Objective: We investigate feasibility of Next Generation Sequencing (NGS) targeted sequencing technique using Ampliseq® technology by Ion PGM® to detect copy number variation mutation in PARK2 gene.

Background: Among causative genes for familial Parkinson’s disease (PD), PARK2 is the most common autosomal recessive gene for familial Parkinson’s disease. In East Asian, while 10% of mutations in PARK2 are single nucleotide variants, 90% of PARK2 mutations are copy number variation (CNV) mutation (i.e., deletion/duplication) caused by exonic rearrangement. Targeted resequencing of a panel of genes using NGS technique has been known to capture single nucleotide variant accurately. However, whether this technique can detect CNV mutation precisely enough to be used in clinical genetics has not been systemically studied.

Methods: Targeted resequencing of five PD-causing genes (PARK2, ATP13A, PLA2G6, PINK1, SNCA) using Ion PGM® was performed in a group of 32 PD patients with early onset PD. Results of copy number change analyses in PARK2 based on depth-based algorithm was compared to those obtained by a gold standard method, real-time PCR (RT-PCR).

Results: An average of 144,767 mapping reads per a sample were on target (99.28%) were obtained with an average coverage depth 1,273X and coverage uniformity of 95.40 %. In unsupervised analyses, the concordances as determined by Cohen’s kappa between depth-based CNV detection and RT-PCR was poor to good (kappa=0.43, 95% CI 0.24-0.63). In a supervised analysis, kappa was higher (kappa=0.77, CI 0.63-0.91), however, results of CNV analyzed by Ampliseq® technology by Ion PGM® using coverage depth-based algorithm were significantly different from those by a gold standard method, RT-PCR (McNemar test, p<0.05).

Conclusions: Our results suggest that depth-based CNV analysis algorithm using data obtained by Ampliseq® technique of NGS is not comparable to RT-PCR in detecting PARK2 exonic rearrangement.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/diagnostic-utility-of-a-targeted-resequencing-technique-of-next-generation-sequencing-in-detecting-copy-number-changes-in-park2/