Mitochondrial genes modulate the phenotypic expression of congenital scoliosis syndrome caused by mutations in the TBXT gene.

BACKGROUND: Congenital scoliosis (CS) is a spinal disorder caused by genetic-congenital vertebral malformations and may be associated with other congenital defects or may occur alone. It is genetically heterogeneous and numerous genes contributing to this disease have been identified. In addition, CS has a wide range of phenotypic and genotypic variability, which has been explained by the intervention of genetic factors like modifiers and environment genes. The aim of the present study was to determine the possible cause of CS in a Tunisian patient and to examine the association between mtDNA mutations and mtDNA content and CS. METHODS: Here we performed Whole-Exome Sequencing (WES) in a patient presenting clinical features suggestive of severe congenital scoliosis syndrome. Direct sequencing of the whole mitochondrial DNA (mtDNA) was also performed in addition to copy number quantification in the blood of the indexed case. In silico prediction tools, 3D modeling and molecular docking approaches were used. RESULTS: The WES revealed the homozygous missense mutation c.512A > G (p.H171R) in the TBXT gene. Bioinformatic analysis demonstrated that the p.H171R variant was highly deleterious and caused the TBXT structure instability. Molecular docking revealed that the p.H171R mutation disrupted the monomer stability which seemed to be crucial for maintaining the stability of the homodimer and consequently to the destabilization of the homodimer-DNA complex. On the other hand, we hypothesized that mtDNA can be a modifier factor, so, the screening of the whole mtDNA showed a novel heteroplasmic m.10150T > A (p.M31K) variation in the MT-ND3 gene. Further, qPCR analyses of the patient's blood excluded mtDNA depletion. Bioinformatic investigation revealed that the p.M31K mutation in the ND3 protein was highly deleterious and may cause the ND3 protein structure destabilization and could disturb the interaction between complex I subunits. CONCLUSION: We described the possible role of mtDNA genetics on the pathogenesis of congenital scoliosis by hypothesizing that the presence of the homozygous variant in TBXT accounts for the CS phenotype in our patient and the MT-ND3 gene may act as a modifier gene.

Expanding the genetic and phenotypic spectrum of TRAPPC9 and MID2-related neurodevelopmental disabilities: report of two novel mutations, 3D-modelling, and molecular docking studies.

Intellectual disabilities (ID) and autism spectrum disorders (ASD) have a variety of etiologies, including environmental and genetic factors. Our study reports a psychiatric clinical investigation and a molecular analysis using whole exome sequencing (WES) of two siblings with ID and ASD from a consanguineous family. Bioinformatic prediction and molecular docking analysis were also carried out. The two patients were diagnosed with profound intellectual disability, brain malformations such as cortical atrophy, acquired microcephaly, and autism level III. The neurological and neuropsychiatric examination revealed that P2 was more severely affected than P1, as he was unable to walk, presented with dysmorphic feature and exhibited self and hetero aggressive behaviors. The molecular investigations revealed a novel TRAPPC9 biallelic nonsense mutation (c.2920 C > T, p.R974X) in the two siblings. The more severely affected patient (P2) presented, along with the TRAPPC9 variant, a new missense mutation c.166 C > T (p.R56C) in the MID2 gene at hemizygous state, while his sister P1 was merely a carrier. The 3D modelling and molecular docking analysis revealed that c.166 C > T variant could affect the ability of MID2 binding to Astrin, leading to dysregulation of microtubule dynamics and causing morphological abnormalities in the brain. As our knowledge, the MID2 mutation (p.R56C) is the first one to be detected in Tunisia and causing phenotypic variability between the siblings. We extend the genetic and clinical spectrum of TRAPPC9 and MID2 mutations and highlights the possible concomitant presence of X-linked as well as autosomal recessive inheritance to causing ID, microcephaly, and autism.

Molecular and in silico investigation of a novel ECHS1 gene mutation in a consanguine family with short-chain enoyl-CoA hydratase deficiency and Mt-DNA depletion: effect on trimer assembly and catalytic activity.

