Amplicon sequencing-based carrier screening for 170 monogenic disorders among children with abnormal LC-MS/MS results

Background: Next-generation sequencing (NGS) has been suggested as a second-tier diagnostic test for newborn screening, which could help identify the carrier status of hundreds monogenic disorders with wider spectrum and earlier stage.

Methods: Among the 1087 children (age from 27 min to 14 years old) underwent liquid chromatography-tandem mass spectrometry (LC-MS/MS), 290 individuals who had at least one abnormal value of LC-MS/MS measurements were sent for amplicon sequencing-based carrier screening (targeting 141 genes for 170 monogenic disorders). Multiplex polymerase chain reaction was used for amplicon capture and library preparation, the NextSeq 500 NGS platform (Illumina PE150) was used for sequencing. The identified clinical significant variants were further validated by Sanger sequencing.

Results: Only 89 children carry none of clinical significant variants, other 201 individuals carry 1-4 variants in 63 genes (132 types; 317 in total: 171 pathogenic, 37 likely pathogenic, 29 variants of unknown significance, and 80 disease-associated functional polymorphisms). Besides the three missing samples with 4 variants, 91.1 % of identified variants (285 variants in 54 genes) were completely validated by Sanger sequencing. The most common genetic variants were in UGT1A1, GJB2, PAH, G6PD, and SLC25A13 (top 5 genes), which corresponding to Gilbert/Crigler-Najjar symdrome (n = 89), autosomal recessive hearing loss type 1A (n = 58), phenylketonuria (n = 12), glucose-6-phosphate dehydrogenease deficiency (n = 11) and Citrin deficiency (n = 9). More than 42 children present higher phenylalanine in LC-MS/MS, but only 12 of them were identified to carry clinical significant variants in PAH gene.

Conclusion: The amplicon sequencing-based carrier screening in our study could further clarify the abnormal LC-MS/MS results, which could also discover more monogenic disorders uncovered by LC-MS/MS screening.