification and prevalence Etiology GENETIC CAUSES OF MENTAL RETARDATION Chromosomal abnormalities Monogenic forms of mental retardation MOLECULAR AND CELLULAR MECHANISMS UNDERLYING MENTAL RETARDATION Neurogenesis Neuronal migration Synaptic function Epigenetic mechanisms METHODS FOR INVESTIGATION OF GENETIC CAUSES OF MENTAL RETARDATION Chromosome analysis and chromosome region specific techniques DNA microarray technology Next-generation sequencing technologies PREVENTION OF MENTAL RETARDATION DUE TO GENETIC FACTORS III. PERSONAL CONTRIBUTIONS PATIENTS AND METHODS General methods Study groups CYTOGENETIC STUDY OF IDIOPATHIC MENTAL RETARDATION Introduction Patients and methods Results Discussion Conclusions OPTIMIZATION OF DIAGNOSIS FOR PATIENTS WITH MENTAL RETARDATION DUE TO GENETIC FACTORS USING MLPA TECHNIQUE... 49

3 3.1. Introduction Patients and methods Results Discussion Conclusions SNP ARRAY TECHNOLOGY BENEFITS FOR DIAGNOSIS OF SPECIFIC MENTAL RETARDATION (A STUDY OF 20 CASES) Introduction Patients and methods Results Discussion Conclusions OPTIMIZATION OF DIAGNOSIS OF AARSKOG SYNDROME AND FRAGILE X SYNDROME Molecular and phenotypic characteristics of patients with Aarskog syndrome Optimization of diagnostic strategy for patients with fragile X syndrome IV. GENERAL CONCLUSIONS REFERENCES ANNEX 1. LIST OF PUBLICATIONS The thesis is illustrated with 73 figures and 27 tables. The summary includes a limited number of figures, retaining their numbering from thesis. The bibliography includes 382 references. Keywords: mental retardation, copy number variations, massively parallel sequencing, Multiplex Ligation-dependent Probe Amplification, microarray technology, Aarskog-Scott syndrome, fragile X syndrome. The research was conducted within the project called Doctoral Scholarships for increasing competitiveness in the medical and pharmaceutical field POSDRU/88/1.5/S/58965, co-funded by the European Social Fund through the Sectoral Operational Program for Human Resources Development

4 SECTION I. INTRODUCTION Mental retardation (MR) is a common disorder and has major consequences for individual, family and society. Causes of MR are extremely heterogeneous, the share of genetic causes being different, according to MR severity. Due to clinical and genetic heterogeneity of MR, in about 50% of cases an etiologic diagnosis cannot be established, despite extensive investigations. Establishing a specific and precise diagnosis of MR, including its etiology and pathogenesis, will have benefits for the patient (eliminating unnecessary and expensive tests, establishing adequate medical and therapeutic actions, presymptomatic detection of complications, educational planning and social intervention), but also for the patient's family (anticipatory guidance, medical, social, and educational support, genetic counseling and reproductive options). The thesis main objective is the optimization of evaluation of MR patients by selecting genetic testing methods which provide the best results (early, complete and correct diagnosis) with convenient costs. Another objective is to determine the frequency of various etiologic types of MR in the studied group, and to update clinical data and mutation spectrum in MR patients. CHAPTER III.2 CYTOGENETIC STUDY OF IDIOPATHIC MENTAL RETARDATION 2.1. Introduction G-banded chromosome analysis is a routine procedure in clinical cytogenetics laboratories and is particularly important in assessing individuals with MR or developmental delay. The percentage of clinically relevant chromosomal abnormalities identified by standard karyotype is difficult to determine accurately, as studies differ in terms of location (pediatric clinic or institution for children with MR), MR severity, presence or absence of congenital anomalies, and cytogenetic study type. The objective of this study was to determine the frequency and types of chromosomal abnormalities identified by standard karyotype in a group of 319 patients with idiopathic syndromic MR. 1