Short-chain enoyl-CoA hydratase deficiency (ECHS1D) is a rare congenital metabolic disorder that follows an autosomal recessive inheritance pattern. It is caused by mutations in the ECHS1 gene, which encodes a mitochondrial enzyme involved in the second step of mitochondrial ß-oxidation of fatty acids. The main characteristics of the disease are severe developmental delay, regression, seizures, neurodegeneration, high blood lactate, and a brain MRI pattern consistent with Leigh syndrome. Here, we report three patients belonging to a consanguineous family who presented with mitochondrial encephalomyopathy. Whole-exome sequencing revealed a new homozygous mutation c.619G > A (p.Gly207Ser) at the last nucleotide position in exon 5 of the ECHS1 gene. Experimental analysis showed that normal ECHS1 pre-mRNA splicing occurred in all patients compared to controls. Furthermore, three-dimensional models of wild-type and mutant echs1 proteins revealed changes in catalytic site interactions, conformational changes, and intramolecular interactions, potentially disrupting echs1 protein trimerization and affecting its function. Additionally, the quantification of mtDNA copy number variation in blood leukocytes showed severe mtDNA depletion in all probands.

Highly efficient, label free, ultrafast plasmonic SERS biosensor (silver nanoarrays/Si) to detect GJB2 gene expressed deafness mutations in real time validated with PCR studies.

Rapid diagnostics of any gene mutations related to organ loss is highly demanded now-a days to consume time as well to reduce cost. Currently, Surface enhanced Raman spectroscopy (SERS) is evolved to be a rapid investigating tool to screen gene mutations down to single molecule sensing with regard to the design and development of substrates used for sensing. The current research focuses on particular towards direct detection of deafness mutations associated with single and dual sites related to GJB2 gene. SERS Sensor construction is achieved with plasmonic silver nanoarrays on Si (SNA/Si) substrate by effortless wet chemical methods (Reaction time: 35 s; Concentration: 20 mM). The fabricated SNA/Si facilitates direct sensing of the deafness mutations of GJB2 gene in single as well dual sites with the enhancement of plasmonic hotspots. Normal DNA DMF-33 (GGGGGG) as well as Mutant DNA at single site DMF-9 (GGGGG) were validated by their guanine fingerprint Raman bands intensity quenching for mutant DNA DMF-9 at 1366 cm-1 and 1595 cm-1 respectively. Likewise, double mutations in DMF-19 are substitutional from G to A, portrayed highly intense fingerprint of Adenine Raman bands at 739 cm-1, 1432 cm-1, 1572 cm-1 in comparison to normal DNA (DMF-33). The findings were well analyzed with Raman mapping data which carries almost 625 scans for each DNA sample. The fabricated sensor exhibited the highest sensitivity towards DNA detection down to 0.1 pg/µL with utmost reproducibility. The current study aims to bring in creation of library files for deafness mutations to facilitate clinical diagnostics in a simple and rapid approach.

A Novel Mutation in the MAP7D3 Gene in Two Siblings with Severe Intellectual Disability and Autistic Traits: Concurrent Assessment of BDNF Functional Polymorphism, X-Inactivation and Oxidative Stress to Explain Disease Severity.

Intellectual disabilities (ID) and autism spectrum disorders (ASD) are characterized by extreme genetic and phenotypic heterogeneity. However, understanding this heterogeneity is difficult due to the intricate interplay among multiple interconnected genes, epigenetic factors, oxidative stress, and environmental factors. Employing next-generation sequencing (NGS), we revealed the genetic cause of ID and autistic traits in two patients from a consanguineous family followed by segregation analysis. Furthermore, in silico prediction methods and 3D modeling were conducted to predict the effect of the variants. To establish genotype-phenotype correlation, X-chromosome inactivation using Methylation-specific PCR and oxidative stress markers were also investigated. By analyzing the NGS data of the two patients, we identified a novel frameshift mutation c.2174_2177del (p.Thr725MetfsTer2) in the MAP7D3 gene inherited from their mother along with the functional BDNF Val66Met polymorphism inherited from their father. The 3D modeling demonstrated that the p.Thr725MetfsTer2 variant led to the loss of the C-terminal tail of the MAP7D3 protein. This change could destabilize its structure and impact kinesin-1's binding to microtubules via an allosteric effect. Moreover, the analysis of oxidative stress biomarkers revealed an elevated oxidative stress in the two patients compared to the controls. To the best of our knowledge, this is the first report describing severe ID and autistic traits in familial cases with novel frameshift mutation c.2174_2177del in the MAP7D3 gene co-occurring with the functional polymorphism Val66M in the BDNF gene. Besides, our study underlines the importance of investigating combined genetic variations, X-chromosome inactivation (XCI) patterns, and oxidative stress markers for a better understanding of ID and autism etiology.

