Expanding deep phenotypic spectrum associated with atypical pathogenic structural variations overlapping 15q11-q13 imprinting region.

BACKGROUND: The 15q11-q13 region is a genetic locus with genes subject to genomic imprinting, significantly influencing neurodevelopment. Genomic imprinting is an epigenetic phenomenon that causes differential gene expression based on the parent of origin. In most diploid organisms, gene expression typically involves an equal contribution from both maternal and paternal alleles, shaping the phenotype. Nevertheless, in mammals, including humans, mice, and marsupials, the functional equivalence of parental alleles is not universally maintained. Notably, during male and female gametogenesis, parental alleles may undergo differential marking or imprinting, thereby modifying gene expression without altering the underlying DNA sequence. Neurodevelopmental disorders, such as Prader-Willi syndrome (PWS) (resulting from the absence of paternally expressed genes in this region), Angelman syndrome (AS) (associated with the absence of the maternally expressed UBE3A gene), and 15q11-q13 duplication syndrome (resulting from the two common forms of duplications-either an extra isodicentric 15 chromosome or an interstitial 15 duplication), are the outcomes of genetic variations in this imprinting region. METHODS: Conducted a genomic study to identify the frequency of pathogenic variants impacting the 15q11-q13 region in an ethnically homogenous population from Bangladesh. Screened all known disorders from the DECIPHER database and identified variant enrichment within this cohort. Using the Horizon analysis platform, performed enrichment analysis, requiring at least >60% overlap between a copy number variation and a disorder breakpoint. Deep clinical phenotyping was carried out through multiple examination sessions to evaluate a range of clinical symptoms. RESULTS: This study included eight individuals with clinically suspected PWS/AS, all previously confirmed through chromosomal microarray analysis, which revealed chromosomal breakpoints within the 15q11-q13 region. Among this cohort, six cases (75%) exhibited variable lengths of deletions, whereas two cases (25%) showed duplications. These included one type 2 duplication, one larger atypical duplication, one shorter type 2 deletion, one larger type 1 deletion, and four cases with atypical deletions. Furthermore, thorough clinical assessments led to the diagnosis of four PWS patients, two AS patients, and two individuals with 15q11-q13 duplication syndrome. CONCLUSION: Our deep phenotypic observations identified a spectrum of clinical features that overlap and are unique to PWS, AS, and Dup15q syndromes. Our findings establish genotype-phenotype correlation for patients impacted by variable structural variations within the 15q11-q13 region.

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.

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 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.

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.

Novel mutations in actionable breast cancer genes by targeted sequencing in an ethnically homogenous cohort.

BACKGROUND: Genetic testing is becoming an essential tool for breast cancer (BC) diagnosis and treatment pathway, and particularly important for early detection and cancer prevention. The purpose of this study was to explore the diagnostic yield of targeted sequencing of the high priority BC genes. METHODS: We have utilized a cost-effective targeted sequencing approach of high priority actionable BC genes (BRCA1, BRCA2, ERBB2 and TP53) in a homogeneous patient cohort from Bangladesh (n = 52) by using tumor and blood samples. RESULTS: Blood derived targeted sequencing revealed 25.58% (11/43) clinically relevant mutations (both pathogenic and variants of uncertain significance (VUS)), with 13.95% (6/43) of samples carrying a pathogenic mutations. We have identified and validated five novel pathogenic germline mutations in this cohort, comprising of two frameshift deletions in BRCA2, and missense mutations in BRCA1, BRCA2 and ERBB2 gene respectively. Furthermore, we have identified three pathogenic mutations and a VUS within three tumor samples, including a sample carrying pathogenic mutations impacting both TP53 (c.322dupG; a novel frameshift insertion) and BRCA1 genes (c.116G > A). 22% of tissue samples had a clinically relevant TP53 mutation. Although the cohort is small, we have found pathogenic mutations to be enriched in BRCA2 (9.30%, 4/43) compare to BRCA1 (4.65%, 2/43). The frequency of germline VUS mutations found to be similar in both BRCA1 (4.65%; 2/43) and BRCA2 (4.65%; 2/43) compared to ERBB2 (2.32%; 1/43). CONCLUSIONS: This is the first genetic study of BC predisposition genes in this population, implies that genetic screening through targeted sequencing can detect clinically significant and actionable BC-relevant mutations.

