Mutations in MINAR2 encoding membrane integral NOTCH2-associated receptor 2 cause deafness in humans and mice.

Discovery of deafness genes and elucidating their functions have substantially contributed to our understanding of hearing physiology and its pathologies. Here we report on DNA variants in MINAR2, encoding membrane integral NOTCH2-associated receptor 2, in four families underlying autosomal recessive nonsyndromic deafness. Neurologic evaluation of affected individuals at ages ranging from 4 to 80 y old does not show additional abnormalities. MINAR2 is a recently annotated gene with limited functional understanding. We detected three MINAR2 variants, c.144G > A (p.Trp48*), c.412_419delCGGTTTTG (p.Arg138Valfs*10), and c.393G > T, in 13 individuals with congenital- or prelingual-onset severe-to-profound sensorineural hearing loss (HL). The c.393G > T variant is shown to disrupt a splice donor site. We show that Minar2 is expressed in the mouse inner ear, with the protein localizing mainly in the hair cells, spiral ganglia, the spiral limbus, and the stria vascularis. Mice with loss of function of the Minar2 protein (Minar2tm1b/tm1b) present with rapidly progressive sensorineural HL associated with a reduction in outer hair cell stereocilia in the shortest row and degeneration of hair cells at a later age. We conclude that MINAR2 is essential for hearing in humans and mice and its disruption leads to sensorineural HL. Progressive HL observed in mice and in some affected individuals and as well as relative preservation of hair cells provides an opportunity to interfere with HL using genetic therapies.

Identification of novel MYH14 variants in families with autosomal dominant sensorineural hearing loss.

Autosomal dominant sensorineural hearing loss (ADSNHL) is a genetically heterogeneous disorder caused by pathogenic variants in various genes, including MYH14. However, the interpretation of pathogenicity for MYH14 variants remains a challenge due to incomplete penetrance and the lack of functional studies and large families. In this study, we performed exome sequencing in six unrelated families with ADSNHL and identified five MYH14 variants, including three novel variants. Two of the novel variants, c.571G > C (p.Asp191His) and c.571G > A (p.Asp191Asn), were classified as likely pathogenic using ACMG and Hearing Loss Expert panel guidelines. In silico modeling demonstrated that these variants, along with p.Gly1794Arg, can alter protein stability and interactions among neighboring molecules. Our findings suggest that MYH14 causative variants may be more contributory and emphasize the importance of considering this gene in patients with nonsyndromic mainly post-lingual severe form of hearing loss. However, further functional studies are needed to confirm the pathogenicity of these variants.

Genome sequencing identifies coding and non-coding variants for non-syndromic hearing loss.

Hearing loss (HL) is a common heterogeneous trait that involves variants in more than 200 genes. In this study, we utilized exome (ES) and genome sequencing (GS) to effectively identify the genetic cause of presumably non-syndromic HL in 322 families from South and West Asia and Latin America. Biallelic GJB2 variants were identified in 58 probands at the time of enrollment these probands were excluded. In addition, upon review of phenotypic findings, 38/322 probands were excluded based on syndromic findings at the time of ascertainment and no further evaluation was performed on those samples. We performed ES as a primary diagnostic tool on one or two affected individuals from 212/226 families. Via ES we detected a total of 78 variants in 30 genes and showed their co-segregation with HL in 71 affected families. Most of the variants were frameshift or missense and affected individuals were either homozygous or compound heterozygous in their respective families. We employed GS as a primary test on a subset of 14 families and a secondary tool on 22 families which were unsolved by ES. Although the cumulative detection rate of causal variants by ES and GS is 40% (89/226), GS alone has led to a molecular diagnosis in 7 of 14 families as the primary tool and 5 of 22 families as the secondary test. GS successfully identified variants present in deep intronic or complex regions not detectable by ES.

H4C5 missense variant leads to a neurodevelopmental phenotype overlapping with Angelman syndrome.