5 2.2. PATIENTS AND METHODS This retrospective study was based on 319 syndromic MR cases referred to our centre during the period , for whom chromosome analysis was performed. Patients referred for suspected Down syndrome were excluded. Standard chromosome analysis was performed on phytohemagglutinin - stimulated peripheral lymphocyte cultures using a method adapted from Moorhead et al. [1]. We analyzed at least 16 metaphases at the resolution level of 550 bands on a Nikon microscope equipped with CCD camera; images were captured and analyzed using the CytoVision software. If two cell lines were detected, the number of analyzed methaphases was 32, and if three cell lines were detected, the number of analyzed methaphases was 64. Chromosomal abnormalities were described according to the international standard nomenclature (International Standard Nomenclature 2005) RESULTS Among the 319 MR cases, a chromosomal abnormality was identified in 84 cases (26.3%). 11.9% of all the analyzed patients (38 of 319) had chromosomal aberrations that are associated with wellestablished syndromes. Numerical abberations were detected in 40% of cases (34 of 84), out of which autosomal abnormalities accounted for 71% of cases (24 of 34). The gender distribution was relatively equal (16 male, 18 females). Structural abberations represented 53% of abnormal cases (44 of 84), the most common being deletions (46%). Complex abnormalities (numerical and structural abnormalities present in the same patient) were detected in 6 patients (7.1% of all abnormalities) DISCUSSION In this study, chromosomal abnormalities were detected in 26.3% of patients with MR (excluding Down syndrome). Various studies (reviewed by Battaglia [2]) reported that pathogenic chromosomal abnormalities detected by first karyotyping account for % of MR cases. Factors influencing the outcome of cytogenetic studies in MR are: selection and ascertainment source of cases (studies on institutionalized children often have a very rigorous selection of patients i.e. patients with severe MR), age of assessment (at a young age information about pre-, peri- and postnatal development is available), severity of MR (IQ<50 increases the proportion of 2

6 established diagnoses), advances in imaging and laboratory techniques, and clinicians experience [3]. The patients included in our study were initially selected in the territory, by the general practitioner (often based on MR association with obvious facial dysmorphism and/or multiple congenital anomalies) and then by the medical geneticist, based on the so-called "chromosomal phenotype" (which is caused by larger chromosomal abnormalities), characterized by moderate-to-severe MR associated to one or more major congenital abnormalities. CHAPTER III.3 OPTIMIZATION OF DIAGNOSIS FOR PATIENTS WITH MENTAL RETARDATION DUE TO GENETIC FACTORS USING MLPA TECHNIQUE 3.1. INTRODUCTION During last decade, the introduction of microarray technology made it possible to detect submicroscopic CNVs with a resolution of up to the size of a single exon, and led to the identification of numerous microdeletion and microduplication syndromes[4], now chromosome microarray being recommended as the first-line diagnostic test in patients with MR and/or multiple congenital anomalies [5]. This technology, however, requires expensive equipment and consumables that are not available to all diagnostic centers. In principle, some of the chromosomal abnormalities identified by microarray can also be detected using different MLPA kits. Given the difference in costs between the platforms for whole genome microarray and MLPA, screening MR patients using MLPA technique is a reasonable option in the diagnostic evaluation of MR patients, especially in developing countries. The aim of this study was to evaluate the ability of a combination of MLPA kits to establish the molecular diagnosis in a group of patients with syndromic MR PATIENTS AND METHODS This prospective study included 307 patients with syndromic idiopathic MR, aged over 3 months. Patients with phenotype suggestive of common aneuploidies (trisomies 21, 13, 18 and Turner syndrome) and fragile X syndrome were not included. All patients were assessed for chromosome imbalance using MLPA technique, with subtelomeric kits, if the phenotype was not suggestive of a microdeletion syndrome (subgroup A patients), or 3

7 microdeletion kits, if the phenotype was suggestive of a microdeletion syndrome (subgroup B patients). Patients diagnosed with Prader Willi syndrome based on consensus criteria established by Holm (subgroup C - 17 patients) were tested with Methylation-specific MLPA kit. Patients in subgroup A were tested with two MLPA kits: SALSA P036 (B1 and E1) and P070-B2. These kits have been developed to screen for subtelomeric copy number changes and contain one MLPA probe for each subtelomeric region, except for the acrocentric chromosomes, for which a probe on the q arm, close to the centromere is included. Abnormal results detected by both MLPA kits were further characterized using follow-up MLPA kits. Rearrangements sizes were determined using UCSC genome browser (NCBI36/hg18, Patients in subgroup B were tested with two MLPA kits (P064 or P096), that have been developed to screen patients with idiopathic MR for multiple microdeletion syndromes simultaneously. The results were interpreted as pathogenic when more than two probes from the same region showed abnormal results. Patients in subgroup C were tested with a Methylation-specific MLPA kit (ME028-B1) for Prader Willi/Angelman syndrome, that can detect copy number changes, but also to analyze CpG island methylation of the 15q11 region RESULTS Subgroup A Subgroup B included 176 patients with syndromic MR. 12 patients showed a singular aberrant telomeric signal (detected by only one of the two MLPA kits), 18 patients showed abnormal results detected by both MLPA kits (~10% of all cases), and 146 patients had normal results. We confirmed and further characterized the chromosome imbalances by follow-up kits for cases 7-18 (15 subtelomeric imbalances) Subgroup B Subgroup B included 114 mentally retarded patients with phenotype suggestive of a microdeletion syndrome. Using (depending on clinical suspicion) one of the two MLPA kits (P064 or P096) we identified 12 microdeletions (10.5%): 6 cases of velocardiofacial syndrome, 5 cases of Williams syndrome and 1 case of Wolf Hirschhorn syndrome. All microdeletions identified were confirmed by FISH test. 4

