Novel variant p.E269K confirms causative role of PLS1 mutations in autosomal dominant hearing loss.

Auditory reception relies on the perception of mechanical stimuli by stereocilia and its conversion to electrochemical signal. Mechanosensory stereocilia are abundant in actin, which provides them with structural conformity necessary for perception of auditory stimuli. Out of three major classes of actin-bundling proteins, plastin 1 encoded by PLS1, is highly expressed in stereocilia and is necessary for their regular maintenance. A missense PLS1 variant associated with autosomal dominant hearing loss (HL) in a small family has recently been reported. Here, we present another PLS1 missense variant, c.805G > A (p.E269K), in a Turkish family with autosomal dominant non-syndromic HL confirming the causative role of PLS1 mutations in HL. We propose that HL due to the p.E269K variant is from the loss of a stable PLS1-ACTB interaction.

Dysfunction of GRAP, encoding the GRB2-related adaptor protein, is linked to sensorineural hearing loss.

We have identified a GRAP variant (c.311A>T; p.Gln104Leu) cosegregating with autosomal recessive nonsyndromic deafness in two unrelated families. GRAP encodes a member of the highly conserved growth factor receptor-bound protein 2 (GRB2)/Sem-5/drk family of proteins, which are involved in Ras signaling; however, the function of the growth factor receptor-bound protein 2 (GRB2)-related adaptor protein (GRAP) in the auditory system is not known. Here, we show that, in mouse, Grap is expressed in the inner ear and the protein localizes to the neuronal fibers innervating cochlear and utricular auditory hair cells. Downstream of receptor kinase (drk), the Drosophila homolog of human GRAP, is expressed in Johnston's organ (JO), the fly hearing organ, and the loss of drk in JO causes scolopidium abnormalities. drk mutant flies present deficits in negative geotaxis behavior, which can be suppressed by human wild-type but not mutant GRAP. Furthermore, drk specifically colocalizes with synapsin at synapses, suggesting a potential role of such adaptor proteins in regulating actin cytoskeleton dynamics in the nervous system. Our findings establish a causative link between GRAP mutation and nonsyndromic deafness and suggest a function of GRAP/drk in hearing.

FOXF2 is required for cochlear development in humans and mice.

Molecular mechanisms governing the development of the human cochlea remain largely unknown. Through genome sequencing, we identified a homozygous FOXF2 variant c.325A>T (p.I109F) in a child with profound sensorineural hearing loss (SNHL) associated with incomplete partition type I anomaly of the cochlea. This variant is not found in public databases or in over 1000 ethnicity-matched control individuals. I109 is a highly conserved residue in the forkhead box (Fox) domain of FOXF2, a member of the Fox protein family of transcription factors that regulate the expression of genes involved in embryogenic development as well as adult life. Our in vitro studies show that the half-life of mutant FOXF2 is reduced compared to that of wild type. Foxf2 is expressed in the cochlea of developing and adult mice. The mouse knockout of Foxf2 shows shortened and malformed cochleae, in addition to altered shape of hair cells with innervation and planar cell polarity defects. Expressions of Eya1 and Pax3, genes essential for cochlear development, are reduced in the cochleae of Foxf2 knockout mice. We conclude that FOXF2 plays a major role in cochlear development and its dysfunction leads to SNHL and developmental anomalies of the cochlea in humans and mice.

Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation.

RIPOR2 (previously known as FAM65B) localizes to stereocilia of auditory hair cells and causes deafness when its function is disturbed by mutations. Here, we demonstrate that during the morphogenesis of the hair cell bundle, absence of Ripor2 affects the orientation of this key subcellular structure. We show that Ripor2 interacts with Myh9, a protein encoded by a known deafness gene. Absence of Ripor2 is associated with low Myh9 abundance in the mouse cochlea despite increased amount of Myh9 transcripts. While Myh9 is mainly expressed in stereocilia, a phosphorylated form of Myh9 is particularly enriched in the kinocilium. In Ripor2-deficient mice, kinocilium shows an aberrant localization which associates with a reduced content of phosphorylated Myh9. Acetylated alpha tubulin, another specific kinociliary protein which contributes to microtubule stabilization, is reduced in the absence of Ripor2 as well. We propose that Ripor2 deficiency influences abundance and/or post-translational modifications of proteins expressed in both stereocilia and kinocilia. This effect may have a negative impact on the structure and function of the auditory hair cell bundle.

