Whole Exome Sequencing Data Research Articles

Early-onset atrial fibrillation is a risk marker for cardiomyopathy: genetic insights from the UK Biobank

Abstract Introduction Atrial fibrillation (AF) is a common cardiac arrhythmia associated with an increased risk of stroke, heart failure and premature death. Genetic associations between AF and rare variants have been identified in several cardiomyopathy-associated genes, and previous studies have shown a high prevalence of deleterious variants among individuals with early onset of AF. Consequently, genetic testing has been suggested in patients with AF onset before 45 years of age. Purpose This study aimed to explore the prevalence and distribution of deleterious genetic variants in cardiomyopathy genes according to age at AF diagnosis. Methods The UK Biobank is a large population-based biobank, comprised of clinical and genetic information on more than 500,000 individuals, enrolled between 2006 and 2010. We analyzed whole-exome sequencing data among unrelated individuals of European ancestry. The cohort was stratified by age at AF onset (AF onset <45 years, AF onset between 45-54 years, AF onset between 55-64 years, AF onset >=65 years, and individuals with no AF diagnosis). We evaluated the presence of rare (minor allele frequency <0.1%) genetic variants in core cardiomyopathy genes, according to guidelines by the American College of Medical Genomics and focused on variants predicted to lead to loss of function. Individuals with a diagnosis of cardiomyopathy prior to AF were excluded. Results Among 374,365 unrelated individuals of European ancestry, 29,119 were diagnosed with AF. Of these, 685 had onset of AF<45 years (2.35%), 2465 had onset between 45-54 years (8.46%), 7778 had onset between 55-64 years (26.71%), and 18191 had onset at or above 65 years of age (62.48%). A total of 884 were carriers of rare loss-of-function variants in cardiomyopathy genes. Among carriers, the majority of variants were in key genes for dilated cardiomyopathy (TTN), hypertrophic cardiomyopathy (MYBPC3), and arrhythmogenic right ventricular cardiomyopathy (PKP2). We observed an inverse dose-response-like relationship between prevalence of the rare genetic variants and age at AF diagnosis. The highest prevalence of carriers was observed among individuals with onset before 45 years (4.96%). In comparison, the proportion of carriers was 3.45% in individuals with AF onset between 45-54 years, 2.96% in individuals with AF onset between 55-64 years, 2.75% in individuals with onset at or after 65 years, and 2.05% among individuals without AF diagnosis. Conclusions Using whole exome sequencing, we identified a high proportion of carriers of deleterious variants in actionable genes of cardiomyopathies among individuals with early onset of AF. We observed an inverse dose-response-like relationship between age of AF diagnosis and prevalence of deleterious genetic variants. Our findings support that very early onset of AF, before 45 years of age, is a potential risk marker for cardiomyopathy and support that genetic screening may be relevant in this patient group.

A homozygous variant in ARHGAP39 is associated with lethal cerebellar vermis hypoplasia in a consanguineous Saudi family

Cerebellar vermis hypoplasia refers to a varying degree of incomplete development of the cerebellum and vermis. A Saudi family with four affected individuals with cerebellar vermis hypoplasia, facial dysmorphology, visual impairment, skeletal, and cardiac abnormalities was ascertained in this study. Three out of four patients could not survive longer and had died in early infancy. Genetic analysis of the youngest affected was performed by genome-wide homozygosity mapping coupled with whole exome sequencing (WES), followed by Sanger validation. Genome-wide genotyping analysis mapped the phenotype to chromosome 8q24.3. Using an autosomal recessive model, considering deleterious variants with minor allele frequency of less than 0.001 in WES data, a homozygous missense variant (NM_025251.2; ARHGAP39; c.1301G > T; p.Cys434Phe) was selected as a potential candidate for the phenotype. The variant (c.1301G > T) in the ARHGAP39 is in the region of homozygosity on chromosome 8q24.3. ARHGAP39 is a Rho GTPase-activating protein 39 and has been known to regulate apoptosis, cell migration, neurogenesis, and cerebral and hippocampal dendritic spine morphology. Mice homozygous for arhgap39 knockouts have shown premature embryonic lethality. Our findings present the first ever human phenotype associated with ARHGAP39 alteration.

Expanded carrier screening for 216 diseases in a cohort of 3 097 healthy Chinese individuals of childbearing age

Objective: To determine the carrier frequency and hot-spot variants of a custom-designed expanded carrier screening (ECS) panel with 216 diseases (216-ECS panel) within a Chinese population of childbearing age. Methods: Whole-exome sequencing data from a cohort of 3 097 unrelated healthy individuals (including 1 424 couples) from Peking Union Medical College Hospital between January 2013 and December 2023 were analyzed. Totally 220 genes which inherited in a recessive manner of 216-ECS panel were included in the analysis. The analysis included variant carrier rate, gene carrier rate, cumulative carrier rate, at-risk couple rates, and variant spectrum. Results: (1) Pathogenic variants were identified in 1 472 (47.53%, 1 472/3 097) individuals, with an average of 0.65 pathogenic variants per individual. The rate of at-risk couples was 3.93% (56/1 424). (2) A total of 180 genes were identified, with 16 genes exhibiting a gene carrier rate of ≥1% and 33 genes having a rate of ≥0.5%, most of which were associated with inherited metabolic diseases. Noteworthy genes with higher gene carrier rates and high-frequency variants included GJB2: c.235del, PAH: c.728G>A, ATP7B: c.2333G>T, SLC26A4: c.919-2A>G, GALC: c.1901T>C, POLG: c.2890C>T, SLC22A5: c.1472C>G, USH2A: c.2802T>G, SLC25A13: c.852_855del, GAA: c.761C>T and c.752C>T. Conclusion: This study offers a focused analysis of carrier frequencies and hot-spot variants of 216 diseases of the ECS panel constructed by our laboratory among the Chinese population, laying a foundation for the development of ECS programs tailored to the Chinese population.

