Spectrum of genetic variants in bilateral sensorineural hearing loss.

Background: Hearing loss (HL) is an impairment of auditory function with identified genetic forms that can be syndromic (30%) or non-syndromic (70%). HL is genetically heterogeneous, with more than 1,000 variants across 150 causative genes identified to date. The genetic diagnostic rate varies significantly depending on the population being tested. Countries with a considerably high rate of consanguinity provide a unique resource for studying rare forms of recessive HL. In this study, we identified genetic variants associated with bilateral sensorineural HL (SNHL) using whole-exome sequencing (WES) in 11 families residing in the United Arab Emirates (UAE). Results: We established the molecular diagnosis in six probands, with six different pathogenic or likely pathogenic variants in the genes MYO15A, SLC26A4, and GJB2. One novel nonsense variant, MYO15A:p.Tyr1962Ter*, was identified in a homozygous state in one family, which has not been reported in any public database. SLC26A4 and GJB2 were found to be the most frequently associated genes in this study. In addition, six variants of uncertain significance (VUS) were detected in five probands in the genes CDH23, COL11A1, ADGRV1, NLRP3, and GDF6. In total, 12 variants were observed in eight genes. Among these variants, eight missense variants (66.7%), three nonsense variants (25.0%), and one frameshift (8.3%) were identified. The overall diagnostic rate of this study was 54.5%. Approximately 45.5% of the patients in this study came from consanguineous families. Conclusion: Understanding the genetic basis of HL provides insight for the clinical diagnosis of hearing impairment cases through the utilization of next-generation sequencing (NGS). Our findings contribute to the knowledge of the heterogeneous genetic profile of HL, especially in a population with a high rate of consanguineous marriage in the Arab population.

Ajay Kumar Parida (1963-2022), an eminent plant biotechnologist with a passion for mangrove biology.

We remember Dr Ajay Parida, a leading plant biotechnologist, whose premature passing has deprived the Indian plant science community of a committed scientist and an able administrator. Born on 12 December 1963 in Bhagabanpur, Cuttack District (now Jajpur district), Odisha, he passed away in Guwahati on 19 July 2022. A collegial scientist, his down-to-earth and approachable nature, as well as his resourcefulness were instrumental in advancing the cause of Indian science and harnessing frontier biotechnological tools as vehicles of social consciousness. His expertise in quantitative DNA variation and molecular marker analysis, paved the way for subsequent research on mangrove molecular diversity at the M. S. Swaminathan Research Foundation (MSSRF), Chennai. His contributions to mangrove biology, genetics and genomics as well as extremophile plant species in the Indian context over two decades are a benchmark in his field. He also provided commendable leadership in his capacity as Director, Institute of Life Sciences (ILS), Bhubaneshwar during the COVID-19 pandemic.

Case report: Birk-Landau-Perez syndrome linked to the SLC30A9 gene-identification of additional cases and expansion of the phenotypic spectrum.

Birk-Landau-Perez syndrome (BILAPES) is an autosomal recessive cerebro-renal syndrome associated with genetic defects in the SLC30A9 gene, initially reported in 2017 in six individuals belonging to a large Bedouin kindred. The SLC30A9 gene encodes a putative mitochondrial zinc transporter with ubiquitous expression, the highest found in the brain, kidney, and skeletal muscle. Since the first report, only one additional affected patient has been described, but there were some inconsistencies, such as hearing loss, failure to thrive, and neuroimaging findings between the clinical presentation of the disease in the Bedouin family and the second patient. Here, we present two more patients from a consanguineous Middle Eastern family with features of chronic kidney disease, neurodevelopmental regression, ataxia, hearing loss, and eye abnormalities, which were largely consistent with BILAPES. Whole-exome sequencing detected a homozygous in-frame deletion c.1049_1051delCAG (p.Ala350del) in the SLC30A9 gene, which was the same variant detected in the patients from the primary literature report and the variant segregated with disease in the family. However, in the patients described here, brain MRI showed cerebellar atrophy, which was not a cardinal feature of the syndrome from the primary report. Our findings provide further evidence for SLC30A9-associated BILAPES and contribute to defining the clinical spectrum.

Novel compound heterozygous variants (c.971delA/c.542C > T) in SLC1A4 causes spastic tetraplegia, thin corpus callosum, and progressive microcephaly: a case report and mutational analysis.

