Novel Locus for Autosomal Recessive Cone–Rod Dystrophy CORD8 Mapping to Chromosome 1q12-Q24

Abstract

purpose. To map the disease locus of a two-generation, consanguineous Pakistani family with autosomal recessive cone–rod dystrophy (arCRD). All affected individuals had night blindness, deterioration of central vision, photophobia, epiphora in bright light, and problems with color distinction. Fundoscopy revealed marked macular degeneration and attenuation of retinal vessels. Mild pigmentary changes were present in the periphery.

methods. Genomic DNA was amplified across the polymorphic microsatellite poly-CA regions identified by markers. Alleles were assigned to individuals that allowed calculation of LOD scores using the Cyrillic (Cherwell Scientific, Oxford, UK) and MLINK (accessed from ftp://linkage.rockefeller.edu/softeware/linkage/) software programs. The cellular retinoic acid-binding protein 2 (CRABP2), cone transducin α-subunit (GNAT2), potassium inwardly rectifying channel, subfamily J, member 10 (KCNJ10), genes were analyzed by heteroduplex analysis and direct sequencing for mutations.

results. A new locus for arCRD (CORD8) has been mapped to chromosome 1q12-q24. A maximum two-point LOD score of 4.22 was obtained with marker D1S2635 at recombination fraction of θ = 0.00. Two critical recombinations in the pedigree positioned this locus to a region flanked by markers D1S457 and D1S2681. A region of homozygosity was observed within the loci D1S442 and D1S2681, giving a probable critical disease interval of 21 cM. Mutation screening of the three candidate genes CRABP2, GNAT2, and KCNJ10 revealed no disease-associated mutations.

conclusions. The findings therefore suggest that this phenotype maps to a new locus and is due to an as yet uncharacterized gene within the 1q12-q24 chromosomal region.

Retinal photoreceptor dystrophies are a clinically and genetically heterogeneous group of retinal degenerations that together form the most frequent cause of inherited visual disorders, with an estimated prevalence of 1 in 4000. ^{1 2}  

Cone-rod dystrophies (CRDs) are a group of severe inherited retinal dystrophies characterized by the simultaneous involvement of both cone and rod photoreceptor cells. ^{3 4 5} Patients are affected from an early age, and there is progressive loss of visual acuity and color vision (cone mediated functions) followed by night blindness and loss of peripheral vision (largely, rod mediated). In later life, vision may be reduced to bare perception of light. Fundus changes in CRDs are typified by macular atrophy and pigmentation in the early stages, with widespread retinal pigmentation resembling classic retinitis pigmentosa (RP) as the disease progresses. In contrast, in RP, the disease process primarily affects the rod photoreceptors and initially manifests as night blindness and reduction in the peripheral visual field, although involvement of the macula with loss of central vision may occur in the later stages. ¹  

CRD may be inherited as autosomal recessive, autosomal dominant, or X-linked traits. Genetic studies on the autosomal disease have so far identified three loci for the autosomal dominant: 6p21.1 (peripherin/RDS), ⁶ 19q13.3 (CRX), ^{7 8} and 17p12-13 (RetGC-1) ⁹ ; and one locus for autosomal recessive CRD: 1p21-p13(ABCR). ¹⁰ Deletion mapping suggests further loci on 6q25-q26, 17q (associated with neurofibromatosis, presumed dominant-like), and 18q21.1-q21.3 (de Grouchy syndrome). ¹¹  

Here we report the linkage of arCRD to a new locus on chromosome 1q12-q24. 

Methods

We studied 16 members of a four-generation, consanguineous Pakistani family in which the inheritance pattern of CRD was consistent with an autosomal recessive trait. Ophthalmic examination of the family members confirmed 6 affected and 10 unaffected individuals (Fig. 1) . Subjects were designated as unaffected if they showed no clinical evidence of CRD by the age of 30 years. The onset of deterioration of central vision was from the ages of 12 to 14 years. Patients also experienced photophobia and epiphora in bright light. Affected subjects already had problems of color distinction in childhood and, at the age of 16 years, loss of central vision was accompanied by complete loss of color vision. Patients did not report nyctalopia. Fundoscopy revealed marked macular degeneration and attenuation of retinal vessels. Mild pigmentary changes were present in the periphery. Because neither electrophysiologic nor peripheral visual field testing was available in Pakistan, these additional tests could not be performed. Based on family history and clinical findings, the disease was classified as (arCRD; Leroy et al. unpublished data, 1999). This project was conducted in line with the Declaration of Helsinki. 