8 Subgroup C Subgroup C included 17 patients phenotype suggestive of Prader Willi syndrome. Using MS-MLPA we detected a 15q11.2-q13 type II heterozygous deletion (between breakpoints 2 and 3) in one patient (figure 3.7) and the presence of two methylated copies and normal dosage at 15q11.2-q13 in 4 patients. The deletion was confirmed by FISH test. Abnormal methylation profile of one of the four patients is illustrated in figure DISCUSSION Subgroup A In three cases (1, 2 and 7) the clinical significance of the anomaly was unclear, and in 15 cases the anomaly was considered pathogenic, being consistent with the phenotype. Taking into account the literature data and location of abnormalities (for case 1 and 2), as well as the size and gene content (for case 7), we considered that the CNV identified in case 1 is possibly pathogen, and those identified in cases 2 and 7 are most likely benign, but to confirm this it is necessary to test the parents. Figure 3.7. Heterozygous deletion on 15q11.2-q13 Type II deletion: ~50% reduced relative peak area of the amplification products from breakpoint 2 (MKRN3 gene) to breakpoint 3 (GABRB3 gene) 5

9 Figure 3.8. Detection of two methylated copies and normal dosage at 15q11.2-q13 Top: normal dosage on 15q11.2-q13 (relative peak area of the amplification products has a value of ~100 %). Bottom: hemimethylated probe-sample DNA hybrids are prevented from being digested by methylation-sensitive restriction enzyme HhaI and the ligated probes generate a signal. Relative peak area of the amplification products has a value of ~100 %, corresponding to two methylated copies. Therefore, by using MLPA kits P036 and P070 we established diagnosis in 15 of 176 individuals with syndromic MR (8.5%). The overall subtelomere rearrangements frequency in children with MR is 6%, ranging from 2 to 29% [6]. The reasons for this differences are the criteria for inclusion in the study, the size of the cohort and the complete exclusion (or not) of the polymorphisms. Taking into account only studies on large groups, the frequency of cryptic subtelomeric abnormalities is estimated at between 2.5% [6] and 2.6% [7]. Selecting patients based on the presence of idiopathic syndromic MR in this study we obtained a detection rate of 8.5%, above the average of 6% reported by Ravnan et al. [6], which illustrates the efficiency of using two subtelomeric screening kits in all patients and further evaluating abnormal results with follow-up kits. 6

10 The use of follow-up kit allows both confirmation of the chromosome imbalance and determining its size. Using different followup kits in 12 patients we determined the approximate size in 9 of 15 identified anomalies (60%). In addition, for two patients, in which subtelomeric screening showed the presence of deletions (5pter, 2qter), the follow-up kits (P358, P264) showed also the presence of proximal duplications. We thus managed to identify the additional material of unknown origin detected by standard karyotype in these two patients. In the literature there is only one study that uses follow-up kits for confirmation of the results and for establishing the size of the imbalances [8]; the authors were able to determine the size in two thirds of subtelomeric imbalances Subgroup B The incidence of microdeletion/microduplication syndromes was 10.5%, the majority (11 of 12) being identified using the P064 kit. Other studies that have used MLPA P064 kit had detection rates of % if patients were selected based on the presence of MR and/or multiple congenital anomalies [9, 10], and of 14,1%, if patients were selected based on phenotype suggestive of a microdeletion syndrome [9]. All these data points that submicroscopic abnormalities are involved in a large number of cases with MR/multiple congenital anomalies, and selection of patients based on phenotype suggestive of a microdeletion syndrome can increase detection rate Subgroup C Methylation specific MLPA (ME028 kit) is able to detect over 99% of cases of Prader Willi syndrome, combining both DNA methylation analysis and dosing analysis across the PWS region, but cannot distinguish between maternal uniparental disomy and imprinting center defects [11]. Using this kit we were able to establish diagnosis in 5 of 17 patients selected according to Holm s criteria (29.4%). Summarizing data from the three groups, the combined use of MLPA kits led to the diagnosis in 32 from 307 patients (10.4%). Other studies that have used a combination of three MLPA kits (subtelomeric screening and P064 kit for multiple microdeletion syndromes) in all patients had detection rates of 14% [8], and 20,7% [10], but the criteria for inclusion in the study were different. In a study of patients with MR and/or dysmorphic features the same three MLPA kits were used, but for separate groups of patients; the detection rate was 7.2% [12]. All these studies show that the combined use of MLPA kits has a relatively high 7

