Early insights into the role of Exoc6B associated with spondyloepimetaphyseal dysplasia with joint laxity type 3 in primary ciliogenesis and chondrogenic differentiation in vitro.

BACKGROUND: Spondyloepimetaphyseal dysplasia with joint laxity type 3 (SEMDJL3) is a rare skeletal dysplasia associated with EXOC6B, a component of the exocyst complex, involved in vesicle tethering and exocytosis at the plasma membrane. So far, EXOC6B and the pathomechanisms underlying SEMDJL3 remain obscure. METHODS AND RESULTS: Exoc6b was detected largely at the perinuclear regions and the primary cilia base in ATDC5 prechondrocytes. Its shRNA lentiviral knockdown impeded primary ciliogenesis. In Exoc6b silenced prechondrocytes, Hedgehog signaling was attenuated, including when stimulated with Smoothened agonist. Exoc6b knockdown deregulated the mRNA and protein levels of Col2a1, a marker of chondrocyte proliferation at 7- and 14-days following differentiation. It led to the upregulation of Ihh another marker of proliferative chondrocytes. The levels of Col10a1, a marker of chondrocyte hypertrophy was enhanced at 14 days of differentiation. Congruently, Axin2, a canonical Wnt pathway modulator that inhibits chondrocyte hypertrophy was repressed. The expression of Mmp13 and Adamts4 that are terminal chondrocyte hypertrophy markers involved in extracellular matrix (ECM) remodelling were downregulated at 7 and 14 days of chondrogenesis. Bglap that encodes for the most abundant non-collagenous bone matrix constituent and promotes ECM calcification was suppressed at 14 days of chondrocyte differentiation. ECM mineralization was assessed by Alizarin Red staining. Gene expression and ciliogenesis were investigated by reverse transcription quantitative real-time PCR, immunoblotting, and immunocytochemistry. CONCLUSIONS: These findings provide initial insights into the potential role of Exoc6b in primary ciliogenesis and chondrogenic differentiation, contributing towards a preliminary understanding of the molecular pathomechanisms underlying SEMDJL3.

The impact of COVID-19 on pulmonary, neurological, and cardiac outcomes: evidence from a Mendelian randomization study.

Background: Long COVID is a clinical entity characterized by persistent health problems or development of new diseases, without an alternative diagnosis, following SARS-CoV-2 infection that affects a significant proportion of individuals globally. It can manifest with a wide range of symptoms due to dysfunction of multiple organ systems including but not limited to cardiovascular, hematologic, neurological, gastrointestinal, and renal organs, revealed by observational studies. However, a causal association between the genetic predisposition to COVID-19 and many post-infective abnormalities in long COVID remain unclear. Methods: Here we employed Mendelian randomization (MR), a robust genetic epidemiological approach, to investigate the potential causal associations between genetic predisposition to COVID-19 and long COVID symptoms, namely pulmonary (pneumonia and airway infections including bronchitis, emphysema, asthma, and rhinitis), neurological (headache, depression, and Parkinson's disease), cardiac (heart failure and chest pain) diseases, and chronic fatigue. Using two-sample MR, we leveraged genetic data from a large COVID-19 genome-wide association study and various disorder-specific datasets. Results: This analysis revealed that a genetic predisposition to COVID-19 was significantly causally linked to an increased risk of developing pneumonia, airway infections, headache, and heart failure. It also showed a strong positive correlation with chronic fatigue, a frequently observed symptom in long COVID patients. However, our findings on Parkinson's disease, depression, and chest pain were inconclusive. Conclusion: Overall, these findings provide valuable insights into the genetic underpinnings of long COVID and its diverse range of symptoms. Understanding these causal associations may aid in better management and treatment of long COVID patients, thereby alleviating the substantial burden it poses on global health and socioeconomic systems.

Primary cilia in skeletal development and disease.

