Performance evaluation of Sanger sequencing for the diagnosis of primary hyperoxaluria and comparison with targeted next generation sequencing

Emma L Williams, Eleanor A L Bagg, Michael Mueller, Jana Vandrovcova, Timothy J Aitman, Gill Rumsby

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)69-78
Number of pages10
JournalMolecular Genetics and Genomic Medicine
Volume3
Issue number1
Early online date5 Nov 2014
DOIs
Publication statusPublished - Jan 2015

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