Mutational spectrum and phenotypic variability of Duchenne muscular dystrophy and related disorders in a Bangladeshi population.

Duchenne muscular dystrophy (DMD) is a severe rare neuromuscular disorder caused by mutations in the X-linked dystrophin gene. Several mutations have been identified, yet the full mutational spectrum, and their phenotypic consequences, will require genotyping across different populations. To this end, we undertook the first detailed genotype and phenotype characterization of DMD in the Bangladeshi population. We investigated the rare mutational and phenotypic spectrum of the DMD gene in 36 DMD-suspected Bangladeshi participants using an economically affordable diagnostic strategy involving initial screening for exonic deletions in the DMD gene via multiplex PCR, followed by testing PCR-negative patients for mutations using whole exome sequencing. The deletion mapping identified two critical DMD gene hotspot regions (near proximal and distal ends, spanning exons 8-17 and exons 45-53, respectively) that comprised 95% (21/22) of the deletions for this population cohort. From our exome analysis, we detected two novel pathogenic hemizygous mutations in exons 21 and 42 of the DMD gene, and novel pathogenic recessive and loss of function variants in four additional genes: SGCD, DYSF, COL6A3, and DOK7. Our phenotypic analysis showed that DMD suspected participants presented diverse phenotypes according to the location of the mutation and which gene was impacted. Our study provides ethnicity specific new insights into both clinical and genetic aspects of DMD.

Neuronal SNCA transcription during Lewy body formation.

Misfolded α-synuclein (α-syn) is believed to contribute to neurodegeneration in Lewy body disease (LBD) based on considerable evidence including a gene-dosage effect observed in relation to point mutations and multiplication of SNCA in familial Parkinson's disease. A contradictory concept proposes early loss of the physiological α-syn as the major driver of neurodegeneration. There is a paucity of data on SNCA transcripts in various α-syn immunoreactive cytopathologies. Here, the total cell body, nuclear, and cytoplasmic area density of SNCA transcripts in neurons without and with various α-syn immunoreactive cytopathologies in the substantia nigra and amygdala in autopsy cases of LBD (n = 5) were evaluated using RNAscope combined with immunofluorescence for disease-associated α-syn. Single-nucleus RNA sequencing was performed to elucidate cell-type specific SNCA expression in non-diseased frontal cortex (n = 3). SNCA transcripts were observed in the neuronal nucleus and cytoplasm in neurons without α-syn, those containing punctate α-syn immunoreactivity, irregular-shaped compact inclusion, and brainstem-type and cortical-type LBs. However, SNCA transcripts were only rarely found in the α-syn immunoreactive LB areas. The total cell body SNCA transcript area densities in neurons with punctate α-syn immunoreactivity were preserved but were significantly reduced in neurons with compact α-syn inclusions both in the substantia nigra and amygdala. This reduction was also observed in the cytoplasm but not in the nucleus. Only single SNCA transcripts were detected in astrocytes with or without disease-associated α-syn immunoreactivity in the amygdala. Single-nucleus RNA sequencing revealed that excitatory and inhibitory neurons, oligodendrocyte progenitor cells, oligodendrocytes, and homeostatic microglia expressed SNCA transcripts, while expression was largely absent in astrocytes and microglia. The preserved cellular SNCA expression in the more abundant non-Lewy body type α-syn cytopathologies might provide a pool for local protein production that can aggregate and serve as a seed for misfolded α-syn. Successful segregation of disease-associated α-syn is associated with the exhaustion of SNCA production in the terminal cytopathology, the Lewy body. Our observations inform therapy development focusing on targeting SNCA transcription in LBD.

Single-cell reconstruction and mutation enrichment analysis identifies dysregulated cardiomyocyte and endothelial cells in congenital heart disease.

