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A novel locus (CORD12) for autosomal dominant cone-rod dystrophy on chromosome 2q24.2-2q33.1
BMC Medical Geneticsvolume 12, Article number: 54 (2011)
Rod-cone dystrophy, also known as retinitis pigmentosa (RP), and cone-rod dystrophy (CRD) are degenerative retinal dystrophies leading to blindness. To identify new genes responsible for these diseases, we have studied one large non consanguineous French family with autosomal dominant (ad) CRD.
Family members underwent detailed ophthalmological examination. Linkage analysis using microsatellite markers and a whole-genome SNP analysis with the use of Affymetrix 250 K SNP chips were performed. Five candidate genes within the candidate region were screened for mutations by direct sequencing.
We first excluded the involvement of known adRP and adCRD genes in the family by genotyping and linkage analysis. Then, we undertook a whole-genome scan on 22 individuals in the family. The analysis revealed a 41.3-Mb locus on position 2q24.2-2q33.1. This locus was confirmed by linkage analysis with specific markers of this region. The maximum LOD score was 2.86 at θ = 0 for this locus. Five candidate genes, CERKL, BBS5, KLHL23, NEUROD1, and SF3B1 within this locus, were not mutated.
A novel locus for adCRD, named CORD12, has been mapped to chromosome 2q24.2-2q33.1 in a non consanguineous French family.
Retinitis pigmentosa (RP, [MIM 268000]) is a genetically heterogeneous group of retinal photoreceptor degeneration characterized by night blindness and loss in the peripheral visual field, slowly progressing towards total blindness after several decades . RP accounts for about 2/3 of the inherited retinal dystrophy cases . In contrast to typical RP, also called rod-cone dystrophies (RCDs) because of primary involvement of rods, inverse RP or cone-rod dystrophies (CRDs) are pigmentary retinopathies characterized by first decrease in visual acuity and loss in the central visual field and lately by night blindness and loss in the peripheral visual field. CRDs are due to the primary degeneration of cone photoreceptors, followed by the secondary, or, sometimes, concomitant loss of rod photoreceptors . Fourty nine genes and loci are responsible for non syndromic RP and 18 for non syndromic CRD (including 6 in common with RP and 4 with Leber congenital amaurosis) http://www.sph.uth.tmc.edu/Retnet. The three types of Mendelian inheritance are encountered in both RP and CRD.
Among the 18 CRD genes, ten (GUCY2D, PITPNM3, GUCA1A, HRG4/UNC119, CRX, AIPL1, RIMS1, SEMA4A, PROM1 and PRPH2/RDS) are found in autosomal dominant (ad) CRD, six (ABCA4, RPGRIP1, RAX2, CORD8, ADAM9 and CERKL) in autosomal recessive (ar) CRD and two (RPGR and CACNA1F) in X-linked CRD http://www.sph.uth.tmc.edu/Retnet. The prevalence of mutations for each gene in the CRD population is highly variable. ABCA4, which causes Stargardt macular dystrophy, is also a major gene for CRD, being responsible for 30-60% of arCRD cases [4–6]. In contrast, the overall prevalence of adCRD genes remains low, many of them being described in only one or a few cases. Only CRX, GUCY2D and PRPH2/RDS have been consistently reported in adCRD [7–10]. Yet, CRX was estimated to account for only 5-10% of adCRD cases and the prevalence of GUCY2D and PRPH2/RDS is unknown [11, 12]. Therefore, there are probably other genes remaining to be discovered in adCRD.
In search for new genes responsible for pigmentary retinopathies, we recruited one large non-consanguineous French family with adCRD. This family was unlinked to any known adRP or adCRD locus and SNP genotyping revealed that it was linked to a new locus on chromosome 2, designated CORD12.
Members of this large French non-consanguineous family (RP470) were identified with CRD which segregated as a dominant trait (adCRD). There were 9 affected patients out of 22 in 4 generations (Figure 1). Examination included assessment of visual acuity, slit lamp biomicroscopy, direct funduscopy and full field electroretinography. There was no evidence of extra-ocular signs of disease indicating that CRD was non syndromic.
