Detection of variations and identifying genomic breakpoints for large deletions in the LDLR by Ion Torrent...

The aims of this study were to 1) compare LDLR variant detection between Ion Torrent Personal Genome Machine (PGM) sequencing and conventional methods used for familial hypercholesterolaemia (FH) diagnosis i.e. exon-by-exon sequence analysis and multiplex ligation-dependent probe amplification (MLPA) and 2) identify genomic breakpoints for 12 cases of large deletions in LDLR previously identified by MLPA.

Thirty FH patient samples were selected, 22 with mutations previously determined. Primers were designed and optimised to generate six amplicons covering the entire LDLR and sequenced on a PGM. An additional twelve samples carrying MLPA variants were sequenced on the PGM followed by Sanger sequencing to establish the breakpoints.

A total of 2179 LDLR variants were identified in the 30 samples, with 383 variants in the region sequenced that was common to both PGM and Sanger methods. Three discrepancies were identified; two of these were identified by visual inspection of the BAM files, whilst the remaining discrepancy was likely an artefact of the PCR approach. Approximate genomic breakpoints for the 12 MLPA variants were identified using PGM sequencing, and Sanger sequencing of these regions established causative breakpoints. Eleven different rearrangements/mutational events were found, with eight out of eleven occurring in Alus. Two of the three samples with exons 2-6del had identical breakpoints. Two samples with exons 11-12del had unique breakpoints, indicating separate ancestral origin or mutational events.

This study showed that Ion Torrent PGM sequencing is an accurate and efficient method to detect LDLR variants while providing additional information such as genomic breakpoints.