Congenital heart disease (CHD) is one of the most prevalent neonatal congenital anomalies. To catalog the putative candidate CHD risk genes, we collected 16,349 variants [single-nucleotide variants (SNVs) and Indels] impacting 8,308 genes in 3,166 CHD cases for a comprehensive meta-analysis. Using American College of Medical Genetics (ACMG) guidelines, we excluded the 0.1% of benign/likely benign variants and the resulting dataset consisted of 83% predicted loss of function variants and 17% missense variants. Seventeen percent were de novo variants. A stepwise analysis identified 90 variant-enriched CHD genes, of which six (GPATCH1, NYNRIN, TCLD2, CEP95, MAP3K19, and TTC36) were novel candidate CHD genes. Single-cell transcriptome cluster reconstruction analysis on six CHD tissues and four controls revealed upregulation of the top 10 frequently mutated genes primarily in cardiomyocytes. NOTCH1 (highest number of variants) and MYH6 (highest number of recurrent variants) expression was elevated in endocardial cells and cardiomyocytes, respectively, and 60% of these gene variants were associated with tetralogy of Fallot and coarctation of the aorta, respectively. Pseudobulk analysis using the single-cell transcriptome revealed significant (P < 0.05) upregulation of both NOTCH1 (endocardial cells) and MYH6 (cardiomyocytes) in the control heart data. We observed nine different subpopulations of CHD heart cardiomyocytes of which only four were observed in the control heart. This is the first comprehensive meta-analysis combining genomics and CHD single-cell transcriptomics, identifying the most frequently mutated CHD genes, and demonstrating CHD gene heterogeneity, suggesting that multiple genes contribute to the phenotypic heterogeneity of CHD. Cardiomyocytes and endocardial cells are identified as major CHD-related cell types.NEW & NOTEWORTHY Congential heart disease (CHD) is one of the most prevalent neonatal congenital anomalies. We present a comprehensive analysis combining genomics and CHD single-cell transcriptome. Our study identifies 90 potential candidate CHD risk genes of which 6 are novel. The risk genes have heterogenous expression suggestive of multiple genes contributing to the phenotypic heterogeneity of CHD. Cardiomyocytes and endocardial cells are identified as major CHD-related cell types.

Cell-specific MAPT gene expression is preserved in neuronal and glial tau cytopathologies in progressive supranuclear palsy.

Microtubule-associated protein tau (MAPT) aggregates in neurons, astrocytes and oligodendrocytes in a number of neurodegenerative diseases, including progressive supranuclear palsy (PSP). Tau is a target of therapy and the strategy includes either the elimination of pathological tau aggregates or reducing MAPT expression, and thus the amount of tau protein made to prevent its aggregation. Disease-associated tau affects brain regions in a sequential manner that includes cell-to-cell spreading. Involvement of glial cells that show tau aggregates is interpreted as glial cells taking up misfolded tau assuming that glial cells do not express enough MAPT. Although studies have evaluated MAPT expression in human brain tissue homogenates, it is not clear whether MAPT expression is compromised in cells accumulating pathological tau. To address these perplexing aspects of disease pathogenesis, this study used RNAscope combined with immunofluorescence (AT8), and single-nuclear(sn) RNAseq to systematically map and quantify MAPT expression dynamics across different cell types and brain regions in controls (n = 3) and evaluated whether tau cytopathology affects MAPT expression in PSP (n = 3). MAPT transcripts were detected in neurons, astrocytes and oligodendrocytes, and varied between brain regions and within each cell type, and were preserved in all cell types with tau aggregates in PSP. These results propose a complex scenario in all cell types, where, in addition to the ingested misfolded tau, the preserved cellular MAPT expression provides a pool for local protein production that can (1) be phosphorylated and aggregated, or (2) feed the seeding of ingested misfolded tau by providing physiological tau, both accentuating the pathological process. Since tau cytopathology does not compromise MAPT gene expression in PSP, a complete loss of tau protein expression as an early pathogenic component is less likely. These observations provide rationale for a dual approach to therapy by decreasing cellular MAPT expression and targeting removal of misfolded tau.

Lack of ethnic diversity in single-cell transcriptomics hinders cell type detection and precision medicine inclusivity.

Perhaps one of the most revolutionary next generation sequencing technologies is single-cell (SC) transcriptomics, which was recognized by Nature in 2013 as the method of the year. SC-technologies delve deep into genomics at the single-cell level, revealing previously restricted, valuable information on the identity of single cells, particularly highlighting their heterogeneity. Understanding the cellular heterogeneity of complex tissue provides insight about the gene expression and regulation across different biological and environmental conditions. This vast heterogeneity of cells and their markers makes identifying populations and sub-clusters especially difficult, even more so in rare cell types limited by the absence of rare sub-population markers. One particularly overlooked challenge is the lack of adequate ethnic representation in single-cell data. As the availability of cell types and their markers grow exponentially through new discoveries, the need to study ethnically driven heterogeneity becomes more feasible, while offering the opportunity to further elaborate ethnicity-related heterogeneity. In this commentary, we will discuss this major single-cell limitation particularly focusing on the repercussions it has on disease research, therapeutic design, and precision medicine.