Genotyping of microsatellite markers and linkage analysis
Informed written consent and peripheral blood samples were obtained from 22 examined family members. The investigators followed the tenets of the Declaration of Helsinki. Genomic DNA was isolated from 10 ml peripheral blood leucocytes using standard salting out procedure . The DNA samples were quantified by a spectrophotometer and diluted to 25 ng/μl for PCR amplification. PCR was carried out in a 25 μl final volume containing 50 ng genomic DNA, 5 picomoles of each primer, 0.2 mM dNTPs (MP Biochemicals), 2 mM MgCl2, PCR buffer and 1 unit of DNA polymerase (AmpliTaq Gold; Applied Biosystems, Foster city, CA). Initial denaturation at 95°C for 10 minutes was followed by 35 cycles of denaturation at 94°C for 30 seconds, specific annealing temperature for 30 seconds, and extension at 72°C for 1 minute. A final extension step was performed at 72°C for 10 minutes. The PCR products were diluted and mixed with Genescan 400HD ROX size standard and subsequently analysed on an Applied Biosystems 3130xL genetic analyser (Applied Biosystems, Foster city, CA).
Genotyping was performed using 2 to 3 polymorphic commercially available microsatellite markers from ABI PRISM Linkage Mapping Set version 2.5 (Applied Biosystems, Foster city, CA), within or contiguous to known adRP and adCRD genes, and within the locus CORD12. Results were analysed with GeneMapper software (version 4.0, Applied Biosystems, Foster city, CA). Segregation of the markers among the family members was examined.
Two-point LOD scores were calculated with Superlink-online http://bioinfo.cs.technion.ac.il/superlink-online/. The phenotype was analyzed as an autosomal dominant and fully penetrant trait with an affected allele frequency of 0.0001. Family and haplotype data were generated using Cyrillic software (version 2.1.3; Cherwell Scientific, Oxford, UK).
SNP genotyping and analysis
To map the disease locus, a genome-wide scan was performed by the Centre National de Génotypage (CNG, http://www.cng.fr) by genotyping 262,264 SNPs (GeneChip Mapping 250 K Nsp Array, Affymetrix, Santa Clara, CA). Results were analyzed using TASE (Transmitted Allele Search Engine) a home-made software which compared every SNP between each individuals in the family.
The first test, named Common Allele to All Affected individuals (C3A), highlighted the common allele to all affected patients within the family. The second test, Transmitted Allele to All Children (TAAC), estimated the specific allele carried by the affected parent in a nuclear family (parents + child) and transmitted to the affected child. Two consecutive mismatched SNPs limited the size of the locus. Only the regions longer than 1 Mb were considered.
Coding exons and adjacent intronic sequences of candidate genes were sequenced with an Applied Biosystems 3130xL genetic analyser (Applied Biosystems, Foster city, CA) using BigDye Terminator cycle sequencing ready reaction kit V3.1 (Applied Biosystems, Foster city, CA) following manufacturer's instructions. Primer pairs and PCR conditions are available on request. Sequence analysis and mutation identification were performed using Collection and Sequence Analysis software package (Applied Biosystems, Foster city, CA).
Statement about Conformity with Author Information: Informed and written consent was obtained for all patients participating to the study. The study was done in adherence to the tenets of the Declaration of Helsinki.
The authors confirm that they are in compliance with their Institutional Review Boards (IRBs) as the Department of Ophthalmology of the Hospital of Montpellier has the authorization # 11018S from the French Ministry of Health for biomedical research in the field of physiology, pathophysiology, epidemiology and genetics in ophthalmology.
The pedigree of the four generations family is shown in Figure 1. The 9 affected patients revealed features of adCRD with intra-familial variable phenotype including progressive loss of the visual acuity, typical bone spicule-shaped pigmentary deposits in the macular area or macular atrophy, moderate night blindness and reduced electroretinogram (ERG) responses (Table 1). The proband (III:1) showed patches of atrophy in the macular area with a few pigment deposits, attenuation of retinal arterioles and temporal pallor of the optic disc (Figure 2).
