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1 Archives of Dermatological Research Phenotypic diversity of patients with LEOPARD syndrome carrying the worldwide recurrent p.tyr9cys PTPN mutation --Manuscript Draft-- Manuscript Number: Full Title: Article Type: Keywords: Corresponding Author: AODR-D-5-00R Phenotypic diversity of patients with LEOPARD syndrome carrying the worldwide recurrent p.tyr9cys PTPN mutation Original Paper LEOPARD syndrome; PTPN gene; p.tyr9cys; worldwide recurrent missense mutation; phenotypic diversity Nikoletta Nagy HUNGARY Corresponding Author Secondary Information: Corresponding Author's Institution: Corresponding Author's Secondary Institution: First Author: Edina Nemes First Author Secondary Information: Order of Authors: Edina Nemes Katalin Farkas Barbara Kocsis-Deák Andrea Drubi Adrienn Sulák Kornélia Tripolszki Piroska Dósa Ferenc Lakatos Nikoletta Nagy Márta Széll Order of Authors Secondary Information: Funding Information: Hungarian Scientific Research Foundation (OTKA) PD02 2-5) not applicable (TÁMOP A-//KONV-2-00 grant) not applicable (TÁMOP-4.2.2/B-0//KONV-0-002) not applicable (TÁMOP A/ ) not applicable (TÁMOP A) Dr. Nikoletta Nagy Not applicable Not applicable Not applicable Not applicable Abstract: LEOPARD syndrome (LS, OMIM 0) is a rare monogenic disorder. The name is an acronym of its major features, including multiple lentigines, electrocardiographic conduction defects, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, growth retardation and sensorineural deafness. LS develops due to mutations in the protein-tyrosine phosphatase nonreceptor-type (PTPN) gene. Here, we report a 5-year-old Hungarian male patient affected by LS. Direct sequencing of the PTPN Powered by Editorial Manager and ProduXion Manager from Aries Systems Corporation

2 gene revealed a worldwide recurrent missense mutation (c.6a/g; p.tyr9cys), which has been previously identified in LS patients. Comparison of the clinical phenotypes of our patient and those reported in the literature revealed great phenotypic diversity, despite the shared genotype. Response to Reviewers: see attachment Powered by Editorial Manager and ProduXion Manager from Aries Systems Corporation

3 Manuscript Click here to download Manuscript: manuscript_nemes et al_5.doc Click here to view linked References Phenotypical diversity of patients with LEOPARD syndrome carrying the worldwide 2 recurrent p.tyr9cys PTPN mutation Edina Nemes *, Katalin Farkas 2*, Barbara Kocsis-Deák, Andrea Drubi, Adrienn Sulák, Kornélia Tripolszki, Piroska Dósa 5, Lakatos Ferenc 4, Nikoletta Nagy 2,,5, Márta Széll 2, 2 4 Dermatology and Venereology Unit of the Orosháza Hospital, Orosháza, HUNGARY MTA-SZTE Dermatological Research Group, University of Szeged, Szeged, HUNGARY 9 Department of Medical Genetics, University of Szeged, Szeged, HUNGARY 4 Cardiology Unit of the Orosháza Hospital, Orosháza, HUNGARY 24 5 Department of Dermatology and Allergology, University of Szeged, Szeged, HUNGARY *These two authors are contributed equally to this work. Concise title: Phenotypical diversity in LEOPARD syndrome Corresponding author: Nikoletta Nagy MD PhD, Department of Medical Genetics, 9 University of Szeged, 6 Somogyi Bela Street, 6724 Szeged, HUNGARY, Tel.: , nikoletta.nagy@gmail.com

4 ABSTRACT 5 LEOPARD syndrome (LS, OMIM 0) is a rare monogenic disorder. The name is an 6 7 acronym of its major features such as multiple lentigines, electrocardiographic conduction defects, ocular hypertelorism, pulmonary stenosis, abnormalities of genitalia, retardation of 2 growth and sensorineural deafness. LS develops due to mutations in the protein-tyrosine 4 phosphatase nonreceptor-type, PTPN. Here we have investigated a 5-year-old Hungarian male patient affected by LS. Direct sequencing of the PTPN gene revealed a 9 worldwide recurrent missense mutation (c.6a/g; p.tyr9cys), which has been previously 2 22 identified in LS patients. Comparison of the clinical phenotypes of our patient and the ones 24 reported in the literature demonstrates great phenotypic diversity despite of the same genotype. Key words: LEOPARD syndrome, PTPN gene, worldwide recurrent missense mutation, 4 phenotypic diversity, p.y9c

