Copy number variant calling

We generated a custom population B-allele frequency (BAF) and GC wave-adjusted log R ratio (LRR) intensity file using GenomeStudio (v2.0.5 Illumina) for all the samples that passed genotyping QC steps and used PennCNV (v1.0.5, Wang et al., 2007) to detect CNVs. Only autosomal CNV were targeted for CNV calling, as calls from sex chromosomes are often of poor quality. Adjacent CNV calls were merged into one single call if the number of overlapping markers between them was less than 20% of the total number when the two segments were combined. We conducted an intensity-based QC to exclude samples with low-quality data. After intensity-based QC, all samples had an LRR standard deviation <0.25, an absolute value of the waviness factor < 0.05 and a BAF drift <0.01. Spurious CNV calls in known problematic genomic regions (provided by PennCNV) were also removed prior the analysis. We excluded additional samples with a total number of CNVs calls greater than 80 (this threshold corresponds to the median + 3 SDs of the total number of CNVs per sample). Called CNVs were removed from the dataset if they spanned <20 SNPs, were < 20 kilobases (kb) in length and had a SNP density < 0.0001 (number of markers/length of CNVs). Additionally, SNP density was not considered for CNVs spanning ≥20 SNPs and ≥ 1 Mb in length. CNVs were then annotated for gene content using refGene including gene name and the corresponding exonic coordinates in the hg19 assembly using ANNOVAR (v 2020-06-08). We then searched for CNVs in the same list of “PD genes” used to screen for rare SNVs. We assessed the frequency of CNVs based on complete overlap with CNVs of the same copy number reported in gnomAD-SV (Collins et al., 2020) and in the Database of Genomic Variants (DGV) (MacDonald et al., 2014). We evaluated the clinical impact of the detected CNVs using the CNV-ClinViewer (Macnee et al., 2023), which integrates clinical interpretation of CNVs according to the ACMG guideline and the ClassifyCNV scores. Selected CNVs were validated using the multiplex ligation-dependent probe amplification (MLPA) assay.