Accurate molecular diagnosis of phenylketonuria and tetrahydrobiopterin-deficient hyperphenylalaninemias using high-throughput targeted sequencing

Abstract

Genetic diagnostics of phenylketonuria (PKU) and tetrahydrobiopterin (BH4) deficient hyperphenylalaninemia (BH4DH) rely on methods that scan for known mutations or on laborious molecular tools that use Sanger sequencing. We have implemented a novel and much more efficient strategy based on high-throughput multiplex-targeted resequencing of four genes (PAH, GCH1, PTS, and QDPR) that, when affected by loss-of-function mutations, cause PKU and BH4DH. We have validated this approach in a cohort of 95 samples with the previously known PAH, GCH1, PTS, and QDPR mutations and one control sample. Pooled barcoded DNA libraries were enriched using a custom NimbleGen SeqCap EZ Choice array and sequenced using a HiSeq2000 sequencer. The combination of several robust bioinformatics tools allowed us to detect all known pathogenic mutations (point mutations, short insertions/deletions, and large genomic rearrangements) in the 95 samples, without detecting spurious calls in these genes in the control sample. We then used the same capture assay in a discovery cohort of 11 uncharacterized HPA patients using a MiSeq sequencer. In addition, we report the precise characterization of the breakpoints of four genomic rearrangements in PAH, including a novel deletion of 899 bp in intron 3. Our study is a proof-of-principle that high-throughputtargeted resequencing is ready to substitute classical molecular methods to perform differential genetic diagnosis of hyperphenylalaninemias, allowing the establishment of specifically tailored treatments a few days after birth.

High‐throughput custom capture sequencing identifies novel mutations in coloboma‐associated genes: Mutation in DNA‐binding domain of retinoic acid receptor beta affects nuclear localization causing ocular coloboma

“…Custom capture or targeted gene sequencing has been a cost‐effective approach to detect mutations in genes known to be associated with a specific disease condition (Aparisi et al, ; Bonachea et al, ; Patel et al, ; Rehm, ; Shang et al, ; Trujillano et al, ; X. Wang et al, ). Previous studies exploring mutations in coloboma‐associated genes have been limited to a subset of genes in affected families (Gonzalez‐Rodriguez et al, ; Williamson & FitzPatrick, ).…”

Tetrahydrobiopterin deficiency among Serbian patients presenting with hyperphenylalaninemia

“…Recently, a novel and efficient strategy based on highthroughput multiplextargeted resequencing of four genes (PAH, GCH1, PTS and QDPR) was implemented (9). This study proved that the approach based on the nextgeneration targeted resequencing is effective and extremely potent in comparison to classical molecular methods based on the subsequent genebygene analysis.…”

Fast clinical molecular diagnosis of hyperphenylalaninemia using next-generation sequencing-based on a custom AmpliSeq™ panel and Ion Torrent PGM sequencing

“…Two other studies had applied NGS to diagnose PKU and BH4D [14,15]. Although the technology process is different, the same problems are unavoidable, such as the low coverage of amplicon with high GC content and missed detection for large heterozygous segment deletion.…”