De novo variants are a common cause of genetic hearing loss.

PURPOSE: De novo variants (DNVs) are a well-recognized cause of genetic disorders. The contribution of DNVs to hearing loss (HL) is poorly characterized. We aimed to evaluate the rate of DNVs in HL-associated genes and assess their contribution to HL. METHODS: Targeted genomic enrichment and massively parallel sequencing were used for molecular testing of all exons and flanking intronic sequences of known HL-associated genes, with no exclusions on the basis of type of HL or clinical features. Segregation analysis was performed, and previous reports of DNVs in PubMed and ClinVar were reviewed to characterize the rate, distribution, and spectrum of DNVs in HL. RESULTS: DNVs were detected in 10% (24/238) of trios for whom segregation analysis was performed. Overall, DNVs were causative in at least ∼1% of probands for whom a genetic diagnosis was resolved, with marked variability based on inheritance mode and phenotype. DNVs of MITF were most common (21% of DNVs), followed by GATA3 (13%), STRC (13%), and ACTG1 (8%). Review of reported DNVs revealed gene-specific variability in contribution of DNV to the mutational spectrum of HL-associated genes. CONCLUSION: DNVs are a relatively common cause of genetic HL and must be considered in all cases of sporadic HL.

Novel loss-of-function mutations in COCH cause autosomal recessive nonsyndromic hearing loss.

COCH is the most abundantly expressed gene in the cochlea. Unsurprisingly, mutations in COCH underly hearing loss in mice and humans. Two forms of hearing loss are linked to mutations in COCH, the well-established autosomal dominant nonsyndromic hearing loss, with or without vestibular dysfunction (DFNA9) via a gain-of-function/dominant-negative mechanism, and more recently autosomal recessive nonsyndromic hearing loss (DFNB110) via nonsense variants. Using a combination of targeted gene panels, exome sequencing, and functional studies, we identified four novel pathogenic variants (two nonsense variants, one missense, and one inframe deletion) in COCH as the cause of autosomal recessive hearing loss in a multi-ethnic cohort. To investigate whether the non-truncating variants exert their effect via a loss-of-function mechanism, we used minigene splicing assays. Our data showed both the missense and inframe deletion variants altered RNA splicing by creating an exon splicing silencer and abolishing an exon splicing enhancer, respectively. Both variants create frameshifts and are predicted to result in a null allele. This study confirms the involvement of loss-of-function mutations in COCH in autosomal recessive nonsyndromic hearing loss, expands the mutational landscape of DFNB110 to include coding variants that alter RNA splicing, and highlights the need to investigate the effect of coding variants on RNA splicing.

A comparative analysis of genetic hearing loss phenotypes in European/American and Japanese populations.

We present detailed comparative analyses to assess population-level differences in patterns of genetic deafness between European/American and Japanese cohorts with non-syndromic hearing loss. One thousand eighty-three audiometric test results (921 European/American and 162 Japanese) from members of 168 families (48 European/American and 120 Japanese) with non-syndromic hearing loss secondary to pathogenic variants in one of three genes (KCNQ4, TECTA, WFS1) were studied. Audioprofile characteristics, specific mutation types, and protein domains were considered in the comparative analyses. Our findings support differences in audioprofiles driven by both mutation type (non-truncating vs. truncating) and ethnic background. The former finding confirms data that ascribe a phenotypic consequence to different mutation types in KCNQ4; the latter finding suggests that there are ethnic-specific effects (genetic and/or environmental) that impact gene-specific audioprofiles for TECTA and WFS1. Identifying the drivers of ethnic differences will refine our understanding of phenotype-genotype relationships and the biology of hearing and deafness.

Genetic Analysis of 400 Patients Refines Understanding and Implicates a New Gene in Atypical Hemolytic Uremic Syndrome.