Androgen production, uptake, and conversion (APUC) genes define prostate cancer patients with distinct clinical outcomes.

BACKGROUNDProstate cancer (PC) is driven by aberrant signaling of the androgen receptor (AR) or its ligands, and androgen deprivation therapies (ADTs) are a cornerstone of treatment. ADT responsiveness may be associated with germline changes in genes that regulate androgen production, uptake, and conversion (APUC).METHODSWe analyzed whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) data from prostate tissues (SU2C/PCF, TCGA, GETx). We also interrogated the Caris Precision Oncology Alliance (POA) DNA (592-gene/whole exome) and RNA (whole transcriptome) next-generation sequencing databases. Algorithm for Linking Activity Networks (ALAN) was used to quantify all pairwise gene-to-gene associations. Real-world overall survival was determined from insurance claims data using Kaplan-Meier estimates.RESULTSSix APUC genes (HSD3B1, HSD3B2, CYP3A43, CYP11A1, CYP11B1, CYP17A1) exhibited coalescent gene behavior in a cohort of metastatic tumors (n = 208). In the Caris POA dataset, the 6 APUC genes (APUC-6) exhibited robust clustering in primary prostate (n = 4,490) and metastatic (n = 2,593) biopsies. Surprisingly, tumors with elevated APUC-6 expression had statically lower expression of AR, AR-V7, and AR signaling scores, suggesting ligand-driven disease biology. APUC-6 genes instead associated with the expression of alternative steroid hormone receptors, ESR1/2 and PGR. We used RNA expression of AR or APUC-6 genes to define 2 subgroups of tumors with differential association with hallmark pathways and cell surface targets.CONCLUSIONSThe APUC-6-high/AR-low tumors represented a subgroup of patients with good clinical outcomes, in contrast with the AR-high or neuroendocrine PCs. Altogether, measuring the aggregate expression of APUC-6 genes in current genomic tests identifies PCs that are ligand (rather than AR) driven and require distinct therapeutic strategies.FUNDINGNCI/NIH 1R37CA288972-01, NCI Cancer Center Support P30 CA077598, DOD W81XWH-22-2-0025, R01 CA249279.

Depletion of loss-of-function germline mutations in centenarians reveals longevity genes

While previous studies identified common genetic variants associated with longevity in centenarians, the role of the rare loss-of-function (LOF) mutation burden remains largely unexplored. Here, we investigated the burden of rare LOF mutations in Ashkenazi Jewish individuals from the Longevity Genes Project and LonGenity study cohorts using whole-exome sequencing data. We found that centenarians had a significantly lower burden (11-22%) of LOF mutations compared to controls. Similar effects were also observed in their offspring. Gene-level burden analysis identified 35 genes with depleted LOF mutations in centenarians, with 14 of these validated in the UK Biobank. Mendelian randomization and multi-omic analyses on these genes identified RGP1, PCNX2, and ANO9 as longevity genes with consistent causal effects on multiple aging-related traits and altered expression during aging. Our findings suggest that a protective genetic background, characterized by a reduced burden of damaging variants, contributes to exceptional longevity, likely acting in concert with specific protective variants to promote healthy aging.

Abstract A017: Identifying proliferative and immune-modulatory drivers of small cell lung cancer (SCLC)

Abstract Small cell lung cancer (SCLC) is a highly aggressive form of lung cancer that causes an estimated 200,000 patient deaths globally every year. Most (>70%) patients present with extensive stage (ES)-SCLC, and the disease primarily presents with a neuroendocrine (NE), immune-desert phenotype, with low expression of antigen-presenting machinery genes. Despite limited immune infiltration in the tumor, SCLC has reproducibly responded to immune checkpoint blockade. IMpower133 was a pivotal Phase III trial that added atezolizumab (anti-PD-L1) to platinum/etoposide chemotherapy for treatment of first-line ES-SCLC, which led to the first approval of an immune checkpoint blockade strategy in SCLC. While the inclusion of atezolizumab to the treatment regimen has extended the survival of some patients, SCLC remains an unmet medical need with a mOS of 12.3 months. Furthermore, we lack a deep molecular understanding of factors mediating response and resistance to standard of care therapies in SCLC. To understand the genetic drivers of SCLC that could be linked to immunotherapy resistance, we analyzed the matched whole-exome sequencing and RNA-seq data from baseline tumor samples collected from the IMpower133 trial. Previously, we applied the non-negative matrix factorization (NMF) approach to the RNA-seq data, which clustered patients into four subsets of SCLC, with varying degrees of immune infiltration, neuroendocrine program activation, and clinical benefit from immunotherapy. Notably, we identified an inflamed-neuroendocrine (I-NE) subset of patients that exhibit longer overall survival with chemoimmunotherapy compared to chemotherapy alone, and an inflamed-non-neuroendocrine (I-nonNE) subset of patients that did not show a similar benefit from the addition of immunotherapy to chemotherapy. Here, we interrogated the matched WES data for recurrent alterations that may identify subsets of tumors with distinct immunological features. We identified several genes whose alterations significantly affected the enrichment of immune-related gene signatures. In particular, we identified MANF and CSK, genes previously shown to be potential tumor suppressors, as genes whose alteration associates with an “immune desert” transcriptional program. Specifically, genetic deletions or low RNA expression was associated with significantly decreased gene expression of gene signatures associated with lymphocytes, macrophages, and antigen presentation machinery. These data suggest a potential mechanism by which commonly inactivated tumor suppressors may shape the “immune desert” phenotype of SCLC. Further work is needed to determine if and how these genes may directly or indirectly modulate the tumor-immune interaction. Citation Format: Myung Chang Lee, Velimir Gayevskiy, Stefanie Morris, Minu K Srivastava, Namrata S Patil, Stephen V Liu, Martin Reck, Sanjeev Mariathasan, David S Shames, David P MacPherson, Robert J Johnston, Barzin Y Nabet. Identifying proliferative and immune-modulatory drivers of small cell lung cancer (SCLC) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A017.

