A novel de novo mutation in COL2A1 gene associated with fetal skeletal dysplasia.

OBJECTIVE: Skeletal dysplasias, caused by genetic mutations, are a heterogenous group of heritable disorders affecting bone development during fetal life. Stickler syndrome, one of the skeletal dysplasias, is an autosomal dominant connective tissue disorder caused by abnormal collagen synthesis owing to a genetic mutation in COL2A1. CASE REPORT: We present the case of a 38-year-old multipara woman whose first trimester screening showed a normal karyotype. However, the bilateral femur and humerus length symmetrically shortened after 20 weeks. Next-generation sequencing for mutations in potential genes leading to skeletal dysplasia detected a novel de novo mutation (c.1438G > A, p.Gly480Arg) in COL2A1, causing Stickler syndrome type 1. This pathogenic mutation might impair or destabilize the collagen structure, leading to collagen type II, IX, and XI dysfunction. CONCLUSION: We identified a novel de novo mutation in COL2A1 related to the STL1 syndrome and delineated the extent of the skeletal dysplasia disease spectrum.

Zika virus can directly infect and damage the auditory and vestibular components of the embryonic chicken inner ear.

BACKGROUND: Sensorineural hearing loss is an understudied consequence of congenital Zika syndrome, and balance disorders are essentially unreported to date. Also lacking is information about the susceptibility and the pathogenesis of the developing inner ear following Zika virus (ZIKV) exposure. To address this, ZIKV was delivered directly into the otic cup/otocyst of chicken embryos and infection of inner ear tissues was evaluated using immunohistochemistry. RESULTS: After injections on embryonic days 2 to 5, ZIKV infection was observed in 90% of the samples harvested 2 to 8 days later; however, the degree of infection was highly variable across individuals. ZIKV was detected in all regions of the inner ear, associated ganglia, and in the surrounding periotic mesenchyme. Detection of virus peaked earlier in the ganglion and vestibular compartments, and later in the cochlea. ZIKV infection increased cell death robustly in the auditory ganglion, and modestly in the auditory sensory organ. Macrophage accumulation was found to overlap with dense viral infection in some tissues. Additionally, dysmorphogenesis of the semicircular canals and ganglion was observed for a subset of injection conditions. CONCLUSIONS: This article presents evidence of direct ZIKV infection of developing inner ear epithelium and shows previously unknown inner ear dysmorphogenesis phenotypes.

[Mutation analysis and prenatal diagnosis for 12 families affected with hereditary hearing loss and enlarged vestibular aqueduct].

OBJECTIVE: To carry out mutation analysis and prenatal diagnosis for 12 families affected with hearing loss and enlarged vestibular aqueduct from southern Zhejiang province. METHODS: Clinical data and peripheral venous blood samples of 38 members from the 12 families were obtained. Mutations of 4 genes, namely SLC26A4, GJB2, c.538C to T and c.547G to A of GJB3, m.1555A to G and m.1494C to T of 12S rRNA, were detected by PCR and Sanger sequencing. Maternal contamination was excluded by application of STR detection during prenatal diagnosis. RESULTS: Among the probands from the 12 families, 11 were found to be compound heterozygotes or homozygotes and 25 were heterozygotes. All of the families were detected with IVS7-2A to G mutations, and 4 had a second heterozygous mutation (c.2168A to G of the SLC26A4 gene). Four rare pathogenic mutations, namely IVS5-1G to A, c.946G to T, c.1607A to G and c.2167C to G, were detected in another four families. In addition, the partner of proband from pedigree 3 was identified with compound heterozygous mutations of c.235delC and c.299-300delAT, and proband of pedigree 5 has carried a mutation of c.109G to A in GJB2. For SLC26A4 gene, prenatal diagnostic testing has revealed heterozygous mutations in 6 fetuses and compound heterozygous mutations in 2 fetuses. CONCLUSION: IVS7-2A to G and c.2168A to G of the SLC26A4 gene were the most common mutations in southern Zhejiang. Such mutations can be found in most families affected with hearing loss and enlarged vestibular aqueduct, which may facilitate genetic counseling and prenatal diagnosis for such families.

Development of novel noninvasive prenatal testing protocol for whole autosomal recessive disease using picodroplet digital PCR.