BACKGROUND: Genetic variation in complement genes is a predisposing factor for atypical hemolytic uremic syndrome (aHUS), a life-threatening thrombotic microangiopathy, however interpreting the effects of genetic variants is challenging and often ambiguous. METHODS: We analyzed 93 complement and coagulation genes in 400 patients with aHUS, using as controls 600 healthy individuals from Iowa and 63,345 non-Finnish European individuals from the Genome Aggregation Database. After adjusting for population stratification, we then applied the Fisher exact, modified Poisson exact, and optimal unified sequence kernel association tests to assess gene-based variant burden. We also applied a sliding-window analysis to define the frequency range over which variant burden was significant. RESULTS: We found that patients with aHUS are enriched for ultrarare coding variants in the CFH, C3, CD46, CFI, DGKE, and VTN genes. The majority of the significance is contributed by variants with a minor allele frequency of <0.1%. Disease-related variants tend to occur in specific complement protein domains of FH, CD46, and C3. We observed no enrichment for multiple rare coding variants in gene-gene combinations. CONCLUSIONS: In known aHUS-associated genes, variants with a minor allele frequency >0.1% should not be considered pathogenic unless valid enrichment and/or functional evidence are available. VTN, which encodes vitronectin, an inhibitor of the terminal complement pathway, is implicated as a novel aHUS-associated gene. Patients with aHUS are not enriched for multiple rare variants in complement genes. In aggregate, these data may help in directing clinical management of aHUS.

Exonic mutations and exon skipping: Lessons learned from DFNA5.

Dysregulation of splicing is a common factor underlying many inherited diseases including deafness. For one deafness-associated gene, DFNA5, perturbation of exon 8 splicing results in a constitutively active truncated protein. To date, only intronic mutations have been reported to cause exon 8 skipping in patients with DFNA5-related deafness. In five families with postlingual progressive autosomal dominant non-syndromic hearing loss, we employed two next-generation sequencing platforms-OtoSCOPE and whole exome sequencing-followed by variant filtering and prioritization based on both minor allele frequency and functional consequence using a customized bioinformatics pipeline to identify three novel and two recurrent mutations in DFNA5 that segregated with hearing loss in these families. The three novel mutations are all missense variants within exon 8 that are predicted computationally to decrease splicing efficiency or abolish it completely. We confirmed their functional impact in vitro using mini-genes carrying each mutant DFNA5 exon 8. In so doing, we present the first exonic mutations in DFNA5 to cause deafness, expand the mutational spectrum of DFNA5-related hearing loss, and highlight the importance of assessing the effect of coding variants on splicing.

High-Throughput Genetic Testing for Thrombotic Microangiopathies and C3 Glomerulopathies.

The thrombotic microangiopathies (TMAs) and C3 glomerulopathies (C3Gs) include a spectrum of rare diseases such as atypical hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, C3GN, and dense deposit disease, which share phenotypic similarities and underlying genetic commonalities. Variants in several genes contribute to the pathogenesis of these diseases, and identification of these variants may inform the diagnosis and treatment of affected patients. We have developed and validated a comprehensive genetic panel that screens all exons of all genes implicated in TMA and C3G. The closely integrated pipeline implemented includes targeted genomic enrichment, massively parallel sequencing, bioinformatic analysis, and a multidisciplinary conference to analyze identified variants in the context of each patient's specific phenotype. Herein, we present our 1-year experience with this panel, during which time we studied 193 patients. We identified 17 novel and 74 rare variants, which we classified as pathogenic (11), likely pathogenic (12), and of uncertain significance (68). Compared with controls, patients with C3G had a higher frequency of rare and novel variants in C3 convertase (C3 and CFB) and complement regulator (CFH, CFI, CFHR5, and CD46) genes (P<0.05). In contrast, patients with TMA had an increase in rare and novel variants only in complement regulator genes (P<0.01), a distinction consistent with differing sites of complement dysregulation in these two diseases. In summary, we were able to provide a positive genetic diagnosis in 43% and 41% of patients carrying the clinical diagnosis of C3G and TMA, respectively.

Comprehensive genetic testing in the clinical evaluation of 1119 patients with hearing loss.

Hearing loss is the most common sensory deficit in humans, affecting 1 in 500 newborns. Due to its genetic heterogeneity, comprehensive diagnostic testing has not previously been completed in a large multiethnic cohort. To determine the aggregate contribution inheritance makes to non-syndromic hearing loss, we performed comprehensive clinical genetic testing with targeted genomic enrichment and massively parallel sequencing on 1119 sequentially accrued patients. No patient was excluded based on phenotype, inheritance or previous testing. Testing resulted in identification of the underlying genetic cause for hearing loss in 440 patients (39%). Pathogenic variants were found in 49 genes and included missense variants (49%), large copy number changes (18%), small insertions and deletions (18%), nonsense variants (8%), splice-site alterations (6%), and promoter variants (<1%). The diagnostic rate varied considerably based on phenotype and was highest for patients with a positive family history of hearing loss or when the loss was congenital and symmetric. The spectrum of implicated genes showed wide ethnic variability. These findings support the more efficient utilization of medical resources through the development of evidence-based algorithms for the diagnosis of hearing loss.