Personalized oncology in pheochromocytomas and paragangliomas: integrating genetic analysis with machine learning.

Pheochromocytomas and paragangliomas (PCCs/PGLs) are uncommon neuroendocrine tumors with a significant genetic tendency. Approximately 35-40% of these tumors are associated with genetic factors. The present study performed a thorough analysis using publicly accessible genetic and clinical data from the Cancer Genome Atlas (TCGA) to examine the involvement of six genes, namely GBP1, KIF13B, GPT, CSDE1, CEP164, and CLCA1, in the development of PCCs/PGLs. By employing multi-omics data, this study investigates the relationship between mutational patterns and the prognosis of tumors, focusing on the possibility of tailoring treatment methods to individual patients. The study utilizes Mutect2 to detect somatic mutations with high confidence in whole-exome sequencing data from PCCG samples. The study uncovers mild effects on protein function caused by particular mutations, including GBP1 (p.Cys12Tyr), KIF13B (p.Arg847Gly), and GPT (p.Gln50Arg). A random forest classifier uses mutational profiles to predict potential drug recommendations, proposing a focused therapy strategy. This study thoroughly analyzes the genetic mutations found in PCCs/PGLs, highlighting the significance of precision medicine in developing specific treatments for these uncommon types of cancer. This study aims to improve the understanding of the development of tumors and identify personalized treatment approaches by combining genetic data with machine learning analyses.

Heterozygous variants in the teashirt zinc finger homeobox 3 (TSHZ3) gene in human congenital anomalies of the kidney and urinary tract.

Around 180 genes have been associated with congenital anomalies of the kidney and urinary tract (CAKUT) in mice, and represent promising novel candidate genes for human CAKUT. In whole-exome sequencing data of two siblings with genetically unresolved multicystic dysplastic kidneys (MCDK), prioritizing variants in murine CAKUT-associated genes yielded a rare variant in the teashirt zinc finger homeobox 3 (TSHZ3) gene. Therefore, the role of TSHZ3 in human CAKUT was assessed. Twelve CAKUT patients from 9/301 (3%) families carried five different rare heterozygous TSHZ3 missense variants predicted to be deleterious. CAKUT patients with versus without TSHZ3 variants were more likely to present with hydronephrosis, hydroureter, ureteropelvic junction obstruction, MCDK, and with genital anomalies, developmental delay, overlapping with the previously described phenotypes in Tshz3-mutant mice and patients with heterozygous 19q12-q13.11 deletions encompassing the TSHZ3 locus. Comparable with Tshz3-mutant mice, the smooth muscle layer was disorganized in the renal pelvis and thinner in the proximal ureter of the nephrectomy specimen of a TSHZ3 variant carrier compared to controls. TSHZ3 was expressed in the human fetal kidney, and strongly at embryonic day 11.5-14.5 in mesenchymal compartments of the murine ureter, kidney, and bladder. TSHZ3 variants in a 5' region were more frequent in CAKUT patients than in gnomAD samples (p < 0.001). Mutant TSHZ3 harboring N-terminal variants showed significantly altered SOX9 and/or myocardin binding, possibly adversely affecting smooth muscle differentiation. Our results provide evidence that heterozygous TSHZ3 variants are associated with human CAKUT, particularly MCDK, hydronephrosis, and hydroureter, and, inconsistently, with specific extrarenal features, including genital anomalies.

The clinical and genetic spectrum of paediatric speech and language disorders.

