Rare SV2C coding variants in Parkinson's disease risk

Abstract

Genome-wide association studies have identified SV2C as a Parkinson's disease (PD) risk locus, with a common missense variant p.Asp543Asn in the synaptic vesicle glycoprotein 2C (SV2C) protein significantly associated with PD. We examined if other rare SV2C variants also influence PD risk. We analyzed sequencing data of 9810 East Asian individuals comprising 4298 patients and 5512 controls and identified 55 rare nonsynonymous variants. There was no significant association of rare nonsynonymous or loss-of-function variants with PD. Our findings show that besides p.Asp543Asn, other rare coding variants in SV2C do not play a major role in PD susceptibility in East Asia.

Plain language summary

Plain language summary: In our previous study, we found a genetic change p.Asp543Asn in the synaptic vesicle glycoprotein 2C (SV2C) protein to be associated with higher Parkinson's disease (PD) risk. Here, we aim to determine if there are other genetic changes, especially those that alter the SV2C protein, that also influence PD risk. We analyzed sequencing data of 9810 East Asian individuals comprising 4298 patients and 5512 controls and identified 55 other genetic changes that were not associated with PD risk. Our findings show that within the SV2C gene, only p.Asp543Asn is associated with PD risk in East Asia.

Introduction

Parkinson's disease (PD) is a common, age-related neurodegenerative disease influenced by multiple rare and common genetic factors. A genome-wide association study in East Asian populations identified SV2C as a novel PD-associated locus. ¹ This association was replicated in European-ancestry populations.^1–3 The lead SNP within the locus rs246814 is in near complete linkage disequilibrium (r² = 1) with a missense variant p.Asp543Asn (rs31244) that occurs within an extracellular/luminal domain of the encoded synaptic vesicle glycoprotein 2C (SV2C) protein. SV2C was previously investigated for a possible role in PD because of its restricted expression in PD-relevant brain regions, whereby knockout mice showed a significant reduction in striatal dopamine release ⁴ and impaired dopamine storage. ⁵ The introduction of a new asparagine residue by this amino acid substitution may affect N-linked glycosylation of this domain via the creation of a new glycosylation site (Asn543-X-Thr545). ⁶ This variant is common, present at allele frequency ∼10% in East Asians. Here, we aim to investigate whether rare loss-of-function variants in SV2C that are not tagged or represented on genome-wide SNP arrays are also associated with PD. We also evaluated whether any other rare missense variants may introduce or abolish other glycosylation sites in the SV2C extracellular/luminal domain.

Methods

We sequenced all coding exons of SV2C in 9810 East Asian individuals comprising 4298 PD patients (age at onset [mean ± standard deviation] 61.1 ± 10.9 years; age at sample collection 66.6 ± 9.8 years; 53.44% males) and 5512 healthy controls (65.2 ± 9.4 years; 46.88% males) from Singapore, Malaysia, Hong Kong, Taiwan and South Korea. ⁷ Samples were 151 bp paired end sequenced on Illumina sequencers (HiSeq 4000 and NovaSeq 6000). Patients were diagnosed with PD using the UK Brain Bank Criteria. Cases and controls were confirmed to be genetically well-matched in a principal component analysis. ⁷ All subjects gave informed consent, and the study received approval from the institutional ethics committee (SingHealth Centralized Institutional Review Board CIRB 2002/008/A and 2019/2334 and Nanyang Technological University Institutional Review Board IRB-2016-08-011). All 12 exons of SV2C were sequenced to a mean coverage of 67X with 97.9% of target bases covered by 10 or more reads. Variant calling was conducted using the Genome Analysis Tool Kit (GATK v3.7) on Hg19/GRCh37 ⁸ following the Best Practices Guidelines. ⁹ Variant quality control was conducted by removal of variants that (i) did not pass Variant Quality Score Recalibration (VQSR), (ii) had low quality genotype calls (DP < 8, GQ < 20), (iii) had call rates < 95% in cases, controls and all samples, (iv) failed Hardy-Weinberg equilibrium test (p < 1 × 10⁻⁶), and (v) had significant differential missingness between cases and controls (p < 1 × 10⁻⁴). Sample quality control was conducted by removal of samples with (i) gender discordance between patient records and sex genetically inferred from whole-exome sequenced data, (ii) poor genotyping concordance (< 95%) between whole-exome sequenced data and previously conducted genome-wide association arrays, ¹ (iii) high heterozygosity (>3.5 standard deviations from the mean), high exome-wide singleton counts, and low genotyping call rates (< 95%), (iv) with the lower call rate in related sample pairs when identity-by-descent analysis (IBD > 0.125), and (v) not of East Asian genetic ancestry from ancestry principal component analysis (PCA) against 2141 samples from the 1000 genomes (1KGP) reference panel. ¹⁰ Variants were also annotated based on canonical transcript with Ensembl Variant Effect Predictor (VEP, version 104). ¹¹