A novel homozygous PIGO mutation associated with severe infantile epileptic encephalopathy, profound developmental delay and psychomotor retardation: structural and 3D modelling investigations and genotype-phenotype correlation.

The PIGO gene encodes the GPI-ethanolamine phosphate transferase 3, which is crucial for the final synthetic step of the glycosylphosphatidylinositol-anchor serving to attach various proteins to their cell surface. These proteins are intrinsic for normal neuronal and embryonic development. In the current research work, a clinical investigation was conducted on a patient from a consanguineous family suffering from epileptic encephalopathy, characterized by severe seizures, developmental delay, hypotonia, ataxia and hyperphosphatasia. Molecular analysis was performed using Whole Exome Sequencing (WES). The molecular investigation revealed a novel homozygous variant c.1132C > T in the PIGO gene, in which a highly conserved Leucine was changed to a Phenylalanine (p.L378F). To investigate the impact of the non-synonymous mutation, a 3D structural model of the PIGO protein was generated using the AlphaFold protein structure database as a resource for template-based tertiary structure modeling. A structural analysis by applying some bioinformatic tools on both variants 378L and 378F models predicted the pathogenicity of the non-synonymous mutation and its potential functional and structural effects on PIGO protein. We also discussed the phenotypic and genotypic variability associated with the PIGO deficiency. To our best knowledge, this is the first report of a patient diagnosed with infantile epileptic encephalopathy showing a high elevation of serum alkaline phosphatase level. Our findings, therefore, widen the genotype and phenotype spectrum of GPI-anchor deficiencies and broaden the cohort of patients with PIGO associated epileptic encephalopathy with an elevated serum alkaline phosphatase level.

Loss-of-function mutations in MYO15A and OTOF cause non-syndromic hearing loss in two Yemeni families.

BACKGROUND: Hearing loss is a rare hereditary deficit that is rather common among consanguineous populations. Autosomal recessive non-syndromic hearing loss is the predominant form of hearing loss worldwide. Although prevalent, hearing loss is extremely heterogeneous and poses a pitfall in terms of diagnosis and screening. Using next-generation sequencing has enabled a rapid increase in the identification rate of genes and variants in heterogeneous conditions, including hearing loss. We aimed to identify the causative variants in two consanguineous Yemeni families affected with hearing loss using targeted next-generation sequencing (clinical exome sequencing). The proband of each family was presented with sensorineural hearing loss as indicated by pure-tone audiometry results. RESULTS: We explored variants obtained from both families, and our analyses collectively revealed the presence and segregation of two novel loss-of-function variants: a frameshift variant, c.6347delA in MYO15A in Family I, and a splice site variant, c.5292-2A > C, in OTOF in Family II. Sanger sequencing and PCR-RFLP of DNA samples from 130 deaf and 50 control individuals confirmed that neither variant was present in our in-house database. In silico analyses predicted that each variant has a pathogenic effect on the corresponding protein. CONCLUSIONS: We describe two novel loss-of-function variants in MYO15A and OTOF that cause autosomal recessive non-syndromic hearing loss in Yemeni families. Our findings are consistent with previously reported pathogenic variants in the MYO15A and OTOF genes in Middle Eastern individuals and suggest their implication in hearing loss.

Case report: Functional analysis of the p.Arg507Trp variant of the PIGT gene supporting the moderate epilepsy phenotype of mutations in the C-terminal region.