Mapping to CORD12
Microsatellite markers for the 21 adRP genes http://www.sph.uth.tmc.edu/Retnet, the 3 most frequent adCRD genes (CRX, GUCY2D and PRPH2/RDS) [7–10] and a fourth adCRD gene, GUCA1A, were used to genotype family members, and to search for co-segregation of the markers with the disease phenotype. All these candidate genes were excluded. We then performed a genome wide scan using Affymetrix 250 K microarrays and genotypes were analysed with the TASE software. No linkage was found for most chromosomal regions except for a large region located on chromosome 2q24.2-2q33.1. The boundaries of the locus were determined by SNP exclusion between SNPs rs174240 and rs4619591 and encompassed a 41.3-Mb region (Figure 3).
Microsatellite markers were then used to confirm linkage with the locus. We genotyped all 22 members of the family with 8 microsatellite markers located on 2q24.2-2q33.1 (Figure 1). All affected patients had a common haplotype and the boundaries of the region were determined by recombination events that occurred in affected individuals III:1, III:4, III:9, IV:4, IV:9 and healthy individual III:7. The proximal boundary was defined by the recombination event between markers D2S142 and D2S306 in affected patient IV:9 and distal boundary by the recombination event between markers D2S117 and D2S325 in healthy individual III:7 (Figure 3). Using Superlink software, we found a maximum LOD score of 2.86 at θ = 0 for the marker D2S118, defining a new locus named CORD12. The markers D2S142, D2S325, D2S2361 and D2S126, outside the locus, gave negative LOD scores (Table 2).
The CORD12 41.3-Mb interval contains 280 genes. None of them were previously reported in adCRD or adRP. However the interval does contain two previously described autosomal recessive RP genes, namely CERKL and BBS5, which cause autosomal recessive RP and Bardet-Biedl syndrome, respectively [14, 15]. All exons and flanking intron regions were sequenced but no mutation was found. Within the CORD12 locus, three other candidate genes were also sequenced. KLHL23 has strong similarities with the recently described gene KLHL7 responsible for adRP . NEUROD1 regulates development and maintenance in the visual system . SF3B1 is a splicing factor . Other essential components of the spliceosome, PRPF31, PRPF3, PRPF8, PAP1 and SNRNP200, have been associated with adRP [19–22]. No disease causing mutations were detected in KLHL7, NEUROD1 and SF3B1.
In this study, a novel locus, CORD12, for autosomal dominant cone-rod dystrophy (adCRD) was identified and localized to chromosome 2q24.2-2q33.1. With CORD8 assigned to chromosome 1q23.1-q23.3, it is the second CRD locus for which the causative gene remains unknown . To date, the total number of known adCRD genes and loci, including CORD12, is eleven.
A maximum two-point LOD score of 2.86 at θ = 0 for the marker D2S118, close to theoretical significance, was obtained. The common haplotype for affected patients in the family was flanked by SNPs between rs174240 and rs4619591, which defined the 41.3-Mb CORD12 locus. Two other retinal dystrophy loci are mapped on chromosome 2. RP54, a 19.98-Mb autosomal recessive RP interval flanked by D2S149 and D2S367 on chromosome 2p22.3-p24.1 and RP28, a 14-Mb autosomal recessive RP interval flanked by D2S1337 and D2S286 on chromosome 2p11-p15 [25, 26]. The causative genes have recently been reported for both regions in September 2010, respectively ZNF513 for RP54 and FAM161A for RP28 [28, 29]. A third gene, C2ORF71, was identified earlier this year next to ZNF513, by homozygosity mapping in two independent studies in an 8-Mb locus on chromosome 2p24.1-p23.1 and in a 6.8-Mb locus on chromosome 2p23.1-p24.1 [30, 31]. None of these 3 regions overlap with CORD12.
The CORD12 41.3-Mb interval contains 280 annotated genes. We sequenced five possible candidate genes. CERKL and BBS5 which cause autosomal recessive RP and Bardet-Biedl syndrome, respectively,[14, 15]KLHL23, which has strong similarities with the recently described gene KLHL7 responsible for adRP,NEUROD1 which regulates development and maintenance in the visual system and the splicing factor SF3B1. No mutation was found in the coding region and splice sites junctions, indicating that these genes do not cause CORD12. However, mutations in other parts of the gene cannot be excluded. Indeed, a single-base substitution in dominant retinitis pigmentosa disease-causing gene, PRPF31, located deep within intron 13 was recently identified . No other obvious candidate genes have been identified in CORD12 based on tissue expression pattern and function of gene products similar to known CRD genes. The comparison with additional families with cone-rod dystrophy showing linkage to this locus will be necessary to narrow the interval and to help the identification of a novel gene.