We then identified SV2C variants (GRCh37 genomic region: chr5:75,378,997-75,649,764) which were rare in East Asians (minor allele frequency (MAF) ≤ 1% in Genome Aggregation Database (gnomAD) EAS) and were predicted to be pathogenic as defined by probably damaging/possibly damaging classification of SNPs by PolyPhen-2 ¹² and high-confidence classification of indels and splice variants by LOFTEE. ¹³ These variants were further assessed for pathogenicity in ClinVar ¹⁴ and Varsome, ¹⁵ with pathogenic variants defined by Varsome classification of “PP3: Pathogenic Very Strong” and “BP4: Supporting”. Visualization of variants across SV2C protein domains was conducted with Protter (https://wlab.ethz.ch/protter/start/).

Gene burden testing of SV2C rare pathogenic variants carrier count between PD cases and controls was conducted using stratified Cochran-Mantel Haenszel test (four strata: Singapore/Malaysia, Hong Kong, Taiwan and South Korea; Supplemental Figure 1) ⁷ without continuity correction. A similar test was done on allele counts for single variant association testing of common variants. Further gene-based tests were also conducted using sequence kernel association tests (SKAT), SKAT-O (optimal) and Burden tests ¹⁶ with adjustment for PCs 1-3.

Results

A total of 4298 PD cases and 5512 matched controls were collected from cohort studies in Singapore, Malaysia, South Korea, Hong Kong, and Taiwan. PCA analysis indicated that these 9810 samples were of East Asian descent (Supplemental Figure 1A). All samples from Singapore, Malaysia, Hong Kong and Taiwan that were retained in our analysis are of Han Chinese Ancestry, with Singapore and Malaysia sharing similar genetic background (Supplemental Figure 1B). ⁷

We identified a total of 92 variants in the coding exons of SV2C out of which 51 were missense variants, 2 were stop-gain variants, 3 were frameshift variants and 1 was a splice site variant. 89 of these variants were rare (MAF ≤ 1%) and 3 variants were common (Figure 1, Supplemental Tables 1 and 2). Of the missense variants, 28 were predicted to be pathogenic by PolyPhen-2 (Table 1). Another 4 variants were predicted to cause loss-of-function at high confidence by LOFTEE (Table 1). Of the 4 variants occurring within the extracellular/luminal domain of SV2C (Figure 1), none occured at the known glycosylation sites within that domain. Similarly, none of the variants introduced new asparagine residues within the extracellular/luminal domain.

Figure 1.

Annotation of rare predicted pathogenic variants and common missense variant (p.Asp543Asn) along the SV2C protein.

Table 1.

Rare variants in the coding exons of SV2C that were predicted to be pathogenic by PolyPhen-2/LOFTEE.

