© American College of Medical Genetics and Genomics ORIGINAL RESEARCH ARTICLE Mutation spectrum in the ABCC6 gene and genotype–phenotype correlations in a French cohort with pseudoxanthoma elasticum Anne Legrand, PharmD1,2,3, Laurence Cornez, PhD4, Wafa Samkari, MD1, Jean-Michael Mazzella1, Annabelle Venisse1, Valérie Boccio1, Karine Auribault, PhD1, Boris Keren, MD, PhD5, Karelle Benistan, MD6, Dominique P. Germain, MD, PhD6,7,8, Michael Frank, MD1,2, Xavier Jeunemaitre, MD, PhD1,2,3 and Juliette Albuisson, MD, PhD1,2,3 Purpose: Pseudoxanthoma elasticum (PXE) is an autosomal reces- distinct variants, of which 66 were novel. Missense variant distribu- sive disorder caused by variants in the ABCC6 gene. Ectopic miner- tion was specific to some regions and residues of ABCC6. For the 220 alization of connective tissues leads to skin, eye, and cardiovascular cases with a complete PS, there was a higher prevalence of eye fea- manifestations with considerable phenotypic variability of unknown tures in Caucasian patients (P = 0.03) and more severe eye and vas- cause. We aimed to identify genotype–phenotype correlations in cular phenotype in patients with loss-of-function variants (P = 0.02 PXE. and 0.05, respectively). Nephrolithiases and strokes, absent from the PS, were prevalent features of the disorder (11 and 10%, respectively). Methods: A molecular analysis was performed on 458 French PXE probands clinically evaluated using the Phenodex score (PS). Variant Conclusion: We propose an updated PS including renal and neuro- topographic analysis and genotype–phenotype correlation analysis logical features and adaptation of follow-up according to the genetic were performed according to the number and type of identified vari- and ethnic status of PXE-affected patients. ants. Genet Med advance online publication 19 January 2017 Results: Complete molecular analysis of 306 cases allowed the iden- Key Words: ABCC6; genotype–phenotype correlation; tification of 538 mutational events (88% detection rate) with 142 Pseudoxanthoma elasticum; Phenodex score INTRODUCTION present in 89–96% of cases.2 AS can be responsible for subreti- Pseudoxanthoma elasticum (PXE; OMIM 264800) is a rare, nal choroidal neovascularization, hemorrhage, and subsequent autosomal recessive disorder characterized by ectopic miner- loss of central vision. Almost all patients present a combination alization and fragmentation of the elastic fibers of select con- of AS and skin lesions.2,4 nective tissues. The estimated prevalence lies between 1/25,000 Cardiovascular symptoms are loss of peripheral pulses, and 1/50,000 (ref. 1). According to this prevalence, the theo- intermittent ischemic claudication, renovascular hyperten- retical frequency of heterozygous subjects in the population lies sion, angina, and, rarely, myocardial infarction. Cerebral isch- between 1/111 and 1/80. emic involvement and gastrointestinal bleeding can also occur. The skin, eye, and cardiovascular system are primarily These manifestations are the consequence of accelerated arte- affected, with considerable interfamilial and intrafamilial riosclerosis with mineralization of the internal elastic lamina of variability. Skin abnormalities begin with yellowish papules medium arteries. The cardiovascular system is less frequently in the flexural areas that progressively coalesce into plaques, affected than the skin and eyes (7 to 30% of patients have inter- which rarely affect nonflexural sites. Skin lesions are the most mittent claudication); symptoms commonly appear in the third prevalent sign of PXE; they are the key features for diagnosis decade of life.2,5 (97–98% of cases).2,3 The most common PXE ocular signs are The clinical diagnosis is commonly made using the consen- peau d’orange and angioid streaks (AS) in the retina, which are sus conference criteria focused on skin and eye manifestations,6 1AP-HP, Hôpital Européen Georges Pompidou, Département de Génétique, Service de Médecine Vasculaire et Centre de Référence des Maladies Vasculaires Rares, Paris, France; 2INSERM, U970, Paris Centre de Recherche Cardiovasculaire–PARCC, Paris, France; 3Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France; 4Commissariat à l’Energie Atomique et aux Energies Alternatives, CEA Saclay, Gif-sur-Yvette, France; 5AP-HP, Groupe Hospitalier Pitié-Salpêtrière, UF de Génomique du Développement, Paris, France; 6AP-HP, Hôpital Raymond Poincaré, Département de Génétique, Garches, France; 7INSERM U1179, Paris-Saclay University, Montigny, France; 8Université de Versailles Saint-Quentin-en-Yvelines, Montigny, France. Correspondence: Juliette Albuisson ([email protected]) Submitted 22 September 2016; accepted 22 November 2016; advance online publication 19 January 2017. doi:10.1038/gim.2016.213 