Speech and language disorders are known to have a substantial genetic contribution. Although frequently examined as components of other conditions, research on the genetic basis of linguistic differences as separate phenotypic subgroups has been limited so far. Here, we performed an in-depth characterization of speech and language disorders in 52 143 individuals, reconstructing clinical histories using a large-scale data-mining approach of the electronic medical records from an entire large paediatric healthcare network. The reported frequency of these disorders was the highest between 2 and 5 years old and spanned a spectrum of 26 broad speech and language diagnoses. We used natural language processing to assess the degree to which clinical diagnoses in full-text notes were reflected in ICD-10 diagnosis codes. We found that aphasia and speech apraxia could be retrieved easily through ICD-10 diagnosis codes, whereas stuttering as a speech phenotype was coded in only 12% of individuals through appropriate ICD-10 codes. We found significant comorbidity of speech and language disorders in neurodevelopmental conditions (30.31%) and, to a lesser degree, with epilepsies (6.07%) and movement disorders (2.05%). The most common genetic disorders retrievable in our analysis of electronic medical records were STXBP1 (n = 21), PTEN (n = 20) and CACNA1A (n = 18). When assessing associations of genetic diagnoses with specific linguistic phenotypes, we observed associations of STXBP1 and aphasia (P = 8.57 × 10-7, 95% confidence interval = 18.62-130.39) and MYO7A with speech and language development delay attributable to hearing loss (P = 1.24 × 10-5, 95% confidence interval = 17.46-infinity). Finally, in a sub-cohort of 726 individuals with whole-exome sequencing data, we identified an enrichment of rare variants in neuronal receptor pathways, in addition to associations of UQCRC1 and KIF17 with expressive aphasia, MROH8 and BCHE with poor speech, and USP37, SLC22A9 and UMODL1 with aphasia. In summary, our study outlines the landscape of paediatric speech and language disorders, confirming the phenotypic complexity of linguistic traits and novel genotype-phenotype associations. Subgroups of paediatric speech and language disorders differ significantly with respect to the composition of monogenic aetiologies.

Copy Number Analysis of Whole Exome Sequencing Data

Abstract Background Genetic alterations implicated in constitutional disorders range in size from single nucleotide substitutions to losses and gains of millions of bases. Whole exome sequencing (WES) captures many clinically relevant variants, but an analytic pipeline optimized for detection of small substitutions, insertions, and deletions may miss pathogenic copy number variants (CNVs). Historically, our laboratory performed separate cytogenetic tests to evaluate for larger genomic imbalances. Addition of copy number analysis to our WES pipeline could improve the assay’s diagnostic yield. This study describes part of our strategy to validate copy number analysis of our constitutional whole exome sequencing data. Methods We identified 18 CNV specimens that underwent both chromosomal microarray analysis (CMA) and WES between 2017 and 2023. Variants included 13 deletions (size range 14Kb-2Mb) and 5 duplications (size range 325Kb-10Mb). The standard method of constitutional copy number analysis in our clinical laboratory is CMA using the Affymetrix Cytoscan HD Array and analysis software. CNVs identified on CMA were reviewed with reference to the Database of Genomic Variants and multiple academic hospital databases. WES was performed on an Illumina sequencing system following capture-based library enrichment with the Integrated DNA Technologies xGen Exome Research, CNV Backbone, Human mtDNA Research, and Human ID Research Panel probes. CNVs identified on WES were called using the Broad Institute’s Genomic Analysis Toolkit (GATK) with a quality score cutoff of 120. Whole exome sequencing data was visualized on the Integrative Genomics Viewer. Results 14 out of 18 CNVs reported on CMA were also detected on WES with a quality score at or above 120 (78%). This includes 10 out of 13 deletions (77%) and 3 out of 5 duplications (60%). For both deletions and duplications, the size ranges of detected variants at or above the cutoff score and undetected variants overlapped. One duplication reported on CMA was detected on WES with a quality score below 120. Visualization of whole exome sequencing data showed variation in probe density that may have affected CNV calling. Conclusions The majority of CNVs reported after CMA (14/18, 78%) were also detected on WES data using a GATK quality score cutoff of 120. The data does not suggest an association between CNV size and detection by WES, although interpretation is limited by the small number of cases. Data visualization may be required to identify some CNVs. Future steps to characterize the limitations of copy number analysis of WES data include assessments of variance in probe density and read depth across the exome. Copy number analysis of WES data from cases with normal CMA results will also be performed for assay validation.

Enhancing Variant Calling in Whole-Exome Sequencing Data Using Population-Matched Reference Genomes.

Whole-exome sequencing (WES) data are frequently used for cancer diagnosis and genome-wide association studies (GWAS), based on high-coverage read mapping, informative variant calling, and high-quality reference genomes. The center position of the currently used genome assembly, GRCh38, is now challenged by two newly published telomere-to-telomere (T2T) genomes, T2T-CHM13 and T2T-YAO, and it becomes urgent to have a comparative study to test population specificity using the three reference genomes based on real case WES data. Here we report our analysis along this line for 19 tumor samples collected from Chinese patients. The primary comparison of the exon regions among the three references reveals that the sequences in up to ∼ 1% target regions in T2T-YAO are widely diversified from GRCh38 and may lead to off-target in sequence capture. However, T2T-YAO still outperforms GRCh38 genomes by obtaining 7.41% more mapped reads. Due to more reliable read-mapping and closer phylogenetic relationship with the samples than GRCh38, T2T-YAO reduces half of variant calls of clinical significance which are mostly benign, while maintaining sensitivity in identifying pathogenic variants. T2T-YAO also outperforms T2T-CHM13 in reducing calls of Chinese-specific variants. Our findings highlight the critical need for employing population-specific reference genomes in genomic analysis to ensure accurate variant analysis and the significant benefits of tailoring these approaches to the unique genetic backgrounds of each ethnic group.

