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Determination of GJB2-related deafness is dependent on the identification of mutations in the DNA of affected individuals. Mutation screening of only exon 2 of GJB2 by any technique is incomplete because there are two common noncoding, noncomplementary DFNBl-causing mutations that must be considered in persons heterozygous for a known GJB2 deafness-causing allele variant. These muta tions are the intron 1 splice donor mutation (IVS1+1G^ A) and the large 5' 342 kilobase (kb) deletion that includes a portion of GJB6 and an additional upstream sequence (A[GJB6-D13S1830]). Based on the relative frequency of GJB2 allele variants in the general population, the frequency of noncoding GJB2 mutations associated with deafness at the DFNB1 locus, and phenotype-genotype correlations,2 the existence of at least one additional mutation associated with the DFNB1 phenotype that is outside the coding region of GJB2 is predicted.

For mutation screening by DHPLC, we use the acetoni-trile gradient and partial denaturing temperature predicted by Wavemaker software, but add analysis 2°C above and below the predicted temperature to detect all possible mutations. To increase column life, cleanup and equilibration durations are extended and hot washing of the column is performed every 200 injections. DHPLC standards are run every 200 injections to confirm column reliability, since the ability of the column to detect standards is directly related to its ability to detect sequence variants in GJB2 and other genes of interest. Water quality is checked by testing resistivity and total organic content. The purity of PCR products is verified by analyzing a sample at 50°C prior to analysis at partial denaturing conditions on the DHPLC.

PENDRED SYNDROME AND DFNBH (SLC26A4) Molecular Basis of Disease

Mutations in SLC26A4 (formerly known as PDS) cause Pendred syndrome (PS; OMIM #274600), an autosomal recessive disorder characterized by sensorineural deafness and goiter.34 The deafness is congenital and associated with temporal bone abnormalities that range from isolated enlargement of the vestibular aqueduct (dilated vestibular aqueduct, DVA) to Mondini dysplasia, a more complex malformation that also includes cochlear hypoplasia, an anomaly in which the normal cochlear spiral of 2.5 turns is replaced by a smaller coil of 1.5 turns. Both DVA and Mondini dysplasia are easily recognized by computed tomography or magnetic resonance imaging.35

The thyromegaly in PS is the result of multinodular goitrous changes in the thyroid gland that develop in the teenage years,36 although affected persons typically remain euthyroid with elevated serum thyroglobulin levels. The perchlorate discharge test is often abnormal. In this test, a person is given radiolabeled iodide and its localization to the thyroid is measured. Potassium perchlorate, a competitive inhibitor of iodide transport into the thyroid, then is administered. Normally, the amount of iodide in the thyroid will remain stable, reflecting rapid oxidation of iodide to iodine as it is incorporated into thyroglobulin. However, in a person with PS, iodide transport into the thyrocyte is delayed and so when perchlorate is administered and blocks the sodium-iodide symporter, cytoplasmic iodide leaks back into the bloodstream. This back leakage is quantifiable as a change in thyroid radioactivity, with a positive result reflecting a drop in radioactivity of greater than 10%.37

In addition to PS, mutations in SLC26A4 cause DFNB4 (OMIM #600791), a type of autosomal recessive nonsyn-dromic deafness in which,by definition, affected persons do not have thyromegaly.38 No other physical abnormalities cosegregate with the deafness, although abnormal inner ear development, and in particular DVA, can be documented by temporal bone imaging. Together, DFNB4 and PS are estimated to account for 1% to 8% of congenital deafness.

Functional studies suggest that some of the observed differences between PS and DFNB4 are due to the degree of residual function of the encoded protein pendrin. While the function of pendrin is not fully determined, by homol-ogy it is thought to function in the transport of negatively charged particles (particularly chloride, iodide, and bicarbonate) across cell membranes. Mutations that abolish all transport function are more likely to be associated with the PS phenotype, while retained minimal transport ability appears to prevent thyroid dysfunction, although sen-sorineural deafness and temporal bone anomalies still occur, as in DFNB4.39

Many clinical studies have demonstrated intrafamilial variability, at times making the distinction between DFNB4 and PS difficult. The perchlorate discharge test is not a reliable test to resolve phenotypic ambiguities and is not consistently positive.36 For example, in one family with two affected siblings, one child demonstrated the classic features of PS with severe-to-profound deafness, goiter, and a positive perchlorate discharge test, but the other child had only mild sensorineural deafness and no goiter.40 In another study in which six individuals had confirmed PS, only three had a positive perchlorate washout of greater than10%.41

In a large clinical study of SLC26A4 mutations in relation to temporal bone abnormalities, deafness-causing mutations were demonstrated in approximately 80% of multiplex families segregating DVA or Mondini dysplasia but in only 30% of simplex families.42 These data suggest that allele variants of SLC26A4 are a major genetic cause of these temporal bone abnormalities.

Since thyroid enlargement is an unreliable clinical indicator of disease and the perchlorate discharge test can be ambiguous, several investigators have recommended genetic testing of SLC26A4 to establish a clinical diagnosis.36,42 To date, 62 mutations have been reported in a total of 116 families.43 Most of these mutations have been reported in only single families; however, 15 mutations are more common, and four (L236P, IVS8+1G^A, E384G, and T416P) account for approximately 60% of the total PS genetic load.42 This broad spectrum of deafness-causing allele variants means that mutation screening of SLC26A4 must include an analysis of all 20 protein-encoding exons (2-21) in addition to the splice donor site of exon 1.

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