Analysis of mitochondrial DNA variations in Indian patients with congenital cataract.

PURPOSE: Identification of mitochondrial DNA (mtDNA) variations in the inherited cataract patients from south India. METHODS: Three families with inherited cataract of maternal origin were evaluated. Clinical and ophthalmologic examinations were performed on available affected as well as unaffected family members. Samples of mtDNA were amplified using 24 pairs of overlapping primers to analyze the entire mitochondrial genome to screen for variations and analyzed for both coding and non-coding regions. Bioinformatic analysis was performed to evaluate the effect of nucleotide variations. RESULTS: DNA sequence analysis of inherited cataract families showed 72 nucleotide variations, of which 15 were observed in the major non-coding D-loop region, 3 in the tRNA genes, 5 in the rRNA genes, and 49 in the protein coding region. Among these variations 56 were reported previously and 16 were novel of which, 12 synonymous substitutions, 2 non-synonymous substitutions along with a frameshift mutation, and one was in the non-coding region. Nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit (ND) gene of mtDNA was highly altered, in general, and found to contain 4 variations specific for cataract patients of the first family, six for the second, and one for the third family. CONCLUSIONS: Seventy-two variations were observed in three inherited cataract families. Four variations were specific for cataract patients of the first family, six for the second, and one for the third family. This is perhaps the first report on the presence of mitochondrial mutations in inherited cataracts.

A novel human CRYGD mutation in a juvenile autosomal dominant cataract.

PURPOSE: Identification of causal mutation in the crystallin, connexin, and paired box gene 6 (PAX6) genes associated with childhood cataract in patients from India. METHODS: In this study, forty eight members from seventeen families and 148 sporadic cases of childhood cataract were evaluated. Clinical and ophthalmologic examinations were performed on available affected and unaffected family members. Samples of genomic DNA were PCR amplified to screen for mutations in the candidate genes viz., alpha-A crystallin (CRYAA), beta- B2 crystallin (CRYBB2), gamma-A crystallin (CRYGA), gamma-B crystallin (CRYGB), gamma-C crystallin (CRYGC), gamma-D crystallin (CRYGD), gap junction alpha-3 (GJA3), gap junction alpha-8 (GJA8), and PAX6 based on polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP) analysis. Samples showing any band mobility shift were subjected to bidirectional sequencing to confirm the variation. Co-segregation of the observed change with the disease phenotype was further tested by restriction fragment length polymorphism (RFLP) for the appropriate restriction site. RESULTS: DNA sequencing analysis of CRYAA, CRYBB2, CRYGA-D, GJA3, GJA8, and PAX6 of the affected members of a family (C-35) showed a novel heterozygous missense mutation C>A at position 229 in CRYGD in three affected members of family C-35 with anterior polar coronary cataract. This variation C229A substitution created a novel restriction site for AluI and resulted in a substitution of highly conserved arginine at position 77 by serine (R77S). AluI restriction site analysis confirmed the transversion mutation. Analysis of the available unaffected members of the family (C-35) and 100 unrelated control subjects (200 chromosomes) of the same ethnic background did not show R77S variation. Data generated using ProtScale and PyMOL programs revealed that the mutation altered the stability and solvent-accessibility of the CRYGD protein. CONCLUSIONS: We describe here a family having anterior polar coronary cataract that co-segregates with the novel allele R77S of CRYGD in all the affected members. The same was found to be absent in the ethnically matched controls (n=100) studied. Interestingly the residue Arg has been frequently implicated in four missense (R15C, R15S, R37S, and R59H) and in one truncation mutation (R140X) of CRYGD. In two of the reported mutations Arg residues have been replaced with Serine. This finding further expands the mutation spectrum of CRYGD in association with childhood cataract and demonstrates a possible mechanism of cataractogenesis. Screening of other familial (n=48) and sporadic (n=148) cases of childhood cataract, did not reveal any previously reported or novel mutation in the candidate genes screened.