Primary cilia are non-motile, microtubule-based sensory organelle present in most vertebrate cells with a fundamental role in the modulation of organismal development, morphogenesis, and repair. Here we focus on the role of primary cilia in embryonic and postnatal skeletal development. We examine evidence supporting its involvement in physiochemical and developmental signaling that regulates proliferation, patterning, differentiation and homeostasis of osteoblasts, chondrocytes, and their progenitor cells in the skeleton. We discuss how signaling effectors in mechanotransduction and bone development, such as Hedgehog, Wnt, Fibroblast growth factor and second messenger pathways operate at least in part at the primary cilium. The relevance of primary cilia in bone formation and maintenance is underscored by a growing list of rare genetic skeletal ciliopathies. We collate these findings and summarize the current understanding of molecular factors and mechanisms governing primary ciliogenesis and ciliary function in skeletal development and disease.

Biallelic loss-of-function variants in EXOC6B are associated with impaired primary ciliogenesis and cause spondylo-epi-metaphyseal dysplasia with joint laxity type 3.

Spondylo-epi-metaphyseal dysplasias with joint laxity, type 3 (SEMDJL3) is a genetic skeletal disorder characterized by multiple joint dislocations, caused by biallelic pathogenic variants in the EXOC6B gene. Only four individuals from two families have been reported to have this condition to date. The molecular pathogenesis related to primary ciliogenesis has not been enumerated in subjects with SEMDJL3. In this study, we report two additional affected individuals from unrelated families with biallelic pathogenic variants, c.2122+15447_2197-59588del and c.401T>G in EXOC6B identified by exome sequencing. One of the affected individuals had an intellectual disability and central nervous system anomalies, including hydrocephalus, hypoplastic mesencephalon, and thin corpus callosum. Using the fibroblast cell lines, we demonstrate the primary evidence for the abrogation of exocytosis in an individual with SEMDLJ3 leading to impaired primary ciliogenesis. Osteogenesis differentiation and pathways related to the extracellular matrix were also found to be reduced. Additionally, we provide a review of the clinical and molecular profile of all the mutation-proven patients reported hitherto, thereby further characterizing SEMDJL3. SEMDJL3 with biallelic pathogenic variants in EXOC6B might represent yet another ciliopathy with central nervous system involvement and joint dislocations.

Exome-Wide Association Study Reveals Host Genetic Variants Likely Associated with the Severity of COVID-19 in Patients of European Ancestry.

Host genetic variability plays a pivotal role in modulating COVID-19 clinical outcomes. Despite the functional relevance of protein-coding regions, rare variants located here are less likely to completely explain the considerable numbers of acutely affected COVID-19 patients worldwide. Using an exome-wide association approach, with individuals of European descent, we sought to identify common coding variants linked with variation in COVID-19 severity. Herein, cohort 1 compared non-hospitalized (controls) and hospitalized (cases) individuals, and in cohort 2, hospitalized subjects requiring respiratory support (cases) were compared to those not requiring it (controls). 229 and 111 variants differed significantly between cases and controls in cohorts 1 and 2, respectively. This included FBXO34, CNTN2, and TMCC2 previously linked with COVID-19 severity using association studies. Overall, we report SNPs in 26 known and 12 novel candidate genes with strong molecular evidence implicating them in the pathophysiology of life-threatening COVID-19 and post-recovery sequelae. Of these few notable known genes include, HLA-DQB1, AHSG, ALOX5AP, MUC5AC, SMPD1, SPG7, SPEG,GAS6, and SERPINA12. These results enhance our understanding of the pathomechanisms underlying the COVID-19 clinical spectrum and may be exploited to prioritize biomarkers for predicting disease severity, as well as to improve treatment strategies in individuals of European ancestry.

The intraflagellar transport protein IFT52 associated with short-rib thoracic dysplasia is essential for ciliary function in osteogenic differentiation in vitro and for sensory perception in Drosophila.

Primary cilia are non-motile sensory cell-organelle that are essential for organismal development, differentiation, and postnatal homeostasis. Their biogenesis and function are mediated by the intraflagellar transport (IFT) system. Pathogenic variants in IFT52, a central component of the IFT-B complex is associated with short-rib thoracic dysplasia with or without polydactyly 16 (SRTD16), with major skeletal manifestations, in addition to other features. Here we sought to examine the role of IFT52 in osteoblast differentiation. Using lentiviral shRNA interference Ift52 was depleted in C3H10T1/2 mouse mesenchymal stem cells. This led to the disruption of the IFT-B anterograde trafficking machinery that impaired primary ciliogenesis and blocked osteogenic differentiation. In Ift52 silenced cells, Hedgehog (Hh) pathway upregulation during osteogenesis was attenuated and despite Smoothened Agonist (SAG) based Hh activation, osteogenic differentiation was incompletely restored. Further we investigated IFT52 activity in Drosophila, wherein the only ciliated somatic cells are the bipolar sensory neurons of the peripheral nervous system. Knockdown of IFT52 in Drosophila neuronal tissues reduced lifespan with the loss of embryonic chordotonal cilia, and produced severe locomotion, auditory and proprioceptive defects in larva and adults. Together these findings improve our knowledge of the role of IFT52 in various physiological contexts and its associated human disorder.

