Search

BackgroundOtitis media (OM) susceptibility has significant heritability; however, the role of rare variants in OM is mostly unknown. Our goal is to identify novel rare variants that confer OM susceptibility.MethodsWe performed exome and Sanger sequencing of >1000 DNA samples from 551 multiethnic families with OM and unrelated individuals, RNA-sequencing and microbiome sequencing and analyses of swabs from the outer ear, middle ear, nasopharynx and oral cavity. We also examined protein localisation and gene expression in infected and healthy middle ear tissues.ResultsA large, intermarried pedigree that includes 81 OM-affected and 53 unaffected individuals cosegregates two known rare A2ML1 variants, a common FUT2 variant and a rare, novel pathogenic variant c.1682A>G (p.Glu561Gly) within SPINK5 (LOD=4.09). Carriage of the SPINK5 missense variant resulted in increased relative abundance of Microbacteriaceae in the middle ear, along with occurrence of Microbacteriaceae in the outer ear and oral cavity but not the nasopharynx. Eight additional novel SPINK5 variants were identified in 12 families and individuals with OM. A role for SPINK5 in OM susceptibility is further supported by lower RNA counts in variant carriers, strong SPINK5 localisation in outer ear skin, faint localisation to middle ear mucosa and eardrum and increased SPINK5 expression in human cholesteatoma.ConclusionSPINK5 variants confer susceptibility to non-syndromic OM. These variants potentially contribute to middle ear pathology through breakdown of mucosal and epithelial barriers, immunodeficiency such as poor vaccination response, alteration of head and neck microbiota and facilitation of entry of opportunistic pathogens into the middle ear.

BackgroundUsher syndrome (USH) is a neurosensory disorder characterised by deafness, variable vestibular areflexia and vision loss. The aim of the study was to identify the genetic defect in a Pakistani family (PKDF1051) segregating USH.MethodsGenome-wide linkage analysis was performed by using an Illumina linkage array followed by Sanger and exome sequencing. Heterologous cells and mouse organ of Corti explant-based transfection assays were used for functional evaluations. Detailed clinical evaluations were performed to characterise the USH phenotype.ResultsThrough homozygosity mapping, we genetically linked the USH phenotype segregating in family PKDF1051 to markers on chromosome 1p36.32-p36.22. The locus was designated USH1M. Using a combination of Sanger sequencing and exome sequencing, we identified a novel homozygous 18 base pair inframe deletion in ESPN. Variants of ESPN, encoding the actin-bundling protein espin, have been previously associated with deafness and vestibular areflexia in humans with no apparent visual deficits. Our functional studies in heterologous cells and in mouse organ of Corti explant cultures revealed that the six deleted residues in affected individuals of family PKDF1051 are essential for the actin bundling function of espin demonstrated by ultracentrifugation actin binding and bundling assays. Funduscopic examination of the affected individuals of family PKDF1051 revealed irregular retinal contour, temporal flecks and disc pallor in both eyes. ERG revealed diminished rod photoreceptor function among affected individuals.ConclusionOur study uncovers an additional USH gene, assigns the USH1 phenotype to a variant of ESPN and provides a 12th molecular component to the USH proteome.

Background Oliver–McFarlane syndrome is characterised by trichomegaly, congenital hypopituitarism and retinal degeneration with choroidal atrophy. Laurence–Moon syndrome presents similarly, though with progressive spinocerebellar ataxia and spastic paraplegia and without trichomegaly. Both recessively inherited disorders have no known genetic cause. Methods Whole-exome sequencing was performed to identify the genetic causes of these disorders. Mutations were functionally validated in zebrafish pnpla6 morphants. Embryonic expression was evaluated via in situ hybridisation in human embryonic sections. Human neurohistopathology was performed to characterise cerebellar degeneration. Enzymatic activities were measured in patient-derived fibroblast cell lines. Results Eight mutations in six families with Oliver–McFarlane or Laurence–Moon syndrome were identified in the PNPLA6 gene, which encodes neuropathy target esterase (NTE). PNPLA6 expression was found in the developing human eye, pituitary and brain. In zebrafish, the pnpla6 curly-tailed morphant phenotype was fully rescued by wild-type human PNPLA6 mRNA and not by mutation-harbouring mRNAs. NTE enzymatic activity was significantly reduced in fibroblast cells derived from individuals with Oliver–McFarlane syndrome. Intriguingly, adult brain histology from a patient with highly overlapping features of Oliver–McFarlane and Laurence–Moon syndromes revealed extensive cerebellar degeneration and atrophy. Conclusions Previously, PNPLA6 mutations have been associated with spastic paraplegia type 39, Gordon–Holmes syndrome and Boucher–Neuhauser syndromes. Discovery of these additional PNPLA6 -opathies further elucidates a spectrum of neurodevelopmental and neurodegenerative disorders associated with NTE impairment and suggests a unifying mechanism with diagnostic and prognostic importance.

