Resolving the genetic heterogeneity of prelingual hearing loss within one family: Performance comparison and application of two targeted next generation sequencing approaches.

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Development of a genotyping microarray for Usher syndrome

BACKGROUND: Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, eight genes have been implicated in the syndrome, together comprising 347 protein-coding exons. METHODS: To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele-specific oligonucleotides corresponding to all 298 Usher syndrome-associated sequence variants known to date, 76 of which are novel, were arrayed. RESULTS: Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. CONCLUSION: The Usher genotyping microarray is a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first-pass screening tool

Hearing loss: A review on molecular genetics and epidemiologic aspects

Background and aims: Hearing loss (HL) happens due to the genetic or environmental causes or both. Risk factors include congenital infections and congenital deformities of auricle and ear duct. The present study was performed to briefly explain the genetics, molecular biology and epidemiology of HL in Middle East especially in Iran. Methods: An intense an comprehensive literature search was prformed through heading journals in the field. All data was organized using Mendeley software and incorporated to the text as required. Results: While the etiology of 25 of HL cases remains indistinct, it is estimated that at least 50 of pre lingual HL cases have a genetic cause. About 70 of genetic HL cases are non Syndromic (NSHL) without anomaly, whereas the remaining 30 are Syndromic. Autosomal recessive non-syndromic hearing loss forms (ARNSHL) are the severest forms of congenital HL with defect in cochlea. In addition to X-linked (DFNX), autosomal dominant (DFNA), autosomal recessive (DFNB) and Y-linked (DFNY) inheritance patterns, HL can be inherited through mitochondrial genes including MT-RNR1 and MT-TS. At least 120 genes have been reported to be associated with HL. Among them, mutations in connexin 26 (GJB2) have been shown to play a very important role in developing ARSNSHL in many populations depending on geographical location and ethnicity. In Caucasians and Spainish/Italian populations, 50 and 79 of HL cases have respectively been reported to be occurred due to mutations in GJB2 gene. Conclusion: In the Middle East, the prevalence seems different as an average of 14-20 of the HL in several region of Iran is due to mutation in GJB2 gene. Alternatively similar studies showed the prevalence of GJB2 mutations around 25 and 6.1 in Turkey and Pakistani populations respectively

Genetic Linkage Analysis of 15 DFNB Loci in a Group of Iranian Families with Autosomal Recessive Hearing Loss

Background: Hearing loss (HL) is the most frequent sensory birth defect in humans. Autosomal recessive non-syndromic HL (ARNSHL) is the most common type of hereditary HL. It is extremely heterogeneous and over 70 loci (known as DFNB) have been identified. This study was launched to determine the relative contribution of more frequent loci in a cohort of ARNSHL families. Methods: Thirty-seven Iranian families including 36 ARNSHL families and 1 family with Pendred syndrome each with >= 4 affected individuals, from seven provinces of Iran, were ascertained. DFNB1 contribution was initially studied by DNA sequencing of GJB2 and linkage analysis using the relative STR markers. The excluded families were then subjected to homozygosity mapping for fifteen ARNSHL loci. Results: Sixteen families were found to be linked to seven different known loci, including DFNB I (6 families), DFNB4 (3 families +1 family with Pendred syndrome), DFNB63 (2 families), DFNB2 (1 family), DFNB7/11 (1 family), DFNB9 (1 family) and DFNB21 (1 family). DNA sequencing of the corresponding genes is in progress to identify the pathogenic mutations. Conclusion: The genetic causes were clarified in 43.2% of the studied families, giving an overview of the causes of ARNSHL in Iran. DFNB4 is ranked second after DFNB1 in the studied cohort. More genetic and epigenetic investigations will have to be done to reveal the causes in the remaining families

Calcium signaling in the cochlea - Molecular mechanisms and physiopathological implications

Calcium ions (Ca2+) regulate numerous and diverse aspects of cochlear and vestibular physiology. This review focuses on the Ca2+ control of mechanotransduction and synaptic transmission in sensory hair cells, as well as on Ca2+ signalling in non-sensory cells of the developing cochlea

Genome-wide association study of kidney function decline in individuals of European descent.

Genome-wide association studies (GWASs) have identified multiple loci associated with cross-sectional eGFR, but a systematic genetic analysis of kidney function decline over time is missing. Here we conducted a GWAS meta-analysis among 63,558 participants of European descent, initially from 16 cohorts with serial kidney function measurements within the CKDGen Consortium, followed by independent replication among additional participants from 13 cohorts. In stage 1 GWAS meta-analysis, single-nucleotide polymorphisms (SNPs) at MEOX2, GALNT11, IL1RAP, NPPA, HPCAL1, and CDH23 showed the strongest associations for at least one trait, in addition to the known UMOD locus, which showed genome-wide significance with an annual change in eGFR. In stage 2 meta-analysis, the significant association at UMOD was replicated. Associations at GALNT11 with Rapid Decline (annual eGFR decline of 3 ml/min per 1.73 m(2) or more), and CDH23 with eGFR change among those with CKD showed significant suggestive evidence of replication. Combined stage 1 and 2 meta-analyses showed significance for UMOD, GALNT11, and CDH23. Morpholino knockdowns of galnt11 and cdh23 in zebrafish embryos each had signs of severe edema 72 h after gentamicin treatment compared with controls, but no gross morphological renal abnormalities before gentamicin administration. Thus, our results suggest a role in the deterioration of kidney function for the loci GALNT11 and CDH23, and show that the UMOD locus is significantly associated with kidney function decline.Kidney International advance online publication, 10 December 2014; doi:10.1038/ki.2014.361