11 detection rate in patients with MR, similar to that of ~19% reported for microarray analysis. A recent study that compared the different approaches for the investigation of MR patients [13] has suggested that the substitution of chromosome analysis with MLPA as the first diagnostic test, followed by microarray may be effective in terms of the detection rate and cost effectiveness. CHAPTER III.4 SNP ARRAY TECHNOLOGY BENEFITS FOR DIAGNOSIS OF SPECIFIC MENTAL RETARDATION (A STUDY OF 20 CASES) 4.1. INTRODUCTION Recently, an international consortium of experts recommended the use of microarray technology as the first diagnostic test in the evaluation of patients with MR and/or multiple congenital anomalies [5]. However, there no consensus regarding the choice of microarray platform, distribution of probes in the genome or resolution that is most appropriate for clinical use. With the increasing resolution and genome coverage represented on the microarray, the number of CNVs of uncertain clinical significance increases disproportionately. Recent estimates indicate that up to 12% of normal human genome may involve CNVs [14], and the number of apparently benign CNVs per person varies from in different studies, depending on the technology and the size range used to define a CNV [15]. Detection of a CNV in a patient with MR/ multiple congenital anomalies raises the question of differentiating between benign CNVs and those that cause the phenotype of the patient [16]. Assessing the clinical relevance of CNVs is difficult, especially in the clinical setting. Here we report the results of whole-genome SNP array testing of 20 mentally retarded patients and discuss the difficulties in determining the clinical significance of detected abnormalities PATIENTS AND METHODS Of the patients evaluated in our center between we selected 20 patients (10 girls and 10 boys, age range from 1 year 9 months to 22 years) with mild to severe MR associated with additional features suggestive of a chromosomal abnormality (dysmorphic face, prenatal or postnatal growth retardation, congenital anomalies and/or positive family history). For patients 1-5 SNP array was performed at the Department of Human Genetics in the Radboud University Nijmegen Medical Centre using CytoScan HD Array (Affymetrix) and 8

12 for patients 6-20 SNP array was performed at Department of Genetics, University Medical Center Groningen using HumanCytoSNP-12 v2.1 BeadChip (Illumina Inc., San Diego, CA). Parents DNA samples were available for 3 patients; the parents were tested by targeted SNP array on the CNV region identified in the child. All CNVs detected were systematically searched in local databases and in Database of Genomic Variants. We applied the following rules to determine if the CNV is benign: a benign CNV has been reported in at least 3 individuals and in the same orientation (deletion / amplification). The CNV found in the patient was considered to be significantly overlapping with a common CNV if the overlap was at least 50% and the non-overlapping genomic segment had less than 100 kb in size. If the CNV has not been reported as a benign variant, we analyzed the relationship between phenotype and genomic position, genes contained and their function. This was done by querying public databases DECIPHER and ECARUCA to identify similar or overlapping CNVs and also the UCSC genome browser and OMIM database for gene functions and inheritance patterns. Literature search on a specific CNV was also performed in order to determine its clinical significance. The detected CNVs were classified in four categories (pathogenic CNVs, clinically significant CNVs unrelated to phenotype, variants of uncertain significance or benign CNVs) according to the guidelines for interpretation and reporting of postnatal constitutional CNVs [17, 18] RESULTS In this study, two SNP array platforms were used to identify pathogenic CNVs in 20 patients with syndromic MR of unknown etiology. Six CNVs in five patients were detected using CytoScan HD platform and 19 CNVs in 15 patients were detected using HumanCytoSNP-12 platform. Six patients had regions of homozygosity DISCUSSION Of the 25 CNVs identified, eight abnormalities were considered pathogenic, 4 CNVs were common polymorphisms, 12 were VOUS (7 likely benign, 5 with uncertain clinical significance) and 1 CNV was clinically significant, unrelated to phenotype. Common polymorphisms were excluded from further investigation. 9

