How VarSeq Powers Research in Inherited Diseases and Cancer

         September 25, 2024

Exome and genome sequencing have advanced genetic research, but data analysis remains complex. VarSeq simplifies this by helping researchers identify key genetic changes in rare diseases and cancer, improving screening and diagnosis. Here are recent customer publications that highlight VarSeq usage.

Expanded carrier screening for inherited genetic disease using exome and genome sequencing

The goal of this study was to assess the feasibility of using exome (ES) and genome sequencing (GS) in guiding preconception genetic screening (PCGS) for couples who are planning to conceive by creating a workflow for identifying risk alleles for autosomal recessive (AR) and X-linked (XL) disorders without the constraints of a predetermined, targeted gene panel. There were several limitations and challenges related to reporting and the technical aspects of ES and GS, which are listed in the discussion. We selected 150 couples from a cohort of families (trios) enrolled in a research protocol where the goal was to define the genetic etiology of disease in an affected child. Pre-existing, de-identified parental sequencing data were analyzed to define variants that would place the couple at risk of having a child affected by an AR or XL disorder. We identified 17 families who would be selected for counseling about risk alleles. We noted that only 3 of these at-risk couples would be identified if we limited ourselves to the current ACMG-recommended expanded carrier screening gene panel. ES and GS successfully identified couples who are at risk of having a child with a rare AR or XL disorder that would have been missed by the current recommended guidelines. Current limitations of this approach include ethical concerns, difficulties in reporting results including variant calling due to the rare nature of some of the variants, determining which disorders to report, as well as technical difficulties in detecting certain variants such as repeat expansions.

The VCF files for this trio, consisting of maternal, paternal, and synthetic proband, was imported into our standard trio template in VarSeq software (Golden Helix). The resulting variant annotation files were filtered for variants with the following parameters (Table 1): retention of variants with a Combined Annotation Dependent Depletion (CADD) score ≥15 (an in silico method used to determine the deleteriousness of SNPs and insertions/deletions), exclusion of variants of uncertain significance (VUS) that were synonymous or found in the 3′-UTR or 5′-UTR of genes, exclusion of variants with more than 5 hemizygotes or homozygotes in the Genome Aggregation Database (gnomAD), exclusion of variants scored as benign or likely benign in the ClinVar database, retention of variants classified as likely pathogenic or pathogenic by ACMG criteria (Richards et al., 2015), and retention of variants only in genes associated with known AR or XL disorders listed in Online Mendelian Inheritance in Man (OMIM) (see Table 1). The retained variants were then manually reviewed to identify risk alleles in the parents that lead to AR (homozygous or compound heterozygous) or XL pathogenic variants in the “synthetic proband.”

Belnap, N., Ramsey, K., Abraham, A., Ryan, A., Rangasamy, S., Bonfitto, A., Naymik, M., Huentelman, M., Strom, S., Perry, D., Subramaniam, A., Grody, W. W., Szelinger, S., & Narayanan, V. (2024). Expanded carrier screening for inherited genetic disease using exome and genome sequencing. Journal of Genetic Counseling, 00, 1–7. https://doi.org/10.1002/jgc4.1964

Universal Genetic Testing for Newly Diagnosed Invasive Breast Cancer

Importance  Between 5% and 10% of breast cancer cases are associated with an inherited germline pathogenic or likely pathogenic variant (GPV) in a breast cancer susceptibility gene (BCSG), which could alter local and systemic therapy recommendations. Traditional genetic testing criteria misses a proportion of these cases.

Objective  To evaluate the prevalence and clinicopathological associations of GPVs in 2 groups of BCSGs among an ethnically diverse cohort of women with newly diagnosed breast cancer.

Design, Setting, and Participants  This cross-sectional study, conducted at 3 Montreal hospitals between September 2019 and April 2022, offered universal genetic counseling and testing to all women with a first diagnosis of invasive breast cancer. Women were offered an obligatory primary panel of BRCA1BRCA2, and PALB2 (B1B2P2) and an optional secondary panel of 14 additional BCSGs. Eligible participants were women 18 years of age or older who received a diagnosis of a first primary invasive breast cancer not more than 6 months before the time of referral to the study. Data were analyzed from November 2023 to June 2024.