Spastic tetraplegia, thin corpus callosum, and progressive microcephaly (SPATCCM) are linked to SLC1A4 genetic variants since the first reported case in 2015. SLC1A4 encodes for the neutral amino acid transporter ASCT1 which is involved in the transportation of serine between astrocytes and neurons. Although most of the reported cases are of Ashkenazi Jewish ancestry, SPATCCM has also been reported in Irish, Italian, Czech, Palestinian, and Pakistani ethnicities. Herein, we report two Pakistani male siblings from a non-consanguineous marriage presented with global developmental delay associated with spastic quadriplegia, microcephaly, and infantile spasm. Since infancy, both siblings suffered from microcephaly with brain MRI demonstrating generalized atrophy of the frontal, temporal, and parietal lobes with a prominence of the subarachnoid spaces, widening of the Sylvian fissures, and enlargement of the ventricular system not compatible with the chronological age of both patients associated with thinning of the corpus callosum. Whole-exome sequencing of both affected brothers revealed novel compound heterozygous variants in the SLC1A4 gene (NM_003038) segregating from their parents. The maternal c.971delA (p.N324Tfs*29) deletion variant disturbs the transcript reading frame leading to the generation of a premature stop codon and its subsequent degradation by nonsense-mediated mRNA decay as detected through expression analysis. The paternal c.542C > T (p.S181F) missense variant was predicted deleterious via multiple in silico prediction tools as the amino acid substitution is speculated to affect the overall ASCT1 structural confirmation due to the loss of an H-bond at the core of the protein at this position which might affect its function as concluded from the simulation analysis. The presented cases expand the genetic and clinical spectrum of ASCT1 deficiency and support the importance of including SLC1A4 gene screening in infants with unexplained global neurodevelopmental delay regardless of ethnicity.

Endoglin Wild Type and Variants Associated With Hereditary Hemorrhagic Telangiectasia Type 1 Undergo Distinct Cellular Degradation Pathways.

Endoglin, also known as cluster of differentiation 105 (CD105), is an auxiliary receptor in the TGFß signaling pathway. It is predominantly expressed in endothelial cells as a component of the heterotetrameric receptor dimers comprising type I, type II receptors and the binding ligands. Mutations in the gene encoding Endoglin (ENG) have been associated with hereditary hemorrhagic telangiectasia type 1 (HHT1), an autosomal dominant inherited disease that is generally characterized by vascular malformation. Secretory and many endomembrane proteins synthesized in the Endoplasmic reticulum (ER) are subjected to stringent quality control mechanisms to ensure that only properly folded and assembled proteins are trafficked forward through the secretory pathway to their sites of action. We have previously demonstrated that some Endoglin variants causing HHT1 are trapped in the ER and fail to traffic to their normal localization in plasma membrane, which suggested the possible involvement of ER associated protein degradation (ERAD) in their molecular pathology. In this study, we have investigated, for the first time, the degradation routes of Endoglin wild type and two mutant variants, P165L and V105D, and previously shown to be retained in the ER. Stably transfected HEK293 cells were treated with proteasomal and lysosomal inhibitors in order to elucidate the exact molecular mechanisms underlying the loss of function phenotype associated with these variants. Our results have shown that wild type Endoglin has a relatively short half-life of less than 2 hours and degrades through both the lysosomal and proteasomal pathways, whereas the two mutant disease-causing variants show high stability and predominantly degrades through the proteasomal pathway. Furthermore, we have demonstrated that Endoglin variants P165L and V105D are significantly accumulated in HEK293 cells deficient in HRD1 E3 ubiquitin ligase; a major ERAD component. These results implicate the ERAD mechanism in the pathology of HHT1 caused by the two variants. It is expected that these results will pave the way for more in-depth research studies that could provide new windows for future therapeutic interventions.