Pathogenic germline variants in the PIGT gene are associated with the "multiple congenital anomalies-hypotonia-seizures syndrome 3" (MCAHS3) phenotype. So far, fifty patients have been reported, most of whom suffer from intractable epilepsy. Recently, a comprehensive analysis of a cohort of 26 patients with PIGT variants has broadened the phenotypical spectrum and indicated that both p.Asn527Ser and p.Val528Met are associated with a milder epilepsy phenotype and less severe outcomes. Since all reported patients are of Caucasian/Polish origin and most harbor the same variant (p.Val528Met), the ability to draw definitive conclusions regarding the genotype-phenotype correlation remains limited. We report a new case with a homozygous variant p.Arg507Trp in the PIGT gene, detected on clinical exome sequencing. The North African patient in question displays a predominantly neurological phenotype with global developmental delay, hypotonia, brain abnormalities, and well-controlled epileptic seizures. Homozygous and heterozygous variants in codon 507 have been reported to cause PIGT deficiency without biochemical confirmation. In this study, FACS analysis of knockout HEK293 cells that had been transfected with wild-type or mutant cDNA constructs demonstrated that the p.Arg507Trp variant leads to mildly reduced activity. Our result confirm the pathogenicity of this variant and strengthen recently reported evidence on the genotype-phenotype correlation of the PIGT variant.

Functional consequences of Genetics variant in TMC1 and TMC2 within a United Arab Emirates family with Pre-lingual hearing loss.

Hearing loss (HL) is the most prevalent sensory disorder whose etiology comes from environmental and/or genetic factors. Approximately 60 % of HL cases are due to mutations in genes responsible for maintaining a normal hearing function. Despite the monogenic inheritance of hereditary hearing loss (HHL), its diagnosis is challenging as both clinical and genetic heterogeneity characterizes it. Through the development of next-generation sequencing (NGS) techniques, the number of identified mutations responsible for HHL has increased exponentially during the last decade. Mutations in the TMC1 have been reported in several patients with nonsyndromic hereditary hearing loss (NSHHL), more precisely in cases with an autosomal recessive inheritance pattern. In this study, we conducted whole-exome sequencing (WES) analysis of a United Arabs Emirates (UAE) family with autosomal recessive nonsyndromic hearing loss (ARNSHL). This analysis revealed segregation of the TMC1 missense mutation c.596A > T (p.Asn199Ile) with the disease. Bioinformatics analysis supported the pathogenic effect of this mutation and predicted its impact at the proteomics level. Molecular docking analysis of TMC2WT, TMC2R123K, TMC2Q205R, and TMC2R123K + Q205R. Finally, protein docking results suggest a role for TMC2 variants in the phenotypic variability observed within the investigated family.

miR-623 Targets Metalloproteinase-1 and Attenuates Extravasation of Brain Metastatic Triple-Negative Breast Cancer Cells.

Background: Most breast cancer-related deaths result from metastasis. Understanding the molecular basis of metastasis is needed for the development of effective targeted and preventive strategies. Matrix metalloproteinase-1 (MMP1) plays an important role in brain metastasis (BM) of triple-negative breast cancer (TNBC) by promoting extravasation of cancer cells across the brain endothelium (BE). MMP1 expression is controlled by endogenous microRNAs. Preliminary bioinformatics analysis has revealed that miR-623, known to target the 3'UTR of MMP1, is significantly downregulated in brain metastatic tumors compared to primary BC tumors. However, the involvement of miR-623 in MMP1 upregulation in breast cancer brain metastatic cells (BCBMC) remains unexplored. Here, we investigated the role of miR-623 in MMP1 regulation and its impact on the extravasation of TNBC cells through the BE in vitro. Materials and Methods: A loss-and-gain of function method was employed to address the effect of miR-623 modulation on MMP1 expression. MMP1 regulation by miR-623 was investigated by real-time PCR, western blot, luciferase and transwell migration assays using an in vitro human BE model. Results: Our results confirmed that brain metastatic TNBC cells express lower levels of miR-623 compared with cells having low propensity to spread toward the brain. miR-623 binds to the 3'-untranslated region of MMP1 transcript and downregulates its expression. Restoring miR-623 expression significantly decreased MMP1 expression, preserved the endothelial barrier integrity, and attenuated transmigration of BCBMC through the BE. Conclusion: Our study elucidates, for the first time, the crucial role of miR-623 as MMP1 direct regulator in BCBMC and sheds light on miR-623 as a novel therapeutic target that can be exploited to predict and prevent brain metastasis in TNBC. Importantly, the presents study helps in unraveling a brain metastasis-specific microRNA signature in TNBC that can be used as a guide to personalized metastasis prediction and preventive approach with better therapeutic outcome.

First description of the MEGDEHL syndrome in the Tunisian population via whole-exome sequencing: Novel nonsense mutation in SERAC1 gene.

