Significant Single Nucleotide Variants Research Articles

Exploring non-coding genetic variability in ACE2: Functional annotation and in vitro validation of regulatory variants

The surge in human whole-genome sequencing data has facilitated the study of non-coding region variations, yet understanding their biological significance remains a challenge. We used a computational workflow to assess the regulatory potential of non-coding variants, with a particular focus on the Angiotensin Converting Enzyme 2 (ACE2) gene. This gene is crucial in physiological processes and serves as the entry point for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing coronavirus disease 19 (COVID-19). In our analysis, using data from the gnomAD population database and functional annotation, we identified 17 significant Single Nucleotide Variants (SNVs) in ACE2, particularly in its enhancers, promoters, and 3′ untranslated regions (UTRs). We found preliminary evidence supporting the regulatory impact of some of these variants on ACE2 expression. Our detailed examination of two SNVs, rs147718775 and rs140394675, in the ACE2 promoter revealed that these co-occurring SNVs, when mutated, significantly enhance promoter activity, suggesting a possible increase in specific ACE2 isoform expression. This method proves effective in identifying and interpreting impactful non-coding variants, aiding in further studies and enhancing understanding of molecular bases of monogenic and complex traits.

Genetic Variants of the Beta-Adrenergic Receptor Pathways as Both Risk and Protective Factors for Retinopathy of Prematurity

PurposeThere is strong evidence that genetic factors influence retinopathy of prematurity (ROP), a neovascular eye disease. It has been previously suggested that polymorphisms in the genes involved in beta-adrenergic receptor (ADRβ) pathways could protect against ROP. Antagonists for the ADRβ are actively tested in clinical trials for ROP treatment but not without controversy and safety concerns. This study was designed to assess if genetic variations in components of the ADRβ signaling pathways associate with risk of developing ROP. DesignAn observational case-control targeted genetic analysis. MethodsA study was carried out on premature participants with (n=30) or without (n=34) ROP and full-term controls (n=20) which were divided into a discovery cohort and a validation cohort. ROP was defined using International Classification of Retinopathy of Prematurity criteria (ICROP). Targeted sequencing of 20 genes in the ADRβ pathways was performed on the discovery cohort. Polymerase chain reaction (PCR)/restriction enzyme analysis for some of the discovered ROP-associated variants was performed for validation of the results using the validation cohort. ResultsThe discovery cohort revealed 543 bi-allelic variants within 20 genes of the ADRβ pathways. Ten single nucleotide variants (SNVs) in five genes including protein kinase A regulatory subunit 1α (PRKAR1A), rap guanine exchange factor 3 (RAPGEF3), adenylyl cyclase 4 (ADCY4), ADCY7, and ADCY9 were associated with ROP (p<0.05). The most significant SNV was found in PRKAR1A (p=0.001). Multiple variants located in the 3’-untranslated region (3’UTR) of RAPGEF3 were also associated with ROP (p<0.05). PCR/restriction enzyme analysis of the 3’UTR of RAPGEF3 methodologically validated these findings. ConclusionSNVs in PRKAR1A may represent protective factors while SNVs in RAPGEF3 may represent risk factors for ROP. PRKAR1α has previously been implicated in retinal vascular development while the RAPGEF3 product has a role in the maintenance of vascular barrier function, two processes important in ROP. Multicenter validation of these newly discovered risk factors could lead to valuable tools for predicting and preventing the development of severe ROP.

Single-Nucleotide Variants in PADI2 and PADI4 and Ancestry Informative Markers in Interstitial Lung Disease and Rheumatoid Arthritis among a Mexican Mestizo Population

Rheumatoid arthritis (RA) is an autoimmune disease mainly characterized by joint inflammation. It presents extra-articular manifestations, with the lungs being one of the affected areas. Among these, damage to the pulmonary interstitium (Interstitial Lung Disease—ILD) has been linked to proteins involved in the inflammatory process and related to extracellular matrix deposition and lung fibrosis establishment. Peptidyl arginine deiminase enzymes (PAD), which carry out protein citrullination, play a role in this context. A genetic association analysis was conducted on genes encoding two PAD isoforms: PAD2 and PAD4. This analysis also included ancestry informative markers and protein level determination in samples from patients with RA, RA-associated ILD, and clinically healthy controls. Significant single nucleotide variants (SNV) and one haplotype were identified as susceptibility factors for RA-ILD development. Elevated levels of PAD4 were found in RA-ILD cases, while PADI2 showed an association with RA susceptibility. This work presents data obtained from previously published research. Population variability has been noticed in genetic association studies. We present data for 14 SNVs that show geographical and genetic variation across the Mexican population, which provides highly informative content and greater intrapopulation genetic diversity. Further investigations in the field should be considered in addition to AIMs. The data presented in this study were analyzed in association with SNV genotypes in PADI2 and PADI4 to assess susceptibility to ILD in RA, as well as with changes in PAD2 and PAD4 protein levels according to carrier genotype, in addition to the use of covariates such as ancestry markers.

Identification of novel oncogenes in oral cancer among elderly nonsmokers.