Characterising the spectrum of autosomal recessive hereditary hearing loss in Iran.

BACKGROUND: Countries with culturally accepted consanguinity provide a unique resource for the study of rare recessively inherited genetic diseases. Although hereditary hearing loss (HHL) is not uncommon, it is genetically heterogeneous, with over 85 genes causally implicated in non-syndromic hearing loss (NSHL). This heterogeneity makes many gene-specific types of NSHL exceedingly rare. We sought to define the spectrum of autosomal recessive HHL in Iran by investigating both common and rarely diagnosed deafness-causing genes. DESIGN: Using a custom targeted genomic enrichment (TGE) panel, we simultaneously interrogated all known genetic causes of NSHL in a cohort of 302 GJB2-negative Iranian families. RESULTS: We established a genetic diagnosis for 67% of probands and their families, with over half of all diagnoses attributable to variants in five genes: SLC26A4, MYO15A, MYO7A, CDH23 and PCDH15. As a reflection of the power of consanguinity mapping, 26 genes were identified as causative for NSHL in the Iranian population for the first time. In total, 179 deafness-causing variants were identified in 40 genes in 201 probands, including 110 novel single nucleotide or small insertion-deletion variants and three novel CNV. Several variants represent founder mutations. CONCLUSION: This study attests to the power of TGE and massively parallel sequencing as a diagnostic tool for the evaluation of hearing loss in Iran, and expands on our understanding of the genetics of HHL in this country. Families negative for variants in the genes represented on this panel represent an excellent cohort for novel gene discovery.

Allelic variants of complement genes associated with dense deposit disease.

The alternative pathway of the complement cascade plays a role in the pathogenesis of dense deposit disease (DDD). Deficiency of complement factor H and mutations in CFH associate with the development of DDD, but it is unknown whether allelic variants in other complement genes also associate with this disease. We studied patients with DDD and identified previously unreported sequence alterations in several genes in addition to allelic variants and haplotypes common to patients with DDD. We found that the likelihood of developing DDD increases with the presence of two or more risk alleles in CFH and C3. To determine the functional consequence of this finding, we measured the activity of the alternative pathway in serum samples from phenotypically normal controls genotyped for variants in CFH and C3. Alternative pathway activity was higher in the presence of variants associated with DDD. Taken together, these data confirm that DDD is a complex genetic disease and may provide targets for the development of disease-specific therapies.

Genetic Causes of Hearing Loss in a Large Cohort of Cochlear Implant Recipients.

Understanding genetic causes of hearing loss can determine the pattern and course of a patient's hearing loss and may also predict outcomes after cochlear implantation. Our goal in this study was to evaluate genetic causes of hearing loss in a large cohort of adults and children with cochlear implants. We performed comprehensive genetic testing on all patients undergoing cochlear implantation. Of the 459 patients included in the study, 128 (28%) had positive genetic testing. In total, 44 genes were identified as causative. The top 5 genes implicated were GJB2 (20, 16%), TMPRSS3 (13, 10%), SLC26A4 (10, 8%), MYO7A (9, 7%), and MT-RNR1 (7, 5%). Pediatric patients had a higher diagnostic rate. This study lays the groundwork for future studies evaluating the relationship between genetic variation and cochlear implant performance.

A synonymous variant in MYO15A enriched in the Ashkenazi Jewish population causes autosomal recessive hearing loss due to abnormal splicing.

