Copy number variation in familial Parkinson disease

Abstract

Copy number variants (CNVs) are known to cause Mendelian forms of Parkinson disease (PD), most notably in SNCA and PARK2. PARK2 has a recessive mode of inheritance; however, recent evidence demonstrates that a single CNV in PARK2 (but not a single missense mutation) may increase risk for PD. We recently performed a genome-wide association study for PD that excluded individuals known to have either a LRRK2 mutation or two PARK2 mutations. Data from the Illumina370Duo arrays were re-clustered using only white individuals with high quality intensity data, and CNV calls were made using two algorithms, PennCNV and QuantiSNP. After quality assessment, the final sample included 816 cases and 856 controls. Results varied between the two CNV calling algorithms for many regions, including the PARK2 locus (genome-wide p = 0.04 for PennCNV and p = 0.13 for QuantiSNP). However, there was consistent evidence with both algorithms for two novel genes, USP32 and DOCK5 (empirical, genome-wide p-values<0.001). PARK2 CNVs tended to be larger, and all instances that were molecularly tested were validated. In contrast, the CNVs in both novel loci were smaller and failed to replicate using real-time PCR, MLPA, and gel electrophoresis. The DOCK5 variation is more akin to a VNTR than a typical CNV and the association is likely caused by artifact due to DNA source. DNA for all the cases was derived from whole blood, while the DNA for all controls was derived from lymphoblast cell lines. The USP32 locus contains many SNPs with low minor allele frequency leading to a loss of heterozygosity that may have been spuriously interpreted by the CNV calling algorithms as support for a deletion. Thus, only the CNVs within the PARK2 locus could be molecularly validated and associated with PD susceptibility.

Figure 1. Scatter plots of raw probe intensities.

A. A good marker, with three distinct clusters and males and females equally distributed in each cluster; B. Complete co-hybridization to sex chromosome, where all females are called as homozygotes and all males are called as heterozygotes; C. A monomorphic marker (i.e. a CNV probe) exhibiting partial co-hybridization to a sex chromosome, such that individuals cluster by gender, but mean Log R ratios do not differ by gender (p = 0.49); D. Polymorphic SNP exhibiting partial hybridization to a sex chromosome, where multiple distinct groups separated by gender.

Figure 2. Plots of large chromosomal rearrangements.

Sample A harbors a large duplication with the region indicated by a blue bar (average Log R ratio is increased, and B allele frequency (BAF; proportion of alleles estimated to be the B allele) match the 4 expected proportions of 0.0 = AAA, 0.33 = AAB, 0.66 = ABB, 1.0 = BBB). Sample B harbors a large deletion with the region indicated by a purple bar (decreased Log R ratio, with no heterozygotes (BAF = 0.50)); however, since BAF are not limited to values of 0 and 1, the deletion appears to be mosaic.

Figure 3. Detecting mosaicism.

Gray dot = normal Log R ratio (LRR) and B allele frequency (BAF) distributions; Green dot = mosaic pattern with a significant enough deviation in mean LRR to be called as a CNV – such chromosomal arms were removed from analyses; Purple dot = mosaic pattern without significant LRR deviation – the CNV calling algorithms did not call these as CNVs; Blue dot = faint mosaics – not called; Red dot = multiple distinct mosaicism events – chromosomal arms removed from analyses; Black dot = normal LRR and BAF distributions – the reason they are outliers is unknown; Pink dot = no mosaicism pattern, but very noisy – all of these samples had already been flagged as having unacceptably high LRR standard deviations and had already been removed from analyses.

Figure 4. MLPA of USP32 exons.

A two color overlay shows a representation of the capillary electrophoresis peak profiles from one sample with a PennCNV deletion call (shown in red) and one sample without a PennCNV deletion call (shown green) in the USP32 region. Internal control probes are also indicated. Every probe in the sample presented a normal amplification pattern, suggesting normal dosage for both copies of USP32.

Figure 5. Gel electrophoresis of DOCK5 VNTR.

This image of 16 samples is representative of all 95 samples run. PennCNV calls are listed for each sample (normal, deletion, duplication). The mean Log R ratio for the 6 monomorphic CNV probes in DOCK5 is listed in parentheses. A few lanes showed evidence of a third band, assumed to be heteroduplexes of the two alleles. Estimated number of copies of the 32 bp repeat did not correlate with PennCNV calls or with mean Log R ratio. The finding is likely a result of artifact due to DNA source (blood versus LCLs).