MPZL2 is a novel gene associated with autosomal recessive nonsyndromic moderate hearing loss.

While recent studies have revealed a substantial portion of the genes underlying human hearing loss, the extensive genetic landscape has not been completely explored. Here, we report a loss-of-function variant (c.72delA) in MPZL2 in three unrelated multiplex families from Turkey and Iran with autosomal recessive nonsyndromic hearing loss. The variant co-segregates with moderate sensorineural hearing loss in all three families. We show a shared haplotype flanking the variant in our families implicating a single founder. While rare in other populations, the allele frequency of the variant is ~ 0.004 in Ashkenazi Jews, suggesting that it may be an important cause of moderate hearing loss in that population. We show that Mpzl2 is expressed in mouse inner ear, and the protein localizes in the auditory inner and outer hair cells, with an asymmetric subcellular localization. We thus present MPZL2 as a novel gene associated with sensorineural hearing loss.

ROR1 is essential for proper innervation of auditory hair cells and hearing in humans and mice.

Hair cells of the inner ear, the mechanosensory receptors, convert sound waves into neural signals that are passed to the brain via the auditory nerve. Little is known about the molecular mechanisms that govern the development of hair cell-neuronal connections. We ascertained a family with autosomal recessive deafness associated with a common cavity inner ear malformation and auditory neuropathy. Via whole-exome sequencing, we identified a variant (c.2207G>C, p.R736T) in ROR1 (receptor tyrosine kinase-like orphan receptor 1), cosegregating with deafness in the family and absent in ethnicity-matched controls. ROR1 is a tyrosine kinase-like receptor localized at the plasma membrane. At the cellular level, the mutation prevents the protein from reaching the cellular membrane. In the presence of WNT5A, a known ROR1 ligand, the mutated ROR1 fails to activate NF-κB. Ror1 is expressed in the inner ear during development at embryonic and postnatal stages. We demonstrate that Ror1 mutant mice are severely deaf, with preserved otoacoustic emissions. Anatomically, mutant mice display malformed cochleae. Axons of spiral ganglion neurons show fasciculation defects. Type I neurons show impaired synapses with inner hair cells, and type II neurons display aberrant projections through the cochlear sensory epithelium. We conclude that Ror1 is crucial for spiral ganglion neurons to innervate auditory hair cells. Impairment of ROR1 function largely affects development of the inner ear and hearing in humans and mice.

Comprehensive analysis via exome sequencing uncovers genetic etiology in autosomal recessive nonsyndromic deafness in a large multiethnic cohort.

PURPOSE: Autosomal recessive nonsyndromic deafness (ARNSD) is characterized by a high degree of genetic heterogeneity, with reported mutations in 58 different genes. This study was designed to detect deafness-causing variants in a multiethnic cohort with ARNSD by using whole-exome sequencing (WES). METHODS: After excluding mutations in the most common gene, GJB2, we performed WES in 160 multiplex families with ARNSD from Turkey, Iran, Mexico, Ecuador, and Puerto Rico to screen for mutations in all known ARNSD genes. RESULTS: We detected ARNSD-causing variants in 90 (56%) families, 54% of which had not been previously reported. Identified mutations were located in 31 known ARNSD genes. The most common genes with mutations were MYO15A (13%), MYO7A (11%), SLC26A4 (10%), TMPRSS3 (9%), TMC1 (8%), ILDR1 (6%), and CDH23 (4%). Nine mutations were detected in multiple families with shared haplotypes, suggesting founder effects. CONCLUSION: We report on a large multiethnic cohort with ARNSD in which comprehensive analysis of all known ARNSD genes identifies causative DNA variants in 56% of the families. In the remaining families, WES allows us to search for causative variants in novel genes, thus improving our ability to explain the underlying etiology in more families.Genet Med 18 4, 364-371.

Whole-exome sequencing and its impact in hereditary hearing loss.

Next-generation sequencing (NGS) technologies have played a central role in the genetic revolution. These technologies, especially whole-exome sequencing, have become the primary tool of geneticists to identify the causative DNA variants in Mendelian disorders, including hereditary deafness. Current research estimates that 1% of all human genes have a function in hearing. To date, mutations in over 80 genes have been reported to cause nonsyndromic hearing loss (NSHL). Strikingly, more than a quarter of all known genes related to NSHL were discovered in the past 5 years via NGS technologies. In this article, we review recent developments in the usage of NGS for hereditary deafness, with an emphasis on whole-exome sequencing.

