Unraveling the genetic complexities of combined retinal dystrophy and hearing impairment.

Usher syndrome, the most prevalent cause of combined hereditary vision and hearing impairment, is clinically and genetically heterogeneous. Moreover, several conditions with phenotypes overlapping Usher syndrome have been described. This makes the molecular diagnosis of hereditary deaf-blindness challenging. Here, we performed exome sequencing and analysis on 7 Mexican and 52 Iranian probands with combined retinal degeneration and hearing impairment (without intellectual disability). Clinical assessment involved ophthalmological examination and hearing loss questionnaire. Usher syndrome, most frequently due to biallelic variants in MYO7A (USH1B in 16 probands), USH2A (17 probands), and ADGRV1 (USH2C in 7 probands), was diagnosed in 44 of 59 (75%) unrelated probands. Almost half of the identified variants were novel. Nine of 59 (15%) probands displayed other genetic entities with dual sensory impairment, including Alström syndrome (3 patients), cone-rod dystrophy and hearing loss 1 (2 probands), and Heimler syndrome (1 patient). Unexpected findings included one proband each with Scheie syndrome, coenzyme Q10 deficiency, and pseudoxanthoma elasticum. In four probands, including three Usher cases, dual sensory impairment was either modified/aggravated or caused by variants in distinct genes associated with retinal degeneration and/or hearing loss. The overall diagnostic yield of whole exome analysis in our deaf-blind cohort was 92%. Two (3%) probands were partially solved and only 3 (5%) remained without any molecular diagnosis. In many cases, the molecular diagnosis is important to guide genetic counseling, to support prognostic outcomes and decisions with currently available and evolving treatment modalities.

Genetic Spectrum of Syndromic and Non-Syndromic Hearing Loss in Pakistani Families.

The current molecular genetic diagnostic rates for hereditary hearing loss (HL) vary considerably according to the population background. Pakistan and other countries with high rates of consanguineous marriages have served as a unique resource for studying rare and novel forms of recessive HL. A combined exome sequencing, bioinformatics analysis, and gene mapping approach for 21 consanguineous Pakistani families revealed 13 pathogenic or likely pathogenic variants in the genes GJB2, MYO7A, FGF3, CDC14A, SLITRK6, CDH23, and MYO15A, with an overall resolve rate of 61.9%. GJB2 and MYO7A were the most frequently involved genes in this cohort. All the identified variants were either homozygous or compound heterozygous, with two of them not previously described in the literature (15.4%). Overall, seven missense variants (53.8%), three nonsense variants (23.1%), two frameshift variants (15.4%), and one splice-site variant (7.7%) were observed. Syndromic HL was identified in five (23.8%) of the 21 families studied. This study reflects the extreme genetic heterogeneity observed in HL and expands the spectrum of variants in deafness-associated genes.

A novel missense variant in MYO3A is associated with autosomal dominant high-frequency hearing loss in a German family.

BACKGROUND: MYO3A, encoding the myosin IIIA protein, is associated with autosomal recessive and autosomal dominant nonsyndromic hearing loss. To date, only two missense variants located in the motor-head domain of MYO3A have been described in autosomal dominant families with progressive, mild-to-profound sensorineural hearing loss. These variants alter the ATPase activity of myosin IIIA. METHODS: Exome sequencing of a proband from a three-generation German family with prelingual, moderate-to-profound, high-frequency hearing loss was performed. Segregation analysis confirmed a dominant inheritance pattern. Regression analysis of mean hearing level thresholds per individual and ear was performed at high-, mid-, and low-frequencies. RESULTS: A novel heterozygous missense variant c.716T>C, p.(Leu239Pro) in the kinase domain of MYO3A was identified that is predicted in silico as disease causing. High-frequency, progressive hearing loss was identified. CONCLUSION: Correlation analysis of pure-tone hearing thresholds revealed progressive hearing loss, especially in the high-frequencies. In the present study, we report the first dominant likely pathogenic variant in MYO3A in a European family and further support MYO3A as an autosomal dominant hearing loss gene.

Small fish, big prospects: using zebrafish to unravel the mechanisms of hereditary hearing loss.