9269 Retrotranscribed sequences, a missing piece in the puzzle of Differences/Disorders of Sex Development with unknown genetic causes

Abstract Disclosure: R.L. Batista: None. N.D. D’Alessandre: None. F.O. Rego: None. G.D. Guardia: None. B. Leitao Braga: None. M.Y. Nishi: None. S. Domenice: None. B.B. Mendonca: None. P.A. Galante: None. Background: The human genome is a complex and dynamic structure. These two characteristics have a major contribution from the Mobile Elements (MEs), which comprise at least 50% of the human genome and are sources of genetic novelties. LINE1 (L1), Alu elements, SVA, and LTR elements (HERVs) are MEs highly frequent in the human genome. MEs can create genomic structural variations with consequent roles in health and diseases. Differences of sex development (DSD) are a heterogeneous group of congenital conditions that result in discordance between an individual’s sex chromosomes, gonads, or anatomic sex. DSDs are caused by genomic variations in genes involved in gonadal or genital development.Objective: We comprehensively examine the potential contributions of MEs as a source of genetic novelties in genes related to sexual development and in the etiology of 46,XY DSD. Subjects and methods: First, we investigate the potential role of fixed MEs, including L1, Alu elements, and retroCNVs, present in DSD genes using whole exome sequencing (WES) data. By applying bioinformatics tools and appropriate pipelines, we parallelly analyzed local WES data from 443 individuals from our lab and a public database (GnomAD). Second, we seek unfixed (polymorphic or somatically acquired) MEs in WES data from a cohort of 41 unrelated DSD patients with undetermined molecular etiology. We found 2,408 L1 elements, 3,587 Alu elements, and 14 SVAs inserted in 75 out of 81 DSD-related genes (92.6%) of the DSD genes. When examining by element class, we identified 3,587 fixed Alus that affected over 70 DSD genes, resulting in an average of approximately 47 Alu element insertions per gene. For L1 elements, 2,408 fixed elements were found in about 60 genes (∼37 insertions per gene). Subsequently, the number of fixed elements identified in SVAs (n=14) and HERVKs (n=8) decreased significantly. Among these insertions, seven genes were identified harboring one retrocopy each and four genes with two or more retrocopies each. In total, we found 19 retrocopies in 11 DSD genes. That massive presence of mobile elements in DSD genes may suggest an evolutionary contribution of MEs incorporation through evolution in human sexual development. Regarding non-fixed elements, we found two polymorphic events: an L1 insertion into WT1 and an Alu insertion into GTF2F1, two key genes to the DSD development. We also found an L1 insertion in the DNAH2 gene (related to spermatogenic failure and male infertility) and an Alu insertion in the TEX15 gene (related to male infertility in mice). Interestingly, we also identified one somatically acquired retroCNV event in a non-classical DSD gene (CHST8). In summary, our study underscores the significance of MEs as potential sources of genetic innovation in human sexual development and as molecular etiologies of DSD. Presentation: 6/1/2024

9269 Retrotranscribed sequences, a missing piece in the puzzle of Differences/Disorders of Sex Development with unknown genetic causes

Abstract Disclosure: R.L. Batista: None. N.D. D’Alessandre: None. F.O. Rego: None. G.D. Guardia: None. B. Leitao Braga: None. M.Y. Nishi: None. S. Domenice: None. B.B. Mendonca: None. P.A. Galante: None. Background: The human genome is a complex and dynamic structure. These two characteristics have a major contribution from the Mobile Elements (MEs), which comprise at least 50% of the human genome and are sources of genetic novelties. LINE1 (L1), Alu elements, SVA, and LTR elements (HERVs) are MEs highly frequent in the human genome. MEs can create genomic structural variations with consequent roles in health and diseases. Differences of sex development (DSD) are a heterogeneous group of congenital conditions that result in discordance between an individual’s sex chromosomes, gonads, or anatomic sex. DSDs are caused by genomic variations in genes involved in gonadal or genital development.Objective: We comprehensively examine the potential contributions of MEs as a source of genetic novelties in genes related to sexual development and in the etiology of 46,XY DSD. Subjects and methods: First, we investigate the potential role of fixed MEs, including L1, Alu elements, and retroCNVs, present in DSD genes using whole exome sequencing (WES) data. By applying bioinformatics tools and appropriate pipelines, we parallelly analyzed local WES data from 443 individuals from our lab and a public database (GnomAD). Second, we seek unfixed (polymorphic or somatically acquired) MEs in WES data from a cohort of 41 unrelated DSD patients with undetermined molecular etiology. We found 2,408 L1 elements, 3,587 Alu elements, and 14 SVAs inserted in 75 out of 81 DSD-related genes (92.6%) of the DSD genes. When examining by element class, we identified 3,587 fixed Alus that affected over 70 DSD genes, resulting in an average of approximately 47 Alu element insertions per gene. For L1 elements, 2,408 fixed elements were found in about 60 genes (∼37 insertions per gene). Subsequently, the number of fixed elements identified in SVAs (n=14) and HERVKs (n=8) decreased significantly. Among these insertions, seven genes were identified harboring one retrocopy each and four genes with two or more retrocopies each. In total, we found 19 retrocopies in 11 DSD genes. That massive presence of mobile elements in DSD genes may suggest an evolutionary contribution of MEs incorporation through evolution in human sexual development. Regarding non-fixed elements, we found two polymorphic events: an L1 insertion into WT1 and an Alu insertion into GTF2F1, two key genes to the DSD development. We also found an L1 insertion in the DNAH2 gene (related to spermatogenic failure and male infertility) and an Alu insertion in the TEX15 gene (related to male infertility in mice). Interestingly, we also identified one somatically acquired retroCNV event in a non-classical DSD gene (CHST8). In summary, our study underscores the significance of MEs as potential sources of genetic innovation in human sexual development and as molecular etiologies of DSD. Presentation: 6/1/2024