Genomic and Ancestral Variation Underlies the Severity of COVID-19 Clinical Manifestation in Individuals of European Descent.

The coronavirus disease (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a wide spectrum of clinical phenotypes ranging from asymptomatic to symptomatic with mild or moderate presentation and severe disease. COVID-19 susceptibility, severity and recovery have demonstrated high variability worldwide. Variances in the host genetic architecture may underlie the inter-individual and population-scale differences in COVID-19 presentation. We performed a genome-wide association analysis employing the genotyping data from AncestryDNA for COVID-19 patients of European descent and used asymptomatic subjects as the control group. We identified 621 genetic variants that were significantly distinct between asymptomatic and acutely symptomatic COVID-19 patients (multiple-testing corrected p-value < 0.001). These variants were found to be associated with pathways governing host immunity, such as interferon, interleukin and cytokine signalling, and known COVID-19 comorbidities, such as obesity and cholesterol metabolism. Further, our ancestry analysis revealed that the asymptomatic COVID-19 patients possess discernibly higher proportions of the Ancestral North Eurasian (ANE) and Eastern Hunter-Gatherer (EHG) ancestry, which was introduced to Europe through Bell Beaker culture (Yamnaya related) and lower fractions of Western Hunter-Gatherer (WHG) ancestry, while severely symptomatic patients have higher fractions of WHG and lower ANE/EHG ancestral components, thereby delineating the likely ancestral differences between the two groups.

Biallelic deep intronic variant c.5457+81T>A in TRIP11 causes loss of function and results in achondrogenesis 1A.

Biallelic loss of function variants in TRIP11 encoding for the Golgi microtubule-associated protein 210 (GMAP-210) causes the lethal chondrodysplasia achondrogenesis type 1A (ACG1A). Loss of TRIP11 activity has been shown to impair Golgi structure, vesicular transport, and results in loss of IFT20 anchorage to the Golgi that is vital for ciliary trafficking and ciliogenesis. Here, we report four fetuses, two each from two families, who were ascertained antenatally with ACG1A. Affected fetuses in both families are homozygous for the deep intronic TRIP11 variant, c.5457+81T>A, which was found in a shared region of homozygosity. This variant was found to cause aberrant transcript splicing and the retention of 77 base pairs of intron 18. The TRIP11 messenger RNA and protein levels were drastically reduced in fibroblast cells derived from one of the affected fetuses. Using immunofluorescence we also detected highly compacted Golgi apparatus in affected fibroblasts. Further, we observed a significant reduction in the frequency of ciliated cells and in the length of primary cilia in subject-derived cell lines, not reported so far in patient cells with TRIP11 null or hypomorphic variants. Our findings illustrate how pathogenic variants in intronic regions of TRIP11 can impact transcript splicing, expression, and activity, resulting in ACG1A.

Bi-allelic missense variant, p.Ser35Leu in EXOSC1 is associated with pontocerebellar hypoplasia.

RNA exosome is a highly conserved ribonuclease complex essential for RNA processing and degradation. Bi-allelic variants in exosome subunits EXOSC3, EXOSC8 and EXOSC9 have been reported to cause pontocerebellar hypoplasia type 1B, type 1C and type 1D, respectively, while those in EXOSC2 cause short stature, hearing loss, retinitis pigmentosa and distinctive facies. We ascertained an 8-months-old male with developmental delay, microcephaly, subtle dysmorphism and hypotonia. Pontocerebellar hypoplasia and delayed myelination were noted on neuroimaging. A similarly affected elder sibling succumbed at the age of 4-years 6-months. Chromosomal microarray returned normal results. Exome sequencing revealed a homozygous missense variant, c.104C > T p.(Ser35Leu) in EXOSC1 (NM_016046.5) as the possible candidate. In silico mutagenesis revealed loss of a polar contact with neighboring Leu37 residue. Quantitative real-time PCR indicated no appreciable differences in EXOSC1 transcript levels. Immunoblotting and blue native PAGE revealed reduction in the EXOSC1 protein levels and EXO9 complex in the proband, respectively. We herein report an individual with the bi-allelic variant c.104C>T p.(Ser35Leu) in EXOSC1 and clinical features of pontocerebellar hypoplasia type 1. Immunoblotting and blue native PAGE provide evidence for the pathogenicity of the variant. Thus, we propose EXOSC1 as a novel candidate gene for pontocerebellar hypoplasia.

