Purpose: To study the correlation of genotype for X-linked red-green gene array with color vision phenotype in 58 subjects with red-green color vision deficiency. Methods: The molecular structure of red and green pigment genes on 58 X chromosomes was studied exon-by-exon by using heteroduplex-SSCP analysis and sequencing. The color vision of these subjects was determined by a Neitz anomaloscope. Results: Variations in the red and green pigment genes were detected in 43 subjects and a hybrid gene was found in 27 subjects. About 50% of the fusion sites occurred at intron 2-3. All 3 anomalous trichromats with intron 4 fusion were mild type but another 3 with intron 2-3 fusion were severe type. No subjects with mild type of color vision defects had a fusion site at intron 2-3 or its upstream. Three subjects with complete deletion of the green pigment gene manifested deuteranomaly. Conclusions: Protans can be differentiated from deutans on the basis of genotype. It is still difficult to establish a clear correlation of different anomalous trichromats with genotype. The fusion site of a hybrid gene affects the phenotype to some degree. Intron 2-3 is the common place for gene crossover.

Purpose: To screen for possible disease-causing mutations in the CRX gene in Chinese patients with Leber congenital amaurosis (LCA) and to enrich the understanding of its mutational phenotype. Methods: Genomic DNA was collected from 27 patients with LCA.The coding sequences of the CRX gene were analyzed using the PCRheteroduplex-SSCP method. Mutations were confirmed by DNA sequencing. Results: We identified two heterozygous variations in the CRX gene in two patients with LCA. One was a deletion (GCCÆ-CC, A181D1bp) leading to a frameshift and protein truncation. This mutation was present in a patient with LCA, but not in his healthy parents. The ocular manifestations of this A181D1bp mutation are described. An intronic variation (IVS1-13G→C) was found in a patient with LCA as well as in his healthy father. Conclusion: A heterozygous A181D1bp mutation in the CRX gene caused an LCA phenotype in a Chinese patient.

Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive retinal dystrophy characterized by multiple glistening intraretinal crystals scattered over the fundus, a characteristic degeneration of the retina, and sclerosis of the choroidal vessels, ultimately resulting in progressive night blindness and constriction of the visual field. The BCD region of chromosome 4q35.1 was refined to an interval flanked centromerically by D4S2924 by linkage and haplotype analysis; mutations were found in the novel CYP450 family member CYP4V2 in 23 of 25 unrelated patients with BCD tested. The CYP4V2 gene, transcribed from 11 exons spanning 19 kb, is expressed widely. Homology to other CYP450 proteins suggests that CYP4V2 may have a role in fatty acid and steroid metabolism, consistent with biochemical studies of patients with BCD.

PURPOSE. To identify the disease locus of autosomal recessive congenital nuclear cataracts in a consanguineous Pakistani family. METHODS. A large Pakistani family with multiple individuals affected by autosomal recessive congenital cataracts was ascertained. Patients were examined, blood samples were collected, and DNA was isolated. A genome-wide scan was performed using 382 polymorphic microsatellite markers on genomic DNA from affected and unaffected family members. Two-point lod scores were calculated, and haplotypes were formed by inspection. RESULTS. In the genome-wide scan, a maximum lod score of 2.89 was obtained for marker D19S414 on 19q13. Fine mapping using D19S931, D19S433, D19S928, D19S225, D19S416, D19S213, D19S425, and D19S220 markers from the Généthon database showed that markers in a 14.3-cM (12.66-Mb) interval flanked by D19S928 and D19S420 cosegregated with the cataract locus. Lack of homozygosity further suggests that the cataract locus may lie in a 7-cM (4.3-Mb) interval flanked by D19S928 proximally and D19S425 distally. On fine mapping, a maximum lod score of 3.09 was obtained with D19S416 at θ=0. CONCLUSIONS. Linkage analysis identified a new locus for autosomal recessive congenital nuclear cataracts on chromosome 19q13 in a consanguineous Pakistani family.

