A comprehensive multiplex PCR based exome-sequencing assay for rapid bloodspot confirmation of inborn errors of metabolism

Wenjie Wang,Weiyue Gu,Wang Wu,Wenjuan Mu,Xiaobing Xie,Jigeng Bai,Xiaogang Zhang,Jianping Yang,Jinjie Xue,David S Cram,Yiqian Liu,Yuyan Gong,Yu Zhang,Mengnan Xu

doi:10.1186/s12881-018-0731-5

Abstract

BackgroundTandem mass spectrometry (MS MS) and simple fluorometric assays are currently used in newborn screening programs to detect inborn errors of metabolism (IEM). The aim of the study was to evaluate the clinical utility of exome sequencing as a second tier screening method to assist clinical diagnosis of the newborn.MethodsA novel PCR-exome amplification and re-sequencing (PEARS) assay was designed and used to detect mutations in 122 genes associated with 101 IEM. Newborn bloodspots positive by biochemical testing were analysed by PEARS assay to detect pathogenic mutations relevant to the IEM.ResultsIn initial validation studies of genomic DNA samples, PEARS assay correctly detected 25 known mutations associated with 17 different IEM. Retrospective gene analysis of newborns with clinical phenylketonuria (PKU), identified compound heterozygote phenylalanine hydroxylase (PAH) gene mutations in eight of nine samples (89%). Prospective analysis of 211 bloodspots correctly identified the two true PKU samples, yielding positive and negative predictive values of 100%. Testing of 8 true positive MS MS samples correctly identified potentially pathogenic compound heterozygote genotypes in 2 cases of citrullinemia type 1 and one case each of methylmalonic acidemia, isobutyryl-CoA dehydrogenase deficiency, short chain acyl-CoA dehydrogenase deficiency and glutaric acid type II and heterozygous genotypes in 2 cases of autosomal dominant methioninemia. Analysis of 11 of 12 false positive MS MS samples for other IEM identified heterozygous carriers in 8 cases for the relevant genes associated with the suspected IEM. In the remaining 3 cases, the test revealed compound heterozygote mutations in other metabolic genes not associated with the suspected IEM, indicating a misinterpretation of the original MS MS data.ConclusionsThe PEARS assay has clinical utility as a rapid and cost effective second-tier test to assist the clinician to accurately diagnose newborns with a suspected IEM.

Highlights

Tandem mass spectrometry (MS Tandem mass spectroscopy (MS)) and simple fluorometric assays are currently used in newborn screening programs to detect inborn errors of metabolism (IEM)
Performance of the PCR-exome amplification and re-sequencing (PEARS)-101 test for detection of pathogenic mutations in metabolic disease genes For initial validation of the PEARS-101 test, the newborn screening laboratory selected 8 peripheral blood genomic DNA samples harbouring known pathogenic variants that were previously identified by Sanger sequencing (Table 1)
Our findings demonstrate that gene testing is a useful method to help verify newborns positive for an Inborn errors of metabolism (IEM) and guide specific biochemical confirmatory testing

Summary

Introduction

Tandem mass spectrometry (MS MS) and simple fluorometric assays are currently used in newborn screening programs to detect inborn errors of metabolism (IEM). Inborn errors of metabolism (IEM) are a heterogeneous group of genetic disorders caused by specific enzyme defects in complex metabolic pathways, resulting in a perturbed metabolic state due to either depletion of essential metabolites or the accumulation of abnormal levels of toxic metabolites. Genotype-phenotype studies of compound heterozygotes have shown that the clinical phenotype of each disease is usually dependent on the expression level of the allele harbouring the less severe pathogenic mutation [6, 7]. PKU is characterised by high levels of blood phenylalanine from birth which results in a state of hyperphenylalaninemia, and if undiagnosed, can lead to severe neurological and neuropsychological symptoms, seizures, ataxia and psychosocial problems [10, 11]. Over 95% of hyperphenylalaninemia is caused by mutations in the PAH gene which is responsible for the conversion of phenylalanine to tyrosine [9]. BH4 deficiency is caused by autosomal recessive mutations in the genes PTS and GCH1 encoding BH4 biosynthesis enzymes, and in genes PCBD1 and QDPR encoding BH4 regeneration enzymes

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