Matthew R. Avenarius

A novel deletion involving the connexin-30 gene, del(GJB6-d13s1854), found in trans with mutations in the GJB2 gene (connexin-26) in subjects with DFNB1 non-syndromic hearing impairment

Hearing impairment is a common and highly heterogeneous sensory disorder. Genetic causes are thought to be responsible for more than 60% of the cases in developed countries.1 In the majority of cases, non-syndromic hearing impairment is inherited in an autosomal recessive pattern.2 Thirty eight different loci and 20 genes for autosomal recessive non-syndromic hearing impairment (ARNSHI) have been identified to date.3 In many populations, up to 50% of all cases of ARNSHI are caused by mutations in the DFNB1 locus (MIM 220290) on 13q12.4 This locus contains the GJB2 gene (MIM 121011), encoding connexin-26 (Cx26),5 which belongs to a family of transmembrane proteins with about 20 members in humans. Hexamers of connexins (connexons) are displayed in the plasma membrane. Docking of connexons on the surfaces of two adjacent cells results in the formation of intercellular gap junction channels.6 Several different connexins, including Cx26, have been shown to participate in the complex gap junction networks of the cochlea.7,8 It has been postulated that these networks play a key role in potassium homeostasis, which is essential for the sound transduction mechanism.9 Given the high prevalence of DFNB1 deafness, molecular testing for GJB2 mutations has become the standard of care for the diagnosis of patients with non-syndromic hearing impairment of unknown cause.10 However, the finding of a large number of affected subjects with only one GJB2 mutant allele complicates the molecular diagnosis of DFNB1 deafness. In different studies, these have accounted for 10–50% of deaf subjects with GJB2 mutations.4 It was hypothesised that there could be other mutations in the DFNB1 locus but outside the GJB2 gene. This hypothesis gained support by the finding of a deletion in the DFNB1 locus outside GJB2 but truncating the neighbouring GJB6 gene (MIM 604418), which …

A Forward Genetics Screen in Mice Identifies Recessive Deafness Traits and Reveals That Pejvakin Is Essential for Outer Hair Cell Function

Deafness is the most common form of sensory impairment in the human population and is frequently caused by recessive mutations. To obtain animal models for recessive forms of deafness and to identify genes that control the development and function of the auditory sense organs, we performed a forward genetics screen in mice. We identified 13 mouse lines with defects in auditory function and six lines with auditory and vestibular defects. We mapped several of the affected genetic loci and identified point mutations in four genes. Interestingly, all identified genes are expressed in mechanosensory hair cells and required for their function. One mutation maps to the pejvakin gene, which encodes a new member of the gasdermin protein family. Previous studies have described two missense mutations in the human pejvakin gene that cause nonsyndromic recessive deafness (DFNB59) by affecting the function of auditory neurons. In contrast, the pejvakin allele described here introduces a premature stop codon, causes outer hair cell defects, and leads to progressive hearing loss. We also identified a novel allele of the human pejvakin gene in an Iranian pedigree that is afflicted with progressive hearing loss. Our findings suggest that the mechanisms of pathogenesis associated with pejvakin mutations are more diverse than previously appreciated. More generally, our findings demonstrate that recessive screens in mice are powerful tools for identifying genes that control the development and function of mechanosensory hair cells and cause deafness in humans, as well as generating animal models for disease.

Human Male Infertility Caused by Mutations in the CATSPER1 Channel Protein

Male infertility, a common barrier that prevents successful conception, is a reproductive difficulty affecting 15% of couples. Heritable forms of nonsyndromic male infertility can arise from single-gene defects as well as chromosomal abnormalities. Although no CATSPER gene has been identified as causative for human male infertility, male mice deficient for members of the CatSper gene family are infertile. In this study, we used routine semen analysis to identify two consanguineous Iranian families segregating autosomal-recessive male infertility. Autozygosity by descent was demonstrated in both families for a approximately 11 cM region on chromosome 11q13.1, flanked by markers D11S1765 and D11S4139. This region contains the human CATSPER1 gene. Denaturing high-performance liquid chromatography (DHPLC) and bidirectional sequence analysis of CATSPER1 in affected family members revealed two separate insertion mutations (c.539-540insT and c.948-949insATGGC) that are predicted to lead to frameshifts and premature stop codons (p.Lys180LysfsX8 and p.Asp317MetfsX18). CATSPER1 is one of four members of the sperm-specific CATSPER voltage-gated calcium channel family known to be essential for normal male fertility in mice. These results suggest that CATSPER1 is also essential for normal male fertility in humans.

OTOF mutations revealed by genetic analysis of hearing loss families including a potential temperature sensitive auditory neuropathy allele

Introduction: The majority of hearing loss in children can be accounted for by genetic causes. Non-syndromic hearing loss accounts for 80% of genetic hearing loss in children, with mutations in DFNB1/GJB2 being by far the most common cause. Among the second tier genetic causes of hearing loss in children are mutations in the DFNB9/OTOF gene. Methods: In total, 65 recessive non-syndromic hearing loss families were screened by genotyping for association with the DFNB9/OTOF gene. Families with genotypes consistent with linkage or uninformative for linkage to this gene region were further screened for mutations in the 48 known coding exons of otoferlin. Results: Eight OTOF pathological variants were discovered in six families. Of these, Q829X was found in two families. We also noted 23 other coding variant, believed to have no pathology. A previously published missense allele I515T was found in the heterozygous state in an individual who was observed to be temperature sensitive for the auditory neuropathy phenotype. Conclusions: Mutations in OTOF cause both profound hearing loss and a type of hearing loss where otoacoustic emissions are spared called auditory neuropathy.

