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Mutations in C8orf37, Encoding a Ciliary Protein, are Associated with Autosomal-Recessive Retinal Dystrophies with Early Macular Involvement

Alejandro Estrada-Cuzcano, Kornelia Neveling, Susanne Kohl, Eyal Banin, Ygal Rotenstreich, Dror Sharon, Tzipora C Falik-Zaccai, Stephanie Hipp, Ronald Roepman, Bernd Wissinger, Stef JF Letteboer, Dorus A Mans, Ellen AW Blokland, Michael P Kwint, Sabine J Gijsen, Ramon AC van Huet, Rob WJ Collin, H Scheffer, Joris A Veltman, Eberhart Zrenner, Anneke I den Hollander, B Jeroen Klevering, Frans PM Cremers | American Journal Human Genetics | 2012 Jan 13 | Vol. 90, Issue 1 | pgs. 102–109 | doi:10.1016/j.ajhg.2011.11.015


Abstract

Cone-rod dystrophy (CRD) and retinitis pigmentosa (RP) are clinically and genetically overlapping heterogeneous retinal dystrophies. By using homozygosity mapping in an individual with autosomal-recessive (ar) RP from a consanguineous family, we identified three sizeable homozygous regions, together encompassing 46 Mb. Next-generation sequencing of all exons, flanking intron sequences, microRNAs, and other highly conserved genomic elements in these three regions revealed a homozygous nonsense mutation (c.497T>A [p.Leu166∗]) in C8orf37, located on chromosome 8q22.1. This mutation was not present in 150 ethnically matched control individuals, single-nucleotide polymorphism databases, or the 1000 Genomes database.


Immunohistochemical studies revealed C8orf37 localization at the base of the primary cilium of human retinal pigment epithelium cells and at the base of connecting cilia of mouse photoreceptors. C8orf37 sequence analysis of individuals who had retinal dystrophy and carried conspicuously large homozygous regions encompassing C8orf37 revealed a homozygous splice-site mutation (c.156−2A>G) in two siblings of a consanguineous family and homozygous missense mutations (c.529C>T [p.Arg177Trp]; c.545A>G [p.Gln182Arg]) in siblings of two other consanguineous families. The missense mutations affect highly conserved amino acids, and in silico analyses predicted that both variants are probably pathogenic. Clinical assessment revealed CRD in four individuals and RP with early macular involvement in two individuals. The two CRD siblings with the c.156−2A>G mutation also showed unilateral postaxial polydactyly. These results underline the importance of disrupted ciliary processes in the pathogenesis of retinal dystrophies.


Main Text

Retinitis pigmentosa (RP [MIM 268000]) is the most common inherited retinal degeneration and has an estimated worldwide prevalence of 1/4,000 individuals.1 RP is initially characterized by rod photoreceptor dysfunction, giving rise to night blindness, which is followed by progressive rod and cone photoreceptor dystrophy, resulting in midperipheral vision loss, tunnel vision, and sometimes blindness. The disease is genetically highly heterogeneous and displays all Mendelian patterns of inheritance. In addition, there are some cases with mitochondrial mutations and digenic inheritance.2, 3 Thus far, mutations in 34 genes have been associated with nonsyndromic autosomal-recessive (ar) RP (RetNet).3


In contrast to RP, cone-rod dystrophy (CRD [MIM 120970]) is characterized by a primary loss of cone photoreceptors and subsequent or simultaneous loss of rod photoreceptors.4, 5 The disease in most cases becomes apparent during primary-school years. The symptoms include photoaversion, a decrease in visual acuity with or without nystagmus, color-vision defects, and decreased sensitivity of the central visual field. Because rods are also involved, night blindness and peripheral vision loss can occur. The diagnosis of CRD is mainly based on electroretinogram (ERG) recordings, in which cone (photopic) responses are more severely reduced than, or equally as reduced as, rod (scotopic) responses.5, 6 CRD occurs in 1/40,000 individuals4, 5 and also displays all types of Mendelian inheritance. Mutations in five genes i.e., ABCA4 (MIM 601691), ADAM9 (MIM 602713), CDHR1 (MIM 609502), CERKL (MIM 608381), and RPGRIP1 (MIM 605446) have thus far been implicated in nonsyndromic arCRD.7, 8, 9, 10, 11


Genes harboring arCRD- and arRP-associated mutations encode proteins that are involved in phototransduction, vitamin A (retinoid) metabolism, transport along the connecting cilium, cell-to-cell signaling or synaptic interaction, gene regulation, and phagocytosis.3 Mutations in these genes are estimated to underlie ∼50% of the cases.


We aimed to identify the genetic defects associated with retinal dystrophies and to clinically investigate individuals with RP and CRD. The tenets of the Declaration of Helsinki were followed, and, in accordance with approvals gathered from the appropriate institutional review boards, informed consent was obtained from all participating individuals prior to the donation of blood samples.


Homozygosity mapping has proven to be a fruitful method of identifying mutations underlying autosomal-recessive retinal diseases12, 13, 14, 15, 16 and of establishing genotype-phenotype correlations.17, 18 To identify the genetic defect in a consanguineous family with RP (family 1; Figure 1A), we analyzed the DNA of individual IV:1 by using an Affymetrix GeneChip Human Mapping 250K SNP array (Affymetrix, Santa Clara, CA, USA) and analyzed the SNP data by using Partek Genomic Suite software (Partek, St. Louis, MO, USA). The analyses showed three large homozygous regions of 7.7 Mb (4q34.3-q35.1, rs2128423–rs59156350), 31.6 Mb (8q22.1-q24.13, rs279475–rs7013593), and 7.0 Mb (11p11.2-q11, rs11039487–rs17494990). Because more than 261 genes were present in these three chromosomal regions, a targeted next-generation sequencing (NGS) approach was used. Sequence capture was done on a 385K sequence-capture array (Roche NimbleGen, Madison, WI, USA). The array design comprised all coding and noncoding exons of these regions, including surrounding sequences that covered the splice sites. The array design harbored additional targeted regions used for similar analyses of homozygous regions in two other families. In total, the design included 4,952 targets, comprising 1,903,789 bp. Sequence capture was done according to the manufacturer's (Roche NimbleGen's) instructions with the Titanium optimized protocol as described by Hoischen et al.19 The enriched DNA regions of individual IV:1 from family 1 were sequenced on one of four lanes of a Roche 454 sequencing run, yielding 86 Mb of sequence data. Approximately 86% of the sequences were mapped back to unique regions of the human genome (hg18, NCBI build 36.1) with the use of the Roche Newbler software (version 2.3). Of all mapped reads, 91% were located on or near the targeted regions (i.e., within 500 bp). This was sufficient to reach an average of 19.3-fold coverage for all target regions. For the regions of interest, fewer than 2.6% of all targeted sequences were not covered, and only 22% of the target sequence was covered fewer than ten times. The Roche 454 software detected a total of 2,755 high-confidence variants, i.e., it identified the variants in at least three reads. We used a custom-made data-analysis pipeline as described elsewhere19 to annotate detected variants with various types of information, including known SNPs, amino acid substitutions, genomic location, and evolutionary conservation. A total of 2,573 variants either were found to represent known SNPs or overlapped with a known polymorphic region (dbSNP129); they were therefore not considered to be likely disease-causing variants.



 

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