INTRODUCTION: MEGDEL syndrome is a rare recessive disorder, with about 100 cases reported worldwide, which is defined by 3-methylglutaconic aciduria (MEG), deafness (D), encephalopathy (E) and Leigh-like syndrome (L). When these manifestations were added to hepatopathy (H), the syndrome was labelled as MEGD(H)EL. Mutations in SERAC1 gene encoding a serine active site containing 1 protein were described in patients affected by this syndrome. PATIENTS AND METHODS: The present study reports the Whole Exome Sequencing (WES) of the first case of MEGDEHL syndrome in Tunisia in a consanguineous family with three affected children. Bioinformatic analysis was also performed in addition to mtDNA deletion screening and mtDNA copy number quantification in the blood of the indexed case, carried out, respectively by Long-Range PCR and qPCR. RESULTS: The WES revealed a novel homozygous nonsense mutation (c.1379G > A; p.W460X) in the SERAC1 gene, which was confirmed by Sanger sequencing. This nonsense mutation was present at a homozygous state in the three affected children and was heterozygous in the parents. In silico analysis using various softwares was performed, and the predictive results supported the pathogenic effect of the identified mutation. Further, long-range PCR and qPCR analyses of the patient's blood excluded any mtDNA deletions or depletions. CONCLUSION: Sequencing results and bioinformatic tools confirmed that the novel mutation (p.W460X) in the SERAC1 gene causes the severe phenotype in the studied family with MEGDEHL syndrome.

Identification of a novel homozygous mutation in NAXE gene associated with early-onset progressive encephalopathy by whole-exome sequencing: in silico protein structure characterization, molecular docking, and dynamic simulation.

Progressive encephalopathy with brain edema and/or leukoencephalopathy, PEBEL1, is a severe neurometabolic disorder characterized by rapidly progressive neurologic deterioration associated with a febrile illness. PEBEL1 is a lethal encephalopathy caused by NAXE gene mutations. Here we report a 6-month-old boy with mitochondrial encephalomyopathy from a consanguineous family. Molecular analysis was performed using whole-exome sequencing followed by segregation analysis. In addition, in silico prediction tools and molecular dynamic approaches were used to predict the structural effect of the mutation. Furthermore, molecular docking of the substrate NADP in both wild-type and mutated NAXE protein was carried out. Molecular analysis revealed the presence of the novel homozygous mutation c.641 T > A (p. Ile214Asn) in the NAXE gene, located at the NAD (P)H hydrate epimerase domain. In addition, bioinformatics analyses and molecular dynamics revealed that p. Ile214Asn mutation could affect the structure, stability, and compactness of the NAXE protein. Moreover, the result of the molecular docking showed that the p. Ile214Asn mutation leads to conformational changes in the catalytic cavity, thus modifying interaction with the substrate and restricting its access. We also compared the phenotype of our patient with those of previously reported cases with PEBEL syndrome. All bioinformatics findings provide evidence that the NAXE variant Asn214 disrupts NAXE protein functionality leading to an insufficient NAD (P)HX repair system and the development of clinical features of PEBEL1 syndrome in our patient. To our knowledge, our case is the 21st case of PEBEL1 patient worldwide and the first case in North Africa.

A novel thymidine phosphorylase mutation in a family with Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE): Molecular docking, dynamic simulation and computational investigations.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE; OMIM 603041) is a rare inherited metabolic disorder mostly caused by mutations in TYMP gene encoding thymidine phosphorylase (TP) protein that affects the mitochondrial nucleotide metabolism. TP, functionally active as a homodimer, is involved in the salvage pathway of pyrimidine nucleosides. MNGIE-like syndrome having an overlapping phenotype of MNGIE was also described and has been associated with mutations in POLG and RRM2B genes. In the present study, we report the molecular investigation of a consanguineous family including two patients with clinical features suggestive of MNGIE syndrome. Bioinformatics analyses were carried out in addition to mtDNA deletion screening and copy number quantification in the blood of the two patients. Whole exome sequencing and Sanger sequencing analyses revealed the segregation in the affected family a novel mutation c.1205T>A (p.L402Q) within the exon 9 of the TYMP gene. In addition, mtDNA analysis revealed the absence of mtDNA deletions and a decrease of the copy number in the blood of the two patients of the studied family. The p.Leu402Gln mutation was located in a conserved amino acid within the α/ß domain of the TP protein and several software supported its pathogenicity. In addition, and based on docking and molecular dynamic simulation analyses, results revealed that L402Q caused a conformational change in TP mutated structure and could therefore alter its flexibility and stability. These changes prevent also the formation of stable homodimer leading to non-functional protein with partial or complete loss of its catalytic activity.

miR-27a-3p regulates expression of intercellular junctions at the brain endothelium and controls the endothelial barrier permeability.