The story of the lost twins: decoding the genetic identities of the Kumhar and Kurcha populations from the Indian subcontinent.

BACKGROUND: The population structure of the Indian subcontinent is a tapestry of extraordinary diversity characterized by the amalgamation of autochthonous and immigrant ancestries and rigid enforcement of sociocultural stratification. Here we investigated the genetic origin and population history of the Kumhars, a group of people who inhabit large parts of northern India. We compared 27 previously published Kumhar SNP genotype data sampled from Uttar Pradesh in north India to various modern day and ancient populations. RESULTS: Various approaches such as Principal Component Analysis (PCA), Admixture, TreeMix concurred that Kumhars have high ASI ancestry, minimal Steppe component and high genomic proximity to the Kurchas, a small and relatively little-known population found ~ 2500 km away in Kerala, south India. Given the same, biogeographical mapping using Geographic Population Structure (GPS) assigned most Kumhar samples in areas neighboring to those where Kurchas are found in south India. CONCLUSIONS: We hypothesize that the significant genomic similarity between two apparently distinct modern-day Indian populations that inhabit well separated geographical areas with no known overlapping history or links, likely alludes to their common origin during or post the decline of the Indus Valley Civilization (estimated by ALDER). Thereafter, while they dispersed towards opposite ends of the Indian subcontinent, their genomic integrity and likeness remained preserved due to endogamous social practices. Our findings illuminate the genomic history of two Indian populations, allowing a glimpse into one or few of numerous of human migrations that likely occurred across the Indian subcontinent and contributed to shape its varied and vibrant evolutionary past.

Phenotypic diversity and genetic complexity of PAX3-related Waardenburg syndrome.

Waardenburg syndrome subtypes 1 and 3 are caused by pathogenic variants in PAX3. We investigated 12 individuals from four unrelated families clinically diagnosed with Waardenburg syndrome type 1/3. Novel pathogenic variants identified in PAX3 included single nucleotide variants (c.166C>T, c.829C>T), a 2-base pair deletion (c.366_367delAA) and a multi-exonic deletion. Two novel variants, c.166C>T and c.829C>T and a previously reported variant, c.256A>T in PAX3 were evaluated for their nuclear localization and ability to activate MITF promoter. The coexistence of two subtypes of Waardenburg syndrome with pathogenic variants in PAX3 and EDNRB was seen in one of the affected individuals. Multiple genetic diagnoses of Waardenburg syndrome type 3 and autosomal recessive deafness 1A was identified in an individual. We also review the phenotypic and genomic spectrum of individuals with PAX3-related Waardenburg syndrome reported in the literature.

Characterization of primary cilia features reveal cell-type specific variability in in vitro models of osteogenic and chondrogenic differentiation.

Primary cilia are non-motile sensory antennae present on most vertebrate cell surfaces. They serve to transduce and integrate diverse external stimuli into functional cellular responses vital for development, differentiation and homeostasis. Ciliary characteristics, such as length, structure and frequency are often tailored to distinct differentiated cell states. Primary cilia are present on a variety of skeletal cell-types and facilitate the assimilation of sensory cues to direct skeletal development and repair. However, there is limited knowledge of ciliary variation in response to the activation of distinct differentiation cascades in different skeletal cell-types. C3H10T1/2, MC3T3-E1 and ATDC5 cells are mesenchymal stem cells, preosteoblast and prechondrocyte cell-lines, respectively. They are commonly employed in numerous in vitro studies, investigating the molecular mechanisms underlying osteoblast and chondrocyte differentiation, skeletal disease and repair. Here we sought to evaluate the primary cilia length and frequencies during osteogenic differentiation in C3H10T1/2 and MC3T3-E1 and chondrogenic differentiation in ATDC5 cells, over a period of 21 days. Our data inform on the presence of stable cilia to orchestrate signaling and dynamic alterations in their features during extended periods of differentiation. Taken together with existing literature these findings reflect the occurrence of not only lineage but cell-type specific variation in ciliary attributes during differentiation. These results extend our current knowledge, shining light on the variabilities in primary cilia features correlated with distinct differentiated cell phenotypes. It may have broader implications in studies using these cell-lines to explore cilia dependent cellular processes and treatment modalities for skeletal disorders centered on cilia modulation.