Nonsyndromic hearing loss is genetically heterogeneous. Despite comprehensive genetic testing, many cases remain unsolved because the clinical significance of identified variants is uncertain or because biallelic pathogenic variants are not identified for presumed autosomal recessive cases. Common synonymous variants are often disregarded. Determining the pathogenicity of synonymous variants may improve genetic diagnosis. We report a synonymous variant c.9861 C > T/p.(Gly3287=) in MYO15A in homozygosity or compound heterozygosity with another pathogenic or likely pathogenic MYO15A variant in 10 unrelated families with nonsyndromic sensorineural hearing loss. Biallelic variants in MYO15A were identified in 21 affected and were absent in 22 unaffected siblings. A mini-gene assay confirms that the synonymous variant leads to abnormal splicing. The variant is enriched in the Ashkenazi Jewish population. Individuals carrying biallelic variants involving c.9861 C > T often exhibit progressive post-lingual hearing loss distinct from the congenital profound deafness typically associated with biallelic loss-of-function MYO15A variants. This study establishes the pathogenicity of the c.9861 C > T variant in MYO15A and expands the phenotypic spectrum of MYO15A-related hearing loss. Our work also highlights the importance of multicenter collaboration and data sharing to establish the pathogenicity of a relatively common synonymous variant for improved diagnosis and management of hearing loss.

Mutations in alternative pathway complement proteins in American patients with atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is characterized by acute renal failure, thrombocytopenia and microangiopathic hemolytic anemia, and occurs with an estimated incidence in the USA of 2 per 1,000,000. Disease pathogenesis is related to dysregulation of the alternative pathway (AP) of the complement cascade at the level of the cell membrane secondary to mutations in a number of complement genes including complement factor H (CFH), complement factor H-related 5 (CFHR5), complement factor I (CFI), CD46 (MCP), complement factor B (CFB), complement component 3 (C3) and thrombomodulin (THBD). Since aHUS is rare, mutation rate data in large patient cohorts are scarce. Here we present the first cohort of American patients in whom mutation screening was completed on all genes currently implicated in aHUS. In addition to identifying a number of novel variants, we provide information on the relative frequency of mutations in these genes in an American aHUS population. Twelve percent (12%) of patients carrying disease-associated genetic variants segregated mutations in more than one gene mandating comprehensive genetic testing in the diagnosis and management of these patients.

Is it Usher syndrome? Collaborative diagnosis and molecular genetics of patients with visual impairment and hearing loss.

Background: Usher syndrome is the most common hereditary syndrome combining deafness and blindness. In the 2017 National Child Count of Children and Youth who are Deaf-Blind, Usher syndrome represented 329 of 10,000 children, but there were also at least 70 other etiologies of deaf-blindness documented. The purpose of this study was to analyze the work-up and ultimate diagnoses of 21 consecutive families who presented to the Genetic Eye-Ear Clinic (GEEC) at the University of Iowa. Our hypothesis was that most families referred to the GEEC would have initial and final diagnoses of Usher syndrome.Materials and Methods: Patients were identified through an IRB approved retrospective chart review of referrals to the GEEC between 2012 and 2019. Details about each patient's history, exam, and clinical and genetic work-up were recorded.Results: From 2012 to 2019, 21 families (25 patients) were referred to the collaborative GEEC. Overall molecular diagnostic rate in this cohort was 14/21 (67%). Evaluation resulted in a change of diagnosis in 11/21 (52%) families. Ultimately, there were eleven unique diagnoses including hereditary, non-hereditary, and independent causes of combined visual impairment and hearing loss. The most common diagnosis was Usher syndrome, which represented 6/21 (29%) families.Conclusions: Providing a correct diagnosis for patients with visual impairment and hearing loss can be challenging for clinicians and their patients, but it can greatly improve clinical care and outcomes. We recommend an algorithm that includes multidisciplinary collaboration, careful clinical evaluation, strategic molecular testing, and consideration of a broad differential diagnosis.

Comprehensive Genetic Testing for Deafness from Fresh and Archived Dried Blood Spots.

Comprehensive genetic testing has become integral in the evaluation of children with deafness, but the amount of blood required to obtain DNA can be prohibitive in newborns. Dried blood spots (DBSs) are routinely collected and would provide an alternative source of DNA. Our objective was to evaluate the use of DBSs for comprehensive genetic testing for deafness. DNA derived from fresh and archived DBS samples was compared with DNA from whole blood. We performed next-generation sequencing of all known deafness genes in 4 DBS samples: 2 positive controls, an unknown sample, and a negative control. The DBS-derived DNA was of sufficient quantity and quality for clinical testing. In the 2 positive control samples, pathogenic variants were identified; in the negative control, no pathogenic variants were found; and in the unknown sample, homozygous deletion of the OTOA gene was identified as the cause of deafness. This pilot study shows that comprehensive genetic testing for deafness is feasible with fresh and/or archived DBSs.

