Fig 1

Restriction enzyme digestion to determine the cis-trans relationship of p.Val27Ile and p.Glu114Gly in GJB2. A, Restriction map of the 431-bp fragment that contains p.Val27Ile and p.Glu114Gly. The WT allele has a unique restriction pattern created by HpyCH4III on p.Val27Ile and ApoI on p.Glu114Gly. The size of each restriction fragment is indicated. After HpyCH4III and ApoI digestion, the WT allele generates 264-, 87-, and 80-bp fragments; the p.Val27Ile (V27I) allele generates 344- and 87-bp fragments; the p.Glu114Gly (E114G) allele produces 351- and 80-bp fragments; alleles with both p.Val27Ile (V27I) and p.Glu114Gly (E114G) abolish both restriction enzyme sites and therefore have the full-length PCR products of 431 bp. B, Restriction enzyme digestion of the 431-bp PCR fragments from patient samples. P1 to P8 were heterozygous for p.Val27Ile and p.Glu114Gly by Sanger sequencing. Ctrl-1 was homozygous for p.Val27Ile and p.Glu114Gly, presenting with only a 431-bp fragment; ctrl-2 was homozygous for p.Val27Ile and heterozygous for p.Glu114Gly, generating fragments of 431, 344, and 87 bp; ctrl-3 was WT for both alleles and generated 264-, 87-, and 80-bp fragments. Uncut represents the 431-bp fragment without restriction digestion. The weak band in between 431 and 264 bp in P1 to P8, and ctrl-1 was explained by star activity of the restriction enzyme.