Live-cell single-molecule fluorescence microscopy for protruding organelles reveals regulatory mechanisms of MYO7A-driven cargo transport in stereocilia of inner ear hair cells.

Stereocilia are unidirectional F-actin-based cylindrical protrusions on the apical surface of inner ear hair cells and function as biological mechanosensors of sound and acceleration. Development of functional stereocilia requires motor activities of unconventional myosins to transport proteins necessary for elongating the F-actin cores and to assemble the mechanoelectrical transduction (MET) channel complex. However, how each myosin localizes in stereocilia using the energy from ATP hydrolysis is only partially understood. In this study, we develop a methodology for live-cell single-molecule fluorescence microscopy of organelles protruding from the apical surface using a dual-view light-sheet microscope, diSPIM. We demonstrate that MYO7A, a component of the MET machinery, traffics as a dimer in stereocilia. Movements of MYO7A are restricted when scaffolded by the plasma membrane and F-actin as mediated by MYO7A's interacting partners. Here, we discuss the technical details of our methodology and its future applications including analyses of cargo transportation in various organelles.

The role of the stria vascularis in neglected otologic disease.

The stria vascularis (SV) has been shown to play a critical role in the pathogenesis of many diseases associated with sensorineural hearing loss (SNHL), including age-related hearing loss (ARHL), noise-induced hearing loss (NIHL), hereditary hearing loss (HHL), and drug-induced hearing loss (DIHL), among others. There are a number of other disorders of hearing loss that may be relatively neglected due to being underrecognized, poorly understood, lacking robust diagnostic criteria or effective treatments. A few examples of these diseases include autoimmune inner ear disease (AIED) and/or autoinflammatory inner ear disease (AID), Meniere's disease (MD), sudden sensorineural hearing loss (SSNHL), and cytomegalovirus (CMV)-related hearing loss (CRHL). Although these diseases may often differ in etiology, there have been recent studies that support the involvement of the SV in the pathogenesis of many of these disorders. We strive to highlight a few prominent examples of these frequently neglected otologic diseases and illustrate the relevance of understanding SV composition, structure and function with regards to these disease processes. In this study, we review the physiology of the SV, lay out the importance of these neglected otologic diseases, highlight the current literature regarding the role of the SV in these disorders, and discuss the current strategies, both approved and investigational, for management of these disorders.

Further confirmation of the association of SLC12A2 with non-syndromic autosomal-dominant hearing impairment.

Congenital hearing impairment (HI) is genetically heterogeneous making its genetic diagnosis challenging. Investigation of novel HI genes and variants will enhance our understanding of the molecular mechanisms and to aid genetic diagnosis. We performed exome sequencing and analysis using DNA samples from affected members of two large families from Ghana and Pakistan, segregating autosomal-dominant (AD) non-syndromic HI (NSHI). Using in silico approaches, we modeled and evaluated the effect of the likely pathogenic variants on protein structure and function. We identified two likely pathogenic variants in SLC12A2, c.2935G>A:p.(E979K) and c.2939A>T:p.(E980V), which segregate with NSHI in a Ghanaian and Pakistani family, respectively. SLC12A2 encodes an ion transporter crucial in the homeostasis of the inner ear endolymph and has recently been reported to be implicated in syndromic and non-syndromic HI. Both variants were mapped to alternatively spliced exon 21 of the SLC12A2 gene. Exon 21 encodes for 17 residues in the cytoplasmatic tail of SLC12A2, is highly conserved between species, and preferentially expressed in cochlear tissues. A review of previous studies and our current data showed that out of ten families with either AD non-syndromic or syndromic HI, eight (80%) had variants within the 17 amino acid residue region of exon 21 (48 bp), suggesting that this alternate domain is critical to the transporter activity in the inner ear. The genotypic spectrum of SLC12A2 was expanded and the involvement of SLC12A2 in ADNSHI was confirmed. These results also demonstrate the role that SLC12A2 plays in ADNSHI in diverse populations including sub-Saharan Africans.

Screening for GJB2-R143W-Associated Hearing Impairment: Implications for Health Policy and Practice in Ghana.

