A next-generation newborn screening pilot study: NGS on dried blood spots detects causal mutations in patients with inherited metabolic diseases

Abstract

The range of applications performed on dried blood spots (DBS) widely broadened during the past decades to now include next-generation sequencing (NGS). Previous publications provided a general overview of NGS capacities on DBS-extracted DNA but did not focus on the identification of specific disorders. We thus aimed to demonstrate that NGS was reliable for detecting pathogenic mutations on genomic material extracted from DBS. Assuming the future implementation of NGS technologies into newborn screening (NBS), we conducted a pilot study on fifteen patients with inherited metabolic disorders. Blood was collected from DBS. Whole-exome sequencing was performed, and sequences were analyzed with a specific focus on genes related to NBS. Results were compared to the known pathogenic mutations previously identified by Sanger sequencing. Causal mutations were readily characterized, and multiple polymorphisms have been identified. According to variant database prediction, an unexplained homozygote pathogenic mutation, unrelated to patient’s disorder, was also found in one sample. While amount and quality of DBS-extracted DNA are adequate to identify causal mutations by NGS, bioinformatics analysis revealed critical drawbacks: coverage fluctuations between regions, difficulties in identifying insertions/deletions, and inconsistent reliability of database-referenced variants. Nevertheless, results of this study lead us to consider future perspectives regarding “next-generation” NBS.

Second-Tier Next Generation Sequencing Integrated in Nationwide Newborn Screening Provides Rapid Molecular Diagnostics of Severe Combined Immunodeficiency

“…NBS in Norway is based on informed consent, and allows for genetic mass screening of selected disorders to be performed without formal genetic counseling. It has been very useful in cystic fibrosis screening (28), in screening for metabolic disorders with limited numbers of disease genes (68)(69)(70)(71), and for SCID screening with multiple disease genes. Analysis of biobanked samples from patients with known PIDs to evaluate the test methods, were performed retrospectively.…”

The Role of Extended CFTR Gene Sequencing in Newborn Screening for Cystic Fibrosis

“…The design should thus be adapted and continuously updated accordingly [50,51]. Low DNA quality and quantity extracted of dried-blot samples are other constraints for the detection of large deletions and insertions [52].…”

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“…This new paradigm generated by the introduction of NGS in NBS must be taken into account by an appropriate public health framework, as recommended by Friedman et al [44]. While some are already proposing that NBS programs incorporate, or even replace their current approach with genomic sequencing [49,50], evidence-based methods should be implemented to inform the decision-making process and consider parameters such as prevalence and penetrance of the condition, false-positive findings, clinical outcome following the interventions, psychological impacts on families, and costs. In the case of NBS, the focus should be the best interest of the child, and thus the identification of conditions where timely interventions or prevention would prevent significant health harms to the child, not on genetic information that may potentially identify later-onset disorders which might need medical attention.…”