13 Pathogenic CNVs Eight CNVs (identified in seven cases: 4 and 15-20) were considered pathogenic because they are associated with known syndromes. These syndromes were subsequently recognized by the medical geneticist, which highlights the difficulty of establishing the diagnosis solely on clinical grounds CNVs of unknown significance (VOUS) Establishing the clinical significance of CNVs not reported in DGV and which have not been associated with microdeletion or microduplication syndromes is challenging, because regions containing coding genes may be present in a variable number of copies without causing clinically evident symptoms. CNVs identified in patients 1, 3 and 5 were considered VOUS- likely benign because they were inherited from an apparently normal parent. However, given that they have not been reported in the literature and public databases, their clinical consequences cannot be determined. Some of the small rearrangements detected in patients with MR and inherited from parents with normal phenotype may be benign variants, while others may represent susceptibility loci for disease [19]. For example, submicroscopic deletion 1q21.1, present in all patients with thrombocytopenia absent radius syndrome, can be inherited from a phenotypically normal parent [20]. Many CNVs (e.g. 22q11.2, deletion, 16p11.2 deletion) have a highly variable phenotype, including phenotypes considered normal. Mechanisms that may explain why some inherited CNVs cause abnormal phenotypes include: incomplete penetrance (CNV is nonpenetrant the parent), variable expressivity (the parent considered "normal" presents subtle abnormalities if a detailed examination is performed), imprinting effects (if the CNV is located in an imprinted region, it will manifest only when inherited from a particular sex), the presence of modifier genes or a second mutation in the same region on homologous chromosome, mosaic CNV or CNV of a different size in the parent, unidentified genetic, epigenetic or environmental factor [17]. Duplications identified in cases 12 and 14 were considered VOUS-likely benign (because CNVs contain no genes). The 13 kb deletion identified in case 2 was also considered a VOUS-likely benign because it was smaller than the minimal critical region that is recurrently deleted in patients with 17q21.31 microdeletion syndrome (160,8 kb) [21]. 10

14 For 5 VOUS we could not determine whether they were de novo because parents DNA probes were not available. To demonstrate the pathogenicity of VOUS we need to identify patients with the same CNV and common phenotype. Thus, databases such as DECIPHER and ECARUCA that gather clinical and molecular cytogenetic data to facilitate understanding of the role of different CNVs in genetic diseases were created. For these 5 VOUS present in patients whose parents were not available for testing there were no cases with complete overlap in DECIPHER and ECARUCA, so they are VOUS with unknown significance (no subclassification). More microarray data are needed in patients with MR and healthy individuals to determine the clinical relevance of these CNVs Regions of homozygosity (ROH) Using a combination of probes that can detect copy number and SNP genotyping, SNP arrays have the ability to detect genomic regions that display an absence of heterozygosity, often in the form of one or more long contiguous stretch of homozygosity. Currently there are no standardized criteria for defining ROH, therefore different studies have used their own criteria in the analysis of homozygosity. The first studies in this direction have focused on continuous DNA sequences without heterozygosity in diploid status at least equal to 1Mb [22, 23], while more recent studies have defined a ROH at a minimum length of 500 kb [24], anticipating the association of shorter ROHs with complex phenotypes. In this study, we identified ROHs in six patients (5-7, 8, 9, and 13). Except patient 7 (institutionalized child), parents reported no consanguinity. Depending on the genomic context, ROHs found in these patients may indicate ancestral homozygosity, uniparental disomy, or parental consanguinity. CHAPTER III.5.1 MOLECULAR AND PHENOTYPIC CHARACTERISTICS OF PATIENTS WITH AARSKOG SYNDROME INTRODUCTION Aarskog Scott syndrome or faciogenital dysplasia (OMIM #305400) is a rare disease, first described by Aarskog [25] and further characterized by Scott [26]. 31 unique mutations in the FGD1 gene have been identified so far [27, 28], but there is no evidence for a correlation between the type and position of mutations and phenotype [28, 29]. In 11

