Identification of rare missense variants in the BSN gene co-segregating with chronic otitis media in a consanguineous Pakistani family.

BACKGROUND: Otitis media (OM) is the most frequent and complex middle ear condition with multifactorial etiology including genetic predisposition. OM depicts a variable clinical spectrum, leading to speech, developmental delay, and hearing loss. Here, we report the clinical and genetic findings of chronic suppurative otitis media (CSOM) segregating in a six-generation consanguineous Pakistani family PKOM08. METHODS: Clinical evaluations, including audio and tympanometry, were conducted to assess OM manifestation and their impact on hearing function. Exome sequencing was performed to identify potential genetic variants underlying CSOM in the study participants. RESULTS: Clinical evaluation of participating individuals revealed varying degrees of disease severity, with mild to moderate hearing loss. All the affected individuals had CSOM with no other apparent comorbidity. Whole exome followed by Sanger sequencing revealed two rare heterozygous variants [c.1867C>T, p.(Pro623Ser) and c.11015G>A, p.(Arg3672Gln)] of BSN gene in most of the affected individuals of family PKOM08. BSN encodes a scaffold bassoon protein involved in synaptic vesicle trafficking. The identified variants replaced evolutionary conserved amino acid residues in the encoded protein and are predicted to impact the ionic interactions in the secondary structure. CONCLUSION: A deep intronic variant of BSN has been previously implicated in the etiology of childhood ear infections. Our study further supports a link between BSN-impaired function and ear infection and CSOM in children.

The role of CDHR3 in susceptibility to otitis media.

Otitis media (OM) is common in young children and can cause hearing loss and speech, language, and developmental delays. OM has high heritability; however, little is known about OM-related molecular and genetic processes. CDHR3 was previously identified as a locus for OM susceptibility, but to date, studies have focused on how the CDHR3 p.Cys529Tyr variant increases epithelial binding of rhinovirus-C and risk for lung or sinus pathology. In order to further delineate a role for CDHR3 in OM, we performed the following: exome sequencing using DNA samples from OM-affected individuals from 257 multi-ethnic families; Sanger sequencing, logistic regression and transmission disequilibrium tests for 407 US trios or probands with OM; 16S rRNA sequencing and analysis for middle ear and nasopharyngeal samples; and single-cell RNA sequencing and differential expression analyses for mouse middle ear. From exome sequence data, we identified a novel pathogenic CDHR3 splice variant that co-segregates with OM in US and Finnish families. Additionally, a frameshift and six missense rare or low-frequency variants were identified in Finnish probands. In US probands, the CDHR3 p.Cys529Tyr variant was associated with the absence of middle ear fluid at surgery and also with increased relative abundance of Lysobacter in the nasopharynx and Streptomyces in the middle ear. Consistent with published data on airway epithelial cells and our RNA-sequence data from human middle ear tissues, Cdhr3 expression is restricted to ciliated epithelial cells of the middle ear and is downregulated after acute OM. Overall, these findings suggest a critical role for CDHR3 in OM susceptibility. KEY MESSAGES: • Novel rare or low-frequency CDHR3 variants putatively confer risk for otitis media. • Pathogenic variant CDHR3 c.1653 + 3G > A was found in nine families with otitis media. • CDHR3 p.Cys529Tyr was associated with lack of effusion and bacterial otopathogens. • Cdhr3 expression was limited to ciliated epithelial cells in mouse middle ear. • Cdhr3 was downregulated 3 h after infection of mouse middle ear.

Morphometric localisation of pterion for lateral neurosurgical planning and approach.