Proteostasis Regulation in the Endoplasmic Reticulum: An Emerging Theme in the Molecular Pathology and Therapeutic Management of Familial Hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal genetic disease characterized by high serum low-density lipoprotein (LDL) content leading to premature coronary artery disease. The main genetic and molecular causes of FH are mutations in low-density lipoprotein receptor gene (LDLR) resulting in the non-clearance of LDL from the blood by hepatocytes and consequently the formation of plaques. LDLR is synthesized and glycosylated in the endoplasmic reticulum (ER) and then transported to the plasma membrane via Golgi. It is estimated that more than 50% of reported FH-causing mutations in LDLR result in misfolded proteins that are transport-defective and hence retained in ER. ER accumulation of misfolded proteins causes ER-stress and activates unfolded protein response (UPR). UPR aids protein folding, blocks further protein synthesis, and eliminates misfolded proteins via ER-associated degradation (ERAD) to alleviate ER stress. Various studies demonstrated that ER-retained LDLR mutants are subjected to ERAD. Interestingly, chemical chaperones and genetic or pharmacological inhibition of ERAD have been reported to rescue the transport defective mutant LDLR alleles from ERAD and restore their ER-Golgi transport resulting in the expression of functional plasma membrane LDLR. This suggests the possibility of pharmacological modulation of proteostasis in the ER as a therapeutic strategy for FH. In this review, we picture a detailed analysis of UPR and the ERAD processes activated by ER-retained LDLR mutants associated with FH. In addition, we discuss and critically evaluate the potential role of chemical chaperones and ERAD modulators in the therapeutic management of FH.

Utility of clinical exome sequencing in a complex Emirati pediatric cohort.

Clinical exome sequencing (CES) has become a routine diagnostic tool in several pediatric subspecialties, with a reported average diagnostic yield of ~25% in this patient poulation. The utility of CES in the United Arab Emirates (UAE) has not been previously investigated, most likely due to the lack of the appropriate tertiary pediatric centers and diagnostic genomic facilities in this country. Here, we report, for the first time, CES findings on a multispecialty pediatric cohort in the UAE (N = 51). This cohort, which was mostly Emirati (86%; 44/51), was followed at Al Jalila Children's Hospital (AJCH), the first and only dedicated tertiary pediatric center in the country. CES demonstrates a high diagnostic yield (41%; 21/51) in this cohort, where 55% (28/51) had previous non-diagnostic genetic testing while for the remaining individuals (45%), CES was the first-tier test. Given the reported high consanguinity rate in this population, 48% of the positive cases (10/21) were due to genes associated with recessive conditions. However, 11 out of 21 positive cases (52%) were due to heterozygous pathogenic variants in genes known to cause dominantly inherited disorders, including a case with a dual diagnosis attributed to two different genes (2%; 1/51), and another case with a novel de novo variant and new phenotypic features for a known gene (2%; 1/51). Overall, we have identified 13 novel clinically significant variants and showed that application of CES as a first-tier test plays a significant role in genetic diagnosis and management of Emirati pediatric patients.

Endoplasmic reticulum quality control of LDLR variants associated with familial hypercholesterolemia.

Loss-of-function mutations in the low-density lipoprotein receptor (LDLR) gene can cause familial hypercholesterolemia (FH), but detailed functional evidence for pathogenicity is limited to a few reported mutations. Here, we investigated the cellular pathogenic mechanisms of three mutations in LDLR causing FH, which are structurally identical to pathogenic mutations in the very low-density lipoprotein receptor (VLDLR). Similar to the VLDLR mutants, LDLR mutants D482H and C667F were found to be localized to the ER, while D445E, which is a conserved amino acid change, did not affect the trafficking of the receptor to the plasma membrane, as confirmed by the N-glycosylation profile. Although the ER-retained mutant proteins were soluble, induction of ER stress was observed as indicated by spliced X-box binding protein-1 (XBP-1) mRNA levels. The mutants were found to associate with ER quality control components, and their stability was enhanced by inhibitors of proteasome. Our results contribute to the growing list of transport-deficient class II LDLR variants leading to FH and provide evidence for the involvement of endoplasmic reticulum-associated degradation in their stability.

Degradation routes of trafficking-defective VLDLR mutants associated with Dysequilibrium syndrome.

Low density lipoprotein receptor (LDLR) family members are involved in signaling in the developing brain. Previously we have reported that missense mutations in the Very Low Density Lipoprotein Receptor gene (VLDLR), causing Dysequilibrium syndrome (DES), disrupt ligand-binding, due to endoplasmic reticulum (ER) retention of the mutants. We explored the degradation routes of these VLDLR mutants in cultured cells. Our results indicate that VLDLR mutants are retained in the ER for prolonged periods which could be facilitated by association with the ER-resident chaperone calnexin. The mutants were prone to aggregation and capable of eliciting ER stress. The VLDLR mutants were found to be degraded predominantly by the proteasomal pathway, since ubiquitinated VLDLR was found to accumulate in response to proteasomal inhibition. Further, the mutants were found to interact with the ER degradation adaptor protein SEL1L. The degradation of VLDLR wild type and mutant were delayed in CRISPR/Cas9 edited SEL1L knock-out cells which was reversed by exogenous expression of SEL1L. In summary, ER retention of pathogenic VLDLR mutants involves binding to calnexin, elevated ER stress, and delayed degradation which is dependent on SEL1L. Since core LDLR family members share common structural domains, common mechanisms may be involved in their ER processing.

