ENLARGED VESTIBULAR AQUEDUCTS (EVA) We completed a study of families with atypical patterns of segregation in multiple affected members. The goal of this study was to characterize the SLC26A4 genotypes and phenotypes of extended families with atypical segregation of EVA. One family had members with EVA caused by different etiologies, and two families had pseudodominant inheritance of recessive mutations of SLC26A4. In five families, the etiology remained unknown and could include inheritance of mutant alleles at another genetic locus, nongenetic influences, or a combination of these factors. We concluded that familial EVA can demonstrate a variety of atypical segregation patterns. Pseudodominant inheritance of SLC26A4 mutations or recessive alleles of other hearing loss genes may be more likely to occur in families in which deaf individuals have intermarried. The etiologic basis of atypical segregation of EVA without detectable SLC26A4 mutations remains unknown. Future studies of these families may reveal novel genes for EVA. We ascertain families with multiple members with nonsyndromic EVA that is not associated with detectable SLC26A4 mutations or Pendred syndrome. Our hypothesis is that these families segregate recessive alleles at one or more other genetic loci that cause nonsyndromic EVA. We are using those families in a linkage-based exome sequencing strategy to identify other genetic causes of EVA. We used recombination breakpoint mapping to define a region of shared linkage overlap containing the SLC26A4 gene on chromosome 7 to search for occult (unidentified) mutations of SLC26A4 in families segregating nonsyndromic EVA with only one detectable mutant allele of SLC26A4. Our hypothesis is that these families segregate a second, unidentified, mutation of SLC26A4. We have used massively parallel sequencing to sequence the entire region. We are using a combination of genetic and functional expression approaches to identify pathogenic variants. We are defining the cellular and molecular transcriptomic architecture of the mouse endolymphatic sac using RNA-seq analysis of single cells isolated from the endolymphatic sac epithelium. DFNA34 HEARING LOSS We mapped a novel nonsyndromic hearing loss locus, DFNA34, in a single large family. We used recombinations to define a critical map interval in which the gene and mutation must be located. We identified a likely mutation in a gene (NLRP3) in which other mutations cause hearing loss associated with autoinflammatory disease. In order to confirm this mutation as causative, we used massively parallel sequencing as well as conventional Sanger dideoxy sequencing to rule out mutations in any of the other genes in the critical map interval. We detect expression of the candidate gene, NLRP3, in the inner ear. We collaborated with Drs. Daniel Kastner. Paola Pinto-Patarroyo and Raphaela Goldbach-Mansky to study the patients for evidence of cochlear and systemic auto-inflammation on magnetic resonance imaging studies at the NIH Clinical Center. We have detected evidence of systemic and cochlear auto-inflammation, providing conclusive proof of the pathogenic nature of the mutation we have detected. We have also shown the existence of macrophage/monocyte-like cells in the normal resting mouse cochlea. We have shown that these cells are capable of expressing NLRP3 and secreting interleukin-1beta. Therefore the mouse cochlea has resident cells capable of mounting an innate immune response. We hypothesize that DFNA34 causes cochlear hearing loss by abnormal activation of the NLRP3 inflammasome pathway within the cochlea. We recently ascertained a second unrelated family with the exact same mutation segregating in the original DFNA34 family. The affected members of this second family also co-segregate signs and symptoms of auto-inflammation although they do not meet criteria for cryopyrin-associated periodic syndromes, a spectrum of auto-inflammatory disorders known to also be caused by NLRP3 mutations. Treatment with an IL-1beta blocker, anakinra, improved or resolved the hearing loss in 3 of 3 family members who were treated. The temporal bone imaging indicated that the cochlear auto-inflammation also improved or resolved, in correlation with their hearing test results. Therefore DFNA34 hearing loss is an example of a genetic form of hearing loss in which precise diagnosis can guide treatment which can stabilize or reverse the loss of hearing. COLLABORATIVE PROJECTS We collaborated with multiple NIDCD Sections and Units to complete a collaborative study of the heritability of auditory processing in children. The results indicate that a majority of the observed variation in many auditory processing abilities is caused by genetic factors.
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