5 INTRODUCTION 5 6 LEOPARD syndrome (LS, OMIM 0) - a rare monogenic disorder belonging to the 7 family of neuro-cardiofacio-cutaneous syndromes [6] - is inherited as an autosomal 9 dominant trait with full penetrance and variable expressivity [, 0]. The major features of LS 2 include multiple lentigines, electrocardiographic conduction abnormalities, ocular 4 hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth and sensorineural deafness, this is why the syndrome is referred with the LEOPARD acronym [4, 9 0]. LS develops as a consequence of mutations of the protein-tyrosine phosphatase 2 22 nonreceptor-type (PTPN) gene encoding a cytoplasmic protein tyrosine phosphatase 24 (SHP-2), which regulates intracellular signaling and controls several distinct developmental processes [5, 9]. In about 5% of the cases a heterozygous missense mutation is detected in 29 the exon 7, 2 or [6, ]. Among the so far identified missense mutations, there are two (p.tyr9cys and p.thr46met), which account for about % of all LS cases worldwide [6, 4 ]. 6 Here we report a Hungarian LS patient carrying the most common p.tyr9cys heterozygous 9 missense mutation and compare his clinical features with the symptoms of previously 4 reported LS patients (n=), in whom the same causative mutation was identified PATIENTS AND METHODS 4 5 Patients 5 A 5-year-old Hungarian male patient was admitted to the cardiology unit of the Orosháza 56 Hospital (Orosháza, Hungary) with dizziness and palpitation. On investigation, facial 5 anomalies including ocular hypertelorism, palpebral ptosis, dysmorphic ear, slight mandibular 6

6 4 prognathism (Fig a) and pigmentation abnormalities such as multiple lentigines (Fig b) and 2 café-au-lait spots (Fig c) were also observed. Cardiology investigations revealed third degree 4 5 atrioventricular block (Fig d). The patient is deaf and dumb since he was born and mild 6 7 growth as well as mental retardations were also present. Urological investigation revealed mild genital abnormalities such as atrophic testes. These clinical symptoms suggested LS, 2 therefore genetic screening of the PTPN gene was also performed. The patient was born out 4 of wedlock, however the family members of his father and his mother were available for the clinical and genetic investigations (Fig e). All the investigated relatives were clinically 9 unaffected suggesting the presence of a de novo mutation in the patient Genetic investigations Blood sample was taken from the patient and genomic DNA has been isolated by a BioRobot 29 EZ DSP Workstation (Qiagen; Godollo, Hungary). All the coding regions of the PTPN gene and the flanking introns were amplified and sequenced (primers were used as displayed 4 on the UCSC Genome Browser The investigation was approved by 6 the Internal Review Board of the University of Szeged. Written informed consent was 9 obtained from the patient, and the study was conducted according to the Principles of the 4 Declaration of Helsinki RESULTS 4 5 Direct sequencing of the coding regions and the flanking introns of the PTPN gene revealed 5 a heterozygous missense mutation (c.6a/g; p.tyr9cys) in the seventh exon (Fig f). The 56 clinically affected patient carried the mutation in heterozygous form, while the unrelated 5 healthy controls carried the wild type sequence (Fig g). 6

7 5 2 DISCUSSION The investigated Hungarian LS patient carries one of the most common missense mutation 9 (p.tyr9cys) of the PTPN gene. Functional studies demonstrated that the p.tyr9cys 0 2 heterozygous missense mutation of the PTPN gene perturbs the switching of the SHP-2 4 protein between its catalytically inactive and active conformation and engenders loss of SHP catalytic activity []. 9 The p.tyr9cys mutation has previously been reported in different LS patients with 2 22 Italian, French, Spanish, German, Estonian, Bosnian, Chinese Han, South-Korean, Japanese 24 and Australian origin [, 5,, 9, 2,, 4, 7,, 2, 22]. Thus this missense mutation is a worldwide recurrent missense mutation. Among the reported LS cases there are ones with de 29 novo mutation [9, 2] and others, in which the disease show familial clustering and affects multiple family members in multiple generations [, 5]. These data suggest a mutational 4 hotspot on the PTPN gene. 6 Previous reports demonstrated that the p.tyr9cys PTPN mutation is associated with 9 short stature, deafness and hypertrophic cardiomyopathy [6, ], the detailed comparison of 4 the clinical symptoms of the 4 LS patients with the same causative mutation further complicated the analysis of the genotype-phenotype correlations of this mutation. 46 The most common symptom, which was present in 46 (96%) out of 4 patients is the presence of multiple lentigines (Fig 2). Further skin abnormality such as the development of café-au- 5 lait spots were observed in only 22 (46%) patients. Besides the characteristic multiple 5 lentigines, some of the facial anomalies were also very common among these patients: ocular 56 hypertelorism was detected in (%) patients, palpebral ptosis in (67%) patients and 5 dysmorphic ears in (%) ones. Besides these common ectodermal abnormalities, patients 6