Results  Of 1017 referred patients, 805 were eligible and offered genetic counseling and testing, and 729 of those 805 (90.6%) consented to be tested. The median age at breast cancer diagnosis was 53 years (range, 23-91 years), and 65.4% were White and of European ancestry. Fifty-four GPVs were identified in 53 patients (7.3%), including 39 patients (5.3%) with B1B2P2 and 15 patients (2.1%) with 6 of the 14 secondary panel BCSGs (ATMBARD1BRIP1CHEK2RAD51D, and STK11). On multivariable analysis, clinical factors independently associated with B1B2P2-positive status included being younger than 40 years of age at diagnosis (odds ratio [OR], 6.83; 95% CI, 2.22-20.90), triple-negative breast cancer (OR, 3.19; 95% CI, 1.20-8.43), high grade disease (OR, 1.68; 95% CI, 1.05-2.70), and family history of ovarian cancer (OR, 9.75; 95% CI, 2.65-35.85). Of 39 B1B2P2-positive patients, 13 (33.3%) were eligible for poly(adenosine diphosphate–ribose) polymerase (PARP) inhibitors.

Conclusions and Relevance  In this cross-sectional universal genetic testing study of women with newly diagnosed invasive breast cancer, the prevalence of GPVs was 7.3%, with 5.3% of patients testing positive for B1B2P2. Among B1B2P2-women women, one-third were eligible for PARP inhibitors.

Variant calling was performed using NextGENe, version 2.4.2.3 and Geneticist Assistant, version 1.8.1 (SoftGenetics) proprietary bioinformatics pipeline. The fastq files from the Illumina MiSeq were aligned to hg19, and variant calling was performed on the resulting BAM files. For copy number variants, we used VarSeq (Golden Helix).

Rezoug Z, Totten SP, Szlachtycz D, et al. Universal Genetic Testing for Newly Diagnosed Invasive Breast Cancer. JAMA Netw Open. 2024;7(9):e2431427. doi:10.1001/jamanetworkopen.2024.31427

Genotype Characterization and MiRNA Expression Profiling in Usher Syndrome Cell Lines

Usher syndrome (USH) is an inherited disorder characterized by sensorineural hearing loss (SNHL), retinitis pigmentosa (RP)-related vision loss, and vestibular dysfunction. USH presents itself as three distinct clinical types, 1, 2, and 3, with no biomarker for early detection. This study aimed to explore whether microRNA (miRNA) expression in USH cell lines is dysregulated compared to the miRNA expression pattern in a cell line derived from a healthy human subject. Lymphocytes from USH patients and healthy individuals were isolated and transformed into stable cell lines using Epstein–Barr virus (EBV). DNA from these cell lines was sequenced using a targeted panel to identify gene variants associated with USH types 1, 2, and 3. Microarray analysis was performed on RNA from both USH and control cell lines using NanoString miRNA microarray technology. Dysregulated miRNAs identified by the microarray were validated using droplet digital PCR technology. DNA sequencing revealed that two USH patients had USH type 1 with gene variants in USH1B (MYO7A) and USH1D (CDH23), while the other two patients were classified as USH type 2 (USH2A) and USH type 3 (CLRN-1), respectively. The NanoString miRNA microarray detected 92 differentially expressed miRNAs in USH cell lines compared to controls. Significantly altered miRNAs exhibited at least a twofold increase or decrease with a p value below 0.05. Among these miRNAs, 20 were specific to USH1, 14 to USH2, and 5 to USH3. Three miRNAs that are known as miRNA-183 family which are crucial for inner ear and retina development, have been significantly downregulated as compared to control cells. Subsequently, droplet digital PCR assays confirmed the dysregulation of the 12 most prominent miRNAs in USH cell lines. This study identifies several miRNA signatures in USH cell lines which may have potential utility in Usher syndrome identification.

Reads for each cell line were aligned and mapped to the human reference genome (GRCh38p.14) [85] using BWA and Samtools, respectively [86,87]. Sequence variants were called using Sentieon’s DNA pipeline for variant detection [88]. Sequencing variant interpretation according to ACMG classification criteria was conducted using the VarSeq software version 2.4.0 (Golden Helix enabling precision medicine, Available online: https://www.goldenhelix.com, accessed on 23 December 2023) [32,89].

Tom, W.A.; Chandel, D.S.; Jiang, C.; Krzyzanowski, G.; Fernando, N.; Olou, A.; Fernando, M.R. Genotype Characterization and MiRNA Expression Profiling in Usher Syndrome Cell Lines. Int. J. Mol. Sci. 2024, 25, 9993. https://doi.org/10.3390/ijms25189993

Whole exome sequencing in relapsed or refractory childhood cancer: case series

Background: The prognosis for relapsed or refractory childhood cancer is approximately 20%. Genetic alterations are one of the significant contributing factors to the prognosis of patients. Objective: To investigate the molecular profile of relapsed or refractory childhood cancers in Thai cases. Methods: The study design is a descriptive study of patients <18 years old, suspected or diagnosed of relapsed or refractory childhood cancer who underwent whole exome sequencing (WES). Results: WES was successfully performed in both the tumor and the blood or saliva samples obtained from 4 unrelated patients. Six different variants were identified in the NCOR2, COL6A3, TP53, and SMAD4 genes. These alterations were found to be associated with tumor aggressiveness. Conclusion: This study is the first one to demonstrate genetic alterations by using WES in relapsed or refractory childhood cancer in Thai cases.

