Combined biochemical profiling and DNA sequencing in the expanded newborn screening for inherited metabolic diseases: the experience in an Italian reference center

Abstract

Background: Newborn screening (NBS) programs have significantly improved the health and outcomes of patients with inherited metabolic disorders (IMDs). Methods based on liquid chromatography/mass spectrometry (LC-MS/MS) analysis are viewed worldwide as the gold standard procedure for the expanded NBS programs for these disorders. Advanced molecular technologies point to genomic sequencing as an alternative and feasible strategy for the screening of genetic diseases, including IMDs. However, each of the two approaches has potential limitations when used as a first-tier analysis. In this study, we tested a workflow-based parallel biochemical and sequencing analyses to determine whether this approach could improve the diagnostic outcome.

Results: For each patient identified by LC-MS/MS as positive, we performed both the biochemical confirmatory tests and next-generation sequencing (NGS) procedures from the same Dried Blood Spot (DBS). NGS analysis was based on applying Exome Sequencing libraries, limiting the analysis to 105 actionable genes involved in IMDs. This allows overtaking the actual limitations of NBS on DBS, enhancing our capacity to identify variants that can drive a genetic disease. Through this approach, we could reach 100% of cases solved, with 37.9% of cases (41/108) for which the combination of the biochemical and NGS analysis was indispensable for a correct diagnosis. In total, we could identify 17 affected, 34 false positives, 12 individuals referred to us for maternal conditions. In 45 newborns the molecular analysis showed heterozygosity for mutations in one or more of the genes analyzed, with results compatible with the biochemical profile indicative of NBS positivity.

Conclusions: In this study, we validated the performance of the proposed workflow. The advantage of this approach is limiting molecular analysis only to positive newborns and using a restricted panel of 105 genes relevant for the expanded NBS, with a 100% rate of diagnosis and potential reduction of the costs related to NBS procedures and reduced impact on patients and families.

Keywords: Inherited metabolic disorders (IMDs); Newborn screening (NBS); Next-generation sequencing (NGS); Whole exome sequencing (WES).

Fig. 1

A standard procedures defined by Campania regional decree n. 30. Positive newborns are referred to the unit of Pediatric Metabolic Diseases for clinical evaluation and for confirmatory biochemical tests and, after evaluation of the results, molecular characterization. B The procedure followed in 108 consecutive newborns referred to us for NBS positivity. In these cases, biochemical and molecular analyses were run in parallel. While in the standard approach the molecular analysis is used as a confirmatory test after the biochemical evaluation, in our study molecular analysis was run in parallel with the biochemical testing. C Workflow of NGS and bioinformatic analysis. When both biochemical and molecular data were available, unless a situation of emergency required immediate evaluation and intervention, individual cases were assessed and discussed by a team of metabolic pediatricians and laboratory staff at the end of the procedure

Fig. 2

A Pie chart representing the amount of case for which biochemical analysis was sufficient to assess the condition and the amount of cases for which NGS was necessary. On the right, barplot showing the percentage of NGS procedures improving or allowing the diagnosis. B Pie chart representing the incidence of each type of diagnosis respect to the total of analyzed infants. For each slice, a barplot represents the percentage of cases for which NGS was critical, helpful or biochemistry alone was sufficient