We developed a protocol of noninvasive prenatal testing (NIPT), employing a higher-resolution picodroplet digital PCR, to detect genetic imbalance in maternal plasma DNA (mpDNA) caused by cell-free fetal DNA (cffDNA). In the present study, this approach was applied to four families with autosomal recessive (AR) congenital sensorineural hearing loss. First, a fraction of the fetal DNA in mpDNA was calculated. Then, we made artificial DNA mixtures (positive and negative controls) to simulate mpDNA containing the fraction of cffDNA with or without mutations. Next, a fraction of mutant cluster signals over the total signals was measured from mpDNA, positive controls, and negative controls. We determined whether fetal DNA carried any paternal or maternal mutations by calculating and comparing the sum of the log-likelihood of the study samples. Of the four families, we made a successful prediction of the complete fetal genotype in two cases where a distinct cluster was identified for each genotype and the fraction of cffDNA in mpDNA was at least 6.4%. Genotyping of only paternal mutation was possible in one of the other two families. This is the first NIPT protocol potentially applicable to any AR monogenic disease with various genotypes, including point mutations.

Spatial and temporal expression patterns of Osbpl2a and Osbpl2b during zebrafish embryonic development.

OBJECTIVE: The mutated OSBPL2 (OMIM: 606731), encoding oxysterol binding protein-like protein 2, was recently identified as a novel causative gene for autosomal dominant nonsyndromic hearing loss (ADNSHL). We reported the expression patterns of Osbpl2 in zebrafish, in order to further understand the role of OSBPL2 in hearing formation and development. METHODS: Zebrafish was used as an animal model, and the expression of Osbpl2 was investigated by whole mount in situ hybridization. RESULTS: Bioinformatics analysis indicates that zebrafish has two homologues of Osbpl2 gene (Osbpl2a and Osbpl2b) and Osbpl2b is the orthologous gene of human OSBPL2. No expression of Osbpl2a and Osbpl2b mRNA is detected at 75% epiboly. The zygotical expression of the two genes has not been started at 11-somite stage. At 24h post-fertilization (hpf), both Osbpl2a and Osbpl2b are found at ventricle zone of brain, however, the expression level of Osbpl2a is higher than that of Osbpl2b. When embryos are 48hpf, the expression level of Osbpl2a and Osbpl2b becomes higher at the ventricle zone. At 72hpf, Osbpl2b is only found at liver primordium, while Osbpl2a is not detected anywhere obviously. At 96hpf, Osbpl2b is found at pharyngeal arches, liver, digestive tract and otic vesicle, while Osbpl2a remains undetected. CONCLUSION: Osbpl2b was demonstrated to be the orthologous gene of human OSBPL2, which has strong maternal expression, while Osbpl2a was detected without obvious maternal expression. This work would contribute to the further study of the molecular mechanism and function of OSBPL2 implicated with ADNSHL.

Ildr1b is essential for semicircular canal development, migration of the posterior lateral line primordium and hearing ability in zebrafish: implications for a role in the recessive hearing impairment DFNB42.

Immunoglobulin-like domain containing receptor 1 (ILDR1) is a poorly characterized gene that was first identified in lymphoma cells. Recently, ILDR1 has been found to be responsible for autosomal recessive hearing impairment DFNB42. Patients with ILDR1 mutations cause bilateral non-progressive moderate-to-profound sensorineural hearing impairment. However, the etiology and mechanism of ILDR1-related hearing loss remains to be elucidated. In order to uncover the pathology of DFNB42 deafness, we used the morpholino injection technique to establish an ildr1b-morphant zebrafish model. Ildr1b-morphant zebrafish displayed defective hearing and imbalanced swimming, and developmental delays were seen in the semicircular canals of the inner ear. The gene expression profile and real-time PCR revealed down-regulation of atp1b2b (encoding Na(+)/K(+) transporting, beta 2b polypeptide) in ildr1b-morphant zebrafish. We found that injection of atp1b2b mRNA into ildr1b-knockdown zebrafish could rescue the phenotype of developmental delay of the semicircular canals. Moreover, ildr1b-morphant zebrafish had reduced numbers of lateral line neuromasts due to the disruption of lateral line primordium migration. In situ hybridization showed the involvement of attenuated FGF signaling and the chemokine receptor 4b (cxcr4b) and chemokine receptor 7b (cxcr7b) in posterior lateral line primordium of ildr1b-morphant zebrafish. We concluded that Ildr1b is crucial for the development of the inner ear and the lateral line system. This study provides the first evidence for the mechanism of Ildr1b on hearing in vivo and sheds light on the pathology of DFNB42.

Assembly of the cochlear gap junction macromolecular complex requires connexin 26.