OBJECTIVE: To localise pterion, as a safe landmark, on dry skulls, for performing various neurosurgical procedures. To analyse the variation in the type and location of pterion among Pakistani male population. METHODS: This cross sectional study was conducted form August 2018 to May 2019 on 50 dry skulls obtained from The anatomy departments of different medical colleges of Rawalpindi and Islamabad. Shape of the pterion was noted and different measurements of the pterion from the two reference points, frontozygomatic suture and superior border of zygomatic arch, were recorded. Mean differences between the right and left sides were compared using SPSS version 23. RESULTS: The pattern of pterion suture was sphenoparietal in 47 skulls, 2 skulls had epipteric type and 1 skull had a stellate type of pterion. The mean distance of pterion, on the right side, from posterolateral aspect of frontozygomatic suture was 2.490±0.596cm, 1.485±0.497cm, 2.922±0.697cm measured as horizontal, vertical and direct respectively. The mean horizontal, vertical and direct frontozygomatic measurements on the left side were 2.265±0.574cm, 1.395±0.548cm, 2.717±0.665cm respectively. The mean frontozygomatic horizontal and direct measurements were significantly greater on the right side as compared to the left side (p value 0.001). The mean distance from superior border of zygomatic arch to the centre of pterion on the right and left sides were 3.744±0.444cm and 3.644±9.473 respectively. CONCLUSIONS: The findings of the study provided important information regarding the probability of type and location of pterion in Pakistani males for lateral skull neurosurgical planning, especially when CT scan facility is not available.

Normal spermatogenesis and fertility in Ddi1 (DNA damage inducible 1) mutant mice.

The ubiquitin proteins play important role in proteasomal degradation and their balanced action is essential for the crucial process of spermatogenesis. The disruption of various ubiquitinating proteins in mice revealed defective spermatogenesis, thus inferring their important function in spermatogenesis. However, the role of some testis-specific ubiquitinating proteins still needs to be discovered. This study was planned to study the in vivo function of testis-specific and evolutionarily conserved ubiquitin shuttle gene, Ddi1 (DNA damage inducible 1). Ddi1 knockout mice were generated by CRISPR/Cas9 technology and we found that Ddi1 knockout mice were fertile without obvious alterations in reproductive parameters, such as sperm number and morphology. Histological examination of testicular tissues manifested compact seminiferous tubule structure along with all type of germ cells in the knockout mice. Moreover, cytological studies of spermatocytes did not exhibit any noteworthy difference in the progression of prophase I which endorse the fact that Ddi1 has not any vital function during meiosis. Overall, these findings suggested that Ddi1 is not critical for mouse fertility under normal laboratory conditions. The outcome of this study will help researchers to avoid overlap that will not only save their resources but also concentrate their focus on indispensable genes in spermatogenesis and fertility.

Multi-omic studies on missense PLG variants in families with otitis media.

Otitis media (OM), a very common disease in young children, can result in hearing loss. In order to potentially replicate previously reported associations between OM and PLG, exome and Sanger sequencing, RNA-sequencing of saliva and middle ear samples, 16S rRNA sequencing, molecular modeling, and statistical analyses including transmission disequilibrium tests (TDT) were performed in a multi-ethnic cohort of 718 families and simplex cases with OM. We identified four rare PLG variants c.112A > G (p.Lys38Glu), c.782G > A (p.Arg261His), c.1481C > T (p.Ala494Val) and c.2045 T > A (p.Ile682Asn), and one common variant c.1414G > A (p.Asp472Asn). However TDT analyses for these PLG variants did not demonstrate association with OM in 314 families. Additionally PLG expression is very low or absent in normal or diseased middle ear in mouse and human, and salivary expression and microbial α-diversity were non-significant in c.1414G > A (p.Asp472Asn) carriers. Based on molecular modeling, the novel rare variants particularly c.782G > A (p.Arg261His) and c.2045 T > A (p.Ile682Asn) were predicted to affect protein structure. Exploration of other potential disease mechanisms will help elucidate how PLG contributes to OM susceptibility in humans. Our results underline the importance of following up findings from genome-wide association through replication studies, preferably using multi-omic datasets.

Novel Mutations in CLPP, LARS2, CDH23, and COL4A5 Identified in Familial Cases of Prelingual Hearing Loss.

We report the underlying genetic causes of prelingual hearing loss (HL) segregating in eight large consanguineous families, ascertained from the Punjab province of Pakistan. Exome sequencing followed by segregation analysis revealed seven potentially pathogenic variants, including four novel alleles c.257G>A, c.6083A>C, c.89A>G, and c.1249A>G of CLPP, CDH23, COL4A5, and LARS2, respectively. We also identified three previously reported HL-causing variants (c.4528C>T, c.35delG, and c.1219T>C) of MYO15A, GJB2, and TMPRSS3 segregating in four families. All identified variants were either absent or had very low frequencies in the control databases. Our in silico analyses and 3-dimensional (3D) molecular modeling support the deleterious impact of these variants on the encoded proteins. Variants identified in MYO15A, GJB2, TMPRSS3, and CDH23 were classified as "pathogenic" or "likely pathogenic", while the variants in CLPP and LARS2 fall in the category of "uncertain significance" based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) variant pathogenicity guidelines. This paper highlights the genetic diversity of hearing disorders in the Pakistani population and reports the identification of four novel mutations in four HL families.