Genetic variants in the peripheral auditory system significantly affect adult cochlear implant performance.

BACKGROUND: Cochlear implantation is an effective habilitation modality for adults with significant hearing loss. However, post-implant performance is variable. A portion of this variance in outcome can be attributed to clinical factors. Recent physiological studies suggest that the health of the spiral ganglion also impacts post-operative cochlear implant outcomes. The goal of this study was to determine whether genetic factors affecting spiral ganglion neurons may be associated with cochlear implant performance. METHODS: Adults with post-lingual deafness who underwent cochlear implantation at the University of Iowa were studied. Pre-implantation evaluation included comprehensive genetic testing for genetic diagnosis. A novel score of genetic variants affecting genes with functional effects in the spiral ganglion was calculated. A Z-scored average of up to three post-operative speech perception tests (CNC, HINT, and AzBio) was used to assess outcome. RESULTS: Genetically determined spiral ganglion health affects cochlear implant outcomes, and when considered in conjunction with clinically determined etiology of deafness, accounts for 18.3% of the variance in postoperative speech recognition outcomes. Cochlear implant recipients with deleterious genetic variants that affect the cochlear sensory organ perform significantly better on tests of speech perception than recipients with deleterious genetic variants that affect the spiral ganglion. CONCLUSION: Etiological diagnosis of deafness including genetic testing is the single largest predictor of postoperative speech outcomes in adult cochlear implant recipients. A detailed understanding of the genetic underpinning of hearing loss will better inform pre-implant counseling. The method presented here should serve as a guide for further research into the molecular physiology of the peripheral auditory system and cochlear implants.

Heterogeneity of Hereditary Hearing Loss in Iran: a Comprehensive Review.

A significant contribution to the causes of hereditary hearing impairment comes from genetic factors. More than 120 genes and 160 loci have been identified to be involved in hearing impairment. Given that consanguine populations are more vulnerable to most inherited diseases, such as hereditary hearing loss (HHL), the genetic picture of HHL among the Iranian population, which consists of at least eight ethnic subgroups with a high rate of intermarriage, is expected to be highly heterogeneous. Using an electronic literature review through various databases such as PubMed, MEDLINE, and Scopus, we review the current picture of HHL in Iran. In this review, we present more than 39 deafness genes reported to cause non-syndromic HHL in Iran, of which the most prevalent causative genes include GJB2, SLC26A4, MYO15A, and MYO7A. In addition, we highlight some of the more common genetic causes of syndromic HHL in Iran. These results are of importance for further investigation and elucidation of the molecular basis of HHL in Iran and also for developing a national diagnostic tool tailored to the Iranian context enabling early and efficient diagnosis of hereditary hearing impairment.

Relevance of mutations in the TLR4 receptor in patients with gram-negative septic shock.

BACKGROUND: Septic shock remains a significant health concern worldwide, and despite progress in understanding the physiological and molecular basis of septic shock, the high mortality rate of patients with septic shock remains unchanged. We recently identified a common polymorphism in toll-like receptor 4 (TLR4) that is associated with hyporesponsiveness to inhaled endotoxin or lipopolysaccharide in humans. METHODS: Since TLR4 is a major receptor for lipopolysaccharide in mammals and gram-negative bacteria are the prevalent pathogen associated with septic shock, we investigated whether these specific TLR4 alleles are associated with a predisposition to a more severe disease outcome for patients with septic shock. We genotyped 91 patients with septic shock as well as 73 healthy blood donor controls for the presence of the TLR4 Asp299Gly and TLR4 Thr399Ile mutations. RESULTS: We found the TLR4 Asp299Gly allele exclusively in patients with septic shock (P =.05). Furthermore, patients with septic shock with the TLR4 Asp299Gly/Thr399Ile alleles had a higher prevalence of gram-negative infections. CONCLUSION: Mutations in the TLR4 receptor may predispose people to develop septic shock with gram-negative microorganisms.