Distribution and phenotype of GJB2 mutations in 102 Sicilian patients with congenital non syndromic sensorineural hearing loss

Objective: To evaluate the frequency of GJB2 mutations and their correlation with phenotype in Sicilian non-syndromic sensorineural hearing loss (NSHL) patients. Design: Sequencing of the coding region, basal promoter, exon 1, and donor splice site of the GJB2 gene; screening for the presence of the two common GJB6 deletions. Study sample: A cohort of 102 Sicilian NSHL patients. Results: Fifteen different mutations in GJB2 and seventeen different genotypes were detected. No GJB6 mutations were found. The hearing impairment was profound in the 64.72% of probands (mean PTA 0.25 – 4 kHz of 88.82 26.52 dB HL). A total of 81.37% of patients harboured at least one c.35delG allele; c.167delT and c.-23 1G A were identifi ed in 10.78% and the 9.8% of patients respectively; c.35delG homozygotes presented more severe hearing impairment (75.59% of profound hearing loss) and a higher mean PTA 0.25 – 4 kHz (96.79 21.11 dB HL) with respect to c.35delG/non-c.35delG and c.35delG/Wt patients (P 0.05). Conclusions: This work underlines the role of c.35delG, c.167delT and c.-23 1G A as the most frequent causes of NSHL in Sicily. The c.35delG frequency found is similar to those reported in other populations of the Mediterranean area. The analysis of genetic and audiologic data confi rmed a variability in the phenotype associated to a single genotype