Gonadal mosaicism of large terminal de novo duplication and deletion in siblings with variable intellectual disability phenotypes.

BACKGROUND: Intellectual disability (ID) is a complex condition that can impact multiple domains of development. The genetic contribution to ID's etiology is significant, with more than 100 implicated genes and loci currently identified. The majority of such variants are rare and de novo genetic mutations. METHODS: We have applied whole-genome microarray to identify large, rare, clinically relevant copy number variants (CNVs). We have applied well-established algorithms for variants call. Quantitative polymerase chain reaction (qPCR) was applied to validate the variants using three technical replicates for each family member. To assess whether the copy number variation was due to balanced translocation or mosaicism, we further conducted droplet digital PCR (ddPCR) on the whole family. We have, as well, applied "critical-exon" mapping, human developmental brain transcriptome, and a database of known associated neurodevelopmental disorder variants to identify candidate genes. RESULTS: Here we present two siblings who are both impacted by a large terminal duplication and a deletion. Whole-genome microarray revealed an 18.82 megabase (MB) duplication at terminal locus (7q34-q36.3) of chromosome 7 and a 3.90 MB deletion impacting the terminal locus (15q26.3) of chromosome 15. qPCR and ddPCR experiments confirmed the de novo origin of the variants and the co-occurrence of these two de novo events among the siblings, but their absence in both parents, implicates an unbalanced translocation that could have mal-segregated among the siblings or a possible germline mosaicism. These terminal events impact IGF1R, CNTNAP2, and DPP6, shown to be strongly associated with neurodevelopmental disorders. Detailed clinical examination of the siblings revealed the presence of both shared and distinct phenotypic features. CONCLUSIONS: This study identified two large rare terminal de novo events impacting two siblings. Further phenotypic investigation highlights that even in the presence of identical large high penetrant variants, spectrum of clinical features can be different between the siblings.

Antibody-drug conjugate T-DM1 treatment for HER2+ breast cancer induces ROR1 and confers resistance through activation of Hippo transcriptional coactivator YAP1.

BACKGROUND: A newly developed drug trastuzumab emtansine (T-DM1) has improved the survival of breast cancer (BC) patients. Despite an impressive initial clinical response, a subgroup of patient develop resistance and present therapeutic challenges. The underlying resistance mechanisms are not fully investigated. We report that T-DM1 treatment modulates the expression of ROR1 (type 1 receptor tyrosine kinase-like orphan receptor) and induces self-renewal of cancer stem cells (CSCs) leading to therapeutic resistance. METHODS: Using BC patient tumor samples, and BC cell lines we gained insight into the T-DM1 treatment induced ROR1 overexpression and resistance. In vitro sphere forming assays and in vivo extreme dilution assays were employed to analyze the stemness and self-renewal capacity of the cells. A series of molecular expression and protein assays including qRT-PCR, FACS-sorting, ELISA, immunostaining, Western blotting were used to provide evidence. FINDINGS: Exposure of cells to T-DM1 shifted ROR1 expression from low to high, enriched within the CSC subpopulation, coincident with increased Bmi1 and stemness factors. T-DM1 induced ROR1 cells showed high spheroid and tumor forming efficiency in vitro and in an animal model exhibiting shorter tumor-free time. Mechanistically, the overexpression of ROR1 is partly induced by the activation of YAP1 and its target genes. Silencing of ROR1 and YAP1 by pharmacologic inhibitors and/or sh/siRNA inhibited spheroid formation, the initiation of tumors and the capacity for self-renewal and ROR1 overexpression. INTERPRETATIONS: The results presented here indicate that simultaneous targeting of ROR1 and YAP1 may suppress CSC self-renewal efficacy and inhibit tumor progression in BC. In this manner such treatments may overcome the T-DM1 mediated therapeutic resistance and improve clinical outcome. FUND: This study was supported by Neurogen Technologies for interdisciplinary research.

An ANKRD26 nonsense somatic mutation in a female with epidermodysplasia verruciformis (Tree Man Syndrome).

Epidermodysplasia verruciformis (EV) is an extremely rare hereditary skin disease characterized by an abnormal susceptibility to the human papilloma virus (HPV) with an increased risk of cutaneous malignancy. Here we report the first female severe EV case in Bangladesh, a 10-year-old girl with a nonsense somatic mutation impacting ANKRD26 gene.