Copy number variants are a common cause of non-syndromic hearing loss.

BACKGROUND: Copy number variants (CNVs) are a well-recognized cause of genetic disease; however, methods for their identification are often gene-specific, excluded as 'routine' in screens of genetically heterogeneous disorders, and not implemented in most next-generation sequencing pipelines. For this reason, the contribution of CNVs to non-syndromic hearing loss (NSHL) is most likely under-recognized. We aimed to incorporate a method for CNV identification as part of our standard analysis pipeline and to determine the contribution of CNVs to genetic hearing loss. METHODS: We used targeted genomic enrichment and massively parallel sequencing to isolate and sequence all exons of all genes known to cause NSHL. We completed testing on 686 patients with hearing loss with no exclusions based on type of hearing loss or any other clinical features. For analysis we used an integrated method for detection of single nucleotide changes, indels and CNVs. CNVs were identified using a previously published method that utilizes median read-depth ratios and a sliding-window approach. RESULTS: Of 686 patients tested, 15.2% (104) carried at least one CNV within a known deafness gene. Of the 38.9% (267) of individuals for whom we were able to determine a genetic cause of hearing loss, a CNV was implicated in 18.7% (50). We identified CNVs in 16 different genes including 7 genes for which no CNVs have been previously reported. CNVs of STRC were most common (73% of CNVs identified) followed by CNVs of OTOA (13% of CNVs identified). CONCLUSION: CNVs are an important cause of NSHL and their detection must be included in comprehensive genetic testing for hearing loss.

Causes of alternative pathway dysregulation in dense deposit disease.

BACKGROUND AND OBJECTIVES: This study was designed to investigate the causes of alternative pathway dysregulation in a cohort of patients with dense deposit disease (DDD). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Thirty-two patients with biopsy-proven DDD underwent screening for C3 nephritic factors (C3Nefs), factor H autoantibodies (FHAAs), factor B autoantibodies (FBAAs), and genetic variants in CFH. C3Nefs were detected by: ELISA, C3 convertase surface assay (C3CSA), C3CSA with properdin (C3CSAP), two-dimensional immunoelectrophoresis (2DIEP), and immunofixation electrophoresis (IFE). FHAAs and FBAAs were detected by ELISA, and CFH variants were identified by Sanger sequencing. RESULTS: Twenty-five patients (78%) were positive for C3Nefs. Three C3Nef-positive patients were also positive for FBAAs and one of these patients additionally carried two novel missense variants in CFH. Of the seven C3Nef-negative patients, one patient was positive for FHAAs and two patients carried CFH variants that may be causally related to their DDD phenotype. C3CASP was the most sensitive C3Nef-detection assay. C3CASP and IFE are complementary because C3CSAP measures the stabilizing properties of C3Nefs, whereas IFE measures their expected consequence-breakdown of C3b. CONCLUSIONS: A test panel that includes C3CSAP, IFE, FHAAs, FBAAs, and genetic testing for CFH variants will identify a probable cause for alternative pathway dysregulation in approximately 90% of DDD patients. Dysregulation is most frequently due to C3Nefs, although some patients test positive for FHAAs, FBAAs, and CFH mutations. Defining the pathophysiology of DDD should facilitate the development of mechanism-directed therapies.

The genetics of the alternative pathway of complement in the pathogenesis of HELLP syndrome.

OBJECTIVE: Recent preliminary evidence suggests that gene mutations in the alternative pathway of complement may play a crucial role in the pathogenesis of HELLP syndrome. To verify this hypothesis, a consecutive series of women who developed the syndrome was screened for variants in alternative pathway genes. METHODS: The coding sequences and intron-exon boundaries of the complement factor H (CFH), complement factor I (CFI), Membrane Cofactor Protein (MCP), complement factor B (CFB) and C3 were sequenced in 33 women with a diagnosis of HELLP syndrome. RESULTS: Three patients carried heterozygotic variants - two in CFI and one in MCP. One of the two CFI mutations, was previously described as an unremarkable polymorphism. Conversely, computational analyses for the remaining two cases suggest that they may have a functional impact. CONCLUSIONS: The present study confirms that the alternative pathway of complement may play a role in the pathogenesis of HELLP syndrome. However, its overall contribution to the determinism of the syndrome is less relevant than initially reported.