Figure 1.
Hearing impairment in family S1599.
A Pedigree of family S1599, indicating the GJB2 genotypes of subjects who were analyzed. B Audiograms (air conduction) of affected individuals II:1 and II:6 at ages 50 and 31 years, respectively (circles, right ear; crosses, left ear). The binaural means of the hearing thresholds for air conduction at frequencies 0.5, 1, and 2 kHz for these subjects are 33.3 and 25.8 dB, respectively, corresponding to mild HI (21-40 dB).
Figure 2.
Sequence of the exon-intron junctions of the single GJB2 intron.
Exonic sequence is shown in capitals, whereas intronic sequence is shown in lowercase italics. The three acceptor splice sites identified in this work appear boxed in bold: KS, known acceptor site; AS1, alternative site 1; AS2, alternative site 2. The ATG start codon appears in bold. Nucleotides affected by the c. -22-2A>C and c.35delG mutations are indicated by arrows. Locations of the primers used for identifying GJB2 splicing products are underlined.
Figure 3.
Analysis of GJB2 splicing products.
Splicing products from transcription of GJB2 were amplified in a fluorescent RT-PCR assay performed with total RNA extracted from saliva samples and were separated by capillary electrophoresis. A GJB2 splicing products from a normal-hearing control (genotype wt/wt). B GJB2 splicing products from c. -22-2A>C/c.35delG compound heterozygous subject II:4.
Figure 4.
Sequence electropherograms of cloned GJB2 transcripts from c.
-22-2A>C/c.35delG compound heterozygous subject II:4. The stretch of six guanine nucleotides affected by the c.35delG deletion appears boxed in red, while the start ATG codon is boxed in black. A Sequence of the transcript derived from the c.35delG allele, generated by use of the known acceptor site. Note that the stretch of guanine nucleotides contains only five guanines. B Sequence of the transcript derived from the c. -22-2A>C allele generated by use of the alternative acceptor site 1. Note the cytosine nucleotide at c. -22-2, boxed in purple.
Figure 5.
Relative expression levels of GJB2 transcripts generated by splicing at different acceptor sites.
Each panel shows results for a normal-hearing control (genotype wt/wt) and for c. -22-2A>C/c.35delG compound heterozygous subject II:4. A Relative expression of GJB2 transcript generated by splicing from the known site (two-tailed p < 0.0001, N=5). B Relative expression of GJB2 transcript generated by splicing from alternative site 1 (two-tailed p= 0.0094, N=3).