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15 cM region, which was further refined to 2 cM on 7p15 and, subsequently,
the critical interval narrowed to 600 to 850 kb (Van Laer et al. 1997). Of the
three expressed sequence tags (ESTs) identified in the DFNA5 candidate
region, one of these genes was found to have 10 exons and was predicted
to encode a 499 amino acid protein containing no domains or motifs in
common with proteins of known function. A Southern blot of genomic
DNA from normal and affected individuals from the DFNA5 family was
prepared and probed with the EST clone derived from this gene. This blot
and subsequent PCR analyses revealed a 1,189 base pair (bp) deletion in
intron 7 of all affected individuals, but not in normal-hearing individuals of
this family.
Why would a deletion of intronic sequence cause a mutant phenotype?
One possibility is that the deletion affected normal mRNA splicing. This
hypothesis was tested by reverse transcription-polymerase chain reaction
(RT-PCR) analysis of mRNA from lymphoblastoid cell lines derived from
affected and unaffected individuals. PCR amplification of the cDNA prod-
ucts with a forward primer in exon 7 and a reverse primer in exon 9 gen-
erated a 314-bp fragment in unaffected controls and a 121-bp fragment
from affected individuals. Sequence analysis indicated that exon 8 was
skipped in DFNA5 mRNA from affected individuals. The function of
DFNA5 in the auditory system and in other tissues where it is expressed is
unknown (Table 6.4).
3.6 DFNA8, DFNA12 and DFNB21 are the Same Locus
DFNA8 and DFNA12 were mapped to an overlapping region on chromo-
some 11q22-q24 in one Austrian and one Belgian family, respectively, each
segregating dominant nonsyndromic hearing loss (Fig. 6.1). CT scans of the
temporal bones revealed no gross abnormalities of the inner ear. Hearing
loss in affected members of the Belgian kindred was prelingual with stable
moderate to severe losses in all frequencies. This is an unusual phenotype
for dominant nonsyndromic hearing loss loci, since most mutant alleles of
DFNA loci cause postlingual progressive hearing loss (Table 6.1).
DFNA12 mapped to a 36 cM interval on chromosome 11q (Verhoeven
et al. 1997). A chromosomal region of this size may encompass hundreds,
if not thousands, of genes. Identification of the responsible gene is a daunt-
ing endeavor unless there is a compelling candidate gene. TECTA is the
homologue of mouse Tecta on chromosome 9. Based on conserved synteny,
TECTA was predicted to be on human chromosome 11q22-24 in the inter-
val to which DFNA12 mapped. TECTA encodes a-tectorin, a noncollage-
nous extracellular protein found exclusively in the inner ear, located
primarily in the tectorial membrane where it is cross-linked to b-tectorin.
The genomic DNA sequence of the 23 exons of TECTA was screened by
SSCP analysis for mutations in affected individuals in the two families used
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