Genetic factors significantly contribute to the burden of hearing impairment (HI) in Ghana as there is a high carrier frequency (1.5%) of the connexin 26 gene founder variant GJB2-R143W in the healthy Ghanaian population. GJB2-R143W mutation accounts for nearly 26% of causes in families segregating congenital non-syndromic HI. With HI associated with high genetic fitness, this indicates that Ghana will likely sustain an increase in the number of individuals living with inheritable HI. There is a universal newborn hearing screening (UNHS) program in Ghana. However, this program does not include genetic testing. Adding genetic testing of GJB2-R143W mutation for the population, prenatal and neonatal stages may lead to guiding genetic counseling for individual and couples, early detection of HI for at-risk infants, and improvement of medical management, including speech therapy and audiologic intervention, as well as provision of the needed social service to enhance parenting and education for children with HI. Based on published research on the genetics of HI in Ghana, we recommend that the UNHS program should include genetic screening for the GJB2-R143W gene variant for newborns who did not pass the initial UNHS tests. This will require an upgrade and resourcing of public health infrastructures to implement the rapid and cost-effective GJB2-R143W testing, followed by appropriate genetic and anticipatory guidance for medical care.

Bi-Allelic Novel Variants in CLIC5 Identified in a Cameroonian Multiplex Family with Non-Syndromic Hearing Impairment.

DNA samples from five members of a multiplex non-consanguineous Cameroonian family, segregating prelingual and progressive autosomal recessive non-syndromic sensorineural hearing impairment, underwent whole exome sequencing. We identified novel bi-allelic compound heterozygous pathogenic variants in CLIC5. The variants identified, i.e., the missense [NM_016929.5:c.224T>C; p.(L75P)] and the splicing (NM_016929.5:c.63+1G>A), were validated using Sanger sequencing in all seven available family members and co-segregated with hearing impairment (HI) in the three hearing impaired family members. The three affected individuals were compound heterozygous for both variants, and all unaffected individuals were heterozygous for one of the two variants. Both variants were absent from the genome aggregation database (gnomAD), the Single Nucleotide Polymorphism Database (dbSNP), and the UK10K and Greater Middle East (GME) databases, as well as from 122 apparently healthy controls from Cameroon. We also did not identify these pathogenic variants in 118 unrelated sporadic cases of non-syndromic hearing impairment (NSHI) from Cameroon. In silico analysis showed that the missense variant CLIC5-p.(L75P) substitutes a highly conserved amino acid residue (leucine), and is expected to alter the stability, the structure, and the function of the CLIC5 protein, while the splicing variant CLIC5-(c.63+1G>A) is predicted to disrupt a consensus donor splice site and alter the splicing of the pre-mRNA. This study is the second report, worldwide, to describe CLIC5 involvement in human hearing impairment, and thus confirms CLIC5 as a novel non-syndromic hearing impairment gene that should be included in targeted diagnostic gene panels.

Incidence and mortality of cancer in the Volta Region of Ghana.

IMPACT STATEMENT: Region-specific cancer reports are essential in knowing the common cancers in specific populations. This study reports all cancer types recorded in the major hospitals in the Volta Region of Ghana, a population that is sparsely captured in the national cancer registries. The study identified the common cancers as well as the death rates in the Volta Region, hence contributing to the national effort to report cancer data. Although not comprising all the elements of a cancer registry, our data will augment the effort of the two national cancer registries in Ghana to provide the national cancer reports.

GJB4 and GJC3 variants in non-syndromic hearing impairment in Ghana.

IMPACT STATEMENT: Although connexins are known to be the major genetic factors associated with HI, only a few studies have investigated GJB4 and GJC3 variants among hearing-impaired patients. This study is the first to report GJB4 and GJC3 variants from an African HI cohort. We have demonstrated that GJB4 and GJC3 genes may not contribute significantly to HI in Ghana, hence these genes should not be considered for routine clinical screening in Ghana. However, it is important to study a larger population to determine the association of GJB4 and GJC3 variants with HI.

