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Skeletal muscle capillary density is correlated with physical performance and whole-body metabolic properties. Thus, we performed a genome-wide association study of skeletal muscle capillary-to-fiber ratio (C:F) (n = 603 males) and found that the rs115660502 G allele was associated (p < 5 × 10^-8) with increased C:F and reduced skeletal muscle expression of RAB3 GTPase-activating non-catalytic protein subunit 2 (RAB3GAP2). The capillary-increasing G allele was more prevalent in elite endurance athletes than in power athletes and non-athlete controls in two independent cohorts. Low-muscle-expressing RAB3GAP2 expression quantitative trait locus (eQTL) alleles were associated with muscle damage in athletes. In healthy individuals, RAB3GAP2 expression was reduced by high-intensity intermittent training. RAB3GAP2 protein was not uniformly expressed in muscle but predominantly expressed in the endothelium and capillaries. RAB3GAP2 expression was lower in endurance compared with power athletes and was negatively associated with type I (oxidative) muscle fiber density. Experimental reduction of RAB3GAP2 in human endothelial cells led to (1) increased proliferation and tube formation in vitro, (2) regulation of secreted factors (e.g., CD70 and TNC) promoting angiogenesis and T cell activation, and (3) increased in vivo endothelial cell density in mice. RAB3GAP2 expression in skeletal muscle was negatively correlated with exercise-induced release of TNC in vivo in humans. In conclusion, RAB3GAP2 is expressed in the microvascular endothelium and is suggested to be a negative regulator of angiogenesis through a decrease in endothelial cell proliferation, possibly mediated by RAB18, with its low-expressing variant associated with higher muscle C:F and elite endurance performance.

The Twist Comprehensive Viral Research Panel (Twist CVRP) is a probe-based hybridization capture enrichment method for whole-genome sequencing, designed to target all known pathogenic viruses. Unlike shotgun metagenomics, where human DNA dominates, this method enriches for viral sequences within samples. This study presents a novel protocol called Twist-ONT, integrating Twist CVRP with Oxford Nanopore Technologies (ONT) long-read sequencing. Using clinical nasopharyngeal/throat swab and plasma samples PCR-positive for a variety of different viruses, the protocol's capability for viral species classification was demonstrated. It is also shown how high-quality whole-genome assemblies and consensus sequences can be generated from the sequencing reads of this protocol. This protocol facilitates further studies into the viromes of clinical samples and viral genomics in general using ONT sequencing.

Background

Genomic profiling of patients with myeloid neoplasms (MN) may detect variants with high variant allele frequency (VAF), indicating potential germline origin. All current guidelines recommend further investigation of such variants.Objective To define the proportion of variants with suspected germline origin that prompted germline investigation in patients evaluated for MN in the clinical setting.

Methods

We included 738 patients investigated for MN in Mid-Sweden between May 2020 and January 2023 with available sequencing data in genes of interest (GOI) according to the Nordic and other international guidelines.

Results

In total, 90 patients (12%) carried pathogenic/likely pathogenic (P/LP) variants in GOI according to the Nordic guidelines, with TP53, RUNX1, and NRAS being the most common (n = 37, n = 27, and n = 9, respectively). The potential germline origin was investigated in 16/90 (18%) patients. Two patients (2/16, 12.5%) with germline variants were identified (DDX41 and RUNX1, n = 1, respectively). In patients treated with allogeneic stem-cells transplantation (n = 15) the investigation rate was higher (7/15, 47%, p < 0.005) compared to the non-transplanted ones. Applying the European Leukemia Net (ELN) guidelines the number of patients with variants in GOI increased up to 177 mainly due to the inclusion of TET2, while application of other international guidelines leads to similar results with the Nordic ones.

Conclusion

We report a low investigation rate for variants of suspected germline origin detected during the genomic profiling of patients investigated for MN in the clinical setting. Our study underscores the need for further awareness regarding germline investigation for hematological malignancies.

This study aimed to elucidate the clonal origin and evolutionary dynamics of T-cell prolymphocytic leukemia (T-PLL) using targeted next generation sequencing (NGS) of paired samples from diagnosis and relapse. DNA from both nonmalignant and tumor cells was extracted from sorted cell fractions obtained from 16 patients with T-PLL. NGS was performed using a customized Haloplex gene panel comprising 19 genes recurrently mutated in T-PLL (ATM and JAK/STAT pathway). Droplet digital polymerase chain reaction was performed to confirm mutations detected by NGS with low variant allele frequencies. Single-cell analysis of genomic DNA combined with cell surface protein markers was performed using the Mission Bio Tapestri Platform. The most frequently mutated gene was ATM (n = 10) followed by STAT5B (n = 7), JAK3 (n = 3), EZH2 (n = 3), BCOR (n = 1), and STAT6 (n = 1). Relapse samples were available for 9 of the 16 patients. Varying patterns of clonal shifts were observed between diagnosis and relapse (increase, decrease, both increase and decrease, and no change). The presence of pathogenic variants in ATM, EZH2, STAT5B, and JAK3 in both normal sorted B cells and clonal T cells was confirmed. Single-cell analysis revealed shared mutations in both nonmalignant B and clonal T cells in 1 case. A pathogenic variant within the ATM gene of potential germ line origin was observed in 1 case. T-PLL exhibits variable patterns of clonal evolution between diagnosis and relapse. Single-cell multiomics analysis reveals shared mutational signatures in both nonmalignant B cells and clonal T cells. The role of germ line ATM mutations in the pathogenesis of T-PLL should be further explored.