Novel mutations confirm that COL11A2 is responsible for autosomal recessive non-syndromic hearing loss DFNB53.

Hearing loss (HL) is a major public health issue. It is clinically and genetically heterogeneous.The identification of the causal mutation is important for early diagnosis, clinical follow-up, and genetic counseling. HL due to mutations in COL11A2, encoding collagen type XI alpha-2, can be non-syndromic autosomal-dominant or autosomal-recessive, and also syndromic as in Otospondylomegaepiphyseal Dysplasia, Stickler syndrome type III, and Weissenbacher-Zweymuller syndrome. However, thus far only one mutation co-segregating with autosomal recessive non-syndromic hearing loss (ARNSHL) in a single family has been reported. In this study, whole exome sequencing of two consanguineous families with ARNSHL from Tunisia and Turkey revealed two novel causative COL11A2 mutations, c.109G > T (p.Ala37Ser) and c.2662C > A (p.Pro888Thr). The variants identified co-segregated with deafness in both families. All homozygous individuals in those families had early onset profound hearing loss across all frequencies without syndromic findings. The variants are predicted to be damaging the protein function. The p.Pro888Thr mutation affects a -Gly-X-Y- triplet repeat motif. The novel p.Ala37Ser is the first missense mutation located in the NC4 domain of the COL11A2 protein. Structural model suggests that this mutation will likely obliterate, or at least partially compromise, the ability of NC4 domain to interact with its cognate ligands. In conclusion, we confirm that COL11A2 mutations cause ARNSHL and broaden the mutation spectrum that may shed new light on genotype-phenotype correlation for the associated phenotypes and clinical follow-up.

Characterization of ANKRD11 mutations in humans and mice related to KBG syndrome.

Mutations in ANKRD11 have recently been reported to cause KBG syndrome, an autosomal dominant condition characterized by intellectual disability (ID), behavioral problems, and macrodontia. To understand the pathogenic mechanism that relates ANKRD11 mutations with the phenotype of KBG syndrome, we studied the cellular characteristics of wild-type ANKRD11 and the effects of mutations in humans and mice. We show that the abundance of wild-type ANKRD11 is tightly regulated during the cell cycle, and that the ANKRD11 C-terminus is required for the degradation of the protein. Analysis of 11 pathogenic ANKRD11 variants in humans, including six reported in this study, and one reported in the Ankrd11 (Yod/+) mouse, shows that all mutations affect the C-terminal regions and that the mutant proteins accumulate aberrantly. In silico analysis shows the presence of D-box sequences that are signals for proteasome degradation. We suggest that ANKRD11 C-terminus plays an important role in regulating the abundance of the protein, and a disturbance of the protein abundance due to the mutations leads to KBG syndrome.

Identification of copy number variants through whole-exome sequencing in autosomal recessive nonsyndromic hearing loss.

Genetic variants account for more than half of the cases with congenital or prelingual onset hearing loss. Autosomal recessive nonsyndromic hearing loss (ARNSHL) is the most common subgroup. Whole-exome sequencing (WES) has been shown to be effective detecting deafness-causing single-nucleotide variants (SNVs) and insertion/deletions (INDELs). After analyzing the WES data for causative SNVs or INDELs involving previously reported deafness genes in 78 families with ARNSHL, we searched for copy number variants (CNVs) through two different tools in 24 families that remained unresolved. We detected large homozygous deletions in STRC and OTOA in single families. Thus, causative CNVs in known deafness genes explain 2 out of 78 (2.6%) families in our sample set. We conclude that CNVs can be reliably detected through WES and should be the part of pipelines used to clarify genetic basis of hearing loss.

FAM65B is a membrane-associated protein of hair cell stereocilia required for hearing.