Enhancing Genetic Medicine: Rapid and Cost-Effective Molecular Diagnosis for a GJB2 Founder Mutation for Hearing Impairment in Ghana.

In Ghana, gap-junction protein ß 2 (GJB2) variants account for about 25.9% of familial hearing impairment (HI) cases. The GJB2-p.Arg143Trp (NM_004004.6:c.427C>T/OMIM: 121011.0009/rs80338948) variant remains the most frequent variant associated with congenital HI in Ghana, but has not yet been investigated in clinical practice. We therefore sought to design a rapid and cost-effective test to detect this variant. We sampled 20 hearing-impaired and 10 normal hearing family members from 8 families segregating autosomal recessive non syndromic HI. In addition, a total of 111 unrelated isolated individuals with HI were selected, as well as 50 normal hearing control participants. A restriction fragment length polymorphism (RFLP) test was designed, using the restriction enzyme NciI optimized and validated with Sanger sequencing, for rapid genotyping of the common GJB2-p.Arg143Trp variant. All hearing-impaired participants from 7/8 families were homozygous positive for the GJB2-p.Arg143Trp mutation using the NciI-RFLP test, which was confirmed with Sanger sequencing. The investigation of 111 individuals with isolated non-syndromic HI that were previously Sanger sequenced found that the sensitivity of the GJB2-p.Arg143Trp NciI-RFLP testing was 100%. All the 50 control subjects with normal hearing were found to be negative for the variant. Although the test is extremely valuable, it is not 100% specific because it cannot differentiate between other mutations at the recognition site of the restriction enzyme. The GJB2-p.Arg143Trp NciI-RFLP-based diagnostic test had a high sensitivity for genotyping the most common GJB2 pathogenic and founder variant (p.Arg143Trp) within the Ghanaian populations. We recommend the adoption and implementation of this test for hearing impairment genetic clinical investigations to complement the newborn hearing screening program in Ghana. The present study is a practical case scenario of enhancing genetic medicine in Africa.

GJB2 and GJB6 Mutations in Non-Syndromic Childhood Hearing Impairment in Ghana.

Our study aimed to investigate GJB2 (connexin 26) and GJB6 (connexin 30) mutations associated with non-syndromic childhood hearing impairment (HI) as well as the environmental causes of HI in Ghana. Medical reports of 1,104 students attending schools for the deaf were analyzed. Families segregating HI, as well as isolated cases of HI of putative genetic origin were recruited. DNA was extracted from peripheral blood followed by Sanger sequencing of the entire coding region of GJB2. Multiplex PCR and Sanger sequencing were used to analyze the prevalence of GJB6-D3S1830 deletion. Ninety-seven families segregating HI were identified, with 235 affected individuals; and a total of 166 isolated cases of putative genetic causes, were sampled from 11 schools for the deaf in Ghana. The environmental factors, particularly meningitis, remain a major cause of HI impairment in Ghana. The male/female ratio was 1.49. Only 59.6% of the patients had their first comprehensive HI test between 6 to 11 years of age. Nearly all the participants had sensorineural HI (99.5%; n = 639). The majority had pre-lingual HI (68.3%, n = 754), of which 92.8% were congenital. Pedigree analysis suggested autosomal recessive inheritance in 96.9% of the familial cases. GJB2-R143W mutation, previously reported as founder a mutation in Ghana accounted for 25.9% (21/81) in the homozygous state in familial cases, and in 7.9% (11/140) of non-familial non-syndromic congenital HI cases, of putative genetic origin. In a control population without HI, we found a prevalent of GJB2-R143W carriers of 1.4% (2/145), in the heterozygous state. No GJB6-D3S1830 deletion was identified in any of the HI patients. GJB2-R143W mutation accounted for over a quarter of familial non-syndromic HI in Ghana and should be investigated in clinical practice. The large connexin 30 gene deletion (GJB6-D3S1830 deletion) does not account for of congenital non-syndromic HI in Ghana. There is a need to employ next generation sequencing approaches and functional genomics studies to identify the other genes involved in most families and isolated cases of HI in Ghana.