A novel locus (CORD12) for autosomal dominant cone-rod dystrophy on chromosome 2q24.2-2q33.1

Gal Manes 0 1 Maxime Hebrard 0 1 Batrice Bocquet 0 1 Isabelle Meunier Delphine Coustes-Chazalette Audrey Snchal 0 1 Anne Bolland-Aug Diana Zelenika Christian P Hamel 0 1 0 INSERM U1051, Institute for Neurosciences of Montpellier , (80 rue Augustin Fliche), Montpellier, (34091) , France 1 Universite Montpellier 1 , (2 rue Ecole de Background: 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. Methods: 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. Results: 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 = 0 for this locus. Five candidate genes, CERKL, BBS5, KLHL23, NEUROD1, and SF3B1 within this locus, were not mutated. Conclusion: A novel locus for adCRD, named CORD12, has been mapped to chromosome 2q24.2-2q33.1 in a non consanguineous French family. - Background 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 [1]. RP accounts for about 2/3 of the inherited retinal dystrophy cases [2]. 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 [3]. 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. Methods Clinical examination 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 extraocular 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 [13]. The DNA samples were quantified by a spectrophotometer and diluted to 25 ng/l for PCR amplification. PCR was carried out in a 25 l final volume containing 50 ng genomic DNA, 5 picomoles of each primer, 0.2 mM dNTPs (MP Biochemicals), 2 mM Figure 1 Pedigree of family RP470 with autosomal dominant inherited cone-rod dystrophy (adCRD). Arrow indicates the index patient. Filled symbols represent members with adCRD and empty symbols represent unaffected patients. Haplotypes of microsatellite markers spanning the locus 2q24.2-2q33.1 are shown. Question marks indicate unknown alleles. Solid bars denote the haplotype that segregated with the disease phenotype. MgCl2, PCR buffer and 1 unit of DNA polymerase (AmpliTaq Gold; Applied Biosystems, Foster city, CA). Initial denaturation at 95C for 10 minutes was followed by 35 cycles of denaturation at 94C for 30 seconds, specific annealing temperature for 30 seconds, and extension at 72C for 1 minute. A final extension step was performed at 72C 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/superlinkonline/. 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 Gnotypage (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 (...truncated)