A novel APC mutation defines a second locus for ...

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Feb 12, 2015 - upregulated, including IGHM, AKAP11, PPP1R2 and FANCL. (figure 3). DISCUSSION. APC is a very well-known gene clinically given its ...

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JMG Online First, published on February 12, 2015 as 10.1136/jmedgenet-2014-102850 New loci

ORIGINAL ARTICLE

A novel APC mutation defines a second locus for Cenani–Lenz syndrome Nisha Patel,1 Eissa Faqeih,2 Shams Anazi,1 Mohammad Alfawareh,3 Salma M Wakil,1 Dilek Colak,4 Fowzan S Alkuraya1,5 ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ jmedgenet-2014-102850). 1

Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia 2 Department of Pediatrics, King Fahad Medical City, Riyadh, Saudi Arabia 3 Spine Department, King Fahad Medical City, Riyadh, Saudi Arabia 4 Department of Biostatistics and Scientific Computing, Epidemiology, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia 5 Department of Anatomy and Cell Biology, College of Medicine, Alfaisal University, Riyadh, Saudi Arabia Correspondence to Dr Fowzan S Alkuraya at Department of Genetics, King Faisal Specialist Hospital and Research Center, MBC-03, PO box 3354, Riyadh 11211, Saudi Arabia; [email protected] Received 27 October 2014 Accepted 26 January 2015

ABSTRACT Background Cenani–Lenz syndrome (CLS) is an autosomal recessive condition characterised by a unique pattern of syndactyly, and variable penetrance of renal agenesis and facial dysmorphism. LRP4 mutations were identified in most, but not all patients with this syndrome, suggesting the presence of at least one additional locus. Materials and methods Clinical characterisation of a new CLS family followed by autozygosity mapping, whole-exome sequencing and global gene expression profiling. Results We describe an extended consanguineous Saudi family with typical CLS features in addition to significant scoliosis. The disease in this family maps to a single autozygous interval on 5q22.2, in which wholeexome sequencing revealed the presence of a novel splicing mutation in APC that results in ∼80% reduction of the wild-type transcript and the creation of an aberrant transcript that predicts a severely truncated APC. This was found to be associated with upregulation of Wnt/β-catenin signalling. Conclusions In a pattern similar to how LRP4 mutations are predicted to negate the protein’s antagonistic effect on Wnt/β-catenin signalling, we propose that reduction of APC may increase the availability of β-catenin by virtue of impaired degradation, leading to a similar phenotypic outcome. This is the first time APC is linked to a human phenotype distinct from its established role in oncology.

INTRODUCTION

To cite: Patel N, Faqeih E, Anazi S, et al. J Med Genet Published Online First: [please include Day Month Year] doi:10.1136/ jmedgenet-2014-102850

In their classification of various syndactyly forms, Mckusick and Temtamy designated the syndactyly described by Cenani and Lenz as a distinct group.1 Cenani–Lenz syndactyly comprises a highly unusual pattern of fusion between the phalanges and metacarpal/metatarsal bones that results in ‘spoon hands’ resembling Apert’s hands.2 The hands are typically more severely affected, and the additional manifestations of radio-ulnar synostosis and mesomelia are frequently encountered. Temtamy et al3 described a highly inbred family in which Cenani–Lenz syndactyly cosegregated with distinct facial features, most notably prominent forehead, hypertelorism, depressed nasal bridge and prominent maxillary incisors, which prompted the authors to suggest that Cenani–Lenz is a syndrome (CLS). Indeed, subsequent reports of renal involvement and facial dysmorphism substantiated the syndromic designation.4

Nearly all multiplex cases of CLS are consistent with autosomal recessive inheritance. This was unequivocally proven when Li et al5 performed autozygosity mapping on 14 CLS families and were able to identify a single locus that harboured mutations in LRP4 in 12 out of the 14 study families. The discovery of LRP4 as the disease gene in CLS was consistent with available mouse model data where Lrp4 −/− had abnormal limb and renal development; the latter was seen in nearly 50% of patients with CLS.6 LRP4 belongs to the lipoprotein receptor protein family known for its role in Wnt signalling. Unlike LRP5 and LRP6, however, LRP4 antagonises Wnt signalling thereby decreasing the availability of β-catenin.7 This was an attractive disease mechanism given the known role of Wnt/β-catenin in chondrocyte differentiation and limb development.8 9 Genetic heterogeneity of CLS was evident by the failure to identify LRP4 mutations in two of the wellphenotyped 14 families used to map the disease locus, and by lack of linkage to LRP4 in at least one of these two.5 In this report, we describe an extended consanguineous CLS family in which we map the disease to a novel locus that harbours a splicing mutation in APC, implicating APC for the first time in the pathogenesis of this syndrome.

MATERIALS AND METHODS Human subjects, and autozygome and whole-exome analysis Patients underwent thorough clinical genetics and orthopaedic evaluation. Affected and unaffected members were recruited after signing a written informed consent as part of an IRB-approved protocol (KFSHRC RAC#2080006). Venous blood was extracted in EDTA and, in two affected members, sodium-heparin-containing tubes for DNA and RNA extraction, respectively. Autozygosity mapping was essentially as described before.10 Briefly, genome-wide SNP genotyping using Axiom SNP chip platform (Affymetrix, Santa Clara, California, USA) was followed by determination of homozygosity intervals using AutoSNPa. Linkage analysis provided statistical confirmation of the candidate autozygous interval and was performed as described before. Whole-exome sequencing was performed by preparing DNA libraries followed by exome capture and sequencing on Illumina HiSeq 2000 Sequencer. The resulting variants were filtered based on frequency (absent in 1000 Genomes, EVS[OUP_CE13 Exome Variant Server] and 549 Saudi exomes), type (coding/splicing) and location (within the

Patel N, et al. J Med Genet 2015;0:1–5. doi:10.1136/jmedgenet-2014-102850

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New loci coordinates of the critical locus). RT-PCR and real-time RT-PCR confirmation of the splicing mutation and its effect were carried out using upstream and downstream exonic primers.

Global expression profiling Lymphoblasts from two affected patients and two unrelated healthy controls ( performed in duplicates) were used for global expression profiling using Affymetrix’s GeneChip Human Genome U133 Plus V.2.0 Arrays. Sample handling, cDNA synthesis, aRNA labelling and fragmentation, hybridisation, washing and scanning of arrays and all related quality controls were performed according to manufacturer’s instructions (Agilent Technologies, Santa Clara, California and Affymetrix, Santa Clara, California, USA). Filtration and elimination of genes displaying as average intensity inferior to the global array background were done using RMA (Robust Multi-Array Analysis). Significantly modulated genes were defined as those with absolute fold change (FC) >2 and adjusted p value

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