In a large consanguineous Turkish kindred with recessive nonsyndromic, prelingual, profound hearing loss, we identified in the gene FAM65B (MIM611410) a splice site mutation (c.102-1G>A) that perfectly cosegregates with the phenotype in the family. The mutation leads to exon skipping and deletion of 52-amino acid residues of a PX membrane localization domain. FAM65B is known to be involved in myotube formation and in regulation of cell adhesion, polarization, and migration. We show that wild-type Fam65b is expressed during embryonic and postnatal development stages in murine cochlea, and that the protein localizes to the plasma membranes of the stereocilia of inner and outer hair cells of the inner ear. The wild-type protein targets the plasma membrane, whereas the mutant protein accumulates in cytoplasmic inclusion bodies and does not reach the membrane. In zebrafish, knockdown of fam65b leads to significant reduction of numbers of saccular hair cells and neuromasts and to hearing loss. We conclude that FAM65B is a plasma membrane-associated protein of hair cell stereocilia that is essential for hearing.

De novo ACTG2 mutations cause congenital distended bladder, microcolon, and intestinal hypoperistalsis.

Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is characterized by prenatal-onset distended urinary bladder with functional intestinal obstruction, requiring extensive surgical intervention for survival. While it is believed to be an autosomal recessive disorder, most cases are sporadic. Through whole-exome sequencing in a child with MMIHS, we identified a de novo mutation, p.R178L, in the gene encoding the smooth muscle gamma-2 actin, ACTG2. We subsequently detected another de novo ACTG2 mutation, p.R178C, in an additional child with MMIHS. Actg2 transcripts were primarily found in murine urinary bladder and intestinal tissues. Structural analysis and functional experiments suggested that both ACTG2 mutants interfere with proper polymerization of ACTG2 into thin filaments, leading to impaired contractility of the smooth muscle. In conclusion, our study suggests a pathogenic mechanism for MMIHS by identifying causative ACTG2 mutations.

DNASE1L3 mutations in hypocomplementemic urticarial vasculitis syndrome.

OBJECTIVE: Hypocomplementemic urticarial vasculitis syndrome (HUVS) is characterized by recurrent urticaria along with dermal vasculitis, arthritis, and glomerulonephritis. Systemic lupus erythematosus (SLE) develops in >50% of patients with HUVS, although the pathogenesis is unknown. The aim of this study was to identify the causative DNA mutations in 2 families with autosomal-recessive HUVS, in order to reveal the pathogenesis and facilitate the laboratory diagnosis. METHODS: Autozygosity mapping was combined with whole-exome sequencing. RESULTS: In a family with 3 affected children, we identified a homozygous frameshift mutation, c.289_290delAC, in DNASE1L3. We subsequently identified another homozygous DNASE1L3 mutation leading to exon skipping, c.320+4delAGTA, in an unrelated family. The detected mutations led to loss of function, via either nonsense-mediated messenger RNA decay or abolished endonuclease activity, as demonstrated by a plasmid nicking assay. CONCLUSION: These results show that HUVS is caused by mutations in DNASE1L3, encoding an endonuclease that previously has been associated with SLE.

SLITRK6 mutations cause myopia and deafness in humans and mice.

Myopia is by far the most common human eye disorder that is known to have a clear, albeit poorly defined, heritable component. In this study, we describe an autosomal-recessive syndrome characterized by high myopia and sensorineural deafness. Our molecular investigation in 3 families led to the identification of 3 homozygous nonsense mutations (p.R181X, p.S297X, and p.Q414X) in SLIT and NTRK-like family, member 6 (SLITRK6), a leucine-rich repeat domain transmembrane protein. All 3 mutant SLITRK6 proteins displayed defective cell surface localization. High-resolution MRI of WT and Slitrk6-deficient mouse eyes revealed axial length increase in the mutant (the endophenotype of myopia). Additionally, mutant mice exhibited auditory function deficits that mirrored the human phenotype. Histological investigation of WT and Slitrk6-deficient mouse retinas in postnatal development indicated a delay in synaptogenesis in Slitrk6-deficient animals. Taken together, our results showed that SLITRK6 plays a crucial role in the development of normal hearing as well as vision in humans and in mice and that its disruption leads to a syndrome characterized by severe myopia and deafness.

First-degree relatives of persons with type 1 diabetes: insulin resistance and enterovirus infection are associated with different patterns of islet cell autoimmunity.