URPOSE. To identify the disease locus for autosomal recessive congenital cataracts in consanguineous Pakistani families. METHODS. Two Pakistani families were ascertained, patients were examined, blood samples were collected, and DNA was isolated. A genome-wide scan was performed using 382 polymorphic microsatellite markers on genomic DNA from affected and unaffected family members. Two-point lod scores were calculated, haplotypes were formed by inspection, and candidate genes were sequenced. Real-time quantitative PCR techniques were used to determine the mRNA levels, and molecular modeling was performed to gain a better understanding of the significance of the disease-causing mutation. RESULTS. In the genome-wide scan, maximum lod scores of 2.67 and 2.77 for family 60004 and 2.02 and 2.04 for family 60006 were obtained for markers D22S539 and D22S315, respectively. The linked region, 22.7 cM (10 Mb) flanked by markers D22S420 and D22S1163, contains the-crystallin gene cluster including the genes CRYBA4, CRYBB1, CRYBB2, and CRYBB3. Sequencing of these genes showed a G3C transition in exon 6 of CRYBB3 resulting in a p.G165R change in the B3-crystallin protein that cosegregates with the disease in both families. Real-time PCR analysis suggested that βB3-crystallin mRNA levels approximate those of other βγ-crystallins. Molecular modeling predicted changes in electrostatic potential that would be expected to reduce the stability of the fourth Greek-key motif, and hence the entire protein, dramatically. CONCLUSIONS. For the first time, a mutation in CRYBB3 is reported in two consanguineous Pakistani families with autosomal recessive congenital cataracts. (Invest Ophthalmol Vis Sci. 2005;46:2100–2106) DOI:10.1167/iovs.04-148

PURPOSE. To localize and identify the gene and mutations causing autosomal recessive retinitis pigmentosa in three consanguineous Pakistani families. METHODS. Blood samples were collected and DNA was extracted. A genome-wide scan was performed by using 382 polymorphic microsatellite markers on genomic DNA from affected and unaffected family members, and lod scores were calculated. RESULTS. A genome-wide scan of 25 families gave an hlod 4.53 with D8S260. Retinitis pigmentosa in all three families mapped to a 14.21-cM (21.19-Mb) region on chromosome 8 at q11, flanked by D8S532 and D8S260. This region harbors RP1, which is known to cause autosomal dominant retinitis pigmentosa. Sequencing of the coding exons of RP1 showed mutations in all three families: two single-base deletions, c.4703delA and c.5400delA, resulting in a frame shift, and a 4-bp insertion, c.1606insTGAA, all causing premature termination of the protein. All affected individuals in these families are homozygous for the mutations. Parents and siblings heterozygous for the mutant allele did not show any signs or symptoms of RP. CONCLUSIONS. These results provide strong evidence that mutations in RP1 can result in recessive as well as dominant retinitis pigmentosa. The findings suggest that truncation of RP1 before the BIF motif or within the terminal portion results in a simple loss of RP1 function, producing a recessive inheritance pattern. In contrast, disruption of RP1 within or immediately after the BIF domain may result in a protein with a deleterious effect and hence a dominant inheritance pattern. (Invest Ophthalmol Vis Sci. 2005;46:2264–2270) DOI:10.1167/iovs.04-1280

Purpose: To localize and identify the gene and mutations causing autosomal recessive retinitis pigmentosa (RP) in consanguineous Pakistani families. Methods: Families were ascertained and patients underwent complete ophthalmological examinations. Blood samples were collected and DNA was extracted. A genome-wide scan was performed using 382 polymorphic microsatellite markers on genomic DNA from affected and unaffected family members, and lod scores were calculated. Results: A genome-wide scan of 50 families gave a lod score of 7.4172 with D5S2015 using HOMOG1. RP in all 4 linked families mapped to a 13.85 cM (14.87 Mb) region on chromosome 5q31-33 flanked by D5S2090 and D5S422. This region harbors the PDE6A gene, which is known to cause autosomal recessive RP. Sequencing of PDE6A showed a homozygous single base pair change; c.889C->T, single base pair insertion; c.2218-2219insT, and single base pair substitution in the splice acceptor site; IVS10-2A->G in each of three families. In the fourth family linked to this region, no disease-causing mutation was identified in the PDE6A gene. Conclusions: These results provide strong evidence that mutations in PDE6A result in recessive RP in three consanguineous Pakistani families. Although a fourth family was linked to markers in the 5q31-33 interval, no mutation was identified in PDE6A.

Purpose: To map the locus for autosomal recessive retinitis pigmentosa in a large Pakistani family and to determine the causative mutation. Methods: A large family with multiple individuals affected by autosomal recessive retinitis pigmentosa was ascertained in the Punjab province of Pakistan as part of an ongoing project between the CEMB, Lahore, Pakistan and the NEI to identify genetic causes of eye diseases. After initial analysis of previously identified autosomal recessive retinitis pigmentosa loci, a genome wide scan was performed using microsatellite markers at about 10cM intervals. Two point lod scores were calculated and haplotypes were analyzed in order to define disease locus. Bidirectional dideoxynucleotide sequencing was used to screen for mutations in candidate genes. Results: In the genome wide scan, autosomal recessive retinitis pigmentosa in this Pakistani family showed linkage to an 11.7 cM region of chromosome 4p12 between D4S405 and D4S1592 with a maximum lod score of 2.90 with D4S405 at θ=0.01 Sequence analysis of CNGA1 identified a 2 bp deletion in exon 8: c.626_627delTA resulting in a frameshift, p.Ser209fsX26 in the translated protein. This mutation results in deletion of the COOH terminal 482 of 690 total amino acids in CNGA1 and their replacement by 25 novel amino acids before a premature termination. The mutation is found in a homozygous state in all 7 affected individuals and was heterozygous in all 15 unaffected family members examined. The mutant allele of CNGA1 itself shows linkage to the disease with maximum lod score of 4.43 at θ=0. Conclusions: The autosomal recessive retinitis pigmentosa in this family is caused by a mutation in CNGA1 gene. To our knowledge, this is the first report in which both linkage analysis and identification of a mutation support CNGA1 as a cause for autosomal recessive retinitis pigmentosa.