Construction of copy number variation landscape and characterization of associated genes in a Bangladeshi cohort of neurodevelopmental disorders.

Introduction: Copy number variations (CNVs) play a critical role in the pathogenesis of neurodevelopmental disorders (NDD) among children. In this study, we aim to identify clinically relevant CNVs, genes and their phenotypic characteristics in an ethnically underrepresented homogenous population of Bangladesh. Methods: We have conducted chromosomal microarray analysis (CMA) for 212 NDD patients with male to female ratio of 2.2:1.0 to identify rare CNVs. To identify candidate genes within the rare CNVs, gene constraint metrics [i.e., "Critical-Exon Genes (CEGs)"] were applied to the population data. Autism Diagnostic Observation Schedule-Second Edition (ADOS-2) was followed in a subset of 95 NDD patients to assess the severity of autism and all statistical tests were performed using the R package. Results: Of all the samples assayed, 12.26% (26/212) and 57.08% (121/212) patients carried pathogenic and variant of uncertain significance (VOUS) CNVs, respectively. While 2.83% (6/212) patients' pathogenic CNVs were found to be located in the subtelomeric regions. Further burden test identified females are significant carriers of pathogenic CNVs compared to males (OR = 4.2; p = 0.0007). We have observed an increased number of Loss of heterozygosity (LOH) within cases with 23.85% (26/109) consanguineous parents. Our analyses on imprinting genes show, 36 LOH variants disrupting 69 unique imprinted genes and classified these variants as VOUS. ADOS-2 subset shows severe social communication deficit (p = 0.014) and overall ASD symptoms severity (p = 0.026) among the patients carrying duplication CNV compared to the CNV negative group. Candidate gene analysis identified 153 unique CEGs in pathogenic CNVs and 31 in VOUS. Of the unique genes, 18 genes were found to be in smaller (<1 MB) focal CNVs in our NDD cohort and we identified PSMC3 gene as a strong candidate gene for Autism Spectrum Disorder (ASD). Moreover, we hypothesized that KMT2B gene duplication might be associated with intellectual disability. Conclusion: Our results show the utility of CMA for precise genetic diagnosis and its integration into the diagnosis, therapy and management of NDD patients.

Overlapping pathogenic de novo CNVs in neurodevelopmental disorders and congenital anomalies impacting constraint genes regulating early development.

Neurodevelopmental disorders (NDDs) and congenital anomalies (CAs) are rare disorders with complex etiology. In this study, we investigated the less understood genomic overlap of copy number variants (CNVs) in two large cohorts of NDD and CA patients to identify de novo CNVs and candidate genes associated with both phenotypes. We analyzed clinical microarray CNV data from 10,620 NDD and 3176 CA cases annotated using Horizon platform of GenomeArc Analytics and applied rigorous downstream analysis to evaluate overlapping genes from NDD and CA CNVs. Out of 13,796 patients, only 195 cases contained 218 validated de novo CNVs. Eighteen percent (31/170) de novo CNVs in NDD cases and 40% (19/48) de novo CNVs in CA cases contained genomic overlaps impacting developmentally constraint genes. Seventy-nine constraint genes (10.1% non-OMIM entries) were found to have significantly enriched genomic overlap within rare de novo pathogenic deletions (P value = 0.01, OR = 1.58) and 45 constraint genes (13.3% non-OMIM entries) within rare de novo pathogenic duplications (P value = 0.01, OR = 1.97). Analysis of spatiotemporal transcriptome demonstrated both pathogenic deletion and duplication genes to be highly expressed during the prenatal stage in human developmental brain (P value = 4.95 X 10-6). From the list of overlapping genes, EHMT1, an interesting known NDD gene encompassed pathogenic deletion CNVs from both NDD and CA patients, whereas FAM189A1, and FSTL5 are new candidate genes from non-OMIM entries. In summary, we have identified constraint overlapping genes from CNVs (including de novo) in NDD and CA patients that have the potential to play a vital role in common disease etiology.

