Incorporating Next‐Generation Sequencing as a Second‐Tier Test for Primary Carnitine Deficiency

ABSTRACT Background Newborn screening (NBS) for primary carnitine deficiency (PCD) has poor performance. This study aimed to evaluate the feasibility of incorporating next‐generation sequencing (NGS) as a second‐tier PCD test. Methods Between March and December 2020, 60,070 newborns were screened for inherited metabolic disorders. Newborns with free carnitine (C0) levels below 8.5 μmol/L were selected for second‐tier genetic testing. Results In total, 130 (0.22%) newborns with low C0 levels underwent second‐tier genetic testing, 87 (66.92%) had positive genetic testing results, and 30 (23.08%) carried pathogenic variants of the SLC22A5 gene. Six newborns were diagnosed with PCD. The incidence of PCD was approximately 1 in 1:10,012 newborns. The PPV reached 20% after combining with second‐tier NGS. Of the eight variants identified in patients with PCD, the three most common variants were c.760C>T (p.Arg254*), c.51C>G (p.Phe17Leu), and c.1400C>G (p.Ser467Cys). The C0 levels of patients with PCD were significantly lower than those of PCD carriers ( p = 0.0026) and PCD‐negative individuals ( p = 0.0005). Conclusions Our results showed that the PPV reached 20% after combining with second‐tier NGS. The MS/MS‐based NBS and second‐tier NGS combination can effectively reduce the false‐positive rate and detect PCD in patients.