Recurrent de novo missense variants in H4 histone genes have recently been associated with a novel neurodevelopmental syndrome that is characterized by intellectual disability and developmental delay as well as more variable findings that include short stature, microcephaly, and facial dysmorphisms. A 4-year-old male with autism, developmental delay, microcephaly, and a happy demeanor underwent evaluation through the Undiagnosed Disease Network. He was clinically suspected to have Angelman syndrome; however, molecular testing was negative. Genome sequencing identified the H4 histone gene variant H4C5 NM_003545.4: c.295T>C, p.Tyr99His, which parental testing confirmed to be de novo. The variant met criteria for a likely pathogenic classification and is one of the seven known disease-causing missense variants in H4C5. A comparison of our proband's findings to the initial description of the H4-associated neurodevelopmental syndrome demonstrates that his phenotype closely matches the spectrum of those reported among the 29 affected individuals. As such, this report corroborates the delineation of neurodevelopmental syndrome caused by de novo missense H4 gene variants. Moreover, it suggests that cases of clinically suspected Angelman syndrome without molecular confirmation should undergo exome or genome sequencing, as novel neurodevelopmental syndromes with phenotypes overlapping with Angelman continue to be discovered.

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.

Genome-Wide Linkage Study Meta-Analysis of Male Sexual Orientation.

Male sexual orientation is a scientifically and socially important trait shown by family and twin studies to be influenced by environmental and complex genetic factors. Individual genome-wide linkage studies (GWLS) have been conducted, but not jointly analyzed. Two main datasets account for > 90% of the published GWLS concordant sibling pairs on the trait and are jointly analyzed here: MGSOSO (Molecular Genetic Study of Sexual Orientation; 409 concordant sibling pairs in 384 families, Sanders et al. (2015)) and Hamer (155 concordant sibling pairs in 145 families, Mustanski et al. (2005)). We conducted multipoint linkage analyses with Merlin on the datasets separately since they were genotyped differently, integrated genetic marker positions, and combined the resultant LOD (logarithm of the odds) scores at each 1 cM grid position. We continue to find the strongest linkage support at pericentromeric chromosome 8 and chromosome Xq28. We also incorporated the remaining published GWLS dataset (on 55 families) by using meta-analytic approaches on published summary statistics. The meta-analysis has maximized the positional information from GWLS of currently available family resources and can help prioritize findings from genome-wide association studies (GWAS) and other approaches. Although increasing evidence highlights genetic contributions to male sexual orientation, our current understanding of contributory loci is still limited, consistent with the complexity of the trait. Further increasing genetic knowledge about male sexual orientation, especially via large GWAS, should help advance our understanding of the biology of this important trait.

Genome-Wide Linkage and Association Study of Childhood Gender Nonconformity in Males.

Male sexual orientation is influenced by environmental and complex genetic factors. Childhood gender nonconformity (CGN) is one of the strongest correlates of homosexuality with substantial familiality. We studied brothers in families with two or more homosexual brothers (409 concordant sibling pairs in 384 families, as well as their heterosexual brothers), who self-recalled their CGN. To map loci for CGN, we conducted a genome-wide linkage scan (GWLS) using SNP genotypes. The strongest linkage peaks, each with significant or suggestive two-point LOD scores and multipoint LOD score support, were on chromosomes 5q31 (maximum two-point LOD = 4.45), 6q12 (maximum two-point LOD = 3.64), 7q33 (maximum two-point LOD = 3.09), and 8q24 (maximum two-point LOD = 3.67), with the latter not overlapping with previously reported strongest linkage region for male sexual orientation on pericentromeric chromosome 8. Family-based association analyses were used to identify associated variants in the linkage regions, with a cluster of SNPs (minimum association p = 1.3 × 10-8) found at the 5q31 linkage peak. Genome-wide, clusters of multiple SNPs in the 10-6 to 10-8 p-value range were found at chromosomes 5p13, 5q31, 7q32, 8p22, and 10q23, highlighting glutamate-related genes. This is the first reported GWLS and genome-wide association study on CGN. Further increasing genetic knowledge about CGN and its relationships to male sexual orientation should help advance our understanding of the biology of these associated traits.

