This month, we saw overwhelming customer publications with great use cases of VarSeq. Explore the publications below of our amazing customers continuing to pioneer precision medicine.
The polygenic implication of clopidogrel responsiveness: Insights from platelet reactivity analysis and next-generation sequencing
Clopidogrel is widely used worldwide as an antiplatelet therapy in patients with acute coronary disease. Genetic factors influence interindividual variability in response. Some studies have explored the polygenic contributions in the drug response, generating pharmacogenomic risk scores (PgxPRS). Importantly, these factors are less explored in underrepresented populations, such as Latin-American countries. Identifying patients at risk of high-on-treatment platelet reactivity (HTPR) is highly valuable in translational medicine. In this study we used a custom next-generation sequencing (NGS) panel composed of 91 single nucleotide polymorphisms (SNPs) and 28 genes related to clopidogrel metabolism, to analyze 70 patients with platelet reactivity values, assessed through closure time (CT). Our results demonstrated the association of SNPs with HTPR and non-HTPR, revealing the strongest associations with rs2286823 (OR: 5,0; 95% CI: 1,02–24,48; p: 0,03), rs2032582 (OR: 4,41; 95% CI: 1,20–16,12; p: 0,019), and rs1045642 (OR: 3,38; 95% CI: 0,96–11,9; p: 0,05). Bivariate regression analysis demonstrated the significant association of several SNPs with the CT value, a “surrogate” biomarker of clopidogrel response. Exploratory results from the LASSO regression model showed a high discriminatory capacity between HTPR and non-HTPR patients (AUC: 0,955), and the generated PgxPRS demonstrated a significant negative association between the risk score, CT value, and the condition of HTPR and non-HTPR. To our knowledge, our study addresses for the first time the analysis of the polygenic contribution in platelet reactivity using NGS and establishes PgxPRS derived from the LASSO model. Our results demonstrate the polygenic implication of clopidogrel response and offer insights applicable to the translational medicine of antiplatelet therapy in an understudied population.
“To determine the pathogenicity of missense variants, scores from in silico predictors within VarSeq software were considered. These predictors included Sorting Intolerant from Tolerant (SIFT), Polyphen 2, Mutation Taster, Mutation Assessor, and Functional Analysis Through Hidden Markov Models (FATHMM and FATHMM-MKL). Missense variants with 3 out of 6 positive predictors were considered as pathogenic. For the final analysis, variants obtained from filters a and b with a total depth of 8X, allelic representation in heterozygotes of ≥0.25 (minimum 25% of the polymorphic allele), and relevance to clopidogrel response as reported in the PharmGKB database were considered.”
On the Boundary of Exploratory Genomics and Translation in Sequential Glioblastoma
OMICS methods brought significant advancements to the understanding of tumor cell biology, which transformed the treatment and prognosis of several cancers. Clinical practice and outcomes, however, changed significantly less in the case of glioblastoma (GBM). In this study, we aimed to assess the utility of whole exome (WES) sequencing in the clinical setting. Ten pairs of formalin-fixed, paraffin-embedded (FFPE) GBM specimens were obtained at onset (GBM-P) and at recurrence (GBM-R). Histopathological and molecular features of all samples supported the diagnosis of GBM based on WHO CNS5. WES data were filtered, applying a strict and custom-made pipeline, and occurrence of oncogenic and likely oncogenic variants in GBM-P, GBM-R or both were identified by using the VarSeq program version 2.5.0 (Golden Helix, Inc.). Characteristics and recurrence of the variants were analyzed in our own cohort and were also compared to those available in the COSMIC database. The lists of oncogenic and likely oncogenic variants corresponded to those identified in other studies. The average number of these variants were 4 and 5 out of all detected 24 and 34 variants in GBM-P and GBM-R samples, respectively. On average, one shared oncogenic/likely oncogenic variant was found in the pairs. We assessed the identified variants’ therapeutic significance, also taking into consideration the guidelines by the Association for Molecular Pathology (AMP). Our data support that a thorough WES analysis is suitable for identifying oncogenic and likely oncogenic variants in an individual clinical sample or a small cohort of FFPE glioma specimens, which concur with those of comprehensive research studies. Such analyses also allow us to monitor molecular dynamics of sequential GBM. In addition, careful evaluation of data according to the AMP guideline reveal that though therapeutic applicability of the variants is generally limited in the clinic, such information may be valuable in selected cases, and can support innovative preclinical and clinical trials.