Purpose: In a multicenter prospective cohort study, we assessed the diagnostic yield of the Nordic guidelines for germline investigation in myeloid neoplasms and mapped the spectrum of inherited and somatic variants.Experimental Design: Eighty-five patients (acute myeloid leukemia, n = 38; myelodysplastic syndromes, n = 26; thrombocytopenia, n = 14; and other, n = 7) fulfilling the Nordic criteria for germline investigation, based on (i) medical history or family history suggestive of a germline condition and (ii) relevant findings from the somatic diagnostic work-up (CytoMol), were recruited. The genetic analysis included enhanced whole-exome sequencing (n = 69) or sequencing of specific variants of interest (n = 16).

Results: Pathogenic or likely pathogenic (P/LP) germline variants were identified in 35% of patients (30/85). The diagnostic yield varied from 6% (1/16) in the family history group to 52% (17/33) in the CytoMol group. Germline DDX41 P/LP variants were the most frequent finding (13/30, 43% of all positive cases) almost exclusively found within the CytoMol group (12/13). Seven variants of unknown significance were also detected (TERT n = 2 and DDX41, RTEL1, ETV6, PARN, and SAMD9 n = 1). Five patients carried a P/LP variant in genes associated with another hereditary cancer syndrome (BRCA1 n = 3; PALB2 n = 1; and CHEK2; n = 1). Survival analysis showed a trend for longer survival among patients with acute myeloid leukemia and confirmed or suspected germline predisposition that underwent allogeneic stem cell transplantation.

Conclusions: The implementation of the Nordic guidelines in a prospective Swedish cohort results in a high overall diagnostic yield (35%), proving the feasibility and utility of these or similar guidelines in a clinical setting.

Background

Noninvasive prenatal testing (NIPT) is increasingly used to screen for fetal chromosomal aneuploidy by analyzing cell-free DNA (cfDNA) in peripheral maternal blood. The method provides an opportunity for early detection of large genetic abnormalities without an increased risk of miscarriage due to invasive procedures. Commercial applications for use at clinical laboratories often take advantage of DNA sequencing technologies and include the bioinformatic workup of the sequence data. The interpretation of the test results and the clinical report writing, however, remains the responsibility of the diagnostic laboratory. In order to facilitate this step, we developed NIPTviewer, a web-based application to visualize and guide the interpretation of NIPT data results.

Results

NIPTviewer has a database functionality to store the NIPT results and a web interface for user interaction and visualization. The application has been implemented as part of a novel analysis pipeline for NIPT in a diagnostic laboratory at Uppsala University Hospital. The validation data set included 84 previously analyzed plasma samples with known results regarding chromosomes 13, 18, 21, X and Y. They were sequenced in six different experiments, uploaded to NIPTviewer and assigned to a clinical laboratory geneticist for interpretation. The results of all previously analyzed samples were replicated.

Conclusion

NIPTviewer facilitates NIPT results interpretation and has been implemented as part of a NIPT analysis routine that was accredited by the national accreditation body for Sweden (Swedac).

The COVID-19 pandemic highlighted the need for a rapid, convenient, and scalable diagnostic method for detecting a novel pathogen amidst a global pandemic. While command-line interface tools offer automation for SARS-CoV-2 Oxford Nanopore Technology sequencing data analysis, they are inapplicable to users with limited programming skills. A solution is to establish such automated workflows within a graphical user interface software. We developed two workflows in the software Geneious Prime 2022.1.1, adapted for data obtained from the Midnight and Artic's nCoV-2019 sequencing protocols. Both workflows perform trimming, read mapping, consensus generation, and annotation on SARS-CoV-2 Nanopore sequencing data. Additionally, one workflow includes phylogenetic assignment using the bioinformatic tools pangolin and Nextclade as plugins. The basic workflow was validated in 2020, adhering to the requirements of the European Centre for Disease Prevention and Control for SARS-CoV-2 sequencing and analysis. The enhanced workflow, providing phylogenetic assignment, underwent validation at Uppsala University Hospital by analysing 96 clinical samples. It provided accurate diagnoses matching the original results of the basic workflow while also reducing manual clicks and analysis time. These bioinformatic workflows streamline SARS-CoV-2 Nanopore data analysis in Geneious Prime, saving time and manual work for operators lacking programming knowledge.