Kluge, Michelle L.

Abstract Background DPYD testing can identify patients at risk for severe toxicity when a standard fluoropyrimidine antineoplastic dose is prescribed. Sequencing-based methods detect rare variants, which require classification. Methods DPYD sequencing results over a 9-year period through to 2024 were analyzed. The dataset was filtered to calculate the positive test rate for different genotyping strategies, including targeted analysis of the Association for Molecular Pathology (AMP) Tier 1 and 2 recommended alleles, and as compared to sequencing. Variant classifications based on the laboratory’s clinical protocol were compared to ClinVar classifications and designations by the Clinical Pharmacogenetics Implementation Consortium (CPIC) expert panel. Results A DPYD variant was reported in 12.79% (896/7003) of those tested, with 2.53% (170/7003) of all reports including a variant of uncertain significance. Had only AMP Tier 1 or both Tier 1 and 2 been tested, 9.30% and 9.44% of patients would have received a positive report, respectively. Among the 281 unique, non-Tier 1/Tier 2 variants encountered during sequencing, >80% were not classified by the CPIC expert panel, while >48% were not reported in ClinVar. Variant classifications by our laboratory and those in ClinVar were generally concordant with the CPIC expert panel, despite the lack of a standardized classification system. Conclusions While AMP Tier 1 genotyping would have identified most patients at risk for fluoropyrimidine toxicity, sequencing is more comprehensive, identifying 26% more reportable variants compared to targeted genotyping. A standard framework for pharmacogenomic variant classification could facilitate consistent variant classification and a transition from targeted genotyping to sequencing.

Abstract Objectives Protein-truncating variants in the TTN gene are a well-established cause of dilated cardiomyopathy (DCM). We report a novel case of DCM caused by a mobile element insertion (MEI) in TTN, through which we highlight the key features of MEIs in next-generation sequencing data. Because of the rarity of MEIs, the next-generation sequencing data features associated with these events may be mistaken as noise, potentially leading to missed diagnoses. Methods Next-generation sequencing gene panel testing for DCM was performed on a 17-year-old male patient presenting with severe left ventricular dilatation and systolic dysfunction. Manta was used for structural variant detection, followed by manual review of NGS data for potential structural variants. Results Manta detected a potential insertion in TTN. Manual review identified hallmark features consistent with a LINE-1 MEI. This finding was orthogonally confirmed by long-range polymerase chain reaction and gel electrophoresis, which indicated an insertion of approximately 4 to 5 kilobase pairs. The insertion disrupted the reading frame of TTN within an A-band exon, resulting in protein truncation that was classified as likely pathogenic. Conclusions This case expands the mutational spectrum of TTN protein-truncating variants. It also underscores the importance of recognizing rarer types of pathogenic variants (eg, MEIs) to produce accurate genetic diagnostics.

Introduction Genotype-phenotype discordance occurs occasionally in congenital adrenal hyperplasia (CAH). Its causes are largely unknown. We describe a case of monochorionic, diamniotic twins with discordant clinical presentations of CAH, and show evidence for this being due to mosaicism resulting from a postzygotic full gene deletion of CYP21A2 prior to twinning. Case description A 7-day-old 36-week gestation female infant (Twin A) presented to the emergency department with elevated 17-hydroxyprogesterone (17-OHP). Her identical twin (Twin B) had normal 17-OHP on newborn screening. Both twins showed signs of virilization, more pronounced in Twin B. Molecular genetic testing of both twins and their parents showed a WT paternally-inherited CYP21A2 and a maternally-inherited copy containing the c.293-13C>G mutation. Both twins were also found to have a 5′-CYP21A1P/CYP21A2-3′ hybrid (product of the common 30-kb deletion), derived from the deletion of the paternally-inherited CYP21A2. Neither mother nor father carried the deletion. Conclusions The genetic findings are consistent with mosaicism for two CYP21A2 cell lines in both twins. The first cell line is expected, based on parental results, while the second line is due to a postzygotic full gene deletion of the paternally-inherited WT CYP21A2. The resultant genotype, compound heterozygosity for c.293-13C>G and a CYP21A2 full gene deletion, is consistent with a salt-wasting CAH phenotype. Differential distribution of the second cell line between the twins is most likely the cause for their discrepant phenotypes. We believe this is the first report of somatic CYP21A2 mosaicism, and represents a novel cause for discrepant CAH phenotypes in monozygotic twins.

Background— Postmortem genetic testing for heritable cardiovascular (CV) disorders is often lacking because ideal specimens (ie, whole blood) are not retained routinely at autopsy. Formalin-fixed paraffin-embedded tissue (FFPET) is ubiquitously collected at autopsy, but DNA quality hampers its use with traditional sequencing methods. Targeted next-generation sequencing may offer the ability to circumvent such limitations, but a method has not been previously described. The primary aim of this study was to develop and evaluate the use of FFPET for heritable CV disorders via next-generation sequencing. Methods and Results— Nineteen FFPET (heart) and blood (whole blood or dried blood spot) specimens underwent targeted next-generation sequencing using a custom panel of 101 CV-associated genes. Nucleic acid yield and quality metrics were evaluated in relation to FFPET specimen age (6 months to 15 years; n=14) and specimen type (FFPET versus whole blood and dried blood spot; n=12). Four FFPET cases with a clinical phenotype of heritable CV disorder were analyzed. Accuracy and precision were 100% concordant between all sample types, with read depths >100× for most regions tested. Lower read depth, as low as 40×, was occasionally observed with FFPET and dried blood spot. High-quality DNA was obtained from FFPET samples as old as 15 years. Genomic analysis of FFPET from the 4 phenotype-positive/genotype unknown cases all revealed putative disease-causing variants. Conclusions— Similar performance characteristics were observed for next-generation sequencing of FFPET, whole blood, and dried blood spot in the evaluation of inherited CV disorders. Although blood is preferable for genetic analyses, this study offers an alternative when only FFPET is available.

We report a 9-year-old Chinese girl with congenital thrombotic thrombocytopenic purpura found to be a compound heterozygote for 2 pathogenic variants in the ADAMTS13 gene, including a novel variation. The girl suffered from recurrent, life-threatening episodes of thrombocytopenia and hemolysis, and laboratory testing showed ADAMST13 enzyme activity of <5%. Sequencing of the ADAMTS13 gene revealed a previously reported missense variant, c.1787C>T (p.Ala596Val), and a novel duplication defined as c.1007_1025dup19 (p.Asp343Leufs*53); the duplication is predicted to result in a premature stop codon and protein truncation. We propose that this novel variant is partly responsible for the patient’s early-onset and severe phenotype.