Hereditary deafness affects approximately 1 in 2,000 children. Mutations in the gene encoding the cochlear gap junction protein connexin 26 (CX26) cause prelingual, nonsyndromic deafness and are responsible for as many as 50% of hereditary deafness cases in certain populations. Connexin-associated deafness is thought to be the result of defective development of auditory sensory epithelium due to connexion dysfunction. Surprisingly, CX26 deficiency is not compensated for by the closely related connexin CX30, which is abundantly expressed in the same cochlear cells. Here, using two mouse models of CX26-associated deafness, we demonstrate that disruption of the CX26-dependent gap junction plaque (GJP) is the earliest observable change during embryonic development of mice with connexin-associated deafness. Loss of CX26 resulted in a drastic reduction in the GJP area and protein level and was associated with excessive endocytosis with increased expression of caveolin 1 and caveolin 2. Furthermore, expression of deafness-associated CX26 and CX30 in cell culture resulted in visible disruption of GJPs and loss of function. Our results demonstrate that deafness-associated mutations in CX26 induce the macromolecular degradation of large gap junction complexes accompanied by an increase in caveolar structures.

Role of p63 and the Notch pathway in cochlea development and sensorineural deafness.

The ectodermal dysplasias are a group of inherited autosomal dominant syndromes associated with heterozygous mutations in the Tumor Protein p63 (TRP63) gene. Here we show that, in addition to their epidermal pathology, a proportion of these patients have distinct levels of deafness. Accordingly, p63 null mouse embryos show marked cochlea abnormalities, and the transactivating isoform of p63 (TAp63) protein is normally found in the organ of Corti. TAp63 transactivates hairy and enhancer of split 5 (Hes5) and atonal homolog 1 (Atoh1), components of the Notch pathway, known to be involved in cochlear neuroepithelial development. Strikingly, p63 null mice show morphological defects of the organ of Corti, with supernumerary hair cells, as also reported for Hes5 null mice. This phenotype is related to loss of a differentiation property of TAp63 and not to loss of its proapoptotic function, because cochleas in mice lacking the critical Bcl-2 homology domain (BH-3) inducers of p53- and p63-mediated apoptosis--Puma, Noxa, or both--are normal. Collectively, these data demonstrate that TAp63, acting via the Notch pathway, is crucial for the development of the organ of Corti, providing a molecular explanation for the sensorineural deafness in ectodermal dysplasia patients with TRP63 mutations.

Mouse model of enlarged vestibular aqueducts defines temporal requirement of Slc26a4 expression for hearing acquisition.

Mutations in human SLC26A4 are a common cause of hearing loss associated with enlarged vestibular aqueducts (EVA). SLC26A4 encodes pendrin, an anion-base exchanger expressed in inner ear epithelial cells that secretes HCO3- into endolymph. Studies of Slc26a4-null mice indicate that pendrin is essential for inner ear development, but have not revealed whether pendrin is specifically necessary for homeostasis. Slc26a4-null mice are profoundly deaf, with severe inner ear malformations and degenerative changes that do not model the less severe human phenotype. Here, we describe studies in which we generated a binary transgenic mouse line in which Slc26a4 expression could be induced with doxycycline. The transgenes were crossed onto the Slc26a4-null background so that all functional pendrin was derived from the transgenes. Varying the temporal expression of Slc26a4 revealed that E16.5 to P2 was the critical interval in which pendrin was required for acquisition of normal hearing. Lack of pendrin during this period led to endolymphatic acidification, loss of the endocochlear potential, and failure to acquire normal hearing. Doxycycline initiation at E18.5 or discontinuation at E17.5 resulted in partial hearing loss approximating the human EVA auditory phenotype. These data collectively provide mechanistic insight into hearing loss caused by SLC26A4 mutations and establish a model for further studies of EVA-associated hearing loss.

Novel mutations in LHX3 are associated with hypopituitarism and sensorineural hearing loss.

Homozygous loss-of-function mutations in the transcription factor LHX3 have been associated with hypopituitarism with structural anterior pituitary defects and cervical abnormalities with or without restricted neck rotation. We report two novel recessive mutations in LHX3 in four patients from two unrelated pedigrees. Clinical evaluation revealed that all four patients exhibit varying degrees of bilateral sensorineural hearing loss, which has not been previously reported in association with LHX3 mutations, in addition to hypopituitarism including adrenocorticotropic hormone deficiency and an unusual skin and skeletal phenotype in one family. Furthermore, re-evaluation of three patients previously described with LHX3 mutations showed they also exhibit varying degrees of bilateral sensorineural hearing loss. We have investigated a possible role for LHX3 in inner ear development in humans using in situ hybridization of human embryonic and fetal tissue. LHX3 is expressed in defined regions of the sensory epithelium of the developing inner ear in a pattern overlapping that of SOX2, which precedes the onset of LHX3 expression and is known to be required for inner ear and pituitary development in both mice and humans. Moreover, we show that SOX2 is capable of binding to and activating transcription of the LHX3 proximal promoter in vitro. This study therefore extends the phenotypic spectrum associated with LHX3 mutations to encompass variable sensorineural hearing loss and suggests a possible interaction between LHX3 and SOX2 likely to be important for development of both the inner ear and the anterior pituitary in human embryonic development.