BACKGROUND: The brain endothelial barrier permeability is governed by tight and adherens junction protein complexes that restrict paracellular permeability at the blood-brain barrier (BBB). Dysfunction of the inter-endothelial junctions has been implicated in neurological disorders such as multiple sclerosis, stroke and Alzheimer's disease. The molecular mechanisms underlying junctional dysfunction during BBB impairment remain elusive. MicroRNAs (miRNAs) have emerged as versatile regulators of the BBB function under physiological and pathological conditions, and altered levels of BBB-associated microRNAs were demonstrated in a number of brain pathologies including neurodegeneration and neuroinflammatory diseases. Among the altered micro-RNAs, miR-27a-3p was found to be downregulated in a number of neurological diseases characterized by loss of inter-endothelial junctions and disruption of the barrier integrity. However, the relationship between miR-27a-3p and tight and adherens junctions at the brain endothelium remains unexplored. Whether miR-27a-3p is involved in regulation of the junctions at the brain endothelium remains to be determined. METHODS: Using a gain-and-loss of function approach, we modulated levels of miR-27a-3p in an in-vitro model of the brain endothelium, key component of the BBB, and examined the resultant effect on the barrier paracellular permeability and on the expression of essential tight and adherens junctions. The mechanisms governing the regulation of junctional proteins by miR-27a-3p were also explored. RESULTS: Our results showed that miR-27a-3p inhibitor increases the barrier permeability and causes reduction of claudin-5 and occludin, two proteins highly enriched at the tight junction, while miR-27a-3p mimic reduced the paracellular leakage and increased claudin-5 and occludin protein levels. Interestingly, we found that miR-27-3p induces expression of claudin-5 and occludin by downregulating Glycogen Synthase Kinase 3 beta (GSK3ß) and activating Wnt/ß-catenin signaling, a key pathway required for the BBB maintenance. CONCLUSION: For the first time, we showed that miR-27a-3p is a positive regulator of key tight junction proteins, claudin-5 and occludin, at the brain endothelium through targeting GSK3ß gene and activating Wnt/ß-catenin signaling. Thus, miR-27a-3p may constitute a novel therapeutic target that could be exploited to prevent BBB dysfunction and preserves its integrity in neurological disorders characterized by impairment of the barrier's function.

Molecular insights into MYO3A kinase domain variants explain variability in both severity and progression of DFNB30 hearing impairment.

Hereditary hearing impairment (HI) is a common disease with the highest incidence among sensory defects. Several genes have been identified to affect stereocilia structure causing HI, including the unconventional myosin3A. Interestingly, we noticed that variants in MYO3A gene have been previously found to cause variable HI onset and severity. Using clinical exome sequencing, we identified a novel pathogenic variant p.(Lys50Arg) in the MYO3A kinase domain (MYO3A-KD). Previous in vitro studies supported its damaging effect as a 'kinase-dead' mutant. We further analyzed this variation through molecular dynamics which predicts that changes in flexibility of MYO3A structure would influence the protein-ATP binding properties. This Lys50Arg mutation segregated with congenital profound non-syndromic HI. To better investigate this variability, we collected previously identified MYO3A-KDs variants, p.(Tyr129Cys), p.(His142Gln) and p.(Pro189Thr), and built both wild type and mutant 3 D MYO3A-KD models to assess their impact on the protein structure and function. Our results suggest that KD mutations could either cause a congenital profound form of HI, when particularly affecting the kinase activity and preventing the auto-phosphorylation of the motor, or a late onset and progressive form, when partially or completely inactivating the MYO3A protein. In conclusion, we report a novel pathogenic variant affecting the ATP-binding site within the MYO3A-KD causing congenital profound HI. Through computational approaches we provide a deeper understanding on the correlation between the effects of MYO3A-KD mutations and the variable hearing phenotypes. To the best of our knowledge this is the first study to correlate mutations' genotypes with the variable phenotypes of DFNB30.Communicated by Ramaswamy H. Sarma.