15 all except two reported mutations for which relatives were available for molecular analyses the mothers of the probands were found to be carriers, which indicates that there are few de novo mutations [28]. Our aim is to update the data on mutations and clinical spectrum of Aarskog syndrome by presenting clinical and genetic data from 15 patients with mutations in FGD1 gene PATIENTS AND METHODS Our study was initiated by four male patients from four different families who had typical phenotype for Aarskog syndrome, with or without a family history suggestive of X-linked transmission. Standard evaluation of all patients included prenatal, perinatal and postnatal history, family history, anthropometric measurements, physical examination, and psychological examination. Clinical diagnosis of Aarskog syndrome was based on primary and secondary diagnostic criteria established by the Teebi et al. [30]. PCR amplification and Sanger sequencing for all 18 exons of the FGD1 gene (including intron-exon boundaries) were conducted at Greenwood Genetic Center, Greenwood, South Carolina. The reference sequence used was NM_ All mutations were named as recommended by the Human Genome Variation Society (HGVS). Possible impact of FGD1 mutations on protein structure and function was analyzed in silico using MutationTaster, PolyPhen-2 and SIFT software RESULTS In the four families studied we identified four mutations: two substitutions (c.1829g>a in exon 10 (family 1) and c.1138g>a in exon 5 (family 2)) and two deletions (c.297_306del10 in exon 1 (family 3) and c.342delc in exon 3 (family 4)) DISCUSSION The mutations identified in our study in families 2-4 have not been reported to date in patients with Aarskog syndrome and the mutation identified in family 1 was reported by Orrico et al. in 2000 [31]. So far, the results of other studies, although demonstrated extensive allelic heterogeneity and a broad spectrum of clinical phenotypes, failed to derive a clear correlation between mutation type and severity of clinical manifestations [29]. In our patients, the mutation c.342delc (which has not been reported so far) resulted in a more severe phenotype than other mutations, and variability within the family was 12

16 reduced. To establish genotype-phenotype correlations, it is necessary to confirm these data by identifying other patients with the same mutation in unrelated families. CHAPTER III.5.2 OPTIMIZATION OF DIAGNOSTIC STRATEGY FOR PATIENTS WITH FRAGILE X SYNDROME INTRODUCTION Fragile X syndrome is the most common inherited cause of MR, with a prevalence of 1 in males [32]. Since fragile X syndrome is a major cause of MR, various screening programs have been developed in different countries to identify affected patients. Although currently FMR1 gene testing to determine the CGG repeat number is recommended in all children with MR, developmental delay or autism [33], only in 1-2% of children a full mutation is identified, due to phenotypic variability of these patients [34]. In this study we aimed to establish the clinical and anamnestic data that may guide the clinician toward a better selection of patients with suspected fragile X syndrome to perform molecular tests PATIENTS AND METHODS The cases consisted of 22 patients with clinical diagnosis of fragile X syndrome established in Medical Genetics Centre Iaşi, for whom PCR assay for the trinucleotide CGG repeats in the FMR1 gene (using primers designed by Burlet et al.[35]) was performed. Medical records were reviewed for the presence/absence of nine criteria: 6 criteria were established by Giangreco et al.[36], to which we added three criteria: high and narrow palate, and folds macroorchidism and plantar crease. For each of the three criteria we used a score of 0 - if the feature was absent, 1 - if the feature was borderline and 2 - if the feature was clearly present. Finally, the total score was used to assess the clinical severity of the disease. Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) version 19 (SPSS, Chicago, IL, USA). Variables were tested using T test for paired samples, with p <0.05 considered as statistically significant RESULTS Using this method we could not amplify the region of interest (trinucleotide repeats) in seven patients, suggesting the presence of more 13

17 than 200 trinucleotide repeats, corresponding to a full mutation. For the cases 1-4 the full mutation was confirmed by Southern blot. Statistical comparison between each criterion and the cumulative score of the checklist features in individuals with and without a positive diagnosis of fragile X syndrome showed that a significant difference (p <0.05) exists for traits: long face, large ears, plantar crease, MR and total clinical score. The difference was highly significant (p<0.001) for MR and total clinical score. The total clinical score for patients with confirmed diagnosis of fragile X syndrome was at least 9 (mean clinical score = 13.42), and for those in which the number of trinucleotide repeats was in the normal range was up to nine (mean clinical score = 5.86). Mean clinical score for patients with molecularly confirmed fragile X syndrome aged over 14 years was 15, while for those below this age was DISCUSSION Seven cases (four index patients) of 22 had clinical and molecular diagnosis consistent with fragile X syndrome, which corresponds to a frequency of fragile X syndrome in the studied group of 18%. The frequency is much higher than those obtained by other studies involving large series of non-selected MR patients (~ 2.8%) [37, 38]. Our study revealed that long face (p< 0.05), large ears (p< 0.05), plantar crease (p< 0.05) and MR (p< 0.001) are the traits that differentiated patients with fragile X syndrome from others. Also, the clinical checklist (p< 0.001) made the difference between patients with and without fragile X syndrome. Macroorchidism has not been considered so far a good clinical marker for prepubertal males [39], and this was supported by our study. In fragile X patients, average clinical score increased with age, therefore severity of clinical manifestations in patients with fragile X syndrome increases with age, as other studies have shown [40]. SECTION IV. GENERAL CONCLUSIONS 1. Routine chromosome analysis provides an overview of the human genome, but the resolution of the test (and therefore the ability to establish the diagnosis in cases with mental retardation and/or congenital anomalies) is very small compared to the new microarray-based technologies. Careful assessment of MR patients 14