Analyzing single cell transcriptome data from severe COVID-19 patients.

We describe the protocol for identifying COVID-19 severity specific cell types and their regulatory marker genes using single-cell transcriptomics data. We construct COVID-19 comorbid disease-associated gene list using multiple databases and literature resources. Next, we identify specific cell type where comorbid genes are upregulated. We further characterize the identified cell type using gene enrichment analysis. We detect upregulation of marker gene restricted to severe COVID-19 cell type and validate our findings using in silico, in vivo, and in vitro cellular models. For complete details on the use and execution of this protocol, please refer to Nassir et al. (2021b).

Detection of copy number variants and genes by chromosomal microarray in an Emirati neurodevelopmental disorders cohort.

Copy number variations (CNVs) are highly implicated in the etiology of neurodevelopmental disorders (NDDs), and chromosomal microarray analysis (CMA) has been recommended as a first-tier test for many NDDs. We undertook a study to identify clinically relevant CNVs and genes in an ethnically homogenous population of the United Arab Emirates. We genotyped 98 patients with NDDs using genome-wide chromosomal microarray analysis, and observed 47.1% deletion and 52.9% duplication CNVs, of which 11.8% are pathogenic, 23.5% are likely pathogenic, and 64.7% VOUS. The average size of copy number losses (3.9 Mb) was generally higher than of gains (738.4 kb). Analysis of VOUS CNVs for constrained genes (enrichment for brain critical exons and high pLI genes) yielded 7 unique genes. Among these 7 constrained genes, we propose FNTA and PXK as potential candidate genes for neurodevelopmental disorders, which warrants further investigation. Thirty-two overlapping CNVs (Decipher and ClinVar) containing the FNTA gene were previously identified in NDD patients and 6 overlapping CNVs (Decipher and ClinVar) containing the PXK gene were previously identified in NDD patients. Our study supports the utility of CMA for CNV profiling which aids in precise genetic diagnosis and its integration into therapeutics and management of NDD patients.

Mutational Landscape of Autism Spectrum Disorder Brain Tissue.

Rare post-zygotic mutations in the brain are now known to contribute to several neurodevelopmental disorders, including autism spectrum disorder (ASD). However, due to the limited availability of brain tissue, most studies rely on estimates of mosaicism from peripheral samples. In this study, we undertook whole exome sequencing on brain tissue from 26 ASD brain donors from the Harvard Brain Tissue Resource Center (HBTRC) and ascertained the presence of post-zygotic and germline mutations categorized as pathological, including those impacting known ASD-implicated genes. Although quantification did not reveal enrichment for post-zygotic mutations compared with the controls (n = 15), a small number of pathogenic, potentially ASD-implicated mutations were identified, notably in TRAK1 and CLSTN3. Furthermore, germline mutations were identified in the same tissue samples in several key ASD genes, including PTEN, SC1A, CDH13, and CACNA1C. The establishment of tissue resources that are available to the scientific community will facilitate the discovery of new mutations for ASD and other neurodevelopmental disorders.

Single-cell transcriptome identifies molecular subtype of autism spectrum disorder impacted by de novo loss-of-function variants regulating glial cells.

BACKGROUND: In recent years, several hundred autism spectrum disorder (ASD) implicated genes have been discovered impacting a wide range of molecular pathways. However, the molecular underpinning of ASD, particularly from the point of view of 'brain to behaviour' pathogenic mechanisms, remains largely unknown. METHODS: We undertook a study to investigate patterns of spatiotemporal and cell type expression of ASD-implicated genes by integrating large-scale brain single-cell transcriptomes (> million cells) and de novo loss-of-function (LOF) ASD variants (impacting 852 genes from 40,122 cases). RESULTS: We identified multiple single-cell clusters from three distinct developmental human brain regions (anterior cingulate cortex, middle temporal gyrus and primary visual cortex) that evidenced high evolutionary constraint through enrichment for brain critical exons and high pLI genes. These clusters also showed significant enrichment with ASD loss-of-function variant genes (p < 5.23 × 10-11) that are transcriptionally highly active in prenatal brain regions (visual cortex and dorsolateral prefrontal cortex). Mapping ASD de novo LOF variant genes into large-scale human and mouse brain single-cell transcriptome analysis demonstrate enrichment of such genes into neuronal subtypes and are also enriched for subtype of non-neuronal glial cell types (astrocyte, p < 6.40 × 10-11, oligodendrocyte, p < 1.31 × 10-09). CONCLUSION: Among the ASD genes enriched with pathogenic de novo LOF variants (i.e. KANK1, PLXNB1), a subgroup has restricted transcriptional regulation in non-neuronal cell types that are evolutionarily conserved. This association strongly suggests the involvement of subtype of non-neuronal glial cells in the pathogenesis of ASD and the need to explore other biological pathways for this disorder.

