Clin Genet 2015: 88: 479–483 Printed in Singapore. All rights reserved

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: 10.1111/cge.12534

Short Report

Intrafamilial variability of ZRS-associated syndrome: characterization of a mosaic ZRS mutation by pyrosequencing Vanlerberghe C., Faivre L., Petit F., Fruchart O., Jourdain A.-S., Clavier F., Gay S., Manouvrier-Hanu S., Escande F. Intrafamilial variability of ZRS-associated syndrome: characterization of a mosaic ZRS mutation by pyrosequencing. Clin Genet 2015: 88: 479–483. © John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2014 During limb development, the spatio-temporal expression of sonic hedgehog (SHH) is driven by the Zone of polarizing activity Regulatory Sequence (ZRS), located 1 megabase upstream from SHH. Gain-of-function mutations of this enhancer, which cause ectopic expression of SHH, are known to be responsible for congenital limb malformations with variable expressivity, ranging from preaxial polydactyly or triphalangeal thumbs to polysyndactyly, which may also be associated with mesomelic deficiency. In this report, we describe a patient affected with mirror-image polydactyly of the four extremities and bilateral tibial deficiency. The proband’s father had isolated preaxial polydactyly type II (PPD2). Using Sanger sequencing, a ZRS point mutation (NC_000007.14, g.156584153A>G, UCSC, Build hg.19) was only identified in the patient. However, pyrosequencing analysis enabled the detection of a 10% somatic mosaic in the blood and saliva from the father. To our knowledge, this is the first description of a ZRS mosaic mutation. This report highlights the complexity of genotype–phenotype correlation in ZRS-associated syndromes and the importance of detecting somatic mosaicism for accurate genetic counselling. Conflict of interest

The authors declare no conflict of interest.

C. Vanlerberghea,b,c , L. Faivred , F. Petitb,c,e , O. Frucharta,c , A.-S. Jourdaina,c , F. Clavierf , S. Gayg , S. Manouvrier-Hanub,c,e,† and F. Escandea,c,† a Institut

de Biochimie et Génétique Moléculaire, Centre de Biologie Pathologie, CHRU, Lille, France, b Clinique de Génétique médicale, Hôpital Jeanne de Flandre, CHRU, Lille, France, c RADEME Research Team for Rare Metabolic and Developmental Diseases, Université Lille 2, Lille, France, d Service de Génétique clinique, Hôpital d’enfants, Dijon, France, e Faculté de Médecine, Université Lille II, Lille, France, f Centre de référence national maladies rares des malformations des membres et de l’arthrogrypose chez l’enfant Saint Maurice, Hôpital Saint Maurice, Saint Maurice, France, and g Service de pédiatrie, Centre Hospitalier William Morey, Chalon sur Saône, France † These

authors contributed equally.

Key words: mirror-image polydactyly – somatic mosaicism – pyrosequencing – sonic hedgehog – tibial aplasia/ hypoplasia – ZRS Corresponding author: Sylvie Manouvrier-Hanu, Génétique Clinique, Hôpital Jeanne de Flandre, 59037 LILLE CEDEX, France. Tel.: +33 320444911; fax: +33 320444901; e-mail: [email protected] Received 5 August 2014, revised and accepted for publication 3 November 2014

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Vanlerberghe et al. During embryogenesis, limb development proceeds simultaneously along three axes: proximodistal, dorsoventral, and anteroposterior. The anteroposterior patterning of the digits requires signalling from the zone of polarizing activity (ZPA), a group of mesenchymal cells localized at the posterior margin of the tetrapod limb bud. The ZPA secretes the morphogen sonic hedgehog (SHH [MIM 600725]), with a gradient of concentration that decreases from the posterior to the anterior border of the limb bud (1). A highly conserved cis-regulatory enhancer called the ZPA regulatory sequence [ZRS (MIM 605522)] lies within intron 5 of the LMBR1 gene on chromosome 7q36, which is located 1 megabase upstream of its target gene, SHH in human (2, 3). ZRS gain-of-function anomalies are responsible for limb malformations (the so-called ZRS-associated syndromes) (4), with phenotypic presentation ranging through preaxial polydactyly and/or triphalangeal thumbs [PPD2 (MIM 174500)] [reviewed in (5)], triphalangeal thumb-polysyndactyly syndrome [TPTPS (MIM 174500)], syndactyly type IV [SD4; Haas type polysyndactyly (MIM 186200)] (4, 6–9) Werner mesomelic syndrome (WMS [MIM 188770]) (4, 10–12), PPD2 with radial deficiency (13) and some cases of Laurin-Sandrow syndrome (LSS [MIM 135750]) (14). Here, we report a child presenting with mirror-image polydactyly of the hands and feet along with tibial hypoplasia, due to a ZRS point mutation inherited from his father with PPD2, who carries this mutation in a mosaic state. Material and methods Patients