In summary, we report on the identification of a novel locus for adCRD in chromosome 2q24.2-2q33. Identification of the disease causing gene in the interval will increase our understanding of the causes of cone-rod dystrophy.
Hartong DT, Berson EL, Dryja TP: Retinitis pigmentosa. Lancet. 2006, 368: 1795-1809. 10.1016/S0140-6736(06)69740-7.
Daiger SP, Bowne SJ, Sullivan LS: Perspective on genes and mutations causing retinitis pigmentosa. Arch Ophthalmol. 2007, 125: 151-158. 10.1001/archopht.125.2.151.
Hamel CP: Cone rod dystrophies. Orphanet J Rare Dis. 2007, 2: 7-10.1186/1750-1172-2-7.
Maugeri A, Klevering BJ, Rohrschneider K, Blankenagel A, Brunner HG, Deutman AF, Hoyng CB, Cremers FP: Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy. Am J Hum Genet. 2000, 67: 960-966. 10.1086/303079.
Ducroq D, Rozet J, Gerber S, Perrault I, Barbet D, Hanein S, Hakiki S, Dufier J, Munnich A, Hamel C, Kaplan J: The ABCA4 gene in autosomal recessive cone-rod dystrophies. Am J Hum Genet. 2002, 71: 1480-1482. 10.1086/344829.
Fishman GA, Stone EM, Eliason DA, Taylor CM, Lindeman M, Derlacki DJ: ABCA4 gene sequence variations in patients with autosomal recessive cone-rod dystrophy. Arch Ophthalmol. 2003, 121: 851-855. 10.1001/archopht.121.6.851.
Nakazawa M, Naoi N, Wada Y, Nakazaki S, Maruiwa F, Sawada A, Tamai M: Autosomal dominant cone-rod dystrophy associated with a Val200Glu mutation of the peripherin/RDS gene. Retina (Philadelphia, Pa.). 1996, 16: 405-410.
Kelsell RE, Gregory-Evans K, Payne AM, Perrault I, Kaplan J, Yang RB, Garbers DL, Bird AC, Moore AT, Hunt DM: Mutations in the retinal guanylate cyclase (RETGC-1) gene in dominant cone-rod dystrophy. Hum Mol Genet. 1998, 7: 1179-1184. 10.1093/hmg/7.7.1179.
Perrault I, Rozet JM, Gerber S, Kelsell RE, Souied E, Cabot A, Hunt DM, Munnich A, Kaplan J: A retGC-1 mutation in autosomal dominant cone-rod dystrophy. Am J Hum Genet. 1998, 63: 651-654. 10.1086/301985.
Swaroop A, Wang QL, Wu W, Cook J, Coats C, Xu S, Chen S, Zack DJ, Sieving PA: Leber congenital amaurosis caused by a homozygous mutation (R90W) in the homeodomain of the retinal transcription factor CRX: direct evidence for the involvement of CRX in the development of photoreceptor function. Hum Mol Genet. 1999, 8: 299-305. 10.1093/hmg/8.2.299.
Freund CL, Gregory-Evans CY, Furukawa T, Papaioannou M, Looser J, Ploder L, Bellingham J, Ng D, Herbrick JA, Duncan A, Scherer SW, Tsui LC, Loutradis-Anagnostou A, Jacobson SG, Cepko CL, Bhattacharya SS, McInnes RR: Cone-rod dystrophy due to mutations in a novel photoreceptor-specific homeobox gene (CRX) essential for maintenance of the photoreceptor. Cell. 1997, 91: 543-553. 10.1016/S0092-8674(00)80440-7.
Swain PK, Chen S, Wang QL, Affatigato LM, Coats CL, Brady KD, Fishman GA, Jacobson SG, Swaroop A, Stone E, Sieving PA, Zack DJ: Mutations in the cone-rod homeobox gene are associated with the cone-rod dystrophy photoreceptor degeneration. Neuron. 1997, 19: 1329-1336. 10.1016/S0896-6273(00)80423-7.
