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

ABSTRACT: BACKGROUND:Tandem 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. METHODS:A 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. RESULTS:In 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. CONCLUSIONS:The 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.

SUBMITTER: Wang W

PROVIDER: S-EPMC6322297 | biostudies-other | 2019 Jan

REPOSITORIES: biostudies-other

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

Wang Wenjie W Yang Jianping J Xue Jinjie J Mu Wenjuan W Zhang Xiaogang X Wu Wang W Xu Mengnan M Gong Yuyan Y Liu Yiqian Y Zhang Yu Y Xie Xiaobing X Gu Weiyue W Bai Jigeng J Cram David S DS

BMC medical genetics 20190106 1

<h4>Background</h4>Tandem 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.<h4>Methods</h4>A novel PCR-exome amplification and re-sequencing (PEARS) assay was designed and used to detect mutations in 122 genes associated with 101 IEM. Newborn ...[more]

PMID: 30612563

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Project description:Massively parallel DNA sequencing (MPS) has the potential to revolutionize diagnostics, in particular for monogenic disorders. Inborn errors of metabolism (IEM) constitute a large group of monogenic disorders with highly variable clinical presentation, often with acute, nonspecific initial symptoms. In many cases irreversible damage can be reduced by initiation of specific treatment, provided that a correct molecular diagnosis can be rapidly obtained. MPS thus has the potential to significantly improve both diagnostics and outcome for affected patients in this highly specialized area of medicine.We have developed a conceptually novel approach for acute MPS, by analysing pulsed whole genome sequence data in real time, using automated analysis combined with data reduction and parallelization. We applied this novel methodology to an in-house developed customized work flow enabling clinical-grade analysis of all IEM with a known genetic basis, represented by a database containing 474 disease genes which is continuously updated. As proof-of-concept, two patients were retrospectively analysed in whom diagnostics had previously been performed by conventional methods. The correct disease-causing mutations were identified and presented to the clinical team after 15 and 18 hours from start of sequencing, respectively. With this information available, correct treatment would have been possible significantly sooner, likely improving outcome.We have adapted MPS to fit into the dynamic, multidisciplinary work-flow of acute metabolic medicine. As the extent of irreversible damage in patients with IEM often correlates with timing and accuracy of management in early, critical disease stages, our novel methodology is predicted to improve patient outcome. All procedures have been designed such that they can be implemented in any technical setting and to any genetic disease area. The strategy conforms to international guidelines for clinical MPS, as only validated disease genes are investigated and as clinical specialists take responsibility for translation of results. As follow-up in patients without any known IEM, filters can be lifted and the full genome investigated, after genetic counselling and informed consent.

Project description:BackgroundThis study reports on the use of whole exome sequencing (WES) to diagnose children with inborn errors of metabolism and other disorders in United Arab Emirates.MethodsFrom January 2012 to December 2014, 85 patients (46 % females) were seen in the metabolic center at Tawam Hospital (Abu Dhabi) and WES testing was requested because definitive diagnoses were not reached by conventional methods.ResultsEighty (93 %) patients were <18 years old and 44 (52 %) were <5 years. Sixty-eight (80 %) patients had neurologic abnormalities. WES facilitated rapid diagnosis in 50 % of the patients, especially those with mitochondrial disorders. Yet, in most cases extensive investigation was required after the results were available. Most patients with confirmed molecular diagnoses had autosomal recessive disorders and were homozygous for the rare alleles. Most patients with autosomal dominant disorders and all patients with X-linked disorders had de novo mutations. WES results were negative (no pathogenic variants related to patient phenotype were identified) in six patients and incorrect in two patients. One patient had a reported "deleterious" hemizygous mutation in SLC35A2, c.617_620del (p.Q206fs), suggesting 'congenital disorder of glycosylation, TYPE IIm', but glycosylation studies were normal and healthy brothers had the same mutation. Another patient had "pathogenic" mutation in MCCC2, c.1015G > A (p.V339M), but urine organic acids was normal. WES confirmed inborn errors of metabolism (five mitochondrial diseases, three lysosomal storage diseases, and six other disorders) in 14 patients and genetic disorders (14 neurological diseases and three non-neurological diseases) in 17 patients. Variants of unknown significance were identified in 48 patients; 12 had "confirmed pathologic variants"and 12 had "likely pathologic variants", based on consistent phenotypes, biochemical/ segregation studies, or reported pathogenicity. In 24 patients, the variants were inconsistent with phenotypes or biochemical/ familial studies.ConclusionsAlthough WES provided molecular diagnoses, the results required careful interpretations and many patients required additional investigations. This tool is useful when conventional diagnostic methods fail. Staff competence in obtaining consent/ permission, interpreting the findings, and providing the proper counseling are essential before incorporating this technology into routine clinical practices.

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

Publications

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

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