Multiplex ligation‐dependent probe amplification assay for simultaneous detection of Parkinson's disease gene rearrangements

Abstract

Parkinson's disease (PD) is a common disorder caused by degeneration of dopaminergic neurons in the substantia nigra and other brain areas. Mutations in several genes have been associated with both autosomal dominant PD and recessive early onset Parkinsonism (EOP). Genomic rearrangements such as deletions or multiplications of one or more exons represent a common mutational mechanism for most of these genes and are not detectable with routine mutation screening techniques. MLPA (Multiplex Ligation-dependent Probe Amplification), is a cheap, simple, rapid, and sensitive tool to detect exon dosage alterations and specific point mutations in selected genes. We tested the recently developed PD-MLPA assay by using 13 positive control samples carrying known mutations in SNCA, LRRK2, Parkin, PINK1, and DJ-1 genes. We then applied this technique to screen 16 EOP patients who were then cross-tested by quantitative PCR (qPCR). All the mutations present in the positive control samples were clearly detected by MLPA. Moreover, three novel Parkin rearrangements were identified among EOP patients and confirmed by qPCR. Only two samples generated false positive duplications of LRRK2 exon 1 and UCH-L1 exon 9, respectively. These results show that PD-MLPA assay can simultaneously and effectively detect rearrangements in most PD genes (SNCA, Parkin, PINK1, and DJ-1) as well as the LRRK2 G2019S common mutation. Thus, the use of this novel platform can improve the analysis of such mutations, facilitating comprehensive genetic testing in PD and EOP.

Analysis of PARK genes in a Korean cohort of early-onset Parkinson disease

“…Gene dosage change of the aforementioned genes due to large exonal rearrangements were analyzed by multiplex ligation-dependent probe amplification (MLPA) assay using commercially available probes (SALSA P051 Parkinson MLPA kits, MRC Holland, Amsterdam, The Netherlands) [10,11]. Sequence-specific probes enclosed in this kit are against all exons of SNCA, PARKIN, and PINK1 and exons 1, 3, 5, 6, and 7 of DJ-1.…”

Isolated limb dystonia as presenting feature of Parkin disease

“…Parkin gene mutations were detected according to standard procedures. 2 Eight had isolated dystonia at onset. These subjects had full neurological evaluation and videotape documentation.…”

A comprehensive analysis of deletions, multiplications, and copy number variations in PARK2

“…22 The large-scale CNV analysis started with a modified semiquantitative PCR protocol 25 that was used for 252 patients and 299 control subjects. With the advent of PDspecific multiplex ligation-dependent probe amplification (MLPA), 26,27 we switched to the SALSA MLPA kit P052B from MRC-Holland (Amsterdam, the Netherlands) for 1,956 patients and 1,477 control subjects. We used more conservative measures than recommended by the MLPA manufacturer and were able to establish false-positive and false-negative rates (appendix e-1).…”