Abstract:

Objective To assess the efficacy of tandem mass spectrometry?specific indicators in diagnosing β?ketothiolase deficiency （BKD） and to elucidate the associated gene variations contributing to the corresponding pathogenic phenotype， thereby facilitating rapid and accurate diagnosis of BKD. Methods Data from tandem mass spectrometry （MS/MS） screening of dried blood spots collected from 16，071 children between January 2018 and December 2021， along with results from gas chromatography?mass spectrometry （GC?MS） analysis and high?throughput sequencing of positive cases， were analyzed retrospectively. The study aimed to evaluate the contribution of specific MS/MS indicators in the clinical diagnosis of BKD and to trace the genetic etiology of this disease. Results Among the 16 071 subjects screened by MS/MS， 37 cases （2.30‰） exhibited elevated C5OH levels， 41 cases （2.55‰） showed increased C5∶1 levels， and 2 cases were diagnosed with BKD based on GC?MS analysis. When diagnosing BKD using C5OH as a single indicator， the false positive rate was 0.22%， which is lower than that of C5∶1 （0.24%）. The positive predictive value for C5OH was 5.40%， higher than that of C5∶1 （4.88%）. Among the 16 071 pediatric patients， only 2 cases were diagnosed with BKD due to elevated C5OH combined with increased C5∶1 levels， resulting in a positive predictive value of 100%. Whole exome sequencing of these two BKD patients revealed that both carried acetyl?CoA acetyltransferase 1 （ACAT1） gene double allele missense heterozygous mutations. The four previously unreported mutation sites were c.949G > C （p.Asp317His）， c.1063G > A （p.Ala355Thr）， c.146G > A （p.Arg49Lys）， and c.700G > A （p.Glu234Lys）. These findings provide novel insights into the genetic basis of BKD. Conclusions The MS/MS screening indicator C5OH demonstrates superior diagnostic efficacy compared to C5∶1 in diagnosing BKD， as evidenced by lower false positive rates and higher positive predictive values. When diagnosing BKD， the use of combined indicators significantly enhances the accuracy of biochemical diagnosis compared to single indicators. Exome sequencing revealed that all BKD patients carried previously unreported mutations in the ACAT1.

Key words: beta-ketothiolase deficiency （BKD）, tandem mass spectrum （MS/MS）, acylcarnitine， protein sequence alignment