TSPEAR variants are primarily associated with ectodermal dysplasia and tooth agenesis but not hearing loss: A novel cohort study.

Biallelic loss-of-function variants in the thrombospondin-type laminin G domain and epilepsy-associated repeats (TSPEAR) gene have recently been associated with ectodermal dysplasia and hearing loss. The first reports describing a TSPEAR disease association identified this gene is a cause of nonsyndromic hearing loss, but subsequent reports involving additional affected families have questioned this evidence and suggested a stronger association with ectodermal dysplasia. To clarify genotype-phenotype associations for TSPEAR variants, we characterized 13 individuals with biallelic TSPEAR variants. Individuals underwent either exome sequencing or panel-based genetic testing. Nearly all of these newly reported individuals (11/13) have phenotypes that include tooth agenesis or ectodermal dysplasia, while three newly reported individuals have hearing loss. Of the individuals displaying hearing loss, all have additional variants in other hearing-loss-associated genes, specifically TMPRSS3, GJB2, and GJB6, that present competing candidates for their hearing loss phenotype. When presented alongside previous reports, the overall evidence supports the association of TSPEAR variants with ectodermal dysplasia and tooth agenesis features but creates significant doubt as to whether TSPEAR variants are a monogenic cause of hearing loss. Further functional evidence is needed to evaluate this phenotypic association.

Targeted broad-based genetic testing by next-generation sequencing informs diagnosis and facilitates management in patients with kidney diseases.

BACKGROUND: The clinical diagnosis of genetic renal diseases may be limited by the overlapping spectrum of manifestations between diseases or by the advancement of disease where clues to the original process are absent. The objective of this study was to determine whether genetic testing informs diagnosis and facilitates management of kidney disease patients. METHODS: We developed a comprehensive genetic testing panel (KidneySeq) to evaluate patients with various phenotypes including cystic diseases, congenital anomalies of the kidney and urinary tract (CAKUT), tubulointerstitial diseases, transport disorders and glomerular diseases. We evaluated this panel in 127 consecutive patients ranging in age from newborns to 81 years who had samples sent in for genetic testing. RESULTS: The performance of the sequencing pipeline for single-nucleotide variants was validated using CEPH (Centre de'Etude du Polymorphism) controls and for indels using Genome-in-a-Bottle. To test the reliability of the copy number variant (CNV) analysis, positive samples were re-sequenced and analyzed. For patient samples, a multidisciplinary review board interpreted genetic results in the context of clinical data. A genetic diagnosis was made in 54 (43%) patients and ranged from 54% for CAKUT, 53% for ciliopathies/tubulointerstitial diseases, 45% for transport disorders to 33% for glomerulopathies. Pathogenic and likely pathogenic variants included 46% missense, 11% nonsense, 6% splice site variants, 23% insertion-deletions and 14% CNVs. In 13 cases, the genetic result changed the clinical diagnosis. CONCLUSION: Broad genetic testing should be considered in the evaluation of renal patients as it complements other tests and provides insight into the underlying disease and its management.

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.

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 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.

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.

DFNA8/12 caused by TECTA mutations is the most identified subtype of nonsyndromic autosomal dominant hearing loss.

The prevalence of DFNA8/DFNA12 (DFNA8/12), a type of autosomal dominant nonsyndromic hearing loss (ADNSHL), is unknown as comprehensive population-based genetic screening has not been conducted. We therefore completed unbiased screening for TECTA mutations in a Spanish cohort of 372 probands from ADNSHL families. Three additional families (Spanish, Belgian, and English) known to be linked to DFNA8/12 were also included in the screening. In an additional cohort of 835 American ADNSHL families, we preselected 73 probands for TECTA screening based on audiometric data. In aggregate, we identified 23 TECTA mutations in this process. Remarkably, 20 of these mutations are novel, more than doubling the number of reported TECTA ADNSHL mutations from 13 to 33. Mutations lie in all domains of the α-tectorin protein, including those for the first time identified in the entactin domain, as well as the vWFD1, vWFD2, and vWFD3 repeats, and the D1-D2 and TIL2 connectors. Although the majority are private mutations, four of them-p.Cys1036Tyr, p.Cys1837Gly, p.Thr1866Met, and p.Arg1890Cys-were observed in more than one unrelated family. For two of these mutations founder effects were also confirmed. Our data validate previously observed genotype-phenotype correlations in DFNA8/12 and introduce new correlations. Specifically, mutations in the N-terminal region of α-tectorin (entactin domain, vWFD1, and vWFD2) lead to mid-frequency NSHL, a phenotype previously associated only with mutations in the ZP domain. Collectively, our results indicate that DFNA8/12 hearing loss is a frequent type of ADNSHL.