In recent years, an increase in oral cancer among elderly nonsmokers has been noted. The aim of this study was to identify novel oncogenes in oral cancer in older nonsmokers. Whole-exome sequencing (WES) data from 324 oral cancer patients were obtained from The Cancer Genome Atlas. Single nucleotide variants (SNVs) and insertions/deletions (INDELs) were extracted from the WES data of older patients. Fisher's exact test was performed to determine the specificity of variants in these genes. Finally, SNVs and INDELs were identified by target enrichment sequencing. Gene ontology analysis of 112 genes with significant SNVs or INDELs in nonsmokers revealed that nonsynonymous SNVs in HECTD4 were significantly more frequent in nonsmokers than in smokers by target enrichment sequencing (p = .02). Further investigation of the function of HECTD4 variants as oncogenes in older nonsmokers is warranted.

Mutation survey in Taiwanese patients with Stickler syndrome.

The purpose of this study was to identify gene mutation and phenotype correlations in a cohort of Taiwanese patients with Stickler syndrome. Patients clinically diagnosed with Stickler syndrome or suspected Stickler syndrome were enrolled. DNA was extracted from venous blood samples. For the targeted next-generation sequencing (NGS) approach, specific primers were designed for all COL2A1, COL11A1, COL11A2, COL9A1, and COL9A2 exons and flanking intron sequences. Twenty-three patients from 12 families were enrolled in this study. The myopia power in these 23 cases (35 eyes) ranged from -4.625 to -25.625 D, with a median of -10.00 D. Four patients had retinal detachment. Fourteen patients had a cleft palate. These 23 patients and 13 healthy controls were enrolled in the NGS study. Three families had significant single nucleotide variants (SNVs) in COL2A1. The mutation rates in this survey were 25% (3/12 families) and 35% (8/23 cases). The SNV of family #1, located at exon 27, c.1753G >T, p. Gly585Val, was novel and has not yet been reported in the ClinVar database. Families #10 and #11 had the same SNV, located in exon 33, c.2101C >T, p. Arg701X. Both variants were classified as likely pathogenic according to the American College of Medical Genetics and Genomics guidelines. Genetic mutations in COL2A1 were found in 25% of Taiwanese families with Stickler syndrome. One novel variant was identified using NGS, which expanded the COL2A1 mutation spectrum. Molecular genetic analysis is helpful to confirm the clinical diagnosis of patients with suspected Stickler syndrome.

Genetic Variants Associated With Intraparenchymal Hemorrhage Progression After Traumatic Brain Injury

Intracerebral hemorrhage progression is associated with unfavorable outcome after traumatic brain injury (TBI). No effective treatments are currently available. This secondary injury process reflects an extreme form of vasogenic edema and blood-brain barrier breakdown. The sulfonylurea receptor 1-transient receptor potential melastatin 4 (SUR1-TRPM4) cation channel is a key underlying mechanism. A phase 2 trial of SUR1-TRPM4 inhibition in contusional TBI is ongoing, and a phase 3 trial is being designed. Targeted identification of patients at increased risk for hemorrhage progression may inform prognostication, trial design (including patient selection), and ultimately treatment response. To determine whether ABCC8 (SUR1) and TRPM4 genetic variability are associated with intraparenchymal hemorrhage (IPH) progression after severe TBI, based on the putative involvement of the SUR1-TRPM4 channel in this pathophysiology. In this genetic association study, DNA was extracted from 416 patients with severe TBI prospectively enrolled from a level I trauma academic medical center from May 9, 2002, to August 8, 2014. Forty ABCC8 and TRPM4 single-nucleotide variants (SNVs) were genotyped (multiplex, unbiased). Data were analyzed from January 7, 2020, to May 3, 2021. Primary analyses addressed IPH progression at 6, 24, and 120 hours in patients without acute craniectomy (n = 321). Multivariable regressions and receiver operating characteristic curves assessed SNV and haplotype associations with progression. Spatial modeling and functional predictions were determined using standard software. Of the 321 patients included in the analysis (mean [SD] age, 37.0 [16.3] years; 247 [76.9%] male), IPH progression occurred in 102. Four ABCC8 SNVs were associated with markedly increased odds of progression (rs2237982 [odds ratio (OR), 2.60-3.80; 95% CI, 1.14-5.90 to 1.80-8.02; P = .02 to P < .001], rs2283261 [OR, 3.37-4.77; 95% CI, 1.07-10.77 to 1.89-12.07; P = .04 to P = .001], rs3819521 [OR, 2.96-3.92; 95% CI, 1.13-7.75 to 1.42-10.87; P = .03 to P = .009], and rs8192695 [OR, 3.06-4.95; 95% CI, 1.02-9.12 to 1.67-14.68]; P = .03-.004). These are brain-specific expression quantitative trait loci (eQTL) associated with increased ABCC8 messenger RNA levels. Regulatory annotations revealed promoter and enhancer marks and strong and/or active brain-tissue transcription, directionally consistent with increased progression. Three SNVs (rs2283261, rs2237982, and rs3819521) in this cohort have been associated with intracranial hypertension. Four TRPM4 SNVs were associated with decreased IPH progression (rs3760666 [OR, 0.40-0.49; 95% CI, 0.19-0.86 to 0.27-0.89; P = .02 to P = .009], rs1477363 [OR, 0.40-0.43; 95% CI, 0.18-0.88 to 0.23-0.81; P = .02 to P = .006], rs10410857 [OR, 0.36-0.41; 95% CI, 0.20-0.67 to 0.20-0.85; P = .02 to P = .001], and rs909010 [OR, 0.27-0.40; 95% CI, 0.12-0.62 to 0.16-0.58; P = .002 to P < .001]). Significant SNVs in both genes cluster downstream, flanking exons encoding the receptor site and SUR1-TRPM4 binding interface. Adding genetic variation to clinical models improved receiver operating characteristic curve performance from 0.6959 to 0.8030 (P = .003). In this genetic association study, 8 ABCC8 and TRPM4 SNVs were associated with IPH progression. Spatial clustering, brain-specific eQTL, and regulatory annotations suggest biological plausibility. These findings may have important implications for neurocritical care risk stratification, patient selection, and precision medicine, including an upcoming phase 3 trial design for SUR1-TRPM4 inhibition in severe TBI.