Purpose: To describe the clinical characteristics of a Y-sutural cataract associated with myopia in a large Chinese family and to identify the causative gene and mutation. Methods: An autosomal dominant Y-sutural cataract and myopia were identified in members of a large family of Han ethnicity living in southern China. Ophthalmological examinations were performed and a medical history was taken. Blood samples were collected for DNA isolation. A genome wide scan was performed using markers spaced at about 10cM intervals for genotyping and two point linkage analysis. Candidate genes were sequenced. Results: Bilateral lens opacities, the only sign of cataract in early childhood and the most prominent sign in all affected individuals, involved the entire anterior Y and posterior inverted Y sutures, showing a feather duster like appearance. The Y-sutural cataract in this family mapped to an 11.4cM (13.5 Mb) region between D3S3606 and D3S1309 on chromosome 3q22 with a maximum lod score of 5.7 at θ=0 for D3S1292. Sequence analysis of the beaded filament structural protein 2 (BFSP2) gene identified a previously described c.697_699delGAA (E233del) mutation which was present in all individuals with Y-sutural cataract but not in unaffected individuals and controls. Myopia, observed in 10 out of 12 cataract patients and significantly higher than that in unaffected offspring and siblings (1 out of 8), was independently mapped to a 61.2 cM (59 Mb) region between D3S3606 and D3S1262 on 3q21.3-q27.2 with maximum lod score of 3.79. Conclusions: This Y-sutural cataract is caused by an E233del mutation in BFSP2 which provides additional evidence supporting mutations in BFSP2 as a cause for cataract and demonstrates phenotypic variability in cataracts caused by BFSP2. The Y-sutural opacity in the lens might be the typical and earliest sign for cataract caused by the BFSP2 mutation. In addition, these results demonstrate a myopia susceptibility locus in this region, which might also be associated with the mutation in BFSP2.

Purpose: The purpose of this paper is to map the locus for a variant form of Oguchi disease in a Pakistani family and to identify the causative mutation. Methods: Family 61029 was ascertained in the Punjab province of Pakistan. It includes three 13-to 19-year-old patients with night blindness and 12 unaffected family members. A complete ophthalmological examination including fundus photography and electroretinography (ERG) was performed on each family member. A genome-wide scan was performed using microsatellite markers at about 10cM intervals, and two-point lod scores were calculated. Polymerase chain reaction (PCR) cycle dideoxynucleotide sequencing was used to screen candidate genes inside the linked region for mutations and to delineate the deletion. Multiplex PCR and long template PCR were used to detect deletions and to define the size of deletions. Evaluation of fundus changes and ERG, lod score estimation, and identification of a mutation in the GRK1 gene were carried out. Results: All patients had night blindness since early childhood. Irregular coarse pigmentation was observed in the peripheral retina of each patient. The fundus appearance before and after 4h of dark adaptation was similar except that the peripheral retinal pigmentary changes were slightly less evident after extended dark adaptation. Minimal or no rod function with normal cone function on ERG recordings were detected in all three affected members. The rod showed slow recovery to nearly normal amplitude after 4h in the dark ERG in one individual but not in two other patients. A genomewide scan showed linkage only to D13S285. Fine mapping defined a region from D13S1315 to 13qter, with a lod score of 2.89 at θ=0 shown by D13S285 and 2.90 at θ=0 by the D13S261-D13S285-D13S1295-D13S293 haplotype. Analysis of the GRK1 gene, which is included in this interval, identified a c.827+623_883del mutation. This intragenic deletion cosegregates with the disease in the family and is only homozygos in affected individuals. This mutation was not detected in 96 controls. Conclusions: The retinal disease in the family reported here has several features differing from typical Oguchi disease, including an atypical Mizuo-Nakamura phenomenon and a non-recordable rod ERG even after 4h of dark adaptation. Normal visual acuity, normal caliber of retinal blood vessels, and normal cone response on ERG recording suggest retinal dysfunction rather than degeneration (i.e., a variant form of Oguchi disease but unlikely to be retinitis pigmentosa). The disease in the Pakistani family localizes to 13q34 and is caused by a novel deletion including Exon 3 of the GRK1 gene.