DNA Extraction

Peripheral blood samples were collected with informed consent from the affected and unaffected members of the family (Fig. 1) . For comparison, 100 ethnically matched individuals with no personal or family history of retinopathy were selected to serve as controls. Genomic DNA was extracted from whole blood by using an extraction kit (Nucleon II; Scotlab Bioscience, Manchester, UK). 

Microsatellite and Linkage Analysis

Genomic DNA was amplified using primers that specifically amplify the polymorphic microsatellite poly-CA regions identified by markers. Polymerase chain reaction (PCR) products were separated by nondenaturing polyacrylamide gel electrophoresis (Protogel; National Diagnostics, Manville, NJ), and visualized under UV illumination after staining with ethidium bromide. Alleles were assigned to individuals, and genotypic data were used to calculate the LOD scores using the Cyrillic (Cherwell Scientific, Oxford, UK) and MLINK (accessed from ftp://linkage.rockefeller.edu/software/linkage/) software programs. Allele frequencies were calculated from the spouses in this family and a control ethnically matched population. The phenotype was analyzed as an autosomal recessive trait with complete penetrance and a frequency of 0.0001 for the affected allele. 

Mutation Screening

Intronic forward and reverse primers were designed for the exons of the candidate genes. Twenty-five- or 50-μl PCR reactions were performed, using standard conditions with Taq DNA polymerase (Bio-Line, London, UK), annealing at the exon-specific temperature. The amplified exons were analyzed by electrophoresis using MDE gel run at 180 V overnight using a commercial system (model 600S; Hoefer, San Francisco, CA) to identify any heteroduplexes. ¹²  

Products of PCR amplification were sequenced using a kit (PRISM Ready Reaction; Perkin–Elmer/Applied Biosystems, Foster City, CA), and the products were analyzed on an automated sequencer (ABI 373A XL; Perkin–Elmer/Applied Biosystems). All PCR products were sequenced in the forward and reverse directions. 

Results

Linkage analysis was performed on this family as described. The known arCRD/RP loci (RP20 RPE65 1p31, RP19 ABCR 1p21-p13, RP12 CRB1 1q31-q32.1, RP26 2q31-q33, RP4 RHO 3q21-q24, PDE6B 4p16.3, CNGC 4p14-q13, PDE6A 5q31.2-q34, RP14 TULP1 6p21.3, RP25 6cen-q15 and CRALBP 15q26, and RP22 16p12.1-p12.3) were tested for linkage to the disease in this family, using two microsatellite markers centered on the critical region of each locus. Linkage was not observed to any of the known arCRD/RP loci. 

A genome-wide search was undertaken using 200 polymorphic markers spanning the entire human genome at 20-cM intervals. Significant exclusion was obtained for all markers except those located on chromosome 1q12-q24 within the RP18-adRP region. Haplotypes for these markers are shown in Figure 1 . Two-point LOD scores between arCRD and the markers in this region (D1S495, D1S457, D1S2746, D1S2881, D1S534, D1S514, D1S442, D1S498, D1S2635, D1S2771, D1S484, D1S2768, D1S2681, and D1S210) are summarized in Table 1 . The maximum LOD score of 4.22 was obtained for the marker D1S2635. Positive LOD scores ranging from 2.13 to 3.97 at θ = 0.00 to 0.20 were also obtained with several other markers within the region. Recombination events involving markers D1S2681 in individual IV:9 and D1S457 in individual IV:1 define the telomeric and centromeric boundaries, respectively, of the disease locus between these markers (Fig. 1) . 

Because 28RP was a consanguineous family, the disease region might be expected to be homozygous for the microsatellites closest to the associated gene. All the patients in both branches of the family were homozygous for markers D1S498, D1S2635, D1S2771, D1S484, and D1S2768. Therefore, it is probable that the disease gene lies between D1S442 and D1S2681, a refined region of approximately 21 cM. 