Single-cell transcriptome identifies FCGR3B upregulated subtype of alveolar macrophages in patients with critical COVID-19.

Understanding host cell heterogeneity is critical for unraveling disease mechanism. Utilizing large-scale single-cell transcriptomics, we analyzed multiple tissue specimens from patients with life-threatening COVID-19 pneumonia, compared with healthy controls. We identified a subtype of monocyte-derived alveolar macrophages (MoAMs) where genes associated with severe COVID-19 comorbidities are significantly upregulated in bronchoalveolar lavage fluid of critical cases. FCGR3B consistently demarcated MoAM subset in different samples from severe COVID-19 cohorts and in CCL3L1-upregulated cells from nasopharyngeal swabs. In silico findings were validated by upregulation of FCGR3B in nasopharyngeal swabs of severe ICU COVID-19 cases, particularly in older patients and those with comorbidities. Additional lines of evidence from transcriptomic data and in vivo of severe COVID-19 cases suggest that FCGR3B may identify a specific subtype of MoAM in patients with severe COVID-19 that may present a novel biomarker for screening and prognosis, as well as a potential therapeutic target.

Single-cell transcriptomics trajectory and molecular convergence of clinically relevant mutations in Brugada syndrome.

Brugada syndrome (BrS) is a rare, inherited arrhythmia with high risk of sudden cardiac death. To evaluate the molecular convergence of clinically relevant mutations and to identify developmental cardiac cell types that are associated with BrS etiology, we collected 733 mutations represented by 16 sodium, calcium, potassium channels, and regulatory and structural genes related to BrS. Among the clinically relevant mutations, 266 are unique singletons and 88 mutations are recurrent. We observed an over-representation of clinically relevant mutations (∼80%) in SCN5A gene and also identified several candidate genes, including GPD1L, TRPM4, and SCN10A. Furthermore, protein domain enrichment analysis revealed that a large proportion of the mutations impacted ion transport domains in multiple genes, including SCN5A, TRPM4, and SCN10A. A comparative protein domain analysis of SCN5A further established a significant (P = 0.04) enrichment of clinically relevant mutations within ion transport domain, including a significant (P = 0.02) mutation hotspot within 1321-1380 residue. The enrichment of clinically relevant mutations within SCN5A ion transport domain is stronger (P = 0.00003) among early onset of BrS. Our spatiotemporal cellular heart developmental (prenatal to adult) trajectory analysis applying single-cell transcriptome identified the most frequently BrS-mutated genes (SCN5A and GPD1L) are significantly upregulated in the prenatal cardiomyocytes. A more restrictive cellular expression trajectory is prominent in the adult heart ventricular cardiomyocytes compared to prenatal. Our study suggests that genomic and proteomic hotspots in BrS converge into ion transport pathway and cardiomyocyte as a major BrS-associated cell type that provides insight into the complex genetic etiology of BrS.NEW & NOTEWORTHY Brugada syndrome is a rare inherited arrhythmia with high risk of sudden cardiac death. We present the findings for a molecular convergence of clinically relevant mutations and identification of a single-cell transcriptome-derived cardiac cell types that are associated with the etiology of BrS. Our study suggests that genomic and proteomic hotspots in BrS converge into ion transport pathway and cardiomyocyte as a major BrS-associated cell type that provides insight into the complex genetic etiology of BrS.

Long-Read Sequencing Improves the Detection of Structural Variations Impacting Complex Non-Coding Elements of the Genome.