Type 1 diabetes (T1D) results from the interaction of genetic and environmental factors. Previous studies indicate an association between detection of Enterovirus (EV) genome in blood and the clinical onset of T1D. Insulin resistance can also represent a risk factor for progression to clinically overt T1D. This study aimed at evaluating whether there is association between both EV infection and insulin resistance with islet autoantibodies in first-degree relatives of persons with type 1 diabetes. We collected sera from 94 first-degree relatives with (32) or without (64) islet cell antibodies (ICA) from the Cuban T1D prediction program. Blood glucose and insulin concentrations were determined. Antibodies to GAD65 and IA-2 were determined by radioimmunoassay. Insulin resistance was estimated by the homeostasis model assessment (HOMA-IR). EV-RNA was detected in serum using a highly sensitive reverse transcriptase-polymerase chain reaction method. The occurrence of EV-RNA was higher in ICA-positive relatives than in ICA-negative ones [15.6% (5/32) vs. 1.6% (1/62), P = 0.016]. GAD65 autoantibodies were more frequent in subjects with insulin resistance [34.5% (10/29) vs. 13.9% (9/65), P = 0.028] as defined by the HOMA-IR value. GAD65 autoantibodies also positively correlated with HOMA-IR (r.bis = 0.28, P < 0.01). IA-2 autoantibodies did correlate neither with EV-RNA nor with insulin resistance. There was no association between the presence of EV-RNA and insulin resistance. Our data suggest that enterovirus infection and insulin resistance are two independent events associated with ICA and GAD65 autoantibodies, respectively. These observations support the multifactorial nature of T1D.

Whole-exome sequencing efficiently detects rare mutations in autosomal recessive nonsyndromic hearing loss.

Identification of the pathogenic mutations underlying autosomal recessive nonsyndromic hearing loss (ARNSHL) is difficult, since causative mutations in 39 different genes have so far been reported. After excluding mutations in the most common ARNSHL gene, GJB2, via Sanger sequencing, we performed whole-exome sequencing (WES) in 30 individuals from 20 unrelated multiplex consanguineous families with ARNSHL. Agilent SureSelect Human All Exon 50 Mb kits and an Illumina Hiseq2000 instrument were used. An average of 93%, 84% and 73% of bases were covered to 1X, 10X and 20X within the ARNSHL-related coding RefSeq exons, respectively. Uncovered regions with WES included those that are not targeted by the exome capture kit and regions with high GC content. Twelve homozygous mutations in known deafness genes, of which eight are novel, were identified in 12 families: MYO15A-p.Q1425X, -p.S1481P, -p.A1551D; LOXHD1-p.R1494X, -p.E955X; GIPC3-p.H170N; ILDR1-p.Q274X; MYO7A-p.G2163S; TECTA-p.Y1737C; TMC1-p.S530X; TMPRSS3-p.F13Lfs*10; TRIOBP-p.R785Sfs*50. Each mutation was within a homozygous run documented via WES. Sanger sequencing confirmed co-segregation of the mutation with deafness in each family. Four rare heterozygous variants, predicted to be pathogenic, in known deafness genes were detected in 12 families where homozygous causative variants were already identified. Six heterozygous variants that had similar characteristics to those abovementioned variants were present in 15 ethnically-matched individuals with normal hearing. Our results show that rare causative mutations in known ARNSHL genes can be reliably identified via WES. The excess of heterozygous variants should be considered during search for causative mutations in ARNSHL genes, especially in small-sized families.

Mutations in OTOGL, encoding the inner ear protein otogelin-like, cause moderate sensorineural hearing loss.

Hereditary hearing loss is characterized by a high degree of genetic heterogeneity. Here we present OTOGL mutations, a homozygous one base pair deletion (c.1430 delT) causing a frameshift (p.Val477Glufs(∗)25) in a large consanguineous family and two compound heterozygous mutations, c.547C>T (p.Arg183(∗)) and c.5238+5G>A, in a nonconsanguineous family with moderate nonsyndromic sensorineural hearing loss. OTOGL maps to the DFNB84 locus at 12q21.31 and encodes otogelin-like, which has structural similarities to the epithelial-secreted mucin protein family. We demonstrate that Otogl is expressed in the inner ear of vertebrates with a transcription level that is high in embryonic, lower in neonatal, and much lower in adult stages. Otogelin-like is localized to the acellular membranes of the cochlea and the vestibular system and to a variety of inner ear cells located underneath these membranes. Knocking down of otogl with morpholinos in zebrafish leads to sensorineural hearing loss and anatomical changes in the inner ear, supporting that otogelin-like is essential for normal inner ear function. We propose that OTOGL mutations affect the production and/or function of acellular structures of the inner ear, which ultimately leads to sensorineural hearing loss.