Each month, we highlight new research from the scientific community that advances our understanding of complex genetic diseases and showcases the tools researchers rely on for precise variant interpretation. January’s customer publications explore pharmacogenomics testing and variants that reveal how specific genetic mechanisms shape human immune responses. Together, these studies highlight the importance of integrated analysis approaches, including VarSeq, in investigating rare variants and refining genotype–phenotype relationships.
Title: Development and Validation of a Multigene Panel for Pharmacogenomics Testing Using Next-Generation Sequencing for Routine Clinical Practice
Background: This study focuses on the rigorous validation of an in-house NGS PGx assay using the Ion AmpliSeq Pharmacogenomics Panel, emphasizing accuracy, reliability, and robust bioinformatics to support clinical implementation.
Objective: To determine if the accuracy and reliability of Next-Generation Sequencing (NGS), coupled with in-house developed bioinformatics pipelines, is sufficient for routine clinical practice.
Subjects and Methods: This study validated an NGS-based pharmacogenomics workflow using 28 reference DNA samples. Clinically relevant variants from nine pharmacogenes were selected based on AMP and CPIC guidelines, with sequencing performed on the Ion S5 XL platform and analyzed using both the standard and a customized BIH bioinformatics protocol. The BIH-protocol incorporated enhanced CNV detection for CYP2D6 and rigorous quality control, enabling robust diplotype calling and comprehensive performance evaluation for clinical implementation.
Results:
- SNVs and indels showed near-perfect accuracy (99.87% and 100%, respectively) with 100% sensitivity and NPV, while diplotype calling achieved 99.38% accuracy; CNV detection using the standard protocol was reliable but limited by a high no-call rate, particularly for CYP2D6.
- Implementing a strict GQ threshold and optimized CNV calling resolved false positives, reduced no-calls, achieved 100% accuracy at both gene- and exon-level CYP2D6 CNV detection, and demonstrated high reproducibility (>99.8%), supporting robust clinical implementation.
Conclusions: Using the Ion AmpliSeq Pharmacogenomics Panel with a customized BIH bioinformatics protocol, this study demonstrated high accuracy, sensitivity, reproducibility, and robust CYP2D6 CNV detection across clinically relevant genes covering over 130 commonly prescribed drugs. Although short-read sequencing and manual curation introduce limitations, the optimized BIH-protocol mitigated false calls and proved suitable for routine clinical PGx testing, supporting its integration into a multidisciplinary personalized medicine workflow
How VarSeq Was Used: The BIH-protocol was developed to overcome the eight individual requirements per batch by using the PGxAnalysis plugin to identify only SNVs and indels, while CNV analysis was performed using the VarSeq program version 2.2.5 (Golden Helix, USA). SNVs and indels were also confirmed concurrently within VarSeq, incorporating a genotype quality (GQ) threshold to ensure accurate and reliable results (Table S5). CNV results were combined with SNVs and indels data to determine the diplotypes, which were interpreted using AlleleTyper v1.0 (Figure 1).
Citation: Hongkaew, Y., Kunadirek, P., Sangtian, M., Pateetin, P., Bunlungsup, S., Panmontha, W., Kawprasertsri, S., & Khunlertkit, T. (2026). Development and Validation of a Multigene Panel for Pharmacogenomics Testing Using Next-Generation Sequencing for Routine Clinical Practice. Clinical and translational science, 19(1), e70442. https://doi.org/10.1111/cts.70442
Title: Functional Characterization of an IL2RG Variant, a Case Report of X‐Linked T‐ B + NK + SCID
Background: X-linked severe combined immunodeficiency (X-SCID) is caused by loss-of-function variants in the IL2RG gene, which encodes the common gamma chain essential for cytokine signaling and T and NK cell development, leading to life-threatening infections without curative therapy. Many variants occur in exon 5, which encodes a conserved extracellular region containing the WSXWS motif required for proper receptor folding, cytokine binding, and JAK–STAT signaling. Although IL2RG variants typically cause a T−B+NK− SCID phenotype, this report describes a patient with an exon 5 variant who lacked T cells but had normal peripheral NK cells, prompting further functional and expression analyses.
Objectives: To illustrate possible mechanisms for pathogenic variants in IL2RG generating a T – B + NK+ phenotype instead of the more common γ chain (γc/CD132),
Subjects and Methods: This study was conducted on a single subject. Whole-genome sequencing was performed, and the genome was analyzed for copy-number variants.
Results:
- Whole genome sequencing revealed the patient to be hemizygous for a maternally derived missense variant (c677G > A; p. Arg 226 His) in exon 5 of the IL2RG gene.
- Both the patient’s and the mother’s neutrophils displayed, when compared to their respective lymphocytes, a right‐shift in BV421 intensity consistent with neutrophil surface expressed IL‐15Rα
Conclusions: An X-linked T-B⁺NK⁺ SCID was caused by an IL2RG missense variant that partially impairs immune signaling. While the variant allows initial STAT5 activation and NK cell degranulation, it fails to sustain normal NK cell activation and proliferation, highlighting the importance of intact IL-15–mediated signaling for maintaining peripheral NK cell numbers.