Modulation of the Promoter Activation Rate Dictates the Transcriptional Response to Graded BMP Signaling Levels in the Drosophila Embryo.

Morphogen gradients specify cell fates during development, with a classic example being the bone morphogenetic protein (BMP) gradient's conserved role in embryonic dorsal-ventral axis patterning. Here, we elucidate how the BMP gradient is interpreted in the Drosophila embryo by combining live imaging with computational modeling to infer transcriptional burst parameters at single-cell resolution. By comparing burst kinetics in cells receiving different levels of BMP signaling, we show that BMP signaling controls burst frequency by regulating the promoter activation rate. We provide evidence that the promoter activation rate is influenced by both enhancer and promoter sequences, whereas Pol II loading rate is primarily modulated by the enhancer. Consistent with BMP-dependent regulation of burst frequency, the numbers of BMP target gene transcripts per cell are graded across their expression domains. We suggest that graded mRNA output is a general feature of morphogen gradient interpretation and discuss how this can impact on cell-fate decisions.

Recurrent 1q21.1 deletion syndrome: report on variable expression, nonpenetrance and review of literature.

The clinical phenotype of 1q21.1 microdeletion syndrome is highly heterogeneous. It is characterized by dysmorphic facial features, microcephaly, and developmental delay. Several congenital defects, including cardiac, ocular, skeletal anomalies, and psychiatric or behavioural abnormalities, have also been described. Here, we report on two siblings with substantial intrafamilial phenotypic variability carrying a heterozygous deletion of the 1q21.1 region spanning a known critical genomic area (~1.35 Mb). The microdeletion was inherited from the unaffected father. Patients described here show a spectrum of clinical features, a portion of which overlap with those previously reported in patients with 1q21.1 microdeletions. In addition, we review the clinical reports of 66 individuals with this condition. These findings extend and substantiate the current clinical understanding of recurrent copy number variations in the 1q21.1 region.

Novel splice site and nonsense variants in INVS cause infantile nephronophthisis.

Nephronophthisis is an autosomal recessive disease characterized by cystic kidney disease with progression to end-stage kidney disease in children and adolescents with or without extra-renal involvement. It is caused by biallelic pathogenic variants in 19 genes including INVS that encodes a ciliary protein essential for renal development and left-right axis establishment. We report a child with bilateral enlarged, echogenic, polycystic kidneys with end-stage renal disease, anemia and metabolic acidosis caused by biallelic novel pathogenic variants, c.796 + 5G > A and c.1789C > T in INVS. We show that the variant, c.796 + 5G > A disrupts the canonical splicing and nonsense variant, c.1789C > T results in nonsense mediated decay.

Investigating the West Eurasian ancestry of Pakistani Hazaras.

The Hazaras are a distinct ethnic group from central Afghanistan and northwestern Pakistan of Mongoloid descent. Here, we sought to dissect the genetic admixture history of the Pakistani Hazaras and investigated their likely affiliation to ancient and extant West Eurasian populations. Our results indicated that the likely proportion of West Eurasian ancestry was low in the Hazaras and could be attributed putatively to a combination of Steppe populations from Early/Middle Bronze Age or Middle/Late Bronze Age and the Neolithic Iranians. Our results expand upon the current understanding and provide an improved resolution into the population history of the Pakistani Hazaras.

Application of the geographic population structure (GPS) algorithm for biogeographical analyses of wild and captive gorillas.