The advent of long-read sequencing offers a new assessment method of detecting genomic structural variation (SV) in numerous rare genetic diseases. For autism spectrum disorders (ASD) cases where pathogenic variants fail to be found in the protein-coding genic regions along chromosomes, we proposed a scalable workflow to characterize the risk factor of SVs impacting non-coding elements of the genome. We applied whole-genome sequencing on an Emirati family having three children with ASD using long and short-read sequencing technology. A series of analytical pipelines were established to identify a set of SVs with high sensitivity and specificity. At 15-fold coverage, we observed that long-read sequencing technology (987 variants) detected a significantly higher number of SVs when compared to variants detected using short-read technology (509 variants) (p-value < 1.1020 × 10-57). Further comparison showed 97.9% of long-read sequencing variants were spanning within the 1-100 kb size range (p-value < 9.080 × 10-67) and impacting over 5000 genes. Moreover, long-read variants detected 604 non-coding RNAs (p-value < 9.02 × 10-9), comprising 58% microRNA, 31.9% lncRNA, and 9.1% snoRNA. Even at low coverage, long-read sequencing has shown to be a reliable technology in detecting SVs impacting complex elements of the genome.

Whole exome sequencing uncovered highly penetrant recessive mutations for a spectrum of rare genetic pediatric diseases in Bangladesh.

Collectively, rare genetic diseases affect a significant number of individuals worldwide. In this study, we have conducted whole-exome sequencing (WES) and identified underlying pathogenic or likely pathogenic variants in five children with rare genetic diseases. We present evidence for disease-causing autosomal recessive variants in a range of disease-associated genes such as DHH-associated 46,XY gonadal dysgenesis (GD) or 46,XY sex reversal 7, GNPTAB-associated mucolipidosis II alpha/beta (ML II), BBS1-associated Bardet-Biedl Syndrome (BBS), SURF1-associated Leigh Syndrome (LS) and AP4B1-associated spastic paraplegia-47 (SPG47) in unrelated affected members from Bangladesh. Our analysis pipeline detected three homozygous mutations, including a novel c. 863 G > C (p.Pro288Arg) variant in DHH, and two compound heterozygous variants, including two novel variants: c.2972dupT (p.Met991Ilefs*) in GNPTAB and c.229 G > C (p.Gly77Arg) in SURF1. All mutations were validated by Sanger sequencing. Collectively, this study adds to the genetic heterogeneity of rare genetic diseases and is the first report elucidating the genetic profile of (consanguineous and nonconsanguineous) rare genetic diseases in the Bangladesh population.

A telomerase with novel non-canonical roles: TERT controls cellular aggregation and tissue size in Dictyostelium.

Telomerase, particularly its main subunit, the reverse transcriptase, TERT, prevents DNA erosion during eukaryotic chromosomal replication, but also has poorly understood non-canonical functions. Here, in the model social amoeba Dictyostelium discoideum, we show that the protein encoded by tert has telomerase-like motifs, and regulates, non-canonically, important developmental processes. Expression levels of wild-type (WT) tert were biphasic, peaking at 8 and 12 h post-starvation, aligning with developmental events, such as the initiation of streaming (~7 h) and mound formation (~10 h). In tert KO mutants, however, aggregation was delayed until 16 h. Large, irregular streams formed, then broke up, forming small mounds. The mound-size defect was not induced when a KO mutant of countin (a master size-regulating gene) was treated with TERT inhibitors, but anti-countin antibodies did rescue size in the tert KO. Although, conditioned medium (CM) from countin mutants failed to rescue size in the tert KO, tert KO CM rescued the countin KO phenotype. These and additional observations indicate that TERT acts upstream of smlA/countin: (i) the observed expression levels of smlA and countin, being respectively lower and higher (than WT) in the tert KO; (ii) the levels of known size-regulation intermediates, glucose (low) and adenosine (high), in the tert mutant, and the size defect's rescue by supplemented glucose or the adenosine-antagonist, caffeine; (iii) the induction of the size defect in the WT by tert KO CM and TERT inhibitors. The tert KO's other defects (delayed aggregation, irregular streaming) were associated with changes to cAMP-regulated processes (e.g. chemotaxis, cAMP pulsing) and their regulatory factors (e.g. cAMP; acaA, carA expression). Overexpression of WT tert in the tert KO rescued these defects (and size), and restored a single cAMP signaling centre. Our results indicate that TERT acts in novel, non-canonical and upstream ways, regulating key developmental events in Dictyostelium.