A novel deletion in the RCA gene cluster causes atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a complex complement-mediated disease that progresses to end-stage renal failure (ESRF) in 50% of cases. Dysregulation of the alternative pathway (AP) of the complement cascade manifests as microangiopathic anaemia and thrombocytopenia. Multiple genes in the AP have been implicated in disease pathogenesis. Here, we report the clinical presentation of an affected patient that was inconsistent with genotype-phenotype data for carriers of CD46 mutations. Tests of AP function in this patient suggested additional genetic factors, and in-depth studies revealed a de novo heterozygous deletion that creates a novel CFH/CFHR1 fusion protein.

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.

Phenotypic variability of patients homozygous for the GJB2 mutation 35delG cannot be explained by the influence of one major modifier gene.

Hereditary hearing loss (HL) is a very heterogeneous trait, with 46 gene identifications for non-syndromic HL. Mutations in GJB2 cause up to half of all cases of severe-to-profound congenital autosomal recessive non-syndromic HL, with 35delG being the most frequent mutation in Caucasians. Although a genotype-phenotype correlation has been established for most GJB2 genotypes, the HL of 35delG homozygous patients is mild to profound. We hypothesise that this phenotypic variability is at least partly caused by the influence of modifier genes. By performing a whole-genome association (WGA) study on 35delG homozygotes, we sought to identify modifier genes. The association study was performed by comparing the genotypes of mild/moderate cases and profound cases. The first analysis included a pooling-based WGA study of a first set of 255 samples by using both the Illumina 550K and Affymetrix 500K chips. This analysis resulted in a ranking of all analysed single-nucleotide polymorphisms (SNPs) according to their P-values. The top 250 most significantly associated SNPs were genotyped individually in the same sample set. All 192 SNPs that still had significant P-values were genotyped in a second independent set of 297 samples for replication. The significant P-values were replicated in nine SNPs, with combined P-values between 3 x 10(-3) and 1 x 10(-4). This study suggests that the phenotypic variability in 35delG homozygous patients cannot be explained by the effect of one major modifier gene. Significantly associated SNPs may reflect a small modifying effect on the phenotype. Increasing the power of the study will be of greatest importance to confirm these results.

Recurrent atypical hemolytic uremic syndrome associated with factor I mutation in a living related renal transplant recipient.

Atypical hemolytic uremic syndrome, or the nondiarrheal form of hemolytic uremic syndrome, is a rare disorder typically classified as familial or sporadic. Recent literature has suggested that approximately 50% of patients have mutations in factor H (CFH), factor I (CFI), or membrane cofactor protein (encoded by CD46). Importantly, results of renal transplantation in patients with mutations in either CFH or CFI are dismal, with recurrent disease leading to graft loss in the majority of cases. We describe an adult renal transplant recipient who developed recurrent hemolytic uremic syndrome 1 month after transplantation. Bidirectional sequencing of CFH, CFI, and CD46 confirmed that the patient was heterozygous for a novel missense mutation, a substitution of a serine reside for a tyrosine residue at amino acid 369, in CFI. This report reemphasizes the importance of screening patients with atypical hemolytic uremic syndrome for mutations in these genes before renal transplantation and shows the challenges in the management of these patients.

