Figure 2. GJA8 mutation in family D. Sequence analysis of GJA8 genomic DNA indicates heterozygosity after position 776 (red arrow) for the proband’s parents, II.2 and II.3 (A) and a homozygous insertion of a “G” at this position in the cDNA from the proband’s lens mRNA (B). C: The comparison of the wild type sequence (NM_005267) with the proband’s sequence (AD) demonstrates that the insertion of the G after position 776 leads to an altered open reading frame. The new amino acid sequence is indicated by red letters in a yellow box. Moreover the mutation causes a loss of the restriction site for BsrD1 but creates a new one (BstX1). In the cDNA sequence, the A of the ATG start codon is counted as #1 and in the amino-acid sequence, the first Met is counted as #1. D: The restriction digest using BstX1 in the members of the family demonstrates that homozygosity of the mutation is present only in both severely affected children. The other family members are heterozygous, independent of the slight nuclear opacity. A control person from outside (Contr) is homozygous wild type, since BstX1 cannot cut.