Evolutionarily conserved and testis-specific gene, 4930524B15Rik, is not essential for mouse spermatogenesis and fertility.

Thousands of genes are involved in spermatogenesis, however, the functional roles of most these genes for male fertility remain to be discovered. This research focused to explore the function of evolutionarily conserved and testis-specific expressed gene 4930524B15Rik, which is known as C5orf47 in human. We generated 4930524B15Rik knockout mice by CRISPR/Cas9 technology and found 4930524B15Rik-/- mice were fertile. Furthermore, no averted abnormalities were observed in testis morphology, epididymal sperm contents and sperm morphology in 4930524B15Rik knockout mice. Subsequently, histological analysis of testicular tissue revealed intact structure of seminiferous tubules along with the presence of all types of germ cells in 4930524B15Rik-/- mice similar to wild type. Additionally, cytological analysis of spermatocytes displayed no significant differences in the prophase I progression of meiosis, further indicating that 4930524B15Rik have no essential function in mammalian spermatogenesis. Altogether, these results indicated that 4930524B15Rik is dispensable for fertility of male mice and these findings will help researchers to avoid future research overlap and to focus on genes that are crucial for spermatogenesis and reproduction.

A DNAH17 missense variant causes flagella destabilization and asthenozoospermia.

Asthenozoospermia is a common cause of male infertility, but its etiology remains incompletely understood. We recruited three Pakistani infertile brothers, born to first-cousin parents, displaying idiopathic asthenozoospermia but no ciliary-related symptoms. Whole-exome sequencing identified a missense variant (c.G5408A, p.C1803Y) in DNAH17, a functionally uncharacterized gene, recessively cosegregating with asthenozoospermia in the family. DNAH17, specifically expressed in testes, was localized to sperm flagella, and the mutation did not alter its localization. However, spermatozoa of all three patients showed higher frequencies of microtubule doublet(s) 4-7 missing at principal piece and end piece than in controls. Mice carrying a homozygous mutation (Dnah17M/M) equivalent to that in patients recapitulated the defects in patients' sperm tails. Further examinations revealed that the doublets 4-7 were destabilized largely due to the storage of sperm in epididymis. Altogether, we first report that a homozygous DNAH17 missense variant specifically induces doublets 4-7 destabilization and consequently causes asthenozoospermia, providing a novel marker for genetic counseling and diagnosis of male infertility.

A TOP6BL mutation abolishes meiotic DNA double-strand break formation and causes human infertility.

Meiosis is pivotal for sexual reproduction and fertility. Meiotic programmed DNA double-strand breaks (DSBs) initiate homologous recombination, ensuring faithful chromosome segregation and generation of gametes. However, few studies have focused on meiotic DSB formation in human reproduction. Here, we report four infertile siblings born to a consanguineous marriage, with three brothers suffering from non-obstructive azoospermia and one sister suffering from unexplained infertility with normal menstrual cycles and normal ovary sizes with follicular activity. An autosomal recessive mutation in TOP6BL was found co-segregating with infertility in this family. Investigation of one male patient revealed failure in programmed meiotic DSB formation and meiotic arrest prior to pachytene stage of prophase I. Mouse models carrying similar mutations to that in patients recapitulated the spermatogenic abnormalities of the patient. Pathogenicity of the mutation in the female patient was supported by observations in mice that meiotic programmed DSBs failed to form in mutant oocytes and oocyte maturation failure due to absence of meiotic recombination. Our study thus illustrates the phenotypical characteristics and the genotype-phenotype correlations of meiotic DSB formation failure in humans.

A2ML1 and otitis media: novel variants, differential expression, and relevant pathways.

