Analysis of mitochondrial DNA variations in Indian patients with congenital cataract

Abstract

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.

Figure 1

Pedigrees of the families with inherited cataract included in the study. All the families showed maternal inheritance. The dark squares and circles represent affected male and female, respectively. The arrow indicates the proband and asterisk indicates the members involved in the study. Both the sexes were affected. Y represents the age in years. The age at onset is represented in bracket next to the affected member.

Figure 2

Cataract phenotypes. The eye images presenting cataract phenotypes in proband of childhood cataract family mt2A (III:4; A) and family mt3A (II:3; B) showing white central dense opacity leading to total cataract. The progression of cataract was observed to be differential in each eye. The zonular cataract phenotype of family mt1A proband (II:2) is not shown.

Figure 3

The distribution of mitochondrial sequence variations identified. The sequence variations identified in the coding and non-coding regions of the mitochondria are shown by arrow heads. The D- loop showed highest number of variation followed by ND1 region and Cytb. The novel mutations observed in inherited cataract families are highlighted in bold and underlined. The circular mitochondrial genome map was retrieved from mitomap.

Figure 4

Mitochondrial haplogroup analysis between childhood cataract families. The map generated for the mitochondrial alterations for each family was analyzed for haplogroups. Family mt1A showed M4 haplogroup and family mt2A, mt3A fall in to M39 haplogroups. The phylogenetic networks and evolutionary branching was constructed using Network 4.5.1.0. The reduced median or RM network algorithm was used providing binary data of the mitochondrial genomic variations. M-Macrohaplogroup, M4’30- Superhaplogroup, M4 haplogroup for family mt1A and M39 haplogroup for families mt2A, mt3A.

Figure 5

Electropherogram and polypeptide of wild type and mutant sequence of ND1 region. A: Electropherogram showing wild type and mutant nucleotide sequence of ND1 region with A>G transition at 3155 nucleotide position. Predicted secondary structure of the wild type and mutant ND1 polypeptide showing the altered amino acid. B: The architecture of the membrane proteins is reflected in the hydropathy profile. The helical wheel diagram of predicted polypeptide show change from polar residue to hydrophobic residue. Arrow mark indicates the altered aminoacid position. The source of the structure prediction is SOOSUI.