Mutation screening of three candidate genes: cellular retinoic acid-binding protein 2 (CRABP2),¹³ cone transducin α-subunit (GNAT2),¹⁴ and potassium inwardly rectifying channel protein (KCNJ10)¹⁵ physically mapped within the disease region, was performed using primers designed to the intronic regions of the genes to permit detection of any splice site mutations. Heteroduplex and direct sequencing revealed no disease-associated mutations. 

Discussion

Linkage analysis in an inbred pedigree has permitted us to identify a novel locus for arCRD (CORD8) at 1q12-q24. This chromosomal region encompasses the RP18 adRP locus ¹⁶ (Fig. 2) . The gene associated with the arCRD in the family described may be distinct from the RP18 gene. Alternatively, the disease may be the result of different mutations in the RP18 gene that are inherited in an autosomal recessive manner and have a detrimental effect on cone as well as the rod photoreceptor cells. A similar phenomenon has been associated with the RetGC-1 gene, the mutations in which can result in either autosomal recessive Leber congenital amaurosis or autosomal dominant CRD, depending on the nature of the mutation. ^{9 17}  

Recently, mutations in ABCR have been reported for arCRD. As shown in Figure 1a , centromeric recombination in individual IV:1 excludes the new locus from the ABCR gene locus on 1p13-22. This second novel autosomal recessive locus illustrates genetic heterogeneity of the arCRD phenotype. 

Although the recombination events seen in this family define a critical disease region between D1S457 and D1S2681, because of the consanguineous nature of the pedigree, it is probable that the disease gene lies in the smaller 21-cM interval between D1S442 and D1S2681 defined by region of homozygosity seen in all the patients of this family. Efforts to identify the disease gene may therefore be preferentially concentrated on this region of chromosome 1. 

³  SK and AH contributed equally to this work.

 Supported by The Wellcome Trust.

 Submitted for publication January 27, 2000; revised April 26 and July 21, 2000; accepted July 28, 2000.

 Commercial relationships policy: N.

 

Figure 1.

 

Pedigree of a family affected by arCRD with genotypic data for microsatellite markers analyzed. Circles: women; squares: men; filled symbols: affected individuals; open symbols: unaffected individuals. Consanguinity is represented by a double line connecting the spouses.

Table 1.

 

Two-Point LOD Scores of Family with arCRD for Chromosome 1q Microsatellite Markers

Markers	Recombination Fraction													Z max	θmax
		0.0	0.01	0.05	0.1	0.2	0.3	0.4
D1S495	−∞	−5.93	−2.55	−1.23	−0.19	0.14	0.15	0.15	0.4
D1S457	−2.17	−0.44	0.36	0.62	0.65	0.44	0.14	0.65	0.2
D1S2746	0.47	2.07	2.50	2.45	1.99	1.30	0.51	2.50	0.05
D1S2881	0.69	2.28	2.70	2.64	2.15	1.42	0.57	2.70	0.05
D1S534	2.88	2.83	2.60	2.32	1.72	1.11	0.51	2.88	0.0
D1S514	−1.18	0.44	0.97	1.06	0.91	0.60	0.23	1.06	0.1
D1S442	−1.46	0.06	0.62	0.75	0.65	0.38	0.06	0.75	0.1
D1S498	2.63	2.58	2.38	2.12	1.59	1.03	0.46	2.63	0.0
D1S2635	4.22	4.13	3.77	3.33	2.41	1.50	0.62	4.22	0.0
D1S2771	1.77	1.73	1.59	1.41	1.03	0.65	0.27	1.77	0.0
D1S484	3.97	3.89	3.58	3.18	2.35	1.50	0.66	3.97	0.0
D1S2768	2.13	2.09	1.92	1.71	1.27	0.82	0.37	2.13	0.0
D1S2681	−∞	−4.04	−1.35	−0.37	0.23	0.26	0.11	0.26	0.3
D1S210	−∞	−3.76	−1.78	−1.01	−0.39	−0.13	−0.02	0.00	0.6

Figure 2.

 

Schematic representation of chromosome 1, showing the novel arCRD locus and relative locations of other known eye disorder loci/genes. References to all loci depicted can be found at RetNet: http://www.sph.uth.tmc.edu/Retnet/disease.htm.