Technical and Clinical Assessment of Custom HaloPlex Target Enrichment and High-Throughput Sequencing Assay In Foetal and Infant Forensic Pathology
Soidinsalo, Pasi (2017-09-27)
Technical and Clinical Assessment of Custom HaloPlex Target Enrichment and High-Throughput Sequencing Assay In Foetal and Infant Forensic Pathology
Soidinsalo, Pasi
(27.09.2017)
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Turun yliopisto
Tiivistelmä
Channelopathies, cardiomyopathies and certain inborn errors of metabolism cause unexpected death during gestation and infancy. The fatality can occur suddenly or after a rapidly progressive disease. Frequently, the diseases present only non-specific symptoms, which confound the diagnosis. Approximately one third of infant fatalities and 20-40% of third trimester foetal deaths are left unexplained after medico-legal postmortem investigation. Mutations in hundreds of genes are associated with potentially fatal monogenetic diseases. Determining the genetic defect post-mortem can facilitate diagnosis and enable genetic counselling, which is crucial in the management of affected family members or future offspring in risk of bearing the genetic disease.
Genetic testing of dozens of genes has remained economically and technically unfeasible until recently due to the advent of high-throughput massive parallel sequencing, which allows multiple genes of multiple patients to be investigated in a single test. In this study, a custom targeted high-throughput sequencing assay was established based on the HaloPlex target enrichment method. The assay targets 64 genes, which are associated with potentially fatal diseases with confounding phenotypes. The performance and clinical utility of the assay was evaluated utilizing Illumina MiSeq sequencer in a retrospective cohort study of 44 unexplained foetal, neonatal and post-neonatal infant death cases. Additionally, 12 positive genomic DNA controls containing 16 known pathogenic mutations were sequenced using the assay to evaluate the assay’s analytical validity.
Post-mortem tissue-derived DNA yielded sequencing libraries with good quality. With 10 samples per run, the HaloPlex assay performed with satisfactory overall quality statistics, predicting high sensitivity and specificity for the assay. Out of the targeted 376 226 base pairs, 99.46% showed coverage over the confident variant call threshold of 15X. However, a few genomic regions suffered from low coverage and remained outside of the assay’s confident detection range. The assay revealed a likely pathogenic mutation in 3 (7%) of the 44 sequenced patients. All 12 positive controls were correctly identified with the assay, and all clinically relevant mutations were true positives after validation with capillary electrophoresis sequencing. The experimental validation of the sensitivity yielded 100% sensitivity, with 95% CI of 83.16% to 100%. The performance of the assay was sufficient for clinical diagnostics and showed clinical utility in finding likely pathogenic mutations contributing to the cause of death. The established targeted sequencing assay can be utilised by neonatologists, geneticists and pathologists in hospitals to reveal potential genetic origins of foetal or infant death in cases in which a genetic disease was suspected, and in cases in which the death was sudden and unexplained.
Genetic testing of dozens of genes has remained economically and technically unfeasible until recently due to the advent of high-throughput massive parallel sequencing, which allows multiple genes of multiple patients to be investigated in a single test. In this study, a custom targeted high-throughput sequencing assay was established based on the HaloPlex target enrichment method. The assay targets 64 genes, which are associated with potentially fatal diseases with confounding phenotypes. The performance and clinical utility of the assay was evaluated utilizing Illumina MiSeq sequencer in a retrospective cohort study of 44 unexplained foetal, neonatal and post-neonatal infant death cases. Additionally, 12 positive genomic DNA controls containing 16 known pathogenic mutations were sequenced using the assay to evaluate the assay’s analytical validity.
Post-mortem tissue-derived DNA yielded sequencing libraries with good quality. With 10 samples per run, the HaloPlex assay performed with satisfactory overall quality statistics, predicting high sensitivity and specificity for the assay. Out of the targeted 376 226 base pairs, 99.46% showed coverage over the confident variant call threshold of 15X. However, a few genomic regions suffered from low coverage and remained outside of the assay’s confident detection range. The assay revealed a likely pathogenic mutation in 3 (7%) of the 44 sequenced patients. All 12 positive controls were correctly identified with the assay, and all clinically relevant mutations were true positives after validation with capillary electrophoresis sequencing. The experimental validation of the sensitivity yielded 100% sensitivity, with 95% CI of 83.16% to 100%. The performance of the assay was sufficient for clinical diagnostics and showed clinical utility in finding likely pathogenic mutations contributing to the cause of death. The established targeted sequencing assay can be utilised by neonatologists, geneticists and pathologists in hospitals to reveal potential genetic origins of foetal or infant death in cases in which a genetic disease was suspected, and in cases in which the death was sudden and unexplained.