Miller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988, 16: 1215-10.1093/nar/16.3.1215.
Tuson M, Marfany G, Gonzàlez-Duarte R: Mutation of CERKL, a novel human ceramide kinase gene, causes autosomal recessive retinitis pigmentosa (RP26). Am J Hum Genet. 2004, 74: 128-138. 10.1086/381055.
Young TL, Penney L, Woods MO, Parfrey PS, Green JS, Hefferton D, Davidson WS: A fifth locus for Bardet-Biedl syndrome maps to chromosome 2q31. Am J Hum Genet. 1999, 64: 900-904. 10.1086/302301.
Friedman JS, Ray JW, Waseem N, Johnson K, Brooks MJ, Hugosson T, Breuer D, Branham KE, Krauth DS, Bowne SJ, Sullivan LS, Ponjavic V, Gränse L, Khanna R, Trager EH, Gieser LM, Hughbanks-Wheaton D, Cojocaru RI, Ghiasvand NM, Chakarova CF, Abrahamson M, Göring HHH, Webster AR, Birch DG, Abecasis GR, Fann Y, Bhattacharya SS, Daiger SP, Heckenlively JR, Andréasson S, Swaroop A: Mutations in a BTB-Kelch protein, KLHL7, cause autosomal-dominant retinitis pigmentosa. Am J Hum Genet. 2009, 84: 792-800. 10.1016/j.ajhg.2009.05.007.
Pennesi ME, Cho J, Yang Z, Wu SH, Zhang J, Wu SM, Tsai M: BETA2/NeuroD1 null mice: a new model for transcription factor-dependent photoreceptor degeneration. J Neurosci. 2003, 23: 453-461.
Isono K, Abe K, Tomaru Y, Okazaki Y, Hayashizaki Y, Koseki H: Molecular cloning, genetic mapping, and expression of the mouse Sf3b1 (SAP155) gene for the U2 snRNP component of spliceosome. Mamm Genome. 2001, 12: 192-198. 10.1007/s003350010258.
Vithana EN, Abu-Safieh L, Allen MJ, Carey A, Papaioannou M, Chakarova C, Al-Maghtheh M, Ebenezer ND, Willis C, Moore AT, Bird AC, Hunt DM, Bhattacharya SS: A human homolog of yeast pre-mRNA splicing gene, PRP31, underlies autosomal dominant retinitis pigmentosa on chromosome 19q13.4 (RP11). Mol Cell. 2001, 8: 375-381. 10.1016/S1097-2765(01)00305-7.
Chakarova CF, Hims MM, Bolz H, Abu-Safieh L, Patel RJ, Papaioannou MG, Inglehearn CF, Keen TJ, Willis C, Moore AT, Rosenberg T, Webster AR, Bird AC, Gal A, Hunt D, Vithana EN, Bhattacharya SS: Mutations in HPRP3, a third member of pre-mRNA splicing factor genes, implicated in autosomal dominant retinitis pigmentosa. Hum Mol Genet. 2002, 11: 87-92. 10.1093/hmg/11.1.87.
van Lith-Verhoeven JJC, van der Velde-Visser SD, Sohocki MM, Deutman AF, Brink HMA, Cremers FPM, Hoyng CB: Clinical characterization, linkage analysis, and PRPC8 mutation analysis of a family with autosomal dominant retinitis pigmentosa type 13 (RP13). Ophthalmic Genet. 2002, 23: 1-12. 10.1076/opge.220.127.116.116.
Zhao C, Bellur DL, Lu S, Zhao F, Grassi MA, Bowne SJ, Sullivan LS, Daiger SP, Chen LJ, Pang CP, Zhao K, Staley JP, Larsson C: Autosomal-dominant retinitis pigmentosa caused by a mutation in SNRNP200, a gene required for unwinding of U4/U6 snRNAs. Am J Hum Genet. 2009, 85: 617-627. 10.1016/j.ajhg.2009.09.020.
Khaliq S, Hameed A, Ismail M, Anwar K, Leroy BP, Mehdi SQ, Payne AM, Bhattacharya SS: Novel locus for autosomal recessive cone-rod dystrophy CORD8 mapping to chromosome 1q12-Q24. Invest Ophthalmol Vis Sci. 2000, 41: 3709-3712.