Human genetic variation in GLS2 is associated with development of complicated Staphylococcus aureus bacteremia.

The role of host genetic variation in the development of complicated Staphylococcus aureus bacteremia (SAB) is poorly understood. We used whole exome sequencing (WES) to examine the cumulative effect of coding variants in each gene on risk of complicated SAB in a discovery sample of 168 SAB cases (84 complicated and 84 uncomplicated, frequency matched by age, sex, and bacterial clonal complex [CC]), and then evaluated the most significantly associated genes in a replication sample of 240 SAB cases (122 complicated and 118 uncomplicated, frequency matched for age, sex, and CC) using targeted sequence capture. In the discovery sample, gene-based analysis using the SKAT-O program identified 334 genes associated with complicated SAB at p<3.5 x 10-3. These, along with eight biologically relevant candidate genes were examined in the replication sample. Gene-based analysis of the 342 genes in the replication sample using SKAT-O identified one gene, GLS2, significantly associated with complicated SAB (p = 1.2 x 10-4) after Bonferroni correction. In Firth-bias corrected logistic regression analysis of individual variants, the strongest association across all 10,931 variants in the replication sample was with rs2657878 in GLS2 (p = 5 x 10-4). This variant is strongly correlated with a missense variant (rs2657879, p = 4.4 x 10-3) in which the minor allele (associated here with complicated SAB) has been previously associated with lower plasma concentration of glutamine. In a microarray-based gene-expression analysis, individuals with SAB exhibited significantly lower expression levels of GLS2 than healthy controls. Similarly, Gls2 expression is lower in response to S. aureus exposure in mouse RAW 264.7 macrophage cells. Compared to wild-type cells, RAW 264.7 cells with Gls2 silenced by CRISPR-Cas9 genome editing have decreased IL1-ß transcription and increased nitric oxide production after S. aureus exposure. GLS2 is an interesting candidate gene for complicated SAB due to its role in regulating glutamine metabolism, a key factor in leukocyte activation.

A founder noncoding GALT variant interfering with splicing causes galactosemia.

Galactosemia is a rare, treatable hereditary disorder of carbohydrate metabolism. We investigated the etiology of decreased GALT enzyme activity in a cohort of newborns referred by the Florida Newborn Screening Program with no detectable GALT variants in diagnostic molecular tests. Six affected individuals from four families with Guatemalan heritage were included. GALT enzyme activity ranged from 20% to 34% of normal. Clinical findings were unremarkable except for speech delay in two children. Via genome sequencing followed by Sanger confirmation we showed that all affected individuals were homozygous for a deep intronic GALT variant, c.1059+390A>G, which segregated as an autosomal recessive trait in all families. The intronic variant disrupts splicing and leads to a premature termination and is associated with a single haplotype flanking GALT, suggesting a founder effect. In conclusion, we present a deep intronic GALT variant leading to a biochemical variant form of galactosemia. This variant remains undiagnosed until it is specifically targeted in molecular testing.

A truncating CLDN9 variant is associated with autosomal recessive nonsyndromic hearing loss.

While the importance of tight junctions in hearing is well established, the role of Claudin- 9 (CLDN9), a tight junction protein, in human hearing and deafness has not been explored. Through whole-genome sequencing, we identified a one base pair deletion (c.86delT) in CLDN9 in a consanguineous family from Turkey with autosomal recessive nonsyndromic hearing loss. Three affected members of the family had sensorineural hearing loss (SNHL) ranging from moderate to profound in severity. The variant is predicted to cause a frameshift and produce a truncated protein (p.Leu29ArgfsTer4) in this single-exon gene. It is absent in public databases as well as in over 1000 Turkish individuals, and co-segregates with SNHL in the family. Our in vitro studies demonstrate that the mutant protein does not localize to cell membrane as demonstrated for the wild-type protein. Mice-lacking Cldn9 have been shown to develop SNHL. We conclude that CLDN9 is essential for proper audition in humans and its disruption leads to SNHL in humans.

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.

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.

Spectrum of DNA variants for non-syndromic deafness in a large cohort from multiple continents.

