RNA analysis of intronic variants in the LAMA2 gene detected by whole genome sequencing confirms a rare dual diagnosis of incontinentia pigmenti with limb‐girdle muscular dystrophy

Abstract We see that a multiple methods approach to diagnosis remains necessary in the era of whole genome sequencing. We also observe that reproductive risk genetic counseling can be a motivating factor for further testing along the diagnostic odyssey

RNA analysis of the GALNS transcript reveals novel pathogenic mechanisms associated with Morquio syndrome A.

Morquio syndrome A (Mucopolysaccharidosis IVA, MPS IVA) is an autosomal recessive lysosomal storage disorder caused by deficiency of N-acetyl-galactosamine-6-sulfatase (GALNS) which catabolizes the glycosaminoglycans (GAG), keratan sulfate and chondroitin-6-sulfate. Homozygous or compound heterozygous pathogenic variants in the GALNS result in the deficiency of the enzyme and consequent GAG accumulations. DNA sequence and copy number analysis of the GALNS coding region fails to identify biallelic causative pathogenic variants in up to 15% of patients with Morquio syndrome A. RNA transcript analysis was performed to identify pathogenic alterations in two unrelated families with Morquio syndrome A in whom a single heterozygous or no pathogenic alteration was detected by standard analysis of the GALNS gene. RNA sequencing and quantitative expression analysis identified the overabundance of an aberrant GALNS transcript isoform and a reduction of the clinically relevant isoform (NM_000512.4) in the Morquio syndrome A patients from both families. The aberrant isoform (ENST00000568613.1) was produced by alternative splicing and contained intronic sequence that was likely a cryptic exon predicted to result in a reading frame shift and generation of a premature termination codon. These findings indicated that the aberrant splicing is likely the novel molecular defect in our patients. RNA transcript analysis could be useful to identify pathogenic alterations and increase the yield of molecular diagnosis in patients with Morquio syndrome A whose genetic variants are not found by standard sequencing or gene dosage analysis

Cross-oncopanel study reveals high sensitivity and accuracy with overall analytical performance depending on genomic regions.

Targeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort, we perform a cross-platform multi-lab evaluation of eight Pan-Cancer panels to assess best practices for oncopanel sequencing. All panels demonstrate high sensitivity across targeted high-confidence coding regions and variant types for the variants previously verified to have variant allele frequency (VAF) in the 5-20% range. Sensitivity is reduced by utilizing VAF thresholds due to inherent variability in VAF measurements. Enforcing a VAF threshold for reporting has a positive impact on reducing false positive calls. Importantly, the false positive rate is found to be significantly higher outside the high-confidence coding regions, resulting in lower reproducibility. Thus, region restriction and VAF thresholds lead to low relative technical variability in estimating promising biomarkers and tumor mutational burden. This comprehensive study provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.All SEQC2 participants freely donated their time, reagents, and computing resources for the completion and analysis of this project. Part of this work was carried out with the support of the Intramural Research Program of the National Institutes of Health (to Mehdi Pirooznia), National Institute of Environmental Health Sciences (to Pierre Bushel), and National Library of Medicine (to Danielle Thierry-Mieg, Jean Thierry-Mieg, and Chunlin Xiao). Leming Shi and Yuanting Zheng were supported by the National Key R&D Project of China (2018YFE0201600), the National Natural Science Foundation of China (31720103909), and Shanghai Municipal Science and Technology Major Project (2017SHZDZX01). Donald J. Johann, Jr. acknowledges the support by FDA BAA grant HHSF223201510172C. Timothy Mercer and Ira Deveson were supported by the National Health and Medical Research Council (NHMRC) of Australia grants APP1108254, APP1114016, and APP1173594 and Cancer Institute NSW Early Career Fellowship 2018/ECF013. This research has also been, in part, financially supported by the MEYS of the CR under the project CEITEC 2020 (LQ1601), by MH CR, grant No. (NV19-03-00091). Part of this work was carried out with the support of research infrastructure EATRIS-CZ, ID number LM2015064, funded by MEYS CR. Boris Tichy and Nikola Tom were supported by research infrastructure EATRIS-CZ, ID number LM2018133 funded by MEYS CR and MEYS CR project CEITEC 2020 (LQ1601).S