Over the past decade, advancements in high-throughput sequencing have greatly enhanced our knowledge of the mutational signatures responsible for hereditary hearing loss. In its present state, the field has a largely uncensored view of protein coding changes in a growing number of genes that have been associated with hereditary hearing loss, and many more that have been proposed as candidate genes. Sequencing data can now be generated using methods that have become widespread and affordable. The greatest hurdles facing the field concern functional validation of uncharacterized genes and rapid application to human diseases, including hearing and balance disorders. To date, over 30 hearing-related disease models exist in zebrafish. New genome editing technologies, including CRISPR/Cas9 will accelerate the functional validation of hearing loss genes and variants in zebrafish. Here, we discuss current progress in the field and recent advances in genome editing approaches.

Exome-wide copy number variation analysis identifies a COL9A1 in frame deletion that is associated with hearing loss.

Pathogenic variants in COL9A1 are primarily associated with autosomal recessive Stickler syndrome. Patients with COL9A1-associated Stickler syndrome (STL) present hearing loss (HL), ophthalmic manifestations and skeletal abnormalities. However, the clinical spectrum of patients with COL9A1 variants can also include multiple epiphyseal dysplasia, as well as non-syndromic HL that was observed in one previously reported proband. Exome sequencing was performed on the genomic DNA of an Iranian patient and his affected brother who both report non-syndromic HL. A 44.6 kb homozygous in-frame deletion spanning exons 6 to 33 of COL9A1 was detected via exome-based copy number variation analysis. The deleted exons were confirmed by PCR in the patient and his affected brother, who both have non-syndromic HL. Segregation analysis via qPCR confirmed the parents as heterozygous deletion carriers. Breakpoint analysis mapped the homozygous deletion spanning introns 5 to 33 (g.70,948,188_70,997,277del, NM_001851.4(COL9A1):c.697-3754_2112+769del, p.(Phe233_Ser704del), with an additional 67 bp of inserted intronic sequence that may have originated due to a fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR) mechanism. This mechanism has not been previously implicated in HL or STL. This is also the first reported copy number variation in COL9A1 that was identified through an exome data set in an Iranian family with apparent non-syndromic HL. The present study emphasizes the importance of exome-wide copy number variation analysis in molecular diagnosis and provides supporting evidence to associate COL9A1 with autosomal recessive non-syndromic HL.

Phenotypic Characterization of DFNB16-associated Hearing Loss.

HYPOTHESIS: We hypothesized that patients with DFNB16 caused hearing loss show characteristical audiological findings depending on genetic results. BACKGROUND: Hearing loss belongs to the most frequent congenital diseases. In 50-70% of individuals, hearing loss is caused by genetic defects. DFNB1 (deafness, neurosensory, autosomal-recessive) is the most frequently affected locus. Despite its great genetic heterogeneity, comprehensive analysis of genes like STRC, encoding stereocilin (DFNB16) is possible. The genetic architecture of the DFNB16 locus is challenging and requires a unique molecular genetic testing assay. The aim of the study is a systematic characterization of the audiological phenotype in DFNB16-positive patients. METHODS: Since 2011, 290 patients with suspicion of inherited hearing loss received a human genetic exploration. Eighty two DFNB1-negative patients advanced to further testing in the DFNB16 locus. STRC-positive patients obtained complete audiological diagnostic workup. Additionally, epidemiological data was collected. RESULTS: Nine of 82 (11%) of the examined patients (mean age 5 yr) showed mutations in the STRC (3 homozygous, 6 compound heterozygous). Aside from a moderate hearing loss in the pure tone audiogram, auditory brainstem response thresholds were 40-50 dB nHL. Otoacoustic emissions were detectable in only one patient. CONCLUSIONS: Examination of the DFNB16-locus should be a standard diagnostic test after negative DFNB1-gene screening result. Notably, DFNB16-associated hearing loss can be audiologically characterized as moderate sensorineural hearing loss in the main speech field with absent otoacoustic emissions. Our study is the first to correlate audiological findings with genetic results in patients with hearing loss due to STRC.

Hereditary hearing loss SNP-microarray pilot study.