Hearing loss is the most common sensory deficit in humans with causative variants in over 140 genes. With few exceptions, however, the population-specific distribution for many of the identified variants/genes is unclear. Until recently, the extensive genetic and clinical heterogeneity of deafness precluded comprehensive genetic analysis. Here, using a custom capture panel (MiamiOtoGenes), we undertook a targeted sequencing of 180 genes in a multi-ethnic cohort of 342 GJB2 mutation-negative deaf probands from South Africa, Nigeria, Tunisia, Turkey, Iran, India, Guatemala, and the United States (South Florida). We detected causative DNA variants in 25 % of multiplex and 7 % of simplex families. The detection rate varied between 0 and 57 % based on ethnicity, with Guatemala and Iran at the lower and higher end of the spectrum, respectively. We detected causative variants within 27 genes without predominant recurring pathogenic variants. The most commonly implicated genes include MYO15A, SLC26A4, USH2A, MYO7A, MYO6, and TRIOBP. Overall, our study highlights the importance of family history and generation of databases for multiple ethnically discrete populations to improve our ability to detect and accurately interpret genetic variants for pathogenicity.

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.

Mutations in ANKRD11 cause KBG syndrome, characterized by intellectual disability, skeletal malformations, and macrodontia.

KBG syndrome is characterized by intellectual disability associated with macrodontia of the upper central incisors as well as distinct craniofacial findings, short stature, and skeletal anomalies. Although believed to be genetic in origin, the specific underlying defect is unknown. Through whole-exome sequencing, we identified deleterious heterozygous mutations in ANKRD11 encoding ankyrin repeat domain 11, also known as ankyrin repeat-containing cofactor 1. A splice-site mutation, c.7570-1G>C (p.Glu2524_Lys2525del), cosegregated with the disease in a family with three affected members, whereas in a simplex case a de novo truncating mutation, c.2305delT (p.Ser769GlnfsX8), was detected. Sanger sequencing revealed additional de novo truncating ANKRD11 mutations in three other simplex cases. ANKRD11 is known to interact with nuclear receptor complexes to modify transcriptional activation. We demonstrated that ANKRD11 localizes mainly to the nuclei of neurons and accumulates in discrete inclusions when neurons are depolarized, suggesting that it plays a role in neural plasticity. Our results demonstrate that mutations in ANKRD11 cause KBG syndrome and outline a fundamental role of ANKRD11 in craniofacial, dental, skeletal, and central nervous system development and function.

Human Organoids for Rapid Validation of Gene Variants Linked to Cochlear Malformations.

Developmental anomalies of the hearing organ, the cochlea, are diagnosed in approximately one-fourth of individuals with congenital deafness. Most patients with cochlear malformations remain etiologically undiagnosed due to insufficient knowledge about underlying genes or the inability to make conclusive interpretations of identified genetic variants. We used exome sequencing for genetic evaluation of hearing loss associated with cochlear malformations in three probands from unrelated families. We subsequently generated monoclonal induced pluripotent stem cell (iPSC) lines, bearing patient-specific knockins and knockouts using CRISPR/Cas9 to assess pathogenicity of candidate variants. We detected FGF3 (p.Arg165Gly) and GREB1L (p.Cys186Arg), variants of uncertain significance in two recognized genes for deafness, and PBXIP1 (p.Trp574*) in a candidate gene. Upon differentiation of iPSCs towards inner ear organoids, we observed significant developmental aberrations in knockout lines compared to their isogenic controls. Patient-specific single nucleotide variants (SNVs) showed similar abnormalities as the knockout lines, functionally supporting their causality in the observed phenotype. Therefore, we present human inner ear organoids as a tool to rapidly validate the pathogenicity of DNA variants associated with cochlear malformations.

Genetic heterogeneity in hereditary hearing loss: Potential role of kinociliary protein TOGARAM2.