A homozygous splicing mutation in ELAC2 suggests phenotypic variability including intellectual disability with minimal cardiac involvement.

BACKGROUND: The group of ELAC2-related encephalomyopathies is a recent addition to the rapidly growing heterogeneous mitochondrial disorders. RESULTS: We describe a highly inbred consanguineous Pakistani family with multiple affected children in 2 branches exhibiting moderately severe global developmental delay. Using homozygosity mapping, we mapped the phenotype in this family to a single locus on chromosome 17. In addition, whole-exome sequencing identified a homozygous splicing mutation (c.1423 + 2 T > A) in ELAC2 gene that disrupted the canonical donor splice site of intron 15 of all known isoforms. A noticeable reduction in ELAC2 expression was observed in patients compared to controls. In addition, patients exhibited significantly increased levels of 5' end unprocessed mt-RNAs compared to the control fibroblast cells. CONCLUSIONS: The only three previously reported families with defects in ELAC2 gene exhibited infantile hypertrophic cardiomyopathy and complex I deficiency. In contrast, our patients exhibited intellectual disability as the main feature with minimal cardiac involvement. Therefore our findings expand the phenotypic spectrum of ELAC2- associated disorders illustrating clinical heterogeneity of mutations in this gene. In addition, ELAC2 mutations should be considered when evaluating patient with mainly intellectual disability phenotypes.

Defective cellular trafficking of the bone morphogenetic protein receptor type II by mutations underlying familial pulmonary arterial hypertension.

Familial pulmonary arterial hypertension (FPAH) is a relatively rare but fatal disorder characterized by elevated arterial pressure caused by abnormal proliferation of endothelial cells of the arteries, which eventually leads to heart failure and death. FPAH is inherited as an autosomal dominant trait and is caused by heterozygous mutations in the BMPR2 gene encoding the bone morphogenetic protein type II receptor (BMPR2). BMPR2 belongs to the TGF ß/BMP super-family of receptors involved in a signal transduction cascade via the SMAD signaling pathway. The BMPR2 polypeptide is composed of 1038 amino acids and consists of a ligand binding domain, a kinase domain and a cytoplasmic tail. To investigate the cellular and functional consequence of BMPR2 mutations, C-terminally FLAG-tagged constructs of eighteen pathogenic BMPR2 missense mutants were generated by site directed mutagenesis and expressed in HeLa and HEK-293T cell lines. The subcellular localizations of the mutant proteins were investigated using immunostaining and confocal microscopy. Post-translational modifications of the proteins were analyzed by Endoglycosidase H deglycosylation assay. Our results indicated that mutations in the ligand binding domain affecting highly conserved cysteine residues resulted in retention of the mutant proteins in the endoplasmic reticulum (ER), as evident from their co-localization with the ER resident protein calnexin. The kinase domain mutants showed both ER and plasma membrane (PM) distributions, while the cytoplasmic tail domain variants were localized exclusively to the PM. The subcellular localizations of the mutants were further confirmed by their characteristic glycosylation profiles. In conclusion, our results indicate that ER quality control (ERQC) is involved in the pathological mechanism of several BMPR2 receptor missense mutations causing FPAH, which can be explored as a potential therapeutic target in the future.

A vacuolar antiporter is differentially regulated in leaves and roots of the halophytic wild rice Porteresia coarctata (Roxb.) Tateoka.

