ENLARGED VESTIBULAR AQUEDUCTS (EVA) Two mutated copies of the SLC26A4 gene are associated with Pendred syndrome, comprised of bilateral hearing loss with enlargement of the vestibular aqueduct (EVA) and thyroid goiter. Other EVA patients have a normal thyroid gland and only one mutated copy of SLC26A4. Our study identified the same combination, or haplotype, of noncoding sequence variants upstream of the non-mutated copy of SLC26A4 in most of these nonsydromic EVA patients. This haplotype defines the most common allele associated with hereditary hearing loss in Caucasians. Testing for this haplotype will facilitate the genetic and prognostic counseling of many patients with EVA. (Ref. 1) We conducted a prospective cohort study of EVA subjects ascertained between 1998 and 2015 at the National Institutes of Health Clinical Center. The objective was to characterize the severity and natural history of hearing loss, and the prevalence of having a cochlear implant in a maturing cohort of individuals with enlarged vestibular aqueduct (EVA) and zero or one mutant allele of SLC26A4. Study subjects were 127 individuals (median age, 8 years; range, 059 years) with EVA in at least one ear. Ears with EVA and zero or one mutant allele of SLC26A4 have less severe hearing loss, no difference in prevalence of fluctuation, and a lower prevalence of cochlear implantation in comparison to ears with two mutant alleles of SLC26A4. (Ref. 2) We have defined the cellular taxonomy and molecular transcriptomic architecture of the mouse endolymphatic sac using RNA-seq analysis of single cells isolated from the endolymphatic sac epithelium. We have identified two primary populations of cells in the mature mouse endolymphatic sac: mitochondria-rich cells (MRCs) and ribosome-rich cells (RRCs). Genes in which mutations cause EVA are differentially expressed in MRCs. The MRC transcriptome indicates that it is the primary cell type mediating sodium chloride absorption in the developing inner ear. This absorption of sodium chloride drives the absorption of water from the lumen of the sac. Disruption of this process leads to EVA and hearing loss. 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 combinatorial linkage-based, functional candidate exome sequencing strategy to identify other genetic causes of EVA. We are prioritizing genes for analysis based upon differential expression in the MRCs of the developing endolymphatic sac . 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 the laboratory of Dominique Eladari who studied the effect of acute genetic ablation of the Slc26a4 on blood pressure in mice. He utilized our mouse line expressing Slc26a4 under the control of doxycycline administration to show that acute loss of Slc26a4 expression lowers blood pressure. This animal study showed that blocking Slc26a4 function or expression in the kidney is a potential therapeutic intervention for hypertension. (Ref. 3) We collaborated with Wade Chien's laboratory to support his study of delivery of the whirlin gene to correct loss of hearing and balance in the whirler mouse model of Usher syndrome. (Ref. 4)
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