Genetic etiology of hereditary hearing loss in the Gulf Cooperation Council countries.

The past 30 years have seen an exponential growth concerning the identification of genes and variants responsible for hereditary hearing loss (HL) worldwide. This has led to a huge gain in our understanding of molecular mechanisms of hearing and deafness, which improved diagnosis for populations with hereditary HL. Many communities around the world, especially in the Middle East and North Africa, have a high prevalence of consanguineous marriages. Congenital monogenic conditions, such as recessive HL, are more common in these populations due to high consanguinity rates. Many studies have shown that high rates of consanguinity, endogamy, and first cousin marriages were observed in the six countries of the Gulf Cooperation Council (GCC). The intent of this study is to investigate the etiology of HL in the GCC region. A deep literature review of genes and variants responsible for HL in this region revealed 89 recessive DNA pathogenic variants reported in 138 cases/familial cases. A total of 21 genes responsible for non-syndromic hearing loss (NSHL) and 17 genes associated with syndromic hearing loss (SHL) were reported in cases from the GCC region. Out of 156 reported affected cases, 112 showed HL only, and 44 showed HL associated with other clinical manifestations. This data suggests that in the GCC region 72% of HL forms are non-syndromic and 28% are syndromic. For individuals with NSHL, 66% of variants were detected in four genes (GJB2, OTOF, TMC1 and CDH23), with a predominance of variants located in the GJB2 gene (37.5%). However, among SHL, Usher syndrome was the more frequent as it has been observed in 41% of the reported syndromic GCC cases. Finally, our analysis showed that HL genetics testing and research in the GCC region took advantage of the next generation sequencing (NGS)-based techniques, as approximately 58% of reported variants were identified using this technology.

Further insights into the spectrum phenotype of TRAPPC9 and CDK5RAP2 genes, segregating independently in a large Tunisian family with intellectual disability and microcephaly.

Intellectual disability (ID) often co-occurs with other neurologic phenotypes making molecular diagnosis more challenging particularly in consanguineous populations with the co-segregation of more than one ID-related gene in some cases. In this study, we investigated the phenotype of three patients from a large Tunisian family with significant ID phenotypic variability and microcephaly and performed a clinical exome sequencing in two cases. We identified, within the first branch, a homozygous variant in the TRAPPC9 gene (p.Arg472Ter) in two cases presenting severe ID, absent speech, congenital/secondary microcephaly in addition to autistic features, supporting the implication of TRAPPC9 in the "secondary" autism spectrum disorders and congenital microcephaly. In the second branch, we identified a homozygous variant (p.Lys189ArgfsTer15) in the CDK5RAP2 gene associated with an heterozygous TRAPPC9 variant (p.Arg472Ter) in one case harbouring primary hereditary microcephaly (MCPH) associated with an inter-hypothalamic adhesion, mixed hearing loss, selective thinning in the retinal nerve fiber layer and parafoveal ganglion cell complex, and short stature. Our findings expand the spectrum of the recently reported neurosensorial abnormalities and revealed the variable phenotype expressivity of CDK5RAP2 defect. Our study highlights the complexity of the genetic background of microcephaly/ID and the efficiency of the exome sequencing to provide an accurate diagnosis and to improve the management and follow-up of such patients.

Whole exome sequencing, in silico and functional studies confirm the association of the GJB2 mutation p.Cys169Tyr with deafness and suggest a role for the TMEM59 gene in the hearing process.

The development of next generation sequencing techniques has facilitated the detection of mutations at an unprecedented rate. These efficient tools have been particularly beneficial for extremely heterogeneous disorders such as autosomal recessive non-syndromic hearing loss, the most common form of genetic deafness. GJB2 mutations are the most common cause of hereditary hearing loss. Amongst them the NM_004004.5: c.506G > A (p.Cys169Tyr) mutation has been associated with varying severity of hearing loss with unclear segregation patterns. In this study, we report a large consanguineous Emirati family with severe to profound hearing loss fully segregating the GJB2 missense mutation p.Cys169Tyr. Whole exome sequencing (WES), in silico, splicing and expression analyses ruled out the implication of any other variants and confirmed the implication of the p.Cys169Tyr mutation in this deafness family. We also show preliminary murine expression analysis that suggests a link between the TMEM59 gene and the hearing process. The present study improves our understanding of the molecular pathogenesis of hearing loss. It also emphasizes the significance of combining next generation sequencing approaches and segregation analyses especially in the diagnosis of disorders characterized by complex genetic heterogeneity.

