title: Performance evaluation of Sanger sequencing for the diagnosis of primary hyperoxaluria and comparison with targeted next generation sequencing.
abstract: Definitive diagnosis of primary hyperoxaluria (PH) currently utilizes sequential Sanger sequencing of the AGXT, GRPHR, and HOGA1 genes but efficacy is unproven. This analysis is time-consuming, relatively expensive, and delays in diagnosis and inappropriate treatment can occur if not pursued early in the diagnostic work-up. We reviewed testing outcomes of Sanger sequencing in 200 consecutive patient samples referred for analysis. In addition, the Illumina Truseq custom amplicon system was evaluated for paralleled next-generation sequencing (NGS) of AGXT,GRHPR, and HOGA1 in 90 known PH patients. AGXT sequencing was requested in all patients, permitting a diagnosis of PH1 in 50%. All remaining patients underwent targeted exon sequencing of GRHPR and HOGA1 with 8% diagnosed with PH2 and 8% with PH3. Complete sequencing of both GRHPR and HOGA1 was not requested in 25% of patients referred leaving their diagnosis in doubt. NGS analysis showed 98% agreement with Sanger sequencing and both approaches had 100% diagnostic specificity. Diagnostic sensitivity of Sanger sequencing was 98% and for NGS it was 97%. NGS has comparable diagnostic performance to Sanger sequencing for the diagnosis of PH and, if implemented, would screen for all forms of PH simultaneously ensuring prompt diagnosis at decreased cost.
authors: Clinical Biochemistry, Imperial College Healthcare London, United Kingdom.
authors: Emma L
authors: EL
authors: Williams
authors: Clinical Biochemistry, Imperial College Healthcare London, United Kingdom.
authors: Eleanor A L
authors: EA
authors: Bagg
authors: Clinical Genome Informatics Facility, Department of Medicine, Imperial College London London, United Kingdom.
authors: Michael
authors: M
authors: Mueller
authors: MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London London, United Kingdom.
authors: Jana
authors: J
authors: Vandrovcova
authors: MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London London, United Kingdom ; Institute of Genetics and Molecular Medicine, University of Edinburgh Edinburgh, United Kingdom.
authors: Timothy J
authors: TJ
authors: Aitman
authors: Clinical Biochemistry, UCL Hospitals London, United Kingdom.
authors: Gill
authors: G
authors: Rumsby
keywords: Primary Hyperoxaluria; genetic diagnosis; next generation sequencing; oxalate
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