Naz S, Riazuddin SA, Li L, Shahid M, Kousar S, Sieving PA, Hejtmancik JF, Riazuddin S: A novel locus for autosomal recessive retinitis pigmentosa in a consanguineous Pakistani family maps to chromosome 2p. Am J Ophthalmol. 2010, 149: 861-866. 10.1016/j.ajo.2009.12.034.
Gu S, Kumaramanickavel G, Srikumari CR, Denton MJ, Gal A: Autosomal recessive retinitis pigmentosa locus RP28 maps between D2S1337 and D2S286 on chromosome 2p11-p15 in an Indian family. J Med Genet. 1999, 36: 705-707.
Kumar A, Shetty J, Kumar B, Blanton SH: Confirmation of linkage and refinement of the RP28 locus for autosomal recessive retinitis pigmentosa on chromosome 2p14-p15 in an Indian family. Mol Vis. 2004, 10: 399-402.
Li L, Nakaya N, Chavali VRM, Ma Z, Jiao X, Sieving PA, Riazuddin S, Tomarev SI, Ayyagari R, Riazuddin SA, Hejtmancik JF: A mutation in ZNF513, a putative regulator of photoreceptor development, causes autosomal-recessive retinitis pigmentosa. Am J Hum Genet. 2010, 87: 400-409. 10.1016/j.ajhg.2010.08.003.
Langmann T, Di Gioia SA, Rau I, Stöhr H, Maksimovic NS, Corbo JC, Renner AB, Zrenner E, Kumaramanickavel G, Karlstetter M, Arsenijevic Y, Weber BHF, Gal A, Rivolta C: Nonsense mutations in FAM161A cause RP28-associated recessive retinitis pigmentosa. Am J Hum Genet. 2010, 87: 376-381. 10.1016/j.ajhg.2010.07.018.
Bandah-Rozenfeld D, Mizrahi-Meissonnier L, Farhy C, Obolensky A, Chowers I, Pe'er J, Merin S, Ben-Yosef T, Ashery-Padan R, Banin E, Sharon D: Homozygosity mapping reveals null mutations in FAM161A as a cause of autosomal-recessive retinitis pigmentosa. Am J Hum Genet. 2010, 87: 382-391. 10.1016/j.ajhg.2010.07.022.
Nishimura DY, Baye LM, Perveen R, Searby CC, Avila-Fernandez A, Pereiro I, Ayuso C, Valverde D, Bishop PN, Manson FDC, Urquhart J, Stone EM, Slusarski DC, Black GCM, Sheffield VC: Discovery and functional analysis of a retinitis pigmentosa gene, C2ORF71. Am J Hum Genet. 2010, 86: 686-695. 10.1016/j.ajhg.2010.03.005.
Collin RWJ, Safieh C, Littink KW, Shalev SA, Garzozi HJ, Rizel L, Abbasi AH, Cremers FPM, den Hollander AI, Klevering BJ, Ben-Yosef T: Mutations in C2ORF71 cause autosomal-recessive retinitis pigmentosa. Am J Hum Genet. 2010, 86: 783-788. 10.1016/j.ajhg.2010.03.016.
Rio Frio T, McGee TL, Wade NM, Iseli C, Beckmann JS, Berson EL, Rivolta C: A single-base substitution within an intronic repetitive element causes dominant retinitis pigmentosa with reduced penetrance. Hum Mutat. 2009, 30: 1340-1347. 10.1002/humu.21071.
The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/12/54/prepub
We thank the family members. The work was supported by private foundations (Information Recherche sur la Rétinite Pigmentaire, Retina France, SOS Rétinite and UNADEV), Centre National de Génotypage and INSERM. Special thanks to UNADEV which supports fellowship for GM and MH.
The authors declare that they have no competing interests.
GM carried out the molecular genetic studies and the sequence alignment, participated in the design of the study and drafted the manuscript. MH performed the genotyping analysis. BB participated in the molecular genetic studies. IM participated in the medical examinations. DCC participated in the molecular genetic studies. AS participated in the sequence alignment. ABA performed the genotyping analysis. DZ performed the genotyping analysis. CPH carried out the medical examinations, conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.