The proband (III-1) is the first child of nonconsanguineous parents (Fig. 1a). His father (II-1) had PPD2 with remaining bilateral triphalangeal thumbs, as the supernumerary thumbs had been removed in infancy. His feet were not affected. His mother (II-2) and his grandparents (I-1, I-2) were healthy. The index subject (III-1) was born at 38 weeks gestation, after an unremarkable pregnancy. Mirror-image polydactyly of the four extremities was observed at birth (Fig. 2). He presented seven fingers on each hand, with complete II–III digit and V–VI digit and partial I–II digit syndactyly on the left-hand side, and complete I–II digit and partial V–VI digit syndactyly on the right-hand side. His feet had eight digit rays, with a mesoaxial large thumb-like structure, three preaxial toes and four postaxial toes. Both legs were short and curved. X-rays showed left tibial agenesis and right tibial hypoplasia. There was no facial dysmorphism and further investigations did not reveal any other congenital malformation. Molecular analysis

DNA was extracted from peripheral blood from the patient, his parents, and paternal grandparents and saliva from his father and grandparents using the EZ1 DNA blood kit (Qiagen, Courtaboeuf, France)

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with the BioRobot® EZ1 (Qiagen). Sanger sequencing of the ZRS region [RefSeq NC_000007.14: g.156,583,690-156,584,790 (hg19); UCSC Genome Browser http://genome.ucsc.edu.] was performed on an ABI Prism 3130XL Genetic Analyser (Applied Biosystems, Foster City, CA). To assess the suspected paternal mosaicism, pyrosequencing (PyroMark MD96 instrument, Qiagen S.A.S., Courtaboeuf, France) was performed following the manufacturer’s instructions. Primers were designed with PSQ Assay Design v1.0 Software (Pyrosequencing AB, Uppsala, Sweden). Pyrogram outputs were analysed by Pyromark MD v1.0 Software (Pyrosequencing AB) using the allelic quantification supplemental software to determine the percentage of mutant vs wild-type alleles according to the percentage relative peak height. All primers used and amplifications conditions are available upon request. Results

Sanger analysis of the ZRS region in the proband revealed a heterozygous point mutation g.156584 153A>G (henceforth called ZRS 417A>G in reference to the sequence published by Lettice et al.) (Fig. 1b), with a normal copy number (data not shown) (3). This mutation had not been previously described and was not found in dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/) or the 1000 Genomes Project Database (http://www. 1000genomes.org/). The mutation altered a highly conserved nucleotide position, and therefore was considered most likely damaging, although it did not disrupt transcription factor binding sites as predicted in silico according to jaspar software (http://jaspardev. genereg.net/). The ZRS 417A>G mutation was not detected in blood samples from the proband’s relatives (Fig. 1b). However, because of the father’s phenotype, paternal somatic mosaicism was suspected. Pyrosequencing analysis permitted detection of the mutation in the father’s DNA derived from either blood or saliva, at an estimated rate of ∼10%, but was not found in the grandparents DNA (Fig. 1c,d). These results suggested a post-zygotic (mosaic) mutation occurred in the father. Discussion

Here, we report a patient with mirror-image polydactyly and tibial hypoplasia due to a heterozygous ZRS point mutation (417A>G) inherited from his father with PPD2, who carried this mutation as a somatic mosaic. This report not only expands the mutational spectrum of ZRS mutations causing mesomelia (position 417) beyond the previously described WMS-associated mutations (position 402, 404, or 406) (4, 11, 12, 15) but also enriches the clinical spectrum of the ‘ZRS-associated phenotypes’ to an association with mesomelia, reminiscent of WMS; and mirror-polydactyly, reminiscent of LSS. Although mirror polydactyly has only been described in animal models (16) and ZRS duplications in humans (14), we report here mirror-image-polydactyly due to ZRS point mutation. This is also, to the best of

