Molecular Analysis Of Human Hereditary Deafness

前庭导水管扩大（伊娃） SLC 26 A4基因的两个突变拷贝与Pendred综合征相关，Pendred综合征包括双侧听力损失伴前庭水管扩大（伊娃）和甲状腺肿。 其他伊娃患者甲状腺正常，只有一个突变的SLC 26 A4拷贝。 我们的研究发现，在大多数非症状性伊娃患者中，SLC 26 A4非突变拷贝上游的非编码序列变异具有相同的组合或单倍型。 该单倍型定义了与高加索人遗传性听力损失相关的最常见等位基因。 检测这种单倍型将有助于许多伊娃患者的遗传和预后咨询。(Ref.第一章 我们对1998年至2015年在美国国立卫生研究院临床中心确定的伊娃受试者进行了一项前瞻性队列研究。 目的是描述听力损失的严重程度和自然史，以及前庭水管扩大（伊娃）和零个或一个SLC 26 A4突变等位基因个体的成熟队列中人工耳蜗植入的患病率。研究受试者为127名至少一只耳朵患有伊娃的个体（中位年龄，8岁;范围，0.59岁）。伊娃和零个或一个突变等位基因的SLC 26 A4的耳朵相比，听力损失的严重程度较低，波动的患病率没有差异，人工耳蜗植入的患病率较低。(Ref.（二） 我们已经定义了细胞分类和分子转录组结构的小鼠内淋巴囊使用RNA-seq分析的单细胞分离的内淋巴囊上皮。 我们已经确定了两个主要群体的细胞在成熟的小鼠内淋巴囊：富含核糖体的细胞（MRC）和富含核糖体的细胞（RRCs）。 突变导致伊娃的基因在MRC中差异表达。 MRC转录组表明它是发育中的内耳中介导氯化钠吸收的主要细胞类型。 这种氯化钠的吸收驱动水从囊腔的吸收。 这一过程的中断导致伊娃和听力损失。 我们确定了多个成员患有非综合征型伊娃的家族，该家族与可检测到的SLC 26 A4突变或Pendred综合征无关。我们的假设是，这些家庭分离隐性等位基因在一个或多个其他遗传基因座，导致非综合征型伊娃。我们正在使用这些家庭在一个组合的连锁为基础的，功能候选外显子组测序策略，以确定其他遗传原因的伊娃。我们正在根据发育中的内淋巴囊的MRC中的差异表达来优先分析基因。 DFNA 34听力损失 我们在一个大家族中定位了一个新的非综合征性听力损失位点DFNA 34。我们使用重组来定义一个关键的图谱间隔，基因和突变必须位于该间隔中。我们确定了一个可能的基因突变（NLRP 3），其中其他突变导致与自身炎症性疾病相关的听力损失。为了确认该突变为致病性，我们使用大规模平行测序以及常规的桑格双脱氧测序来排除在关键图谱间隔中的任何其他基因中的突变。我们检测候选基因NLRP 3在内耳中的表达。我们与丹尼尔·卡斯特纳博士合作。保拉Pinto-Patarroyo和Raphaela Goldbach-Mansky在NIH临床中心的磁共振成像研究中研究患者的耳蜗和全身自身炎症证据。我们已经检测到全身和耳蜗自身炎症的证据，为我们检测到的突变的致病性提供了确凿的证据。我们还表明，在正常的休息小鼠耳蜗中存在巨噬细胞/单核细胞样细胞。我们已经证明这些细胞能够表达NLRP 3并分泌白细胞介素-1 β。因此，小鼠耳蜗具有能够产生先天免疫应答的常驻细胞。我们假设DFNA 34通过耳蜗内NLRP 3炎性体通路的异常激活引起耳蜗听力损失。 我们最近确定了第二个不相关的家庭与完全相同的突变分离在原来的DFNA 34家庭。第二个家族的受影响成员也共同分离自身炎症的体征和症状，尽管它们不符合cryopyrin相关周期性综合征的标准，cryopyrin相关周期性综合征是已知也由NLRP 3突变引起的一系列自身炎症性疾病。IL-1 β受体阻滞剂阿那白滞素治疗改善或消除了3名接受治疗的家庭成员中的3名听力损失。颞骨成像表明耳蜗自身炎症也得到改善或解决，与他们的听力测试结果相关。因此，DFNA 34听力损失是遗传形式的听力损失的一个例子，其中精确的诊断可以指导治疗，可以稳定或逆转听力损失。 合作项目 我们与Dominique Eladari的实验室合作，他研究了Slc 26 a4的急性基因消融对小鼠血压的影响。 他利用我们的小鼠系在多西环素施用的控制下表达Slc 26 a4，以显示Slc 26 a4表达的急性丧失降低血压。 该动物研究表明，阻断Slc 26 a4在肾脏中的功能或表达是高血压的潜在治疗干预。(Ref.第三章 我们与Wade Chien的实验室合作，以支持他的研究，即在Usher综合征的whirler小鼠模型中传递whirlin基因以纠正听力和平衡的丧失。(Ref.四、

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)