GJB2 MUTATIONS: PASSAGE THROUGH IRAN

Hereditary hearing loss (HHL) is a very common disorder. When inherited in an autosomal recessive manner, it typically presents as an isolated finding. Interestingly and unexpectedly, in spite of extreme heterogeneity, mutations in one gene, GJB2, are the most common cause of congenital severe‐to‐profound deafness in many different populations. In this study, we assessed the contributions made by GJB2 mutations and chromosome 13 g.1777179_2085947del (the deletion more commonly known as del (GJB6‐D13S1830) that includes a portion of GJB6 and is hereafter called Δ(GJB6‐D13S1830)) to the autosomal recessive non‐syndromic deafness (ARNSD) genetic load in Iran. Probands from 664 different nuclear families were investigated. GJB2‐related deafness was found in 111 families (16.7%). The carrier frequency of the 35delG mutation showed a geographic variation that is supported by studies in neighboring countries. Δ(GJB6‐D13S1830) was not found. Our prevalence data for GJB2‐related deafness reveal a geographic pattern that mirrors the south‐to‐north European gradient and supports a founder effect in southeastern Europe.

Genetic male infertility and mutation of CATSPER ion channels

A clinically significant proportion of couples experience difficulty in conceiving a child. In about half of these cases male infertility is the cause and often genetic factors are involved. Despite advances in clinical diagnostics ∼50% of male infertility cases remain idiopathic. Based on this, further analysis of infertile males is required to identify new genetic factors involved in male infertility. This review focuses on cation channel of sperm (CATSPER)-related male infertility. It is based on PubMed literature searches using the keywords ‘CATSPER’, ‘male infertility’, ‘male contraception’, ‘immunocontraception’ and ‘pharmacologic contraception’ (publication dates from January 1979 to December 2009). Previously, contiguous gene deletions including the CATSPER2 gene implicated the sperm-specific CATSPER channel in syndromic male infertility (SMI). Recently, we identified insertion mutations of the CATSPER1 gene in families with recessively inherited nonsyndromic male infertility (NSMI). The CATSPER channel therefore represents a novel human male fertility factor. In this review we summarize the genetic and clinical data showing the role of CATSPER mutation in human forms of NSMI and SMI. In addition, we discuss clinical management and therapeutic options for these patients. Finally, we describe how the CATSPER channel could be used as a target for development of a male contraceptive.

Mutations in Grxcr1 Are The Basis for Inner Ear Dysfunction in the Pirouette Mouse

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.

Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like

Hair cells tightly control the dimensions of their stereocilia, which are actin-rich protrusions with graded heights that mediate mechanotransduction in the inner ear. Two members of the myosin-III family, MYO3A and MYO3B, are thought to regulate stereocilia length by transporting cargos that control actin polymerization at stereocilia tips. We show that eliminating espin-1 (ESPN-1), an isoform of ESPN and a myosin-III cargo, dramatically alters the slope of the stereocilia staircase in a subset of hair cells. Furthermore, we show that espin-like (ESPNL), primarily present in developing stereocilia, is also a myosin-III cargo and is essential for normal hearing. ESPN-1 and ESPNL each bind MYO3A and MYO3B, but differentially influence how the two motors function. Consequently, functional properties of different motor-cargo combinations differentially affect molecular transport and the length of actin protrusions. This mechanism is used by hair cells to establish the required range of stereocilia lengths within a single cell.

The Coxsackievirus and Adenovirus Receptor: A new adhesion protein in cochlear development

The Coxsackievirus and Adenovirus Receptor (CAR) is an essential regulator of cell growth and adhesion during development. The gene for CAR, CXADR, is located within the genomic locus for Usher syndrome type 1E (USH1E). Based on this and a physical interaction with harmonin, the protein responsible for USH1C, we hypothesized that CAR may be involved in cochlear development and that mutations in CXADR may be responsible for USH1E. The expression of CAR in the cochlea was determined by PCR and immunofluorescence microscopy. We found that CAR expression is highly regulated during development. In neonatal mice, CAR is localized to the junctions of most cochlear cell types but is restricted to the supporting and strial cells in adult cochlea. A screen of two populations consisting of non-syndromic deaf and Usher 1 patients for mutations in CXADR revealed one haploid mutation (P356S). Cell surface expression, viral receptor activity, and localization of the mutant form of CAR were indistinguishable from wild-type CAR. Although we were unable to confirm a role for CAR in autosomal recessive, non-syndromic deafness, or Usher syndrome type 1, based on its regulation, localization, and molecular interactions, CAR remains an attractive candidate for genetic deafness.

Correlation of Actin Crosslinker and Capper Expression Levels with Stereocilia Growth Phases

During development of the chick cochlea, actin crosslinkers and barbed-end cappers presumably influence growth and remodeling of the actin paracrystal of hair cell stereocilia. We used mass spectrometry to identify and quantify major actin-associated proteins of the cochlear sensory epithelium from E14 to E21, when stereocilia widen and lengthen. Tight actin crosslinkers (i.e. fascins, plastins, and espin) are expressed dynamically during cochlear epithelium development between E7 and E21, with FSCN2 replacing FSCN1 and plastins remaining low in abundance. Capping protein, a barbed-end actin capper, is located at stereocilia tips; it is abundant during growth phase II, when stereocilia have ceased elongating and are increasing in diameter. Capping protein levels then decline during growth phase III, when stereocilia reinitiate barbed-end elongation. Although actin crosslinkers are readily detected by electron microscopy in developing chick cochlea stereocilia, quantitative mass spectrometry of stereocilia isolated from E21 chick cochlea indicated that tight crosslinkers are present there in stoichiometric ratios relative to actin that are much lower than their ratios for vestibular stereocilia. These results demonstrate the value of quantitation of global protein expression in chick cochlea during stereocilia development.