8 6 with the p.tyr9cys PTPN mutation have a great chance to develop cardiovascular 2 anomalies. Hypertrophic cardiomyopathy was diagnosed in (%) patients. Although short 4 5 stature was previously reported to be frequently associated with the p.tyr9cys phenotype, 6 7 it was present only in 9 (%) patients out of 4. 9 Regarding the other side of the spectrum, the p.tyr9cys mutation is rarely associated with 0 2 deafness, which was reported in 2 (%) patients (Fig 2). Moreover our analysis identified 4 that certain symptoms - such as cryptorchidism, macrocephaly, horse kidney, hydrothorax, myelodysplasia and umbilical hernia - are rarely associated with the p.tyr9cys phenotype. 9 In one of the 4 LS patients with the recurrent p.tyr9cys PTPN mutation, Marfan 2 22 syndrome was also present, which is probably a rare independent association [7]. 24 The observed differences in the clinical symptoms of the 4 LS patients carrying the same missense mutation clearly demonstrate the wide phenotypic diversity and the variable 29 expressivity of the disease. In general, multiple lentigines, café-au-lait macules, ocular hypertelorism, palpebral ptosis, dysmorphic ears and hertrophic cardiomyopathy are 4 hallmarks of the p.tyr9cys PTPN mutation related phenotype. However, there is no 6 similar analysis investigating the most frequently associated clinical features in LS patients 9 carrying the other common, recurrent missense mutation (p.thr46met) of the PTPN gene, 4 the published reports suggest that the p.thr46met mutation is also associated with high phenotypic diversity [2, 7]. 46 Further studies are needed to identify putative genetic, environmental or life style factors, which can modify the development of the clinical symptoms and responsible for the observed 5 phenotypic diversity. The availability of the extended clinical findings about the p.tyr9cys 5 mutation carriers, as provided by this study, is critical for promoting both our understanding 56 of the disease and the development of causative therapies that will be more specific and 5 effective than the symptomatic treatments currently available for LS patients. 6

9 7 ACKNOWLEDGEMENTS 2 This study was supported by the Hungarian TÁMOP A-//KONV-2-00 grant, TÁMOP-4.2.2/B-0//KONV grant, TÁMOP A/ grant 7 and TÁMOP A grant. Nikoletta Nagy is supported by the Hungarian Scientific Research Foundation (OTKA) PD grant. CONFLICT OF INTEREST 5 6 The authors declare that they have no conflict of interest

10 REFERENCES 5 6. Begić F, Tahirović H, Kardašević M, Kalev I, Muru K (4) Leopard syndrome: a 7 report of five cases from one family in two generations. Eur J Pediatr 7: Carcavilla A, Pinto I, Muñoz-Pacheco R, Barrio R, Martin-Frías M, Ezquieta B () 2 LEOPARD syndrome (PTPN, T46M) in three boys fulfilling neurofibromatosis 4 type clinical criteria. Eur J Pediatr 70: Digilio MC, Conti E, Sarkozy A, Mingarelli R, Dottorini T, Marino B, Pizzuti A, 9 Dallapiccola B (02) Grouping of multiple-lentigines/leopard and Noonan 2 22 syndromes on the PTPN gene. Am J Hum Genet 7: Digilio MC, Sarkozy A, De Zorzi A, Pacileo G, Limongelli G, Mingarelli R, Calabrò R, Marino B, Dallapiccola B (06) LEOPARD syndrome: clinical diagnosis in the 29 first year of life. Am J Med Genet A : Froster UG, Glander HJ, Heinritz W (0) Molecular genetic mutation analysis of the 4 PTPN gene in the multiple lentigines (LEOPARD) syndrome. Hautarzt : Kalev I, Muru K, Teek R, Zordania R, Reimand T, Köbas K, Ounap K (0) 4 LEOPARD syndrome with recurrent PTPN mutation Y9C and different 46 69:469- cutaneous manifestations: two case reports and a review of the literature. Eur J Pediatr 7. Kato H, Yoshida R, Tsukamoto K, Suga H, Eto H, Higashino T, Araki J, Ogata T, 5 Yoshimura K (0) Familial cases of atypical clinical features genetically diagnosed 5 as LEOPARD syndrome (multiple lentigines syndrome). Int J Dermatol : Keren B, Hadchouel A, Saba S, Sznajer Y, Bonneau D, Leheup B, Boute O, Gaillard 5 D, Lacombe D, Layet V, Marlin S, Mortier G, Toutain A, Beylot C, Baumann C, 6