Abstract 4747: Genomic and transcriptomic landscape of AFP-producing gastrointestinal adenocarcinoma

Abstract AFP-producing gastrointestinal adenocarcinoma (APA-GI) is a rare type of cancer, typically with hepatoid differentiation, chemotherapy resistance and poor prognosis. As the mechanisms of its AFP production and hepatoid differentiation remain unclear, the present study aims to reveal the pathogenesis of APA-GI through genomic and transcriptomic profile analysis. We firstly performed 200 × whole exome sequencing of 29 APA-GI and 8 non-APA-GI cases from our hospital, but found no significant difference in single nucleotide variants (SNV) or insertion/deletion(indels). Additionally, there was no overlap gene when compared with the known genes harbored at 20q11.21-13.12 reported in gastric hepatoid adenocarcinoma. We then performed single-cell mRNA sequencing in one APA-GI, and observed several hepatoid differentiation markers in AFP positive cells including HNF4A and HNF1A. To further determine the specific alterations of APA-GI, we divided 339 gastric cancer patients from TCGA database into AFP-low group and AFP-high group by the level of AFP transcription through a dynamic grouping method named grid search. We performed a series differentially expressed genes detections within different grouping strategies and no significant SNV or indels difference was recognized. We identified hepatoid differentiation markers (i.e. HNF4A, HNF1A and ALB, et al) in AFP-high patients in mRNA level. As the difference of hepatoid differentiation markers only occurred in the mRNA level without DNA alteration, we hypothesized that it was probably caused by epigenetic regulation. We further compared these genes with an epigenetic regulators database named EpiFactors. Finally, it showed CCCTC-Binding Factor Like (CTCFL) was stably related with AFP producing within the grid search. In conclusion, comprehensive genomic analysis of APA-GI shows significant hepatoid differentiation at the transcriptomic level without disease-specific DNA alteration. The pathogenesis of APA-GI may be associated with other mechanisms like epigenetic regulation. Further study was needed to confirm the role of epigenetic regulators we detected in the present study in the development of APA-GI. Citation Format: Jun Li, Xinlin Li, Qiancheng Ye, Yu Tian, Xiangxing Kong, Yue Liu, Xiaoming Xu, Lina Zhang, Qian Xiao, Yeting Hu, Rongzhen Xu, Jingsong Li. Genomic and transcriptomic landscape of AFP-producing gastrointestinal adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4747.

A novel GIT2-BRAF fusion in pilocytic astrocytoma

BackgroundKIAA1549-BRAF fusion is the most common genetic event in pilocytic astrocytoma (PA), and leads to activation of the mitogen activated protein kinase (MAPK) signaling pathway. Fusions of BRAF with other partner genes, as well as other genetic alterations not involving BRAF but also leading to MAPK pathway activation have been described rarely.Case presentationWe present a new fusion partner in the low-grade glioma of a 10-year-old male, who presented with headaches and recent episodes of seizures. Magnetic resonance imaging (MRI) demonstrated a right temporal lobe tumor. Histological and immunohistochemical evaluation, and a next generation sequencing assay (Oncopanel, Illumina, 500 genes) including breaKmer analysis for chromosomal rearrangements were performed.Histology was remarkable for a low-grade glioma composed of mildly atypical astrocytes with piloid processes, in a focally microcystic background. Mitoses were not seen; unequivocal Rosenthal fibers or eosinophilic granular bodies were absent. The tumor was positive for OLIG2 and GFAP and negative for BRAF V600E and IDH1 R132H mutant protein immunostains. Oncopanel showed low SOX2 (3q26.33) copy number gain, and no gains at 7q34. There were no significant single nucleotide variants. BreaKmer detected a GIT2-BRAF fusion with loss of BRAF exons 1–8. The integrated diagnosis was low-grade glioma with piloid features, most consistent with pilocytic astrocytoma, WHO grade I.ConclusionGIT2-BRAF fusion has not been reported in the literature in any tumor. Given that the BRAF sequence deleted is identical to that seen in other fusion events in PA, it most likely acts as tumor driver by activation of the MAPK pathway.