OBJECTIVES: Despite recent advancements in diagnostic tools, the genomic landscape of hereditary hearing loss remains largely uncharacterized. One strategy to understand genome-wide aberrations includes the analysis of copy number variation that can be mapped using SNP-microarray technology. A growing collection of literature has begun to uncover the importance of copy number variation in hereditary hearing loss. This pilot study underpins a larger effort that involves the stage-wise analysis of hearing loss patients, many of whom have advanced to high-throughput sequencing analysis. DATA DESCRIPTION: Our data originate from the Infinium HumanOmni1-Quad v1.0 SNP-microarrays (Illumina) that provide useful markers for genome-wide association studies and copy number variation analysis. This dataset comprises a cohort of 108 individuals (99 with hearing loss, 9 normal hearing family members) for the purpose of understanding the genetic contribution of copy number variations to hereditary hearing loss. These anonymized SNP-microarray data have been uploaded to the NCBI Gene Expression Omnibus and are intended to benefit other investigators interested in aggregating platform-matched array patient datasets or as part of a supporting reference tool for other laboratories to better understand recurring copy number variations in other genetic disorders.

The conserved p.Arg108 residue in S1PR2 (DFNB68) is fundamental for proper hearing: evidence from a consanguineous Iranian family.

BACKGROUND: Genetic heterogeneity and consanguineous marriages make recessive inherited hearing loss in Iran the second most common genetic disorder. Only two reported pathogenic variants (c.323G>C, p.Arg108Pro and c.419A>G, p.Tyr140Cys) in the S1PR2 gene have previously been linked to autosomal recessive hearing loss (DFNB68) in two Pakistani families. We describe a segregating novel homozygous c.323G>A, p.Arg108Gln pathogenic variant in S1PR2 that was identified in four affected individuals from a consanguineous five generation Iranian family. METHODS: Whole exome sequencing and bioinformatics analysis of 116 hearing loss-associated genes was performed in an affected individual from a five generation Iranian family. Segregation analysis and 3D protein modeling of the p.Arg108 exchange was performed. RESULTS: The two Pakistani families previously identified with S1PR2 pathogenic variants presented profound hearing loss that is also observed in the affected Iranian individuals described in the current study. Interestingly, we confirmed mixed hearing loss in one affected individual. 3D protein modeling suggests that the p.Arg108 position plays a key role in ligand receptor interaction, which is disturbed by the p.Arg108Gln change. CONCLUSION: In summary, we report the third overall mutation in S1PR2 and the first report outside the Pakistani population. Furthermore, we describe a novel variant that causes an amino acid exchange (p.Arg108Gln) in the same amino acid residue as one of the previously reported Pakistani families (p.Arg108Pro). This finding emphasizes the importance of the p.Arg108 amino acid in normal hearing and confirms and consolidates the role of S1PR2 in autosomal recessive hearing loss.

Confirmation of PDZD7 as a Nonsyndromic Hearing Loss Gene.

OBJECTIVE: PDZD7 was identified in 2009 in a family with apparent nonsyndromic sensorineural hearing loss. However, subsequent clinical reports have associated PDZD7 with digenic Usher syndrome, the most common cause of deaf-blindness, or as a modifier of retinal disease. No further reports have validated this gene for nonsyndromic hearing loss, intuitively calling correct genotype-phenotype association into question. This report describes a validating second case for biallelic mutations in PDZD7 causing nonsyndromic mild to severe sensorineural hearing loss. It also provides detailed audiometric and ophthalmologic data excluding Usher syndrome in both the present proband (proband 1) and the first proband described in 2009 (proband 2). DESIGN: Proband 1 was sequenced using a custom-designed next generation sequencing panel consisting of 151 deafness genes. Bioinformatics analysis and filtering disclosed two PDZD7 sequence variants (c.1648C>T, p.Q550* and c.2107del, p.S703Vfs*20). Segregation testing followed in the family. For both probands, audiograms were collected and analyzed for progressive hearing loss and detailed ophthalmic evaluations were performed including electroretinography. RESULTS: Proband 1 demonstrated a prelingual, nonsyndromic, sensorineural hearing loss that progressed in the higher frequencies between 4 and 9 years old. PDZD7 segregation analysis confirmed biallelic inheritance (compound heterozygosity). Mutation analysis determined the c.1648C>T mutation as novel and reported the c.2107del deletion as rs397516633 with a calculated minor allele frequency of 0.000018. Clinical evaluation spanning well over a decade in proband 2 disclosed bilateral, nonprogressive hearing loss. Both probands showed healthy retinas, excluding Usher syndrome-like changes in the eye. CONCLUSIONS: PDZD7 is confirmed as a bona fide autosomal recessive nonsyndromic hearing loss gene. In both probands, there was no evidence of impaired vision or ophthalmic pathology. As the current understanding of PDZD7 mutations bridge Mendelian and complex phenotypes, the authors recommend careful variant interpretation, since PDZD7 is one of many genes associated with both Usher syndrome and autosomal recessive nonsyndromic hearing loss. Additional reports are required for understanding the complete phenotypic spectrum of this gene, including the possibility of high-frequency progression, as well as noise-induced hearing loss susceptibility in adult carriers. This report rules out all forms of Usher syndrome with an onset before 12 and 15 years old in probands 1 and 2, respectively. However, due to the young ages of the probands, this report is uninformative regarding older patients.