How VarSeq Was Used: For each proband, paired‐end sequencing (Illumina NextSeq 500 platform, Illumina, San Diego, California, United States) was done after exome capturing by the Agilent SureSelectXT2 Kit (Version 6) (Agilent Technologies, Inc., Santa Clara, California). Quality control analysis was conducted using the FastQC toolkit, and the data were aligned to the human reference genome build GRCh37 (hg19) utilizing the Illumina DRAGEN Bio‐IT Platform. Identification of single‐nucleotide variants, small deletions, and insertions was performed by the Illumina DRAGEN haplotype variant calling system. The found variants were then annotated using VarSeq v2 software (Golden Helix, Inc., Bozeman, Montana, http://www.goldenhelix.com).
Citation: Assing, K., Christensen, E. B., Dellgren, C., Soelberg, K. K., Fagerberg, C., Elle, I. C., Larsen, B. D., Grosen, D., Booth, C., Marquart, H. V., Masmas, T. N., & Hartling, H. J. (2025). Functional Characterization of an IL2RG Variant, a Case Report of X-Linked T- B + NK + SCID. Immunity, inflammation and disease, 13(12), e70307. https://doi.org/10.1002/iid3.70307
Title: Viral Infection and Brain Inflammation with Seizures in PARK7 Deficiency
Background: This study investigates a child with unusually severe RSV infection and encephalopathy, linked to a novel loss-of-function PARK7 mutation that disrupts inflammation, stress responses, cell death, and autophagy, suggesting alternative immune pathways can predispose to severe RSV disease.
Objectives: To understand why a previously healthy four-year-old patient was experiencing fever and long-lasting tonic-clonic seizures after developing an RSV infection.
Subjects and Methods: A previously healthy 4-year-old boy developed severe RSV-associated acute encephalopathy with biphasic seizures (AESD), progressive cerebral edema, and prolonged viral persistence, resulting in profound long-term neurological impairment despite intensive treatment. To investigate underlying susceptibility, the patient and parents underwent whole-genome sequencing and extensive functional studies using patient-derived cells and PARK7-deficient cellular models to assess immune responses, viral susceptibility, and cellular stress pathways.
Results: RSV-induced AESD was found to have a homozygous loss-of-function variant in PARK7, and patient cells exhibited dysregulated oxidative stress responses, impaired autophagy and cell death, and exaggerated inflammation. These findings suggest that PARK7 deficiency predisposes to hyperinflammatory CNS complications during viral infection, identifying PARK7 as a potential novel inborn error of immunity beyond its established role in early-onset Parkinson’s disease.
- Whole-genome sequencing revealed a rare homozygous loss-of-function PARK7 (DJ-1) variant causing the complete absence of the protein.
- PARK7-deficient patient cells showed exaggerated type I IFN and proinflammatory cytokine responses, elevated stress-kinase signaling (ASK1), reduced apoptosis, and impaired autophagy, while RSV replication was unchanged—indicating pathology driven by dysregulated inflammatory responses rather than impaired antiviral immunity.
Conclusions: Although RSV rarely invades the CNS directly, severe infections are associated with neurological symptoms and hyperinflammatory responses, suggesting that immune-mediated pathology rather than viral replication drives brain injury.
How VarSeq Was Used: Genomic DNA from the patient’s PBMCs and his parents’ PBMCs was purified using the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer’s instructions. WGS was performed using Illumina TruSeq PCR-free library prep according to the procedures from the manufacturer (Illumina). The libraries were sequenced paired-end on a NovaSeq 6000 platform (Illumina) to a mean depth of at least 30×. Alignment and variant calling were performed using the Burrows-Wheeler Aligner (BWA) and the Genome Analysis Toolkit (GATK), with the hg19 human genome as a reference. VarSeq (Golden Helix) was used for annotation and filtering of genomic variants, and CNVkit, CNVnator, Manta, and Lumpy were used for copy number analysis. Data from patients and their parents were filtered for variants in all known disease genes, primarily using the Online Mendelian Inheritance in Man database.
Citation: Jonas Lønskov, Annika Sünderhauf, Sisse Andersen, Caroline Bækmann Jeppesen, Franziska Winzig, Daniëla Maria Hinke, Johanna L. Heinz, Kenneth Thomsen, Mette B. Thorup, Thomas Zillinger, Bettina Bundgaard, Kerstin De Keukeleere, Sofie Eg Jørgensen, Jakob Ek, Elsebet Østergaard, Jakob Christensen, Mette Møller Handrup, Renee M. van der Sluis, Trine H. Mogensen; Viral infection and brain inflammation with seizures in PARK7 deficiency. J Hum Immun 2 March 2026; 2 (2): e20250044. doi: https://doi.org/10.70962/jhi.20250044
This month’s publications highlight how diverse clinical questions, ranging from pharmacogenomics to immunology, can benefit from precise, structured variant analysis. Across these different phenotypes, the analytical needs remain consistent: robust filtering, reproducible workflows, and clear evidence frameworks. VarSeq supports this range by giving teams a unified environment to assess variants from exome, genome, or targeted data with confidence and consistency. To learn more about how VarSeq supports clinical and research programs by contacting our team today.
