Figure 6. .

Allele-specific expression assay for GJB6. A, Schematic of design of HpyCH4IV digestion assay for allele-specific expression of GJB6 mRNA performed using 1,550-bp PCR product amplified from cDNA only. A forward primer (F) located in exon 5 of GJB6 is separated on genomic DNA from the reverse primer (R), located in the GJB6 3′ UTR, by a >6-kb intron in addition to ∼1,500 bp of the final exon. B, Pedigree showing rs7333214 genotypes of assayed subjects. DF5-65 bears the novel pathogenic allele (black bar with star) and is heterozygous at rs7333214. C, Results of cDNA-specific assay for GJB6 expression. Digestion of product amplified from DF5-72 yields 460-bp as well as 292- and 168-bp products, indicating that both GJB6 alleles are expressed. Digestion of product amplified from DF5-65 yields a robust 460-bp band, but the band at 292 bp is only barely visible. This indicates underrepresentation of the novel allele among the amplified product.