8703 Retrotranscribed sequences, a missing piece in the puzzle of Differences/Disorders of Sex Development with unknown genetic causes

Abstract Disclosure: R.L. Batista: None. N.D. D’Alessandre: None. F.O. Rego: None. G.D. Guardia: None. B. Leitao Braga: None. M.Y. Nishi: None. S. Domenice: None. B.B. Mendonca: None. P.A. Galante: None. Background: The human genome is a complex and dynamic structure. These two characteristics have a major contribution from the Mobile Elements (MEs), which comprise at least 50% of the human genome and are sources of genetic novelties. LINE1 (L1), Alu elements, SVA, and LTR elements (HERVs) are MEs highly frequent in the human genome. MEs can create genomic structural variations with consequent roles in health and diseases. Differences of sex development (DSD) are a heterogeneous group of congenital conditions that result in discordance between an individual’s sex chromosomes, gonads, or anatomic sex. DSDs are caused by genomic variations in genes involved in gonadal or genital development.Objective: We comprehensively examine the potential contributions of MEs as a source of genetic novelties in genes related to sexual development and in the etiology of 46,XY DSD. Subjects and methods: First, we investigate the potential role of fixed MEs, including L1, Alu elements, and retroCNVs, present in DSD genes using whole exome sequencing (WES) data. By applying bioinformatics tools and appropriate pipelines, we parallelly analyzed local WES data from 443 individuals from our lab and a public database (GnomAD). Second, we seek unfixed (polymorphic or somatically acquired) MEs in WES data from a cohort of 41 unrelated DSD patients with undetermined molecular etiology. We found 2,408 L1 elements, 3,587 Alu elements, and 14 SVAs inserted in 75 out of 81 DSD-related genes (92.6%) of the DSD genes. When examining by element class, we identified 3,587 fixed Alus that affected over 70 DSD genes, resulting in an average of approximately 47 Alu element insertions per gene. For L1 elements, 2,408 fixed elements were found in about 60 genes (∼37 insertions per gene). Subsequently, the number of fixed elements identified in SVAs (n=14) and HERVKs (n=8) decreased significantly. Among these insertions, seven genes were identified harboring one retrocopy each and four genes with two or more retrocopies each. In total, we found 19 retrocopies in 11 DSD genes. That massive presence of mobile elements in DSD genes may suggest an evolutionary contribution of MEs incorporation through evolution in human sexual development.Regarding non-fixed elements, we found two polymorphic events: an L1 insertion into WT1 and an Alu insertion into GTF2F1, two key genes to the DSD development. We also found an L1 insertion in the DNAH2 gene (related to spermatogenic failure and male infertility) and an Alu insertion in the TEX15 gene (related to male infertility in mice). Interestingly, we also identified one somatically acquired retroCNV event in a non-classical DSD gene (CHST8). In summary, our study underscores the significance of MEs as potential sources of genetic innovation in human sexual development and as molecular etiologies of DSD. Presentation: 6/1/2024

8703 Retrotranscribed sequences, a missing piece in the puzzle of Differences/Disorders of Sex Development with unknown genetic causes

Abstract Disclosure: R.L. Batista: None. N.D. D’Alessandre: None. F.O. Rego: None. G.D. Guardia: None. B. Leitao Braga: None. M.Y. Nishi: None. S. Domenice: None. B.B. Mendonca: None. P.A. Galante: None. Background: The human genome is a complex and dynamic structure. These two characteristics have a major contribution from the Mobile Elements (MEs), which comprise at least 50% of the human genome and are sources of genetic novelties. LINE1 (L1), Alu elements, SVA, and LTR elements (HERVs) are MEs highly frequent in the human genome. MEs can create genomic structural variations with consequent roles in health and diseases. Differences of sex development (DSD) are a heterogeneous group of congenital conditions that result in discordance between an individual’s sex chromosomes, gonads, or anatomic sex. DSDs are caused by genomic variations in genes involved in gonadal or genital development.Objective: We comprehensively examine the potential contributions of MEs as a source of genetic novelties in genes related to sexual development and in the etiology of 46,XY DSD. Subjects and methods: First, we investigate the potential role of fixed MEs, including L1, Alu elements, and retroCNVs, present in DSD genes using whole exome sequencing (WES) data. By applying bioinformatics tools and appropriate pipelines, we parallelly analyzed local WES data from 443 individuals from our lab and a public database (GnomAD). Second, we seek unfixed (polymorphic or somatically acquired) MEs in WES data from a cohort of 41 unrelated DSD patients with undetermined molecular etiology. We found 2,408 L1 elements, 3,587 Alu elements, and 14 SVAs inserted in 75 out of 81 DSD-related genes (92.6%) of the DSD genes. When examining by element class, we identified 3,587 fixed Alus that affected over 70 DSD genes, resulting in an average of approximately 47 Alu element insertions per gene. For L1 elements, 2,408 fixed elements were found in about 60 genes (∼37 insertions per gene). Subsequently, the number of fixed elements identified in SVAs (n=14) and HERVKs (n=8) decreased significantly. Among these insertions, seven genes were identified harboring one retrocopy each and four genes with two or more retrocopies each. In total, we found 19 retrocopies in 11 DSD genes. That massive presence of mobile elements in DSD genes may suggest an evolutionary contribution of MEs incorporation through evolution in human sexual development.Regarding non-fixed elements, we found two polymorphic events: an L1 insertion into WT1 and an Alu insertion into GTF2F1, two key genes to the DSD development. We also found an L1 insertion in the DNAH2 gene (related to spermatogenic failure and male infertility) and an Alu insertion in the TEX15 gene (related to male infertility in mice). Interestingly, we also identified one somatically acquired retroCNV event in a non-classical DSD gene (CHST8). In summary, our study underscores the significance of MEs as potential sources of genetic innovation in human sexual development and as molecular etiologies of DSD. Presentation: 6/1/2024