18 and selection of cases with severe phenotype improves the rate of identifying chromosome abnormalities by routine karyotype. 2. Using a combination of MLPA kits (for subtelomeric rearrangements, common microdeletion syndromes and methylation specific kit for the detection of Prader-Willi Syndrome) allowed us to establish the etiologic diagnosis in 10.4% of patients included in the study. For laboratories that do not have yet access to more sophisticated technologies based on microarray, using several MLPA kits represents an effective strategy for establishing the diagnosis in MR patients. Using follow-up MLPA kits (containing more probes per telomere), allowed us both to confirm abnormalities and to determine their size, which facilitates the interpretation of the clinical significance of subtelomeric rearrangements. 3. SNP array analysis has helped in elucidating the diagnosis in 7 of the 20 patients investigated. The results of this study clearly illustrate the ability of SNP array technology to detect submicroscopic CNVs and precisely describe cytogenetic visible defects associated with MR. Collecting genotypic and phenotypic information in public databases from a large number of patients with MR or multiple congenital anomalies is essential in interpreting CNVs, especially for platforms with high resolution or when the DNA samples of the parents are not available. Although criteria have been established to assist in establishing the clinical significance of VOUS, only the recurrent association of chromosomal imbalances with specific phenotypic traits will clarify the causal relationship. 4. Study of patients with Aarskog syndrome enlarges the collection of mutation data in this syndrome, providing new insight into the types and frequency of mutations in FGD1. Three mutations have not been reported so far, and the fourth has been reported in a single family. One of the newly reported mutations (c.342delc) caused a severe phenotype, relatively constant in all family members, but further studies are needed to establish a genotype-phenotype correlation. 15

19 Aarskog syndrome has a great intra- and inter-familial phenotypic variability, suggesting the involvement of other factors in determining the phenotype (such as modifier genes or environmental factors) beside FGD1 gene. Long-term follow-up of the patients showed that dysmorphic features change with age, becoming more difficult to recognize, and in some patients cognitive deficits and short stature improves over time. 5. Clinical features of fragile X syndrome are extremely variable and the rate of establishing the diagnosis by molecular techniques in unselected individuals with MR is low. The checklist used in this study may help clinicians in screening individuals with MR before performing molecular tests for fragile X syndrome. Thus, the vast majority of cases with fragile X syndrome can be identified in a cost-effective manner. Using a clinical checklist as an initial screening tool is needed especially in countries where molecular diagnostic is available in a few center. Our study showed that the following clinical signs are most important for preselection of mentally retarded subjects for fragile X testing: long face, large ears, plantar crease and MR. Selective bibliography 1. Moorhead, P.S., et al., Chromosome preparations of leukocytes cultured from human peripheral blood. Exp Cell Res, : p Battaglia, A. and J.C. Carey, Diagnostic evaluation of developmental delay/mental retardation: An overview. Am J Med Genet C Semin Med Genet, C(1): p Moog, U., The outcome of diagnostic studies on the etiology of mental retardation: considerations on the classification of the causes. Am J Med Genet A, (2): p Slavotinek, A.M., Novel microdeletion syndromes detected by chromosome microarrays. Hum Genet, (1): p Miller, D.T., 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, (5): p Ravnan, J.B., 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, (6): p

20 7. Yu, S., et al., Frequency of truly cryptic subtelomere abnormalities--a study of 534 patients and literature review. Clin Genet, (5): p Pohovski, L.M., et al., Multiplex ligation-dependent probe amplification workflow for the detection of submicroscopic chromosomal abnormalities in patients with developmental delay/intellectual disability. Mol Cytogenet, (1): p Kirchhoff, M., et al., 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, (1): p Jehee, F.S., 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, (4): p. e Cassidy, S.B., et al., Prader-Willi syndrome. Genet Med, (1): p Ahn, J.W., et al., Submicroscopic chromosome imbalance in patients with developmental delay and/or dysmorphism referred specifically for Fragile X testing and karyotype analysis. Mol Cytogenet, : p Kriek, M., et al., Diagnosis of genetic abnormalities in developmentally delayed patients: a new strategy combining MLPA and array-cgh. Am J Med Genet A, (6): p Redon, R., et al., Global variation in copy number in the human genome. Nature, (7118): p Friedman, J., et al., Detection of pathogenic copy number variants in children with idiopathic intellectual disability using 500 K SNP array genomic hybridization. BMC Genomics, : p Hehir-Kwa, J.Y., et al., Accurate distinction of pathogenic from benign CNVs in mental retardation. PLoS Comput Biol, (4): p. e Kearney, H.M., et al., American College of Medical Genetics standards and guidelines for interpretation and reporting of postnatal constitutional copy number variants. Genet Med, (7): p Vermeesch, J.R., et al., Genome-wide arrays: quality criteria and platforms to be used in routine diagnostics. Hum Mutat, (6): p de Ravel, T.J., et al., Molecular karyotyping of patients with MCA/MR: the blurred boundary between normal and pathogenic variation. Cytogenet Genome Res, (3-4): p Klopocki, E., et al., Complex inheritance pattern resembling autosomal recessive inheritance involving a microdeletion in thrombocytopeniaabsent radius syndrome. Am J Hum Genet, (2): p Dubourg, C., et al., Clinical and molecular characterization of 17q21.31 microdeletion syndrome in 14 French patients with mental retardation. Eur J Med Genet, (2): p