Background Recessive mutant alleles of MYO7A , USH1C , CDH23 , and PCDH15 cause non-syndromic deafness or type 1 Usher syndrome (USH1) characterised by deafness, vestibular areflexia, and vision loss due to retinitis pigmentosa. For CDH23 , encoding cadherin 23, non-syndromic DFNB12 deafness is associated primarily with missense mutations hypothesised to have residual function. In contrast, homozygous nonsense, frame shift, splice site, and some missense mutations of CDH23 , all of which are presumably functional null alleles, cause USH1D. The phenotype of a CDH23 compound heterozygote for a DFNB12 allele in trans configuration to an USH1D allele is not known and cannot be predicted from current understanding of cadherin 23 function in the retina and vestibular labyrinth. Methods and results To address this issue, this study sought CDH23 compound heterozygotes by sequencing this gene in USH1 probands, and families segregating USH1D or DFNB12. Five non-syndromic deaf individuals were identified with normal retinal and vestibular phenotypes that segregate compound heterozygous mutations of CDH23 , where one mutation is a known or predicted USH1 allele. Conclusions One DFNB12 allele in trans configuration to an USH1D allele of CDH23 preserves vision and balance in deaf individuals, indicating that the DFNB12 allele is phenotypically dominant to an USH1D allele. This finding has implications for genetic counselling and the development of therapies for retinitis pigmentosa in Usher syndrome. Accession numbers The cDNA and protein Genbank accession numbers for CDH23 and cadherin 23 used in this paper are AY010111.2 and AAG27034.2, respectively.

Background: Approximately half the cases of prelingual hearing loss are caused by genetic factors. Identification of genes causing deafness is a crucial first step in understanding the normal function of these genes in the auditory system. Recently, a mutant allele of Tmhs was reported to be associated with deafness and circling behaviour in the hurry-scurry mouse. Tmhs encodes a predicted tetraspan protein of unknown function, which is expressed in inner ear hair cells. The human homologue of Tmhs is located on chromosome 6p. Objective: To determine the cause of deafness in four consanguineous families segregating recessive deafness linked to markers on chromosome 6p21.1-p22.3 defining a novel DFNB locus. Results: A novel locus for non-syndromic deafness DFNB67 was mapped in an interval of approximately 28.51 cM on human chromosome 6p21.1-p22.3. DNA sequence analysis of TMHS revealed a homozygous frameshift mutation (246delC) and a missense mutation (Y127C) in affected individuals of two families segregating non-syndromic deafness, one of which showed significant evidence of linkage to markers in the DFNB67 interval. The localisation of mTMHS in developing mouse inner ear hair cells was refined and found to be expressed briefly from E16.5 to P3. Conclusions: These findings establish the importance of TMHS for normal sound transduction in humans.

Familial hypertrophic cardiomyopathy (FHC) is typically characterised by left ventricular hypertrophy, diastolic dysfunction, and hypercontractility, and is often associated with disabling symptoms, arrhythmias, and sudden death.1 FHC shows both non-allelic and allelic genetic heterogeneity, and results from any one of more than 100 mutations in genes encoding sarcomeric proteins.2 Identified genes include those encoding β myosin heavy chain, the myosin regulatory and essential light chains, myosin binding protein C, troponin I, troponin C, α cardiac actin, and titin.2,3 The FHC phenotype is characterised by hypertrophy, myocyte disarray and fibrosis, and results from the dominant negative expression of one of these (mainly missense) mutations. The resulting sarcomeric dysfunction leads ultimately, through mechanisms that remain obscure, to pathological left ventricular remodelling. However, as molecular defects are identified in only half the cases, it is likely that non-sarcomeric genes may also be responsible. Non-sarcomeric causes of FHC are largely uncharacterised, and may be associated with distinct or compound phenotypes. Similarly, hereditary sensorineural hearing loss shows a great degree of non-allelic and allelic genetic heterogeneity, and can be dominant, recessive, X linked, or mitochondrial.4–6 Hereditary sensorineural hearing loss is classified according to mode of inheritance and the presence of clinically detectable extra-auditory manifestations (syndromic deafness) or their absence (non-syndromic).6,7 The distributions of mutant gene expression are not necessarily restricted to clinically affected organ systems, and mutant genes associated with “non-syndromic” deafness may therefore have subtle extra-auditory manifestations. Genetic syndromes restricted to a cardio-auditory phenotype include the long QT syndrome (LQTS) caused by mutations in KvLQT1 ( KCNQ1 ) or in KCNE1 , where QT prolongation has autosomal dominant expression (Romano Ward syndrome), but congenital sensorineural hearing loss with LQTS is autosomal recessive (Jervell and Lange-Nielsen syndrome).8 In the LQTS, cardiac structure is normal. Recently, Schonberger et al …