Mosaic ZRS mutation

Fig. 1. (a) Pedigree. (b) ZRS Sanger sequencing – proband (III-1): presence of the heterozygous mutation ZRS 417A>G; father (II-1) and paternal grandparents (I-1, I-2): no mutation detected. (c) and (d) Pyrogram of ZRS 417A>G mutation – proband (III-1): presence of the mutated allele at a rate of 50% in blood (c) (reverse complementary sequence: T/CGGTCAT); father (II-1): presence of the mutated allele at a rate of ≈10% in blood (c) and saliva (d) (reverse complementary sequence: T/CGGTCAT); paternal grandparents (I-1 and I-2) in blood (c) and saliva (d): no mutation detected (reverse complementary sequence: TGGTCAT).

our knowledge, the first description of a somatic mosaic mutation in ZRS-associated syndromes, which suggests a possible role of mosaicism as an additional cause of phenotypic variation in these disorders. Furthermore, it raises the question of the risk to mosaic carriers of apparently de novo ZRS mutations, and consequences in genetic counselling. Somatic mosaicism also changes the recommendation for molecular analysis in patients and specific relatives. Extra- and intra-familial variabilities, as well as rare incomplete penetrance, have been described in ZRS-associated syndromes (11, 15, 17–21). This may be largely due to the complex interaction between SHH and

its regulatory elements in limb development (ZRS, and others, as yet unidentified) (11, 18). Confounding factors may also explain the difficulties experienced with confirming the suggested genotype–phenotype correlations in ZRS-associated syndromes (4). For example, PPD2 due to a heterozygous ZRS 402C>T point mutation, and WMS due to the same mutation in a homozygous form have been described in the same family (15). In the family described in this report, we assumed that the wide variability of expression between the father and his son was likely due to paternal mosaicism of the ZRS mutation in the ZPA. Indeed, it is well understood that the period during embryonic development at which a

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Fig. 2. Patients’ clinical and radiological features. (a–c) Both feet and left hand of the proband: mirror-image polydactyly; (d) upper limbs X-rays: polydactyly without mesomelic anomaly (e) lower limbs X-rays: right tibial hypoplasia, left tibial agenesis and bilateral mirror-image polydactyly; (f, g) hands of the proband’s father: bilateral triphalangeal thumbs.

mutation first occurred, the degree of mosaicism, and its tissue distribution all influence clinical expression and transmission risk (22). Given that the mutation was present in at least three distinct tissues [it was identified in blood (lymphocytes) and saliva (epithelial cells), and was present in the germline as it was transmitted to the child], we expect that the mutation appeared very early during the father’s development (probably before gastrulation). Therefore, the mutation is likely present in numerous mesodermal tissues, including the ZPA, which could explain the father’s phenotype, although the degree of mosaicism varies considerably between different tissues and could not be predicted in ZPA. The identification of mosaicism has a considerable impact on the family in guiding clinical screening and genetic counselling. As mosaicism may be associated with a very mild phenotype, obtaining the appropriate diagnosis can be complicated. This situation highlights the importance of a meticulous physical examination of a proband’s parents and good communication between clinicians and biologists to guide the molecular investigations. Furthermore, recent publications suggest that the overall prevalence of mosaicism is largely

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under-explored and inadequately considered during genetic counselling (23). If an unaffected parent harbours the mutation in a mosaic state, the recurrence risk remains unknown, but can be as high as 50%, while this risk is estimated to be ∼5% in gonadal mosaicism (24). Accordingly, we suggest systematic genetic screening of clinically unaffected parents of patients harbouring a ZRS mutation to detect cryptic mosaicism. The molecular testing for mosaicism requires appropriate and sensitive techniques. Pyrosequencing and SnapShot are easily accessible techniques capable of detecting mutant alleles comprising as little as 5% of mutant alleles for most mutation types, whereas conventional Sanger sequencing requires the presence of at least 20% mutant alleles to allow identification of mutations. Next generation sequencing with high-depth coverage (×5000) may also constitute a valuable alternative for the detection of mutational mosaicism at levels approaching 1% (25). Therefore, given the risk of recurrence, we suggest that mosaic screening technologies be used not only on the parents of patients carrying ZRS mutations, but also in patients with a ‘ZRS-associated phenotype’ in whom no mutations have been detected by Sanger sequencing.

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