21 22. Gibson, J., N.E. Morton, and A. Collins, Extended tracts of homozygosity in outbred human populations. Hum Mol Genet, (5): p Li, L.H., et al., Long contiguous stretches of homozygosity in the human genome. Hum Mutat, (11): p Yang, T.L., et al., Runs of homozygosity identify a recessive locus 12q21.31 for human adult height. J Clin Endocrinol Metab, (8): p Aarskog, D., A familial syndrome of short stature associated with facial dysplasia and genital anomalies. J Pediatr, (5): p Scott, C.I., Unusual facies, joint hypermobility, genital anomaly and short stature: a new dysmorphic syndrome. Birth Defects Orig Artic Ser, (6): p Bedoyan, J.K., et al., First case of deletion of the faciogenital dysplasia 1 (FGD1) gene in a patient with Aarskog-Scott syndrome. Eur J Med Genet, (4): p Orrico, A., et al., Aarskog-Scott syndrome: clinical update and report of nine novel mutations of the FGD1 gene. Am J Med Genet A, A(2): p Orrico, A., et al., Phenotypic and molecular characterisation of the Aarskog-Scott syndrome: a survey of the clinical variability in light of FGD1 mutation analysis in 46 patients. Eur J Hum Genet, (1): p Teebi, A.S., J.K. Rucquoi, and M.S. Meyn, Aarskog syndrome: report of a family with review and discussion of nosology. Am J Med Genet, (5): p Orrico, A., et al., A mutation in the pleckstrin homology (PH) domain of the FGD1 gene in an Italian family with faciogenital dysplasia (Aarskog- Scott syndrome). FEBS Lett, (3): p Turner, G., et al., Prevalence of fragile X syndrome. Am J Med Genet, (1): p Sherman, S., B.A. Pletcher, and D.A. Driscoll, Fragile X syndrome: diagnostic and carrier testing. Genet Med, (8): p Rauch, A., et al., Diagnostic yield of various genetic approaches in patients with unexplained developmental delay or mental retardation. Am J Med Genet A, (19): p Burlet, P., et al., Multiple displacement amplification improves PGD for fragile X syndrome. Mol Hum Reprod, (10): p Giangreco, C.A., et al., A simplified six-item checklist for screening for fragile X syndrome in the pediatric population. J Pediatr, (4): p Zhong, N., et al., Frequency of the fragile X syndrome in Chinese mentally retarded populations is similar to that in Caucasians. Am J Med Genet, (3): p Puusepp, H., et al., Prevalence of the fragile X syndrome among Estonian mentally retarded and the entire children's population. J Child Neurol, (12): p

22 39. Lachiewicz, A.M. and D.V. Dawson, Do young boys with fragile X syndrome have macroorchidism? Pediatrics, (6 Pt 1): p Visootsak, J., et al., Fragile X syndrome: an update and review for the primary pediatrician. Clin Pediatr (Phila), (5): p ANNEX 1 LIST OF PUBLICATIONS 1. Braha E, Sireteanu A, Vulpoi C, Gorduza C, Branisteanu D, Popescu R, Badiu C, Rusu C. Clinical and Endocrine Aspects of Five Prader Willi Patients. Acta Endo (Buc) 2013; 9: Sireteanu A, Braha E, Popescu R, Gramescu M, Gorduza V, Rusu C. Inverted duplication deletion of 8p: characterization by standard cytogenetic and SNP array analyses. Rev Med Chir Soc Med Nat Iasi 2013; 117(3): Sireteanu A, Covic M, Gorduza V. Hibridizarea genomică comparativă pe microreţele: consideraţii tehnice şi aplicaţii. Rev Med Chir Soc Med Nat Iasi 2012, 116(2):