Validation of a Targeted Next-Generation Sequencing workflow for CPT1A Gene Analysis Using Dried Blood Spots from Pediatric Patients Suspected of Carnitine Palmitoyl transferase 1A Deficiency in Vietnam
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Abstract
Carnitine palmitoyltransferase 1A (CPT1A) deficiency is a rare inherited disorder of fatty acid oxidation, biochemically characterized by elevated free carnitine (C0) and reduced long-chain acylcarnitines (C16, C18). This study aimed to establish a targeted next-generation sequencing (NGS) workflow for the CPT1A gene and to identify disease-causing variants in pediatric patients with suspected CPT1A deficiency detected by newborn screening. Twenty-one primer pairs covering all coding regions of CPT1A were designed and validated. PCR amplification was performed on dried blood spot samples and controls, followed by sequencing on the DNBSEQ-G200 platform. Sequencing quality metrics exceeded clinical standards, with Q30 ≥ 84.5%, total reads of 14.65 million, duplication rate of 9.8%, mean depth ranging from 127.77× to 6,932.05×, and 100% target coverage at ≥ 20×. In the suspected patient (S01), nine heterozygous variants were detected, all classified as benign according to ClinVar and gnomAD. Despite a strongly suggestive biochemical profile (C0 = 207.67 µmol/L; C0/(C16+C18) ≈ 407), no pathogenic variant was identified. These findings confirm the robustness of the PCR–NGS workflow and highlight the necessity of integrating biochemical screening, genomic analysis, and second-tier tests to resolve biochemical–genetic discordance in CPT1A deficiency.
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Keywords
CPT1A deficiency, CPT1A gene, next-generation sequencing, NGS, fatty acid oxidation disorders, newborn screening
References
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