The association between short stature and sensorineural hearing loss.

In order to test the Thrifty Phenotype Hypothesis on hearing, data from two cross-sectional studies on hearing were re-evaluated. The data sets comprised 500 18-year-old conscripts, and 483 noise-exposed male employees. Sensorineural hearing loss (SNHL) was over-represented among conscripts with a short stature (odds ratio=2.2) or hearing loss in the family (odds ration=4.2), but not among noise-exposed conscripts (odds ratio=0.9-1.3). Among noise-exposed short employees, hypertension and age exhibited a negative impact on high frequency hearing thresholds, while among tall employees hypertension had no effect on hearing and the influence of age was less pronounced (p<0.01 for body height; p<0.02 for age, hypertension and the interaction between body height and hypertension; p<0.05 for the interaction between body height and age). This suggests that mechanisms linked to fetal programming and growth retardation and/or insulin-like growth factor 1 levels during fetal life, such as a delayed cell cycle during the time window when the cochlea develops, may cause SNHL in adulthood.

Aetiology of bilateral sensorineural hearing impairment in children: a 10 year study.

The study was carried out on children born over a 10 year period from 1981 to 1990 in a defined area known as Greater Manchester and referred to the Centre for Audiology or the Manchester Royal Infirmary for specialist audiological assessment. The children were investigated for possible congenital or intrauterine infection. Perinatal assessment was carried out in conjunction with paediatricians for adverse aetiological factors. Full medical histories were obtained with detailed family history relevant to hearing impairment and any associated condition or syndrome. Parents and siblings were examined and hearing assessed. A total of 339 cases was studied. Children with positive family history of deafness in parents or siblings, or both, constituted 23.3% of the cases (genetic group). Other aetiological groups showed the following distribution: cause unknown 33.9%; perinatal group 12.8%; congenital infections 8.2%; bacterial meningitis 6.5%; chromosomal anomalies 5.3%; syndromal group 5.3%; and miscellaneous group 4.7%. The high incidence of genetic causes indicates that steps should be taken to facilitate genetic counselling and conceivably to reduce the numbers affected.

Effects of mutations at the W locus (c-kit) on inner ear pigmentation and function in the mouse.

The W locus encodes a tyrosine kinase receptor, c-kit, which affects survival of melanoblasts from the neural crest. The primary cochlear defect in Viable Dominant Spotting (Wv/Wv) mutants is a lack of melanocytes within the stria vascularis (SV) associated with an endocochlear potential (EP) close to zero and hearing impairment. In this study, we compare inner ear pigmentation with cochlear potentials in three other W alleles (Wx, Wsh, and W41) and reveal an unequivocal correlation between presence of strial melanocytes and presence of an EP. Asymmetry was common, and 8.3% of Wsh/Wx, 25% of Wsh/Wsh, 60% of W41/Wx, and 69.2% of W41/W41 ears had a pigmented stria and an EP, while the remainder had no strial melanocytes and no EP. In those mutants that partially escaped the effects of the mutation, strial melanocytes rarely extended the entire length of the stria, but were confined to the middle and/or basal turns of the cochlea. The extent of strial pigmentation was unrelated to the EP value, which was measured from the basal turn only. Compound action potential (CAP) responses recorded from ears with an EP were variable and they showed greatly raised thresholds or were absent in all ears where the EP was close to zero. In controls, melanocytes in the vestibular part of the ear were found in the utricle, crus commune, and ampullae, whereas in many mutants only one or two of these regions were pigmented. There was a broad correlation between pigmentation of the stria and pigmentation of the vestibular region but this was not absolute. All W41/Wx, Wsh/Wsh, and W41/W41 mutants had some pigment on the pinna but, in contrast to controls where melanocytes were found in the epidermis and dermis of the pinna, pigment cells were reduced in number and generally restricted to the dermis. Injection of normal neural crest cells into 9.5-day-old mutant embryos increased the extent of skin pigmentation on the head and coat of adult chimeras and was associated with a small increase in the proportion of pigmented strias.