Association between rare, genetic variants linked to autism and ultrasonography fetal anomalies in children with autism spectrum disorder

BackgroundRecent evidence suggests that certain fetal anomalies detected upon prenatal ultrasound screenings are associated with autism spectrum disorder (ASD). In this cross-sectional study, we aimed to identify genetic variants associated with fetal ultrasound anomalies (UFAs) in children with ASD.MethodsThe study included all children with ASD who are registered in the database of the Azrieli National Center of Autism and Neurodevelopment and for whom both prenatal ultrasound and whole exome sequencing (WES) data were available. We applied our in-house integrative bioinformatics pipeline, AutScore, to these WES data to prioritize rare, gene-disrupting variants (GDVs) probably contributing to ASD susceptibily. Univariate statistics and multivariable regression were used to assess the associations between UFAs and GDVs identified in these children.ResultsThe study sample comprised 126 children, of whom 43 (34.1%) had at least one UFA detected in the prenatal ultrasound scan. A total of 87 candidate ASD genetic variants were detected in 60 children, with 24 (40%) children carrying multiple variants. Children with UFAs were more likely to have loss-of-function (LoF) mutations (aOR = 2.55, 95%CI: 1.13–5.80). This association was particularly noticeable when children with structural anomalies or children with UFAs in their head and brain scans were compared to children without UFAs (any mutation: aOR = 8.28, 95%CI: 2.29–30.01; LoF: aOR = 5.72, 95%CI: 2.08–15.71 and any mutation: aOR = 6.39, 95%CI: 1.34–30.47; LoF: aOR = 4.50, 95%CI: 1.32–15.35, respectively). GDVs associated with UFAs were enriched in genes highly expressed across all tissues (aOR = 2.76, 95%CI: 1.14–6.68). There was a weak, but significant, correlation between the number of mutations and the number of abnormalities detected in the same children (r = 0.21, P = 0.016).ConclusionsThe results provide valuable insights into the potential genetic basis of prenatal organogenesis abnormalities associated with ASD and shed light on the complex interplay between genetic factors and fetal development.

A heterozygous SPRY4 variant identified in female infertility characterized by reduced oocyte potential and early embryonic arrest.

Can novel genetic factors contributing to early embryonic arrest in infertile patients be identified, along with the underlying mechanisms of the pathogenic variant? We identified a heterozygous variant in the SPRY4 (sprouty RTK signaling antagonist 4) in infertile patients and conducted in vitro and in vivo studies to investigate the effects of the variant/deletion, highlighting its critical role in female reproductive health. SPRY4 acts as a negative regulator of receptor tyrosine kinases (RTKs) and functions as a tumor suppressor. Its abnormal expression can lead to recurrent miscarriage by affecting trophoblast function. In mice, Spry4 knockout (KO) leads to craniofacial anomalies and growth defects. A human study links the SPRY4 variant to a male patient with isolated hypogonadotropic hypogonadism (IHH), hypothetically impacting gonadotropin-releasing hormone (GnRH) neurons, and causing reproductive dysfunctions. SPRY4 is thus potentially integral in regulating endocrine homeostasis and reproductive function. To date, no study has reported SPRY4 variants associated with female fertility, and a causal relationship has not been established with functional evidence. Whole-exome sequencing (WES) was performed in 392 infertile women who suffered from primary infertility of unknown reason, and the heterozygous SPRY4 variant were identified in one independent family. The infertile patients presenting were recruited from July 2017 to November 2023. Women diagnosed with primary infertility were recruited from the Reproduction Center of Zhongshan Hospital, Fudan University. Genomic DNA was extracted from peripheral blood for WES analysis. The SPRY4 variant were identified through WES, in silico analysis, and variant screening. All variants were confirmed by Sanger sequencing. The effects of the variants were investigated in human embryonic kidney (HEK) 293T (HEK293T) cells via western blotting, and in mouse oocytes and embryos through complementary RNA (cRNA) injection, RNA sequencing, fluorescence, absorbance, and RT-qPCR assays. Gene function was further examined in Spry4 KO mice via histology, western blotting, ELISA, and RT-qPCR assays. We identified a missense heterozygous pathogenic variant in SPRY4 (GRCh38, GenBank: NM_030964.5, c.157C>T p.(Arg53Trp), rs200531302) that reduces SPRY4 protein levels in HEK293T cells and disrupts the redox system and mitochondrial function in mouse oocyte, and perturbs developmental potential in mouse embryos. These phenotypes could be partially reversed by the exogenous addition of Nrf1 cRNA. Additionally, Spry4-/- mice exhibit ovarian oxidative stress and decreased ovarian function. Due to the limited WES data and population, we identified only one family with a SPRY4 mutation. The deeper mechanism and therapeutic strategy should be further investigated through mutant mice and recovery experiment. Our study has identified a pathogenic variant in SPRY4 associated with early embryonic arrest in humans. These findings enhance our understanding of the role of SPRY4 in early embryonic development and present a new genetic marker for female infertility. This work was supported by the National Natural Science Foundation of China (82071643 and 82171655) and Natural Science Foundation of Shanghai (22ZR1456200). None of the authors have any competing interests. N/A.

