Laboratory Issues

To ensure a high level of accuracy and reliability and to optimize cost-effectiveness and turnaround time, a number of testing parameters must be considered. Ampli-cons should be maintained between 200 and 1,000 base pairs for accurate DHPLC analysis, intronic primers should be selected sufficiently far upstream and downstream of splice sites, and PCR conditions must be optimized for high-stringency exon amplification. Although the optimal acetonitrile gradient and partial denaturing temperature are determined using Wavemaker software, additional testing must be performed 2°C above and below the predicted temperature to detect all possible mutations.

While other investigators have been able to correlate DHPLC chromatogram profile with mutation type,33 this correlation may be unreliable. The profile for a given SLC26A4 allele variant differs from column to column and even in the same column, based on column life and buffer constitution. However, all heterozygote mutations are distinguished from wild-type samples. Because DHPLC does not distinguish homozygote allele variants due to the limitations inherent in HA, homozygotes can be detected by analysis following pooling of the unknown samples with sequence-verified wild-type DNA at a ratio of 2:1. By coupling additional automated instrumentation with DHPLC, high-throughput, accurate, reliable, and cost-effective mutation screening of persons with a Pendred syn-drome/DNFB4 phenotype is possible.

Detection of only a single mutation is more common for testing of simplex families.42,46 In three multiplex families segregating single mutations, Southern hybridization, and real-time PCR have failed to identify abnormalities in the "normal" allele, although all affected persons within a family had the same parental "normal" allele.

WOLFRAM SYNDROME AND DFNA6/14 (WFS1) Molecular Basis of Disease

Mutations in WFS1 cause autosomal dominant low-frequency sensorineural hearing impairment (LFSNHI) at the DFNA6/14 loci (OMIM #600965).47,48 Deafness is bilaterally symmetrical and affects frequencies below 4,000 Hz; hearing is most impaired at the lowest frequencies, giving the DFNA6/14 audiogram an upsloping configuration.49-52 DFNA1 also is characterized by LFSNHI, but in contrast to DFNA6/14 deafness, DFNA1 deafness is rapidly progressive and ultimately affects all frequencies.53 Progression of DFNA6/14 deafness is minimal, although with aging the consequences of presbycusis result in flattening of the audiogram.

Mutations in WFS1 also cause Wolfram syndrome, an autosomal recessive disease characterized by diabetes insipidus, diabetes mellitus, optic atrophy, and deafness, giving rise to the acronym DIDMOAD for this disease.54,55 Minimal diagnostic criteria are diabetes mellitus and optic atrophy, and in addition to diabetes insipidus and sen-sorineural deafness, peripheral neuropathy, urinary-tract atony, and psychiatric illness can occur. Remarkably, the hearing loss in DIDMOAD syndrome is in the high frequencies.56,57

The observed phenotypic differences between DIDMOAD and DFNA6/14 appear to have a genotypic correlate. Sixty-five percent of persons with DIDMOAD carry inactivating mutations in WFS1, suggesting that loss of function of the encoded protein is causally related to Wolfram syndrome; most of these mutations lie in predicted transmembrane domains. In contrast, all disease-causing DFNA6/14 allele variants have missense mutations, and with one exception these amino acid changes are in the fifth intracellular domain of WFS1.58,59 The protein lacks significant homology to published DNA or protein sequences, but secondary structure predictions suggest that it is has nine helical transmembrane segments. Biochemical studies suggest that wolframin is an endoglycosi-dase H-sensitive membrane glycoprotein predominantly located in the endoplasmic reticulum (ER).60 Although the function of wolframin within the inner ear is unknown, it may play a role in the canalicular reticulum, a specialized ER that maintains intracellular ion homeostasis. Functional studies are necessary to test this hypothesis and to determine how different mutations in WFS1 give rise to different phenotypes.

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