Vacuolar NHX-type antiporters play a role in Na(+)/K(+) uptake that contributes to growth, nutrition and development. Under salt/osmotic stress they mediate the vacuolar compartmentalization of K(+)/Na(+), thereby preventing toxic Na(+)K(+) ratios in the cytosol. Porteresia coarctata (Roxb.) Tateoka, a mangrove associate, is a distant wild relative of cultivated rice and is saline as well as submergence tolerant. A vacuolar NHX homolog isolated from a P. coarctata cDNA library (PcNHX1) shows 96 % identity (nucleotide level) to OsNHX1. Diurnal PcNHX1 expression in leaves was found to be largely unaltered, though damped by salinity. PcNHX1 promoter directed GUS expression is phloem-specific in leaves, stem and roots of transgenic plants in the absence of stress. Under NaCl stress, GUS expression was also seen in the epidermal and sub-epidermal layers (mesophyll, guard cells and trichomes) of leaves, root tip. The salinity in the rhizosphere of P. coarctata varies considerably due to diurnal/semi-diurnal tidal inundation. The diurnal expression of PcNHX1 in leaves and salinity induced expression in roots may have evolved in response to dynamic changes in salinity of in the P. coarctata rhizosphere. Despite high sequence conservation between OsNHX1 and PcNHX1, the distinctive expression pattern of PcNHX1 exemplifies how variation in expression is fine tuned to suit the halophytic growth habitat of a plant.

Impaired trafficking of the very low density lipoprotein receptor caused by missense mutations associated with dysequilibrium syndrome.

Dysequilibrium syndrome (DES, OMIM 224050) is a genetically heterogeneous condition that combines autosomal recessive non-progressive cerebellar ataxia with mental retardation. The subclass dysequilibrium syndrome type 1 (CAMRQ1) has been attributed to mutations in the VLDLR gene encoding the very low density lipoprotein receptor (VLDLR). This receptor is involved in the Reelin signaling pathway that guides neuronal migration in the cerebral cortex and cerebellum. Three missense mutations (c.1459G>T; p.D487Y, c.1561G>C; p.D521H and c.2117G>T; p.C706F) have been previously identified in VLDLR gene in patients with DES. However, the functional implications of those mutations are not known and therefore we undertook detailed functional analysis to elucidate the cellular mechanisms underlying their pathogenicity. The mutations have been generated by site-directed mutagenesis and then expressed in cultured cell lines. Confocal microscopy and biochemical analysis have been employed to examine the subcellular localization and functional activities of the mutated proteins relative to wild type. Our results indicate that the three missense mutations lead to defective intracellular trafficking and ER retention of the mutant VLDLR protein. This trafficking impairment prevents the mutants from reaching the plasma membrane and binding exogenous Reelin, the initiating event in Reelin signaling. Collectively, our results provide evidence that ER quality control is involved in the functional inactivation and underlying pathogenicity of these DES-associated mutations in the VLDLR.

A novel mutation in DDR2 causing spondylo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications (SMED-SL) results in defective intra-cellular trafficking.

BACKGROUND: The rare autosomal genetic disorder, Spondylo-meta-epiphyseal dysplasia with short limbs and abnormal calcifications (SMED-SL), is reported to be caused by missense or splice site mutations in the human discoidin domain receptor 2 (DDR2) gene. Previously our group has established that trafficking defects and loss of ligand binding are the underlying cellular mechanisms of several SMED-SL causing mutations. Here we report the clinical characteristics of two siblings of consanguineous marriage with suspected SMED-SL and identification of a novel disease-causing mutation in the DDR2 gene. METHODS: Clinical evaluation and radiography were performed to evaluate the patients. All the coding exons and splice sites of the DDR2 gene were sequenced by Sanger sequencing. Subcellular localization of the mutated DDR2 protein was determined by confocal microscopy, deglycosylation assay and Western blotting. DDR2 activity was measured by collagen activation and Western analysis. RESULTS: In addition to the typical features of SMED-SL, one of the patients has an eye phenotype including visual impairment due to optic atrophy. DNA sequencing revealed a novel homozygous dinucleotide deletion mutation (c.2468_2469delCT) on exon 18 of the DDR2 gene in both patients. The mutation resulted in a frameshift leading to an amino acid change at position S823 and a predicted premature termination of translation (p.S823Cfs*2). Subcellular localization of the mutant protein was analyzed in mammalian cell lines, and it was found to be largely retained in the endoplasmic reticulum (ER), which was further supported by its N-glycosylation profile. In keeping with its cellular mis-localization, the mutant protein was found to be deficient in collagen-induced receptor activation, suggesting protein trafficking defects as the major cellular mechanism underlying the loss of DDR2 function in our patients. CONCLUSIONS: Our results indicate that the novel mutation results in defective trafficking of the DDR2 protein leading to loss of function and disease. This confirms our previous findings that DDR2 missense mutations occurring at the kinase domain result in retention of the mutant protein in the ER.