Mugen-UMAP: UMAP visualization and clustering of mutated genes in single-cell DNA sequencing data

BackgroundThe application of Uniform Manifold Approximation and Projection (UMAP) for dimensionality reduction and visualization has revolutionized the analysis of single-cell RNA expression and population genetics. However, its potential in single-cell DNA sequencing data analysis, particularly for visualizing gene mutation information, has not been fully explored.ResultsWe introduce Mugen-UMAP, a novel Python-based program that extends UMAP’s utility to single-cell DNA sequencing data. This innovative tool provides a comprehensive pipeline for processing gene annotation files of single-cell somatic single-nucleotide variants and metadata to the visualization of UMAP projections for identifying clusters, along with various statistical analyses. Employing Mugen-UMAP, we analyzed whole-exome sequencing data from 365 single-cell samples across 12 non-small cell lung cancer (NSCLC) patients, revealing distinct clusters associated with histological subtypes of NSCLC. Moreover, to demonstrate the general utility of Mugen-UMAP, we applied the program to 9 additional single-cell WES datasets from various cancer types, uncovering interesting patterns of cell clusters that warrant further investigation. In summary, Mugen-UMAP provides a quick and effective visualization method to uncover cell cluster patterns based on the gene mutation information from single-cell DNA sequencing data.ConclusionsThe application of Mugen-UMAP demonstrates its capacity to provide valuable insights into the visualization and interpretation of single-cell DNA sequencing data. Mugen-UMAP can be found at https://github.com/tengchn/Mugen-UMAP

HAPLN3 p.T34A contributes to incomplete penetrance of moyamoya disease in Chinese carrying RNF213 p.R4810K.

The penetrance of the RNF213 p.R4810K, a founder mutation of moyamoya disease (MMD), is estimated to be only 1/150-1/300. However, the factors affecting its penetrance remain unclear. Therefore, our study aims to identify modifier genes associated with the incomplete penetrance of this founder mutation. Whole-exome sequencing (WES) was performed on 36 participants, including 22 MMD patients and 14 non-MMD controls with RNF213 p.R4810K mutation. Fisher's exact test was used to assess the presence of genetic variants that differed significantly between MMD patients and non-MMD controls. In order to exclude false-positive results, another 55 carriers were included to perform Fisher's exact test for the selected sites in the WES discovery stage. Subsequently, human brain microvascular endothelial cells were transfected with wild-type and mutant HAPLN3 for tube formation assays and western blotting to explore the impact of candidate genes on angiogenesis. Analysis of variants from WES data revealed a total of 12 non-synonymous variants. Through bioinformatics analysis, the focus was on the HAPLN3 p.T34A variant with a significant p value of 0.00731 in Fisher's exact test. Validation through Sanger sequencing confirmed the presence of this variant in the WES data. Invitro experiments revealed that silencing HAPLN3 and transfecting HAPLN3 p.T34A significantly enhanced tube formation and increased the relative protein expression of vascular endothelial growth factor in endothelial cells. These results suggest that HAPLN3 may function as a modifier gene of RNF213 p.R4810K, promoting the development of MMD and contributing to the incomplete penetrance of MMD founder mutations.

Abstract B010: Design of personalized neoantigen mRNA vaccines against breast cancer patients in Pakistan based on next-generation sequencing data

Abstract Breast cancer is the most common malignancy among women in Pakistan, with an outrageous increase in the incidence and mortality rate. Because of decreased effectiveness and lower survival rates, there is an urgent need for developing novel, personalized vaccines with enhanced activity. The development of a personalized mRNA vaccine illustrates a promising approach to combating the prevalence of breast cancer. We used the whole exome sequencing (WES) data of Pakistani tumor samples to predict immunogenic neoantigens. The WES data was aligned to the GRCM39 reference genome using BWA. The genome analysis toolkit (GATK) was employed for variant calling to identify somatic mutations. Using immune-bioinformatics tools, we predicted cytotoxic CD8+ T cell epitopes and helper CD4+ T cell epitopes within the identified neoantigens and designed vaccines by assembling the fusion of CTL neoepitopes, helper sequences, and adjuvants together with linkers. The Insilco ImmSim algorithm was used to examine the immune responses of the vaccine. The vaccine design was further explored by checking its simulation effect on the immune system, its physiochemical characteristics, its binding to specific immune system molecules, and cloning into an appropriate vector. In addition, molecular docking, MD simulation, and binding free energies were performed to investigate the interaction mechanism between the construct vaccine and Toll-like receptors (TLR2, TLR4, TLR7, and TLR9).By examining these factors, we aimed to ensure the vaccine's safety, stability, and ability to bind tightly to specific immune cells (class I MHC molecules), ultimately triggering a comprehensive immune response against breast cancer cells. Citation Format: Kayode Yomi Raheem, Muhammad Muddassar. Design of personalized neoantigen mRNA vaccines against breast cancer patients in Pakistan based on next-generation sequencing data [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr B010.