Molecular genetics of Usher syndrome -inherited deafness and blindness

Usher syndrome (USH) is an inherited blindness and deafness disorder with variable vestibular dysfunction. The syndrome is divided into three subtypes according to the progression and severity of clinical symptoms. The gene mutated in Usher syndrome type 3 (USH3), clarin 1 (CLRN1), was identified in Finland in 2001 and two mutations were identified in Finnish patients at that time. Prior to this thesis study, the two CLRN1 gene mutations were the only USH mutations identified in Finnish USH patients. To further clarify the Finnish USH mutation spectrum, all nine USH genes were studied. Seven mutations were identified: one was a previously known mutation in CLRN1, four were novel mutations in myosin VIIa (MYO7A) and two were a novel and a previously known mutation in usherin (USH2A). Another aim of this thesis research was to further study the structure and function of the CLRN1 gene, and to clarify the effects of mutations on protein function. The search for new splice variants resulted in the identification of eight novel splice variants in addition to the three splice variants that were already known prior to this study. Studies of the possible promoter regions for these splice variants showed the most active region included the 1000 bases upstream of the translation start site in the first exon of the main three exon splice variant. The 232 aa CLRN1 protein encoded by the main (three-exon) splice variant was transported to the plasma membrane when expressed in cultured cells. Western blot studies suggested that CLRN1 forms dimers and multimers. The CLRN1 mutant proteins studied were retained in the endoplasmic reticulum (ER) and some of the USH3 mutations caused CLRN1 to be unstable. During this study, two novel CLRN1 sequence alterations were identified and their pathogenicity was studied with cell culture protein expression. Previous studies with mice had shown that Clrn1 is expressed in mouse cochlear hair cells and spiral ganglion cells, but the expression profile in mouse retina remained unknown. The Clrn1 knockout mice display cochlear cell disruption/death, but do not have a retinal phenotype. The zebrafish, Danio rerio, clrn1 was found to be expressed in hair cells associated with hearing and balance. Clrn1 expression was also found in the inner nuclear layer (INL), photoreceptor layer and retinal pigment epithelium layer (RPE) of the zebrafish retina. When Clrn1 production was knocked down with injected morpholino oligonucleotides (MO) targeting Clrn1 translation or correct splicing, the zebrafish larvae showed symptoms similar to USH3 patients. These larvae had balance/hearing problems and reduced response to visual stimuli. The knowledge this thesis research has provided about the mutations in USH genes and the Finnish USH mutation spectrum are important in USH patient diagnostics. The extended information about the structure and function of CLRN1 is a step further in exploring USH3 pathogenesis caused by mutated CLRN1 as well as a step in finding a cure for the disease.Usherin oireyhtymään (USH) kuuluu perinnöllinen kuuro-sokeus ja vaihteleva tasapainon häiriö. Oireyhtymä jaetaan kolmeen alatyyppiin kliinisten oireiden etenemisen ja vaikeusasteen mukaan. Oireyhtymän alatyyppi 3 (USH3) tunnistettiin alkujaan suomalaisissa potilaissa. Geeni, klariini, jonka mutatoituminen aiheuttaa USH3:n löydettiin Suomessa vuonna 2001 ja klariinista löytyi kaksi mutaatiota. Näistä toinen on suomalainen päämutaatio ja löytyy jokaiselta suomalaiselta USH3 potilaalta. Usherin oireyhtymän eri alatyyppejä aiheuttavat kuitenkin mutaatiot yhdeksässä geenissä, joiden tuottamien proteiinien uskotaan toimivan yhdessä niin silmän kuin korvan soluissa. Tämän väitöskirjan tekemisen aikana suomalaisilta potilailta löytyivät ensimmäiset USH1 ja USH2 geenien mutaatiot. Näistä neljä USH1 mutaatiota sekä yksi USH2 mutaatio on aikaisemmin tuntemattomia ja vain yksi Suomesta tunnistettu USH2 mutaatio on jo aikaisemmin raportoitu muualla. Suomalaisten USH -mutaatioiden kirjo onkin tyypillinen Suomen väestölle. Mutaatioiden kartoitus on tärkeää potilaiden diagnostiikassa koska mutatoituneen geenin tietäminen helpottaa mm. potilaiden taudin etenemisen ennusteessa ja hoitotoimenpiteiden päättämisessä. Tutkimus keskittyi klariini -geeniin ja sen geenivirheiden aiheuttamaan, suomalaiseen tautiperimään kuuluvaan USH3 alatyyppiin. Tutkimuksen aikana klariinista löydettiin kahdeksan uutta silmukointimuotoa. Tällöin geenin osat yhdistellään versioiksi, jotka mahdollisesti luetaan erilaisiksi proteiineiksi. Klariinin toiminnan säätelystä vastaava alue pystyttiin kartoittamaan ja sen toimintaa tutkimaan eri solutyypeissä. Lisäksi klariini -proteiinia tutkimalla nähtiin proteiinin kulkeutuvan solukalvolle ja mutatoituneiden proteiinien huomattiin jäävän solun sisäisiin kalvostoihin. Mutatoituessaan klariini muuttuu lisäksi usein epävakaaksi. Tiedämme, että klariini on kalvoproteiini ja klariinit muodostavat keskenään rykelmiä kalvoilla. Emme kuitenkaan tiedä mikä tehtävä klariinin mutatoituessa häiriintyy. Tiedämme vain sen että tuo tehtävä on elintärkeä verkkokalvolla että sisäkorvassa. Klariinia tutkittiin myös silmässä ja korvassa, joissa USH oireet esiintyvät. Hiirillä, joilla ei ole toimivaa klariinia, klariinin puutos aiheuttaa pelkkää kuuroutta muttei sokeutta. Seeprakaloissa klariinia tuotetaan korvan karvasoluissa sekä verkkokalvolla. Kun klariinin tuotto estetään seeprakalan kehityksen aikana, niille kehittyy kuulon/tasapainoaistin häiriöitä ja kalojen näkökyky heikentyy. Tutkimus on kuitenkin vasta alussa. USH3 -kalamalli alkaa tuottaa toimivaa klariinia parin päivän ikäisenä. Osalla kaloista kyky nähdä ja kuulla alkaa palautua. Kysymys siitä, voiko jo alkaneen USH3 -oireyhtymän pysäyttää tai jopa parantaa tuomalla normaalin klariini -geenin verkkokalvolle tai sisäkorvaan, on yhäkin vastaamatta. Onko klariinin tehtävä tärkeä kehityksen aikana, vai voiko sen korvata myöhemmin, vaikka silmä ja korva ovat kehittyneet ilman klariinia, ovat tärkeitä kysymyksiä geeniterapian onnistumisen kannalta

The Genetic Architecture of Noise-Induced Hearing Loss: Evidence for a Gene-by-Environment Interaction.

The discovery of environmentally specific genetic effects is crucial to the understanding of complex traits, such as susceptibility to noise-induced hearing loss (NIHL). We describe the first genome-wide association study (GWAS) for NIHL in a large and well-characterized population of inbred mouse strains, known as the Hybrid Mouse Diversity Panel (HMDP). We recorded auditory brainstem response (ABR) thresholds both pre and post 2-hr exposure to 10-kHz octave band noise at 108 dB sound pressure level in 5-6-wk-old female mice from the HMDP (4-5 mice/strain). From the observation that NIHL susceptibility varied among the strains, we performed a GWAS with correction for population structure and mapped a locus on chromosome 6 that was statistically significantly associated with two adjacent frequencies. We then used a "genetical genomics" approach that included the analysis of cochlear eQTLs to identify candidate genes within the GWAS QTL. In order to validate the gene-by-environment interaction, we compared the effects of the postnoise exposure locus with that from the same unexposed strains. The most significant SNP at chromosome 6 (rs37517079) was associated with noise susceptibility, but was not significant at the same frequencies in our unexposed study. These findings demonstrate that the genetic architecture of NIHL is distinct from that of unexposed hearing levels and provide strong evidence for gene-by-environment interactions in NIHL