Hearing loss (HL) is a heterogenous trait with pathogenic variants in more than 200 genes that have been discovered in studies involving small and large HL families. Over one-third of families with hereditary HL remain etiologically undiagnosed after screening for mutations in the recognized genes. Genetic heterogeneity complicates the analysis in multiplex families where variants in more than one gene can be causal in different individuals even in the same sibship. We employed exome or genome sequencing in at least two affected individuals with congenital or prelingual-onset, severe to profound, non-syndromic, bilateral sensorineural HL from four multiplex families. Bioinformatic analysis was performed to identify variants in known and candidate deafness genes. Our results show that in these four families, variants in a single HL gene do not explain HL in all affected family members, and variants in another known or candidate HL gene were detected to clarify HL in the entire family. We also present a variant in TOGARAM2 as a potential cause underlying autosomal recessive non-syndromic HL by showing its presence in a family with HL, its expression in the cochlea and the localization of the protein to cochlear hair cells. Conclusively, analyzing all affected family members separately can serve as a good source for the identification of variants in known and novel candidate genes for HL.

Genome sequencing reveals novel noncoding variants in PLA2G6 and LMNB1 causing progressive neurologic disease.

Neurodegenerative disorders and leukodystrophies are progressive neurologic conditions that can occur following the disruption of intricately coordinated patterns of gene expression. Exome sequencing has been adopted as an effective diagnostic tool for determining the underlying genetic etiology of Mendelian neurologic disorders, however genome sequencing offer advantages in its ability to identify and characterize copy number, structural, and sequence variants in noncoding regions. Genome sequencing from peripheral leukocytes was performed on two patients with progressive neurologic disease of unknown etiology following negative genetic investigations including exome sequencing. RNA sequencing from peripheral blood was performed to determine gene expression patterns in one of the patients. Potential causative variants were matched to the patients' clinical presentation. The first proband was found to be heterozygous for a likely pathogenic missense variant in PLA2G6 (c.386T>C; p.Leu129Pro) and have an additional deep intronic variant in PLA2G6 (c.2035-926G>A). RNA sequencing indicated this latter variant created a splice acceptor site leading to the incorporation of a pseudo-exon introducing a premature termination codon. The second proband was heterozygous for a 261 kb deletion upstream of LMNB1 that included an enhancer region. Previous reports of copy number variants spanning this region of cis-acting regulatory elements corroborated its pathogenicity. When combined with clinical presentations, these findings led to a definitive diagnosis of autosomal recessive infantile neuroaxonal dystrophy and autosomal dominant adult-onset demyelinating leukodystrophy, respectively. In patients with progressive neurologic disease of unknown etiology, genome sequencing with the addition of RNA analysis where appropriate should be considered for the identification of causative noncoding pathogenic variants.

Genomic evidence consistent with antagonistic pleiotropy may help explain the evolutionary maintenance of same-sex sexual behaviour in humans.

Human same-sex sexual behaviour (SSB) is heritable, confers no immediately obvious direct reproductive or survival benefit and can divert mating effort from reproductive opportunities. This presents a Darwinian paradox: why has SSB been maintained despite apparent selection against it? We show that genetic effects associated with SSB may, in individuals who only engage in opposite-sex sexual behaviour (OSB individuals), confer a mating advantage. Using results from a recent genome-wide association study of SSB and a new genome-wide association study on number of opposite-sex sexual partners in 358,426 individuals, we show that, among OSB individuals, genetic effects associated with SSB are associated with having more opposite-sex sexual partners. Computer simulations suggest that such a mating advantage for alleles associated with SSB could help explain how it has been evolutionarily maintained. Caveats include the cultural specificity of our UK and US samples, the societal regulation of sexual behaviour in these populations, the difficulty of measuring mating success and the fact that measured variants capture a minority of the total genetic variation in the traits.

Response to Comment on "Large-scale GWAS reveals insights into the genetic architecture of same-sex sexual behavior".

Hamer et al argue that the variable "ever versus never had a same-sex partner" does not capture the complexity of human sexuality. We agree and said so in our paper. But Hamer et al neglect to mention that we also reported follow-up analyses showing substantial overlap of the genetic influences on our main variable and on more nuanced measures of sexual behavior, attraction, and identity.