The variants were annotated and filtered using the Golden Helix VarSeq analysis workflow implementing the ACMG guidelines for the interpretation of sequence variants. This includes a comparison against the gnomAD population catalog of variants in 123,136 exomes, the 1000 Genomes Project Consortium’s publication of 2,500 genomes, the NCBI ClinVar database of clinical assertions on variant’s pathogenicity and multiple lines of computational evidence on conservation and functional impact

Thangpong, Rungroj, et al. “Whole exome sequencing in relapsed or refractory childhood cancer: case series” Asian Biomedicine, vol. 18, no. 4, Sciendo, 2024, pp. 186-191. https://doi.org/10.2478/abm-2024-0025

Germline DNA Damage Repair Gene Alterations in Patients with Metachronous Breast and Colorectal Cancer

A hereditary component of breast (BC) and colorectal cancer (CRC) has been described in approximately one-third of these tumor types. BC patients have an increased risk of developing CRC as a second primary tumor and vice versa. Germline genomic variants (NextSeq550, Illumina) were investigated in 24 unrelated BC and/or CRC patients and 7 relatives from 3 index patients. Fifty-six pathogenic or likely pathogenic variants were identified in 19 of 24 patients. We detected single-nucleotide variants (SNVs) in CRC predisposition genes (MLH1 and MUTYH) and other promising candidates (CDK5RAP3MAD1L1NOS3, and POLM). Eighteen patients presented SNVs or copy number variants (CNVs) in DNA damage repair genes. We also identified SNVs recently associated with BC or CRC predisposition (PABPC1TYRO3MAP3K1SLC15A4, and LAMA1). The PABPC1c.1255C>T variant was detected in nine unrelated patients. Each patient presented at least one SNV/CNV in a candidate gene, and most had alterations in more than one gene, reinforcing a polygenic model for BC/CRC predisposition. A significant fraction of BC/CRC patients with a family history of these tumors harbored deleterious germline variants in DNA repair genes. Our findings can lead to strategies to improve the diagnosis, genetic counseling, and treatment of patients and their relatives.

We used VarSeq™ software version 2.2.2 (Golden Helix, Inc., Bozeman, MT, US) to annotate and select germline variants. To select novel and/or rare variants, we filtered all candidates with alternate allele frequencies less than 1% in the Genome Aggregation Database (GnomAD v2.1) [81], Genome (v3), Exome Variants (v2), and Online Archive of Brazilian Mutations (ABRaOM): Brazilian Genomic variants [82]. Variants were selected with a threshold strand Bias Fisher’s < 50, quality by depth >1.5, and a Variant Allele Frequency > 0.2. The variants were filtered out if they resulted in synonymous alterations, potentially presented homopolymer artifacts (homopolymer sequence length > 6), or were predicted as benign/likely benign by the American College of Medical Genetics and Genomics (ACMG) or ClinVar classification [83,84]. Sequence data supporting the remaining variants were evaluated in paired BAMs using the Golden Helix GenomeBrowse visualization tool v.2.2.2. We removed variants in the presence of artifacts resulting from PCR amplification, sequencing, or alignment.

Villacis, R.A.R.; Côrtes, L.; Basso, T.R.; do Canto, L.M.; Souza, J.S.; Aagaard, M.M.; da Cruz Formiga, M.N.; Aguiar, S., Jr.; Achatz, M.I.; Rogatto, S.R. Germline DNA Damage Repair Gene Alterations in Patients with Metachronous Breast and Colorectal Cancer. Int. J. Mol. Sci. 2024, 25, 10275. https://doi.org/10.3390/ijms251910275

VarSeq is essential for analyzing genetic data, focusing on relevant changes to enhance screening and diagnosis. As genetics advances, VarSeq will continue to support precision medicine and disease understanding. If you are interested in learning more, please visit our website at the link below.

About Casey Fullem

Casey Fullem, Marketing Coordinator at Golden Helix, joined team in October of 2021. He is an analytics driven digital marketer that values tactile marketing efforts that yield return. Building a cohesive brand and executing performative marketing campaigns is his passion, alongside the slew of outdoor activities that Bozeman has to offer. Casey is largely passionate about skiing, rock climbing, whitewater kayaking and rafting, fly fishing, backpacking, and other outdoor activities that excite.

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