“Subsequently, three variant categories were distinguished based on the VarSeq Cancer Classifier algorithm and scoring system: oncogenic (5+ ≥ score), likely oncogenic (3+ ≥ score) and variant of uncertain significance (VUS) (2+ ≤ score). The software analyses were performed as “duos” of GBM-P and GBM-R samples, and variants were simultaneously evaluated and checked as to whether they exclusively appeared in one of the pairs (GBM-P or GBM-R) or in both (GBM-S).”
“In addition, we applied the COSMIC filter card, identified oncogenic and likely oncogenic variants based on the new VarSeq Cancer Classifier criteria system (2023.11.15, version 1.0) and implemented a custom-made glioma gene panel based on published data [27,28,29,30,31,32,33] (Table S2).”
Vascular calcification in chronic kidney disease associated with pathogenic variants in ABCC6
Vascular calcification is prevalent in chronic kidney disease (CKD). Genetic causes of CKD account for 10–20% of adult-onset disease. Vascular calcification is thought to be one of the most important risk factors for increased cardiovascular morbidity and mortality in CKD patients and is detectable in 80% of patients with end stage kidney disease (ESKD). Despite the high prevalence of vascular calcification in CKD, no single gene cause has been described. We hypothesized that variants in vascular calcification genes may contribute to disease pathogenesis in CKD, particularly in families who exhibit a predominant vascular calcification phenotype.
We developed a list of eight genes that are hypothesized to play a role in vascular calcification due to their involvement in the ectopic calcification pathway: ABCC6, ALPL, ANK1, ENPP1, NT5E, SLC29A1, SLC20A2, and S100A12. With this, we assessed exome data from 77 CKD patients, who remained unsolved following evaluation for all known monogenic causes of CKD. We also analyzed an independent cohort (Ontario Neurodegenerative Disease Research Initiative (ONDRI), n = 520) who were screened for variants in ABCC6 and compared this to a control cohort of healthy adults (n = 52).
We identified two CKD families with heterozygous pathogenic variants (R1141X and A667fs) in ABCC6. We identified 10 participants from the ONDRI cohort with heterozygous pathogenic or likely pathogenic variant in ABCC6. Replication in a healthy control cohort did not reveal any variants.
Our study provides preliminary data supporting the hypothesis that ABCC6 may play a role in vascular calcification in CKD. By screening CKD patients for genetic causes early in the diagnostic pathway, patients with genetic causes associated with vascular calcification can potentially be preventatively treated with new therapeutics with aims to decrease mortality.
“We also completed copy number variant (CNV) analysis using VarSeq CNV Caller (VarSeq v2.2.1, Golden Helix, Bozeman, MT) (Iacocca et al., 2017).”
Genomic Landscape of Rare and Common Variants in a Longitudinal Cohort Indicates Modifiers of Disease Pathogenesis
To understand the genomic landscape of rare and common glaucoma and anterior segment dysgenesis (ASD)-associated variants in the longitudinal Andhra Pradesh Eye Disease Study (APEDS) cohort for identifying their appropriateness as “controls” and as potential modifiers of disease pathogenesis.
Methods : The APEDS is a population-based rural cohort that was enrolled (1996-2000) from the State of Andhra Pradesh in Southern India and followed up at 10 and 15 years. The final follow up included 5395 subjects who underwent comprehensive clinical examination, deep phenotyping and assessments of risk factors. They were screened using a targeted panel comprising genes involved in glaucoma and ASD along with their interacting partners by deep sequencing. The data was analysed by GATK and VarSeq softwares and included subjects who had no visual impairments from baseline until the last follow up (n=2500). The APEDS cohort was assessed in conjunction with data on five ethnic populations of Indian origin (EPIO) represented in the gnomAD database (n=489) and compared to the overall mutation spectrum across all forms of primary glaucoma and ASD. The observed multi-allelic interactions in a subject were functionally assessed.