A Novel de novo Mutation in CEACAM16 Associated with Postlingual Hearing Impairment.

Mutations in CEACAM16 cause autosomal dominant nonsyndromic hearing loss (DFNA4B). So far, 2 families have been reported with segregating missense mutations, both in the immunoglobulin constant domain A of the CEACAM16 protein. In this study, we used the TruSight One panel to investigate a parent-child trio without familial history of hearing loss and one affected child. When filtering for recessive inheritance and de novo events, we discovered a de novo CEACAM16 mutation (c.1094T>G, p.Leu365Arg) as the sole likely pathogenic variant. The de novo mutation was confirmed by Sanger sequencing and STR analysis. The proband's hearing loss closely matches the described onset and severity for DFNA4B. We present the third CEACAM16 variant and the first de novo mutation in CEACAM16. This de novo mutation is robustly described as a pathogenic mutation according to in silico mutation prediction tools and affects a highly conserved amino acid in the most strongly conserved CEACAM16 N2 domain. Our strategy of screening family trios enhances de novo mutation discovery and the exclusion of other variants of potential interest through pedigree filtering.

Non-syndromic hearing loss gene identification: A brief history and glimpse into the future.

From the first identified non-syndromic hearing loss gene in 1995, to those discovered in present day, the field of human genetics has witnessed an unparalleled revolution that includes the completion of the Human Genome Project in 2003 to the $1000 genome in 2014. This review highlights the classical and cutting-edge strategies for non-syndromic hearing loss gene identification that have been used throughout the twenty year history with a special emphasis on how the innovative breakthroughs in next generation sequencing technology have forever changed candidate gene approaches. The simplified approach afforded by next generation sequencing technology provides a second chance for the many linked loci in large and well characterized families that have been identified by linkage analysis but have presently failed to identify a causative gene. It also discusses some complexities that may restrict eventual candidate gene discovery and calls for novel approaches to answer some of the questions that make this simple Mendelian disorder so intriguing.

Targeted next-generation sequencing of deafness genes in hearing-impaired individuals uncovers informative mutations.

PURPOSE: Targeted next-generation sequencing provides a remarkable opportunity to identify variants in known disease genes, particularly in extremely heterogeneous disorders such as nonsyndromic hearing loss. The present study attempts to shed light on the complexity of hearing impairment. METHODS: Using one of two next-generation sequencing panels containing either 80 or 129 deafness genes, we screened 30 individuals with nonsyndromic hearing loss (from 23 unrelated families) and analyzed 9 normal-hearing controls. RESULTS: Overall, we found an average of 3.7 variants (in 80 genes) with deleterious prediction outcome, including a number of novel variants, in individuals with nonsyndromic hearing loss and 1.4 in controls. By next-generation sequencing alone, 12 of 23 (52%) probands were diagnosed with monogenic forms of nonsyndromic hearing loss; one individual displayed a DNA sequence mutation together with a microdeletion. Two (9%) probands have Usher syndrome. In the undiagnosed individuals (10/23; 43%) we detected a significant enrichment of potentially pathogenic variants as compared to controls. CONCLUSION: Next-generation sequencing combined with microarrays provides the diagnosis for approximately half of the GJB2 mutation-negative individuals. Usher syndrome was found to be more frequent in the study cohort than anticipated. The conditions in a proportion of individuals with nonsyndromic hearing loss, particularly in the undiagnosed group, may have been caused or modified by an accumulation of unfavorable variants across multiple genes.