GENETICS in MEDICINE | Volume 19 | Number 8 | August 2017 909 ORIGINAL RESEARCH ARTICLE LEGRAND et al | Mutation spectrum in pseudoxanthoma elasticum with sensitivity and specificity close to 100%.4 The severity of insufficiency (C1-attributed). Age at diagnosis was approxi- the disease can be evaluated with a scoring system called the mated by the age of the molecular analysis request. The PS was Phenodex score (PS), which was proposed at the consensus calculated for each case by the primary investigator. conference in 2007 (Supplementary Table S1 online).3 Written informed consent for the genetic study was obtained, PXE is caused mainly by loss-of-function variants in the and genetic testing was performed in accordance with French ABCC6 gene7 encoding an ATP-binding cassette (ABC) protein, legislation regarding genetics diagnostics tests (French bio- which functions as an efflux transporter. Its specific substrate ethics law 2004-800). The study was approved by the Ethics remains unknown, although ABCC6 was recently demon- Committee of the Hôpitaux Universitaires Paris Ouest, Paris, strated to be a major regulator of plasmatic inorganic pyro- France. phosphate, a strong ectopic mineralization inhibitor with low plasmatic inorganic pyrophosphate plasmatic levels associated Genetic analysis with PXE.8 Up to 300 causative variants have been identified The Qiamp DNA Blood Midi kit (Qiagen, Hilden, Germany) in ABCC6 and gathered in a dedicated database (CLINVAR: was used according to the manufacturer’s instructions to extract https://www.ncbi.nlm.nih.gov/clinvar/?term=ABCC6). These genomic DNA from leukocyte pellets. ABCC6 exons and the include missense, nonsense, and splice-site variants and dele- flanking intronic sequences were amplified by PCR with spe- tions and insertions. Although most of them are private, two cific primers to avoid amplification of ABCC6 pseudogenes Ψ1 recurrent variants of high frequency have been identified: a and Ψ2 (ref. 14), sequenced using BigDye Terminator kit v3.1 nonsense variant in exon 24, c.3421C>T, p.Arg1141Ter, with a cycle sequencing kits, and run on an ABI Prism 3730XL DNA 25% prevalence in cases of various ethnic backgrounds,3 and Analyzer (Life Technologies, Foster City, CA). DNA variants Alu1-mediated deletion of exons 23 to 29 (del23-29), which is were identified using Sequencher software. Sanger sequencing the most common variant in Americans of European descent, was completed by a multiplex PCR targeting the recurrent dele- with a prevalence of 28%.9 tion including exons 23 to 29 (ref. 15). In previous case series, attempts were made to identify spe- ABCC6 multiplex ligation-dependent probe amplification cific variant distributions along the protein as well as genotype– (MLPA) was performed to detect large rearrangements in a sub- phenotype correlations.3,10–12 Schulz et al.12 related the severity set of cases with single heterozygous point variants or suspected of the disorder to complete loss of the protein and established hemizygosity based on sequencing results. The SALSA reagent a significant correlation between the PXE phenotype and vari- set P092B kit (MRC-Holland, Amsterdam, The Netherlands) ants in ABCC6. No specific variant clustering could be found in was used according to the manufacturer’s recommendations the ABCC6 protein. (http://www.mlpa.com). Fragments were detected using an ABI Here, we present the results of phenotypic and molecular 3730XL Genetic Analyzer (Life Technologies, Foster City, CA) characterization of ABCC6 in a series of 306 French PXE pro- with ROX500 (PerkinElmer Applied Biosystems, Foster City, bands. In 220 cases, the phenotype based on the PS was well CA) as an internal size standard. Genemapper (PerkinElmer characterized, allowing genotype–phenotype correlation analy- Applied Biosystems, Foster City, CA) and Coffalyzer (MRC- sis and providing significant results. Holland, Amsterdam, The Netherlands) software were used to analyze the MLPA fragments. MATERIALS AND METHODS Array comparative genomic hybridization (array-CGH) Study population was performed to confirm cases with large deletions with the Unrelated PXE probands from diverse backgrounds were Illumina HumanCytoSNP-12 v2.1 BeadChip array, Illumina examined by dermatologists or geneticists across France from GenomeStudio v2011.1, and Illumina cnvPartition v3.1.6 soft- 1 January 2000 to 30 November 2015, and were referred to ware (Illumina, San Diego, CA). the Genetics Department of the Georges Pompidou European Unreported variants were interpreted with in silico pre- Hospital (Paris, France) for molecular analysis. The clinical diction tools (SIFT: http://www.Blocks.fhcrc.org/sift/SIFT. diagnosis was based on the consensus criteria.6 A detailed ques- html,