Genotype-phenotype correlations for SLC26A4-related deafness.

Pendred syndrome (PS) and non-syndromic enlarged vestibular aqueduct (EVA) are two recessive disorders characterized by the association of sensorineural hearing loss (SNHL) with inner ear malformations that range from isolated EVA to Mondini Dysplasia, a complex malformation that includes a cochlear dysplasia and EVA. Mutations in the SLC26A4 gene, coding for the protein pendrin, have been implicated in the pathophysiology of both disorders. In order to determine whether SLC26A4 genotypes can be correlated to the complexity and severity of the phenotypes, we ascertained 1,506 deaf patients. Inner ear abnormalities were present in 474 patients (32%). Mutation screening of SLC26A4 detected two mutations in 16% of patients, one mutation in 19% of patients and zero mutation in 65% of patients. When the distribution of SLC26A4 genotypes was compared across phenotypes, a statistically significant difference was found between PS patients and non-syndromic EVA-Mondini patients (P = 0.005), as well as between EVA patients and Mondini patients (P = 0.0003). There was a correlation between phenotypic complexity of inner ear malformations and genetic heterogeneity--PS patients have the most severe phenotype and the most homogeneous etiology while EVA patients have the least severe phenotype and the most heterogeneous etiology. For all patients, variability in the degree of hearing loss is seen across genotypes implicating other genetic and/or environmental factors in the pathogenesis of the PS-Mondini-EVA disease spectrum.

Identification of three novel TECTA mutations in Iranian families with autosomal recessive nonsyndromic hearing impairment at the DFNB21 locus.

Forty-five consanguineous Iranian families segregating autosomal recessive nonsyndromic hearing loss (ARNSHL) and negative for mutations at the DFNB1 locus were screened for allele segregation consistent with homozygosity by descent (HBD) at the DFNB21 locus. In three families demonstrating HBD at this locus, mutation screening of TECTA led to the identification of three novel homozygous mutations: one frameshift mutation (266delT), a transversion of a cytosine to an adenine (5,211C > A) leading to a stop codon, and a 9.6 kb deletion removing exon 10. In total, six mutations in TECTA have now been described in families segregating ARNSHL. All of these mutations are inactivating and produce a similar phenotype that is characterized by moderate-to-severe hearing loss across frequencies with a mid frequency dip. The truncating nature of these mutations is consistent with loss-of-function, and therefore the existing TECTA knockout mouse mutant represents a good model in which to study DFNB21-related deafness.

Genetic association studies of cleft lip and/or palate with hypodontia outside the cleft region.

OBJECTIVE: The purpose of this study was to determine whether the candidate genes previously studied in subjects with cleft lip, cleft palate, or both are associated with hypodontia outside the region of the cleft. SUBJECTS: One hundred twenty subjects from the Iowa Craniofacial Anomalies Research Center were selected based on the availability of both dental records and genotype information. METHOD: The type of orofacial clefting and type and location of dental anomalies (missing teeth, supernumerary teeth, or peg laterals) were assessed by dental chart review and radiographic examination. Genotype analysis of candidate genes was performed using polymerase chain reaction/single-strand conformation polymorphism analysis. RESULTS: The prevalence of hypodontia in this sample was 47.5%, with 30.0% of subjects having missing teeth outside the cleft. There was a positive association between subjects with cleft lip or cleft lip and palate who had hypodontia outside the cleft region (compared with noncleft controls) and both muscle segment homeo box homolog 1 (MSX1) (p =.029) and transforming growth factor beta 3 (TGFB3) (p =.024). It was not possible in this analysis to determine whether this association was specifically associated with orofacial clefting combined with hypodontia or whether it was due primarily to the clefting phenotype. CONCLUSIONS: In this sample, there was a significantly greater incidence of hypodontia outside the cleft region in subjects with cleft lip and palate, compared with cleft lip only or cleft palate only. Cleft lip and/or palate with hypodontia outside the cleft region was positively associated with both TGFB3 and MSX1, compared with noncleft controls.