A DNA resequencing array for genes involved in Parkinson’s disease

https://doi.org/10.1016/j.parkreldis.2011.12.012Get rights and content

Abstract

Parkinson’s disease (PD) is aetiologically complex with both familial and sporadic forms. Familial PD results from rare, highly penetrant pathogenic mutations whereas multiple variants of low penetrance may contribute to the risk of sporadic PD. Common variants implicated in PD risk appear to explain only a minor proportion of the familial clustering observed in sporadic PD. It is therefore plausible that combinations of rare and/or common variants in genes already implicated in disease pathogenesis may help to explain the genetic basis of PD.

We have developed a CustomSeq Affymetrix resequencing array to enable high-throughput sequencing of 13 genes (44 kb) implicated in the pathogenesis of PD. Using the array we sequenced 269 individuals, including 186 PD patients and 75 controls, achieving an overall call rate of 96.5% and 93.6%, for two respective versions of the array, and >99.9% accuracy for five samples sequenced by capillary sequencing in parallel. We identified modest associations with common variants in SNCA and LRRK2 and a trend suggestive of an overrepresentation of rare variants in cases compared to controls for several genes. We propose that this technology offers a robust and cost-effective alternative to targeted sequencing using traditional sequencing methods, and here we demonstrate the potential of this approach for either routine clinical investigation or for research studies aimed at understanding the genetic aetiology of PD.

Introduction

Although most cases of Parkinson’s disease (PD) present as sporadic disease, between 5 and 10% of these cases have first- or second-degree relatives with PD, suggesting a Mendelian inheritance pattern and a broader role for genetics in the heritability of PD [1]. Pathological sequence variants have been identified in genes underlying familial PD [2], [3], [4], [5], [6], [7]. Candidate gene association studies and genome-wide association studies demonstrate that risk of PD [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17] is conferred by common variants in genes implicated in familial disease as well as other genes. However, the small effect sizes observed in these association studies fail to explain much of the familial clustering of PD, leading to the hypothesis that rare variants in known and unknown genes may account for the missing genetic component. Public-access genomic databases of human sequence variants are heavily biased towards common variants, so deep and comprehensive resequencing of genes already implicated in PD is likely to provide further insights into the genetic basis of PD.

The high cost and low-throughput of conventional sequencing methods has inhibited large-scale targeted sequencing of genes implicated in PD and often limiting studies to a small number of genes or genotyping of known variants. Therefore, a higher throughput, less expensive alternative to conventional sequencing would be invaluable for both researcher and clinician. To this end, we developed the PD GeneChip®, a custom designed resequencing array that produces 44 kilobase (kb) of accurate and reproducible sequence data from genes implicated in PD, with significantly less labour and reagent costs than conventional techniques [18].

We have screened 269 samples (186 PD cases, 75 controls, one asymptomatic sibling of a PD case, and 7 individuals with other neurological diseases) using the PD GeneChip® to evaluate the accuracy and reproducibility of the array and to provide insights into the genetic basis of PD. We demonstrate that even with a relatively small sample, it is possible to detect pathogenic alterations and common sequence variants associated with disease.

Section snippets

Materials and methods

The study was approved by ethics committees of the Royal Melbourne, St Vincent’s, Austin and Royal North Shore Hospitals and adhered to the National Health and Medical Research Council code of practice. All subjects provided written informed consent.

Results

Two versions of the PD GeneChip® were designed and specific sequences such as repetitive elements and internal duplications were excluded where necessary. The two designs resequenced 44,020 base pair (bp) and 44,471 bp of human genomic sequence, respectively. The resequencing data for 269 individuals, or 280 arrays (11 samples processed in duplicate) were analysed using GSEQ and data from the two versions of the array were analysed separately (Table 1). After accounting for PCR failures (no

Discussion

We developed the PD GeneChip®, a custom designed resequencing array that delivers 44 kb of accurate and reproducible sequence data with significantly less labour and reagent costs than conventional techniques [18]. In addition to validating the technology for use in high-throughput resequencing, we have demonstrated that even with relatively small sample numbers, full resequencing of genes implicated in PD can identify pathogenic sequence alterations and provide for genotype–phenotype

Acknowledgements

We thank Laura Johnson for sample handling and for funding of this research we are grateful to The Australian Research Council (Linkage Project: LPO776735), The Australian Brain Foundation and the Rebecca L. Cooper Medical Research Foundation.

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