11 9 Verloes A, Cavé H; French Collaborative Noonan Study Group (04) PTPN 2 mutations in patients with LEOPARD syndrome: a French multicentric experience. J Med Genet 4: Kim J, Kim MR, Kim HJ, Lee KA, Lee MG () LEOPARD Syndrome with 9 PTPN Gene Mutation Showing Six Cardinal Symptoms of LEOPARD. Ann 0 2 Dermatol : Legius E, Schrander-Stumpel C, Schollen E, Pulles-Heintzberger C, Gewillig M, Fryns JP (02) PTPN mutations in LEOPARD syndrome. J Med Genet 9: Limongelli G, Sarkozy A, Pacileo G, Calabrò P, Digilio MC, Maddaloni V, Gagliardi 24 G, Di Salvo G, Iacomino M, Marino B, Dallapiccola B, Calabrò R (0) Genotype phenotype analysis and natural history of left ventricular hypertrophy in LEOPARD 29 syndrome. Am J Med Genet A 46: Martínez-Quintana E, Rodríguez-González F (2) LEOPARD Syndrome Caused by 4 Tyr9Cys Mutation in the PTPN Gene. Mol Syndromol 2:- 6. Paradisi M, Pedicelli C, Ciasulli A, Pinto F, Conti E, Sarkozy A, Angelo C (05) 9 PTPN gene mutation in LEOPARD syndrome. Minerva Pediatr : Sarkozy A, Conti E, Digilio MC, Marino B, Morini E, Pacileo G, Wilson M, Calabrò R, Pizzuti A, Dallapiccola B (04) Clinical and molecular analysis of patients 46 with multiple lentigines LEOPARD syndrome. J Med Genet 4:6 5 Dis : 5 5. Sarkozy A, Digilio MC, Dallapiccola B (0) Leopard syndrome. Orphanet J Rare 6. Spatola M, Wider C, Kuntzer T, Croquelois A (5) PTPN mutation manifesting 56 as leopard syndrome associated with hypertrophic plexi and neuropathic pain. BMC 5 Neurol 5: 6

12 0 7. Tang S, Hoshida H, Kamisago M, Yagi H, Momma K, Matsuoka R (09) Phenotype- 2 genotype correlation in a patient with co-occurrence of Marfan and LEOPARD syndromes. Am J Med Genet A : Tartaglia M, Martinelli S, Stella L, Bocchinfuso G, Flex E, Cordeddu V, Zampino G, van der Burgt I, Palleschi A, Petrucci TC, Sorcini M, Schoch C, Foa R, Emanuel PD, 2 Gelb BD (06) Diversity and functional consequences of germline and somatic 4 PTPN mutations in human disease. Am J Hum Genet 7: Tartaglia M, Mehler EL, Goldberg R, Zampino G, Brunner HG, Kremer H, Van Der 9 Burgt I, Crosby AH, Ion A, Jeffery S, Kalidas K, Patton MA, Kucherlapati RS, Gelb 2 22 BD (0) Mutations in PTPN, encoding the protein tyrosine phosphatase SHP-2, 24 cause Noonan syndrome. Nat Genet 29:4-46. Uçar C, Calýskan U, Martini S, Heinritz W (06) Acute myelomonocytic leukemia in 29 a boy with LEOPARD syndrome (PTPN gene mutation positive). J Pediatr Hematol Oncol : Wang Y, Chen C, Wang DW (4) Leopard syndrome caused by heterozygous 6 missense mutation of Tyr 9 Cys in the PTPN gene in a sporadic case of Chinese 9 Han. Int J Cardiol 74: Yoshida R, Nagai T, Hasegawa T, Kinoshita E, Tanaka T, Ogata T (04) Two novel 4 46 : and one recurrent PTPN mutations in LEOPARD syndrome. Am J Med Genet A

13 FIGURE LEGENDS 2 4 Fig. The clinical symptoms, the pedigree of the patient and the identified recurrent 5 6 mutation of the PTPN gene. On examination facial dysmorphims including ocular 7 9 hypertelorism, palpebral ptosis, slight mandibular prognathism and dysmorphic ears were 0 observed (Fig. a). Pigmentational abnormalities such as multiple lentigines (Fig. b) and 2 4 café-au-lait spots (Fig. c) were also present on the patient skin. On electrocardiography a 5 6 third degree antrioventricular block was present (Fig. d). The patient was born outside 7 9 marriage. The family members of his father and his mother were all clinically unaffected 2 individuals suggesting (Fig. e). Direct sequencing revealed a heterozygous missense mutation (c.6a/g; p.tyr9cys) in the seventh exon of the PTPN gene. The patient 26 carried the mutation in heterozygous form (Fig. f), while the unrelated controls carried the wild type sequence (Fig. g). 29 Fig. 2 Comparison of the frequency of the different symptoms detected in the 4 6 p.tyr9cys phenotype. The most common symptoms were the pigmentational abnormalities including multiple lentigines and café-au-lait spots and the mild facial 9 dysmorphisms such as ocular hypertelorism, palpebral ptosis and dysmorphic ears. In contrast 4 with these, cryptorchidism, mental retardation, macrocephaly, horse kidney, hydrothorax, myelodysplasia and umbilical hernia were rarely associated with the p.tyr9cys phenotype

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