Results : The share of rare pathogenic alleles reported across the 25 glaucoma associated genes (n=221) were significantly higher (p<0.01; OR=3.01; 95%CI, 1.82-4.91) in the APEDS cohort (n=65; 29.45%) compared to EPIO (n=27; 12.21%) that contributed to a population attributable risk percent (PAR%) of 26.08. Similarly, among the ASD-associated pathogenic alleles (n=66) across 15 genes, the number of shared alleles were significantly higher (p<0.05; OR=2.57, 95%CI, 1.15-5.73) in the APEDS cohort compared to EPIO with a PAR% of 22.92. The common variants did not exhibit much variability across these datasets. Co-inheritance of multiple alleles contributed to 10.08% of all pathogenic variations and subsets of these indicated perturbation of their physical interactions, which could act as potential modifiers.
Conclusions : The overall landscape of gene variants in the APEDS cohort suggested that genetic assessments should be based on geographical and local ethnicity towards assessing the role of pathogenic alleles and potential modifiers in glaucoma and ASD cases. The limited variability across EPIO dataset limits their use as random “controls” in the Indian context.
Unveiling cystinosis in India
Cystinosis, a rare autosomal recessive disease, stems from genetic alterations in the CTNS gene, leading to a malfunction of lysosomal ‘cystinosin’ protein. This dysfunction causes intracellular cystine accumulation, resulting in nephropathic and ocular abnormalities. Cystinosis is relatively rare in Asian countries, partly due to underreporting and lack of awareness, and cases often lack sufficient genetic evidence to support their diagnosis. This study presents a descriptive case series involving four Indian patients with cystinosis, elucidating clinical and genetic aspects.
All four patients underwent comprehensive ophthalmic evaluations. The corneal cystine crystal (CCC) score was determined using anterior segment optical coherence tomography (AS-OCT) and in vivo confocal microscopy (IVCM). Genetic testing was performed using whole exome sequencing (WES).
Corneal crystal deposition, a hallmark of cystinosis, was evident in all cases. Systemic analysis revealed manifestations such as polyuria, bony abnormalities, growth retardation, hypothyroidism, and developmental delay. Genetic testing in two patients identified a homozygous pathogenic variant c.18_21delGACT (p.Thr7PhefsX7) in the CTNS gene, previously reported to cause cystinosis in different ethnic population.
Our case series sheds light on underrepresented cases of cystinosis in the Indian population. The rarity of this condition poses diagnostic challenges, leading to delayed or inaccurate diagnoses. AS-OCT can serve as a viable alternative to IVCM for assessing corneal crystal density status in cystinosis. Timely recognition and management are crucial in preventing complications, and the inclusion of genetic testing can expedite cystinosis diagnosis.
“Variants were annotated and categorized using Golden Helix VarSeq and Varsome analysis workflow implementing ACMG (American College of Medical Genetics and Genomics) guidelines.”
Detection of Spondyloepiphyseal Dysplasia Phenotype with CHST3 Mutation in an Asian Indian Family and Outcomes of Index and Subsequent Pregnancy
CHST3-related skeletal dysplasia is an autosomal recessive disorder with a prenatal onset. Variations in the CHST3 gene are associated with spondyloepiphyseal dysplasia with short stature of prenatal onset; dislocation of the knees, hips or elbows; club feet; postnatal limitation of range of motion of large joints; and progressive kyphosis, occasional scoliosis and minor heart valve dysplasia. The identification of pathogenic variants in the family is helpful for carrier and prenatal testing. We describe the prenatal identification of spondyloepiphyseal dysplasia with CHST3 mutation in the index pregnancy of an Asian Indian woman and the outcome of the subsequent pregnancy.
“The extracted deoxyribonucleic acid libraries were sequenced[4] [5] and copy number variants were detected from VarSeq 2.2.3 in addition to single nucleotide variants and small indels.[6] Clinically relevant mutations were annotated using published variants in literature and disease databases that included ClinVar, Online Mendelian Inheritance in Man, Genome-Wide Association Studies, Human Gene Mutation Database (v2020.2) and SwissVar.”
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