A genetic basis for otitis media is established, however, the role of rare variants in disease etiology is largely unknown. Previously a duplication variant within A2ML1 was identified as a significant risk factor for otitis media in an indigenous Filipino population and in US children. In this report exome and Sanger sequencing was performed using DNA samples from the indigenous Filipino population, Filipino cochlear implantees, US probands, Finnish, and Pakistani families with otitis media. Sixteen novel, damaging A2ML1 variants identified in otitis media patients were rare or low-frequency in population-matched controls. In the indigenous population, both gingivitis and A2ML1 variants including the known duplication variant and the novel splice variant c.4061 + 1 G>C were independently associated with otitis media. Sequencing of salivary RNA samples from indigenous Filipinos demonstrated lower A2ML1 expression according to the carriage of A2ML1 variants. Sequencing of additional salivary RNA samples from US patients with otitis media revealed differentially expressed genes that are highly correlated with A2ML1 expression levels. In particular, RND3 is upregulated in both A2ML1 variant carriers and high-A2ML1 expressors. These findings support a role for A2ML1 in keratinocyte differentiation within the middle ear as part of otitis media pathology and the potential application of ROCK inhibition in otitis media.

FUT2 Variants Confer Susceptibility to Familial Otitis Media.

Non-secretor status due to homozygosity for the common FUT2 variant c.461G>A (p.Trp154∗) is associated with either risk for autoimmune diseases or protection against viral diarrhea and HIV. We determined the role of FUT2 in otitis media susceptibility by obtaining DNA samples from 609 multi-ethnic families and simplex case subjects with otitis media. Exome and Sanger sequencing, linkage analysis, and Fisher exact and transmission disequilibrium tests (TDT) were performed. The common FUT2 c.604C>T (p.Arg202∗) variant co-segregates with otitis media in a Filipino pedigree (LOD = 4.0). Additionally, a rare variant, c.412C>T (p.Arg138Cys), is associated with recurrent/chronic otitis media in European-American children (p = 1.2 × 10-5) and US trios (TDT p = 0.01). The c.461G>A (p.Trp154∗) variant was also over-transmitted in US trios (TDT p = 0.01) and was associated with shifts in middle ear microbiota composition (PERMANOVA p < 10-7) and increased biodiversity. When all missense and nonsense variants identified in multi-ethnic US trios with CADD > 20 were combined, FUT2 variants were over-transmitted in trios (TDT p = 0.001). Fut2 is transiently upregulated in mouse middle ear after inoculation with non-typeable Haemophilus influenzae. Four FUT2 variants-namely p.Ala104Val, p.Arg138Cys, p.Trp154∗, and p.Arg202∗-reduced A antigen in mutant-transfected COS-7 cells, while the nonsense variants also reduced FUT2 protein levels. Common and rare FUT2 variants confer susceptibility to otitis media, likely by modifying the middle ear microbiome through regulation of A antigen levels in epithelial cells. Our families demonstrate marked intra-familial genetic heterogeneity, suggesting that multiple combinations of common and rare variants plus environmental factors influence the individual otitis media phenotype as a complex trait.

Whole exome sequencing identifies a novel dominant missense mutation underlying leukonychia in a Pakistani family.

Hereditary leukonychia (also known as porcelain nails or white nails) is a genetic disorder. It may exist as an isolated feature or associated with other cutaneous or systemic disorders. Although a number of genes have been described to cause leukonychia, still the underlying genetic etiologies of many cases remain unknown. Here, we report a Pakistani family presenting leukonychia and koilonychia nails in mother and five of her kids. All the affected individuals had white to pale nails in appearance exhibiting complete and partial leukonychia, respectively. Similarly, nails of finger and toe appeared brittle and concave, showing the characteristics features of koilonychia. Whole exome sequencing and subsequent Sanger sequencing identified a pathogenic novel missense mutation (c.1390G>A, p.Glu464Lys) in PLCD1, co-segregating with the disorder in an autosomal dominant pattern. In silico prediction tools supported the pathogenicity of the identified mutation. Literature review determined that mutations in PLCD1 only cause leukonychia. Therefore, our findings add another pathogenic variant to the PLCD1 mutation pool causing leukonychia that would help to understand the underlying molecular mechanism.