Novel pathogenic mutations and further evidence for clinical relevance of genes and variants causing hearing impairment in Tunisian population.

Introduction: Hearing impairment (HI) is characterized by complex genetic heterogeneity. The evolution of next generation sequencing, including targeted enrichment panels, has revolutionized HI diagnosis. Objectives: In this study, we investigated genetic causes in 22 individuals with non-GJB2 HI. Methods: We customized a HaloplexHS kit to include 30 genes known to be associated with autosomal recessive nonsyndromic HI (ARNSHI) and Usher syndrome in North Africa. Results: In accordance with the ACMG/AMP guidelines, we report 11 pathogenic variants; as follows; five novel variants including three missense (ESRRB-Tyr295Cys, MYO15A-Phe2089Leu and MYO7A-Tyr560Cys) and two nonsense (USH1C-Gln122Ter and CIB2-Arg104Ter) mutations; two previously reported mutations (OTOF-Glu57Ter and PNPT1-Glu475Gly), but first time identified among Tunisian families; and four other identified mutations namely WHRN-Gly808AspfsX11, SLC22A4-Cys113Tyr and two MYO7A compound heterozygous splice site variants that were previously described in Tunisia. Pathogenic variants in WHRN and CIB2 genes, in patients with convincing phenotype ruling out retinitis pigmentosa, provide strong evidence supporting their association with ARNSHI. Moreover, we shed lights on the pathogenic implication of mutations in PNPT1 gene in auditory function providing new evidence for its association with ARNSHI. Lack of segregation of a previously identified causal mutation OTOA-Val603Phe further supports its classification as variant of unknown significance. Our study reports absence of otoacoustic emission in subjects using bilateral hearing aids for several years indicating the importance of screening genetic alteration in OTOF gene for proper management of those patients. Conclusion: In conclusion, our findings do not only expand the spectrum of HI mutations in Tunisian patients, but also improve our knowledge about clinical relevance of HI causing genes and variants.

Combinatorial targeting of microRNA-26b and microRNA-101 exerts a synergistic inhibition on cyclooxygenase-2 in brain metastatic triple-negative breast cancer cells.

PURPOSE: Extravasation of triple-negative (TN) metastatic breast cancer (BC) cells through the brain endothelium (BE) is a critical step in brain metastasis (BM). During extravasation, metastatic cells induce alteration in the inter-endothelial junctions and transmigrate through the endothelial barrier. Transmigration of metastatic cells is mediated by the upregulation of cyclooxygenase-2 (COX-2) that induces matrix metalloproteinase-1 (MMP-1) capable of degrading inter-endothelial junctional proteins. Despite their important role in BM, the molecular mechanisms upregulating COX-2 and MMP-1 in TNBC cells remain poorly understood. In this study, we unraveled a synergistic effect of a pair of micro-RNAs (miR-26b-5p and miR-101-3p) on COX-2 expression and the brain transmigration ability of BC cells. METHODS: Using a gain-and-loss of function approach, we modulated levels of miR-26b-5p and miR-101-3p in two TNBC cell lines (the parental MDA-MB-231 and its brain metastatic variant MDA-MB-231-BrM2), and examined the resultant effect on COX-2/MMP-1 expression and the transmigration of cancer cells through the BE. RESULTS: We observed that the dual inhibition of miR-26b-5p and miR-101-3p in BC cells results in higher increase of COX-2/MMP-1 expression and a higher trans-endothelial migration compared to either micro-RNA alone. The dual restoration of both micro-RNAs exerted a synergistic inhibition on COX-2/MMP-1 by targeting COX-2 and potentiated the suppression of trans-endothelial migration compared to single micro-RNA. CONCLUSION: These findings provide new insights on a synergism between miR-26-5p and miR-101-3p in regulating COX-2 in metastatic TNBC cells and shed light on miR-26-5p and miR-101-3p as prognostic and therapeutic targets that can be exploited to predict or prevent BM.