Analysis of genotypes and biochemical phenotypes of neonates with abnormal metabolism of butyrylcarnitine

［ Abstract ］ Objective : To investigate the genotypes and biochemical phenotypes of neonates with abnormal metabolism of butyrylcarnitine (C4). Methods : One hundred and twenty neonates with increased C4 levels detected by tandem mass spectrometry in the neonatal screening at Children ’ s Hospital of Zhejiang University School of Medicine from January 2018 to June 2023. The initial screening data and recalled data of C4 and C4/C3 were collected and converted into multiples of C4 reference range. The next generation sequencing was performed and the exons with adjacent 50 bp regions of ACAD8 and ACADS genes were captured by liquid phase capture technique. The variants were obtained by bioinformatic analysis and the pathogenicity were classified according to the ACMG criteria. The neonates with increased C4 levels were divided into ACAD8 biallelic variants group, ACADS biallelic variants group and ACADS monoallelic variants group. The Wilcoxon rank sum test was used to analyze the differences among the groups. Results : In total, 32 variants in ACAD8 gene were detected, of which 7 variants were first discovered; while 41 variants of ACADS gene were detected, of which 17 variants had not been reported yet. There were 39 cases with ACAD8 biallelic variants, 3 cases with ACAD8 monoallelic variants; 34 cases with ACADS biallelic variants and 36 cases with ACADS monoallelic variants. Furthermore, 5 cases were detected with both ACAD8 and ACADS gene variants. Inter group comparison showed that the multiples of C4 reference range in initial screening and reexamination of the ACAD8 biallelic variants and ACADS biallelic variants groups were significantly higher than those of the ACADS monoallelic variants group (all P <0.01), while the multiples in the ACAD8 biallelic variants group were significantly higher than those in the ACADS biallelic variants group (all P <0.01). The multiples of C4 reference range in initial screening greater than 1.5 times were observed in all neonates carrying ACAD8 or ACADS biallelic variants, while only 25.0 % (9/36) in neonates carrying ACADS monoallelic variants. Conclusion : ACAD8 and/or ACADS gene variants are the main genetic causes of elevated C4 in newborns in Zhejiang region with high genotypic heterogeneity. The C4 levels of neonates with biallelic variants are significantly higher than those of neonates with monoallelic variants. If the cutoff value of C4 level could be modestly elevated, false positive rate would be reduced in tandem mass spectrometry neonatal screening.