BACKGROUND: The utilization of high resolution genome data has important implications for the phylogeographical evaluation of non-human species. Biogeographical analyses can yield detailed understanding of their population biology and facilitate the geo-localization of individuals to promote their efficacious management, particularly when bred in captivity. The Geographic Population Structure (GPS) algorithm is an admixture based tool for inference of biogeographical affinities and has been employed for the geo-localization of various human populations worldwide. Here, we applied the GPS tool for biogeographical analyses and localization of the ancestral origins of wild and captive gorilla genomes, of unknown geographic source, available in the Great Ape Genome Project (GAGP), employing Gorillas with known ancestral origin as the reference data. RESULTS: Our findings suggest that GPS was successful in recapitulating the population history and estimating the geographic origins of all gorilla genomes queried and localized the wild gorillas with unknown geographical origin < 150 km of National Parks/Wildlife Reserves within the political boundaries of countries, considered as prominent modern-day abode for gorillas in the wild. Further, the GPS localization of most captive-born gorillas was congruent with their previously presumed ancestral homes. CONCLUSIONS: Currently there is limited knowledge of the ancestral origins of most North American captive gorillas, and our study highlights the usefulness of GPS for inferring ancestry of captive gorillas. Determination of the native geographical source of captive gorillas can provide valuable information to guide breeding programs and ensure their appropriate management at the population level. Finally, our findings shine light on the broader applicability of GPS for protecting the genetic integrity of other endangered non-human species, where controlled breeding is a vital component of their conservation.

Phenotyping and genotyping of skeletal dysplasias: Evolution of a center and a decade of experience in India.

Genetic heterogeneity, high burden and the paucity of genetic testing for rare diseases challenge genomic healthcare for these disorders in India. Here we report our experience over the past decade, of establishing the genomic evaluation of skeletal dysplasia at a tertiary university hospital in India. Research or clinical genomic testing was carried out by Sanger sequencing and next-generation sequencing. Close national and international collaborations aided phenotyping and genotyping. We report 508 families (557 affected individuals) with the definitive molecular diagnosis of skeletal dysplasia. Dysostoses multiplex (n = 196), genetic inflammatory/rheumatoid-like osteoarthropathies (n = 114) and osteogenesis imperfecta and decreased bone density (n = 58) were the most common diagnoses. We enumerate the processes, clinical diagnoses and causal variants in the cohort with 48 novel variants in 21 genes. We summarize scientific contributions of the center to the description of clinical and mutation profiles and discovery of new phenotypes and genetic etiology. Our study illustrates the establishment and application of genomic testing tools for genetic disorders of skeleton in a large cohort. We believe this could be a model to emulate for other developing genetic centers.

Correction to: Application of geographic population structure (GPS) algorithm for biogeographical analyses of populations with complex ancestries: a case study of South Asians from 1000 genomes project.

Following publication of the original article [1], the authors flagged that acknowledgment of their equal contribution is omitted in the article [1].

An Ancestry Informative Marker Set Which Recapitulates the Known Fine Structure of Populations in South Asia.

The inference of genomic ancestry using ancestry informative markers (AIMs) can be useful for a range of studies in evolutionary genetics, biomedical research, and forensic analyses. However, the determination of AIMs for highly admixed populations with complex ancestries has remained a formidable challenge. Given the immense genetic heterogeneity and unique population structure of the Indian subcontinent, here we sought to derive AIMs that would yield a cohesive and faithful understanding of South Asian genetic origins. To discern the most optimal strategy for extracting AIMs for South Asians we compared three commonly used AIMs-determining methods namely, Infocalc, FST, and Smart Principal Component Analysis with ADMIXTURE, using previously published whole genome data from the Indian subcontinent. Our findings suggest that the Infocalc approach is likely most suitable for delineation of South Asian AIMs. In particular, Infocalc-2,000 (N = 2,000) appeared as the most informative South Asian AIMs panel that recapitulated the finer structure within South Asian genomes with high degree of sensitivity and precision, whereas a negative control with an equivalent number of randomly selected markers when used to interrogate the South Asian populations, failed to do so. We discuss the utility of all approaches under evaluation for AIMs derivation and interpreting South Asian genomic ancestries. Notably, this is the first report of an AIMs panel for South Asian ancestry inference. Overall these findings may aid in developing cost-effective resources for large-scale demographic analyses and foster expansion of our knowledge of human origins and disease, in the South Asian context.