Variant (Hg19/ GRCh37)	Ref/Alt	Amino Acid Substitution	Variant Type	Pathogenicity predictors		MAF in EAS (gnomAD v2.1.1)		Cases (N = 4298)		Control (N = 5512)		Protein Domain c	ClinVar Germline classification	Varsome
				Polyphen-2 a	LoF b	exomes	genomes	n	%	n	%
chr5:75427717	C/T	p.Arg48Trp	Missense	D	NA	5.57E-05	NA	0	0%	2	0.036%	CD1	-	BP4: Supporting
chr5:75427801	A/T	p.Ser76Cys	Missense	D	NA	NA	NA	1	0.023%	0	0%	CD1	-	BP4: Supporting
chr5:75427933	C/T	p.Arg120Trp	Missense	D	NA	0	NA	1	0.023%	0	0%	CD1	-	BP4: Supporting
chr5:75427936	C/T	p.Arg121Trp	Missense	P	NA	0.0017	0.0013	15	0.350%	18	0.328%	CD1	Likely benign	BP4: Benign Strong
chr5:75428010	C/A	p.Cys145Ter	Nonsense	NA	HC	0	NA	1	0.023%	0	0%	CD1	-	Not conclusive / -
chr5:75428021	G/A	p.Arg149His	Missense	D	NA	2.00E-04	0	0	0%	1	0.018%	CD1	-	BP4: Benign Moderate
chr5:75428059	C/T	p.Leu162Phe	Missense	D	NA	NA	NA	0	0%	2	0.036%	TM1	-	BP4: Supporting
chr5:75428062	A/C	p.Met163Leu	Missense	P	NA	0	NA	1	0.023%	0	0%	TM1	-	BP4: Supporting
chr5:75428089	G/A	p.Val172Ile	Missense	D	NA	5.57E-05	NA	1	0.023%	0	0%	TM1	Uncertain significance	BP4: Supporting
chr5:75428098	G/A	p.Val175Met	Missense	D	NA	0	0	0	0%	1	0.018%	TM1	-	BP4: Supporting
chr5:75490761	G/A	p.Gly200Arg	Missense	D	NA	0	NA	0	0%	1	0.018%	TM1	-	BP4: Supporting
chr5:75505578	C/G	p.Pro260Arg	Missense	D	NA	5.55E-05	NA	0	0%	1	0.018%	TM1	-	BP4: Supporting
chr5:75505590	C/T	p.Ser264Leu	Missense	P	NA	0	0	1	0.023%	0	0%	TM1	-	BP4: Supporting
chr5:75505613	C/T	p.Arg272Trp	Missense	D	NA	5.51E-05	0	1	0.023%	0	0%	CD2	-	BP4: Supporting
chr5:75505622	C/T	p.Arg275Trp	Missense	D	NA	0	0	3	0.070%	0	0%	CD2	-	BP4: Supporting
chr5:75505631	C/T	p.His278Tyr	Missense	D	NA	NA	NA	1	0.023%	0	0%	CD2	-	BP4: Supporting
chr5:75505697	A/G	p.Ile300Val	Missense	P	NA	0	NA	1	0.023%	0	0%	TM5	Uncertain significance	BP4: Supporting
chr5:75505704	C/T	p.Pro302Leu	Missense	D	NA	0	0	1	0.023%	1	0.018%	LD3	-	BP4: Supporting
chr5:75505712	G/A	p.Gly305Arg	Missense	D	NA	0	NA	0	0%	1	0.018%	LD3	-	PP3: Pathogenic Very Strong
chr5:75581627	TG/T	p.Trp357Ter	Nonsense	NA	HC	NA	NA	0	0%	2	0.036%	CD3	-	-
chr5:75581669	C/T	p.Arg371Trp	Missense	D	NA	0	0	0	0%	1	0.018%	CD3	-	BP4: Supporting
chr5:75581694	C/T	p.Thr379Met	Missense	D	NA	3.00E-04	NA	5	0.117%	4	0.073%	CD3	-	PP3: Pathogenic Strong
chr5:75587133	C/T	p.Arg409Trp	Missense	D	NA	5.62E-05	NA	1	0.023%	0	0%	CD3	-	BP4: Supporting
chr5:75587154	G/A	p.Gly416Arg	Missense	D	NA	0	NA	0	0%	1	0.018%	CD3	-	BP4: Supporting
chr5:75587155	GAGTA/G	c.1248 + 5_1248 + 8delGTAA	Splice Donor	NA	HC	6.04E-05	NA	0	0%	2	0.036%	CD3	-	-
chr5:75591754	T/C	p.Tyr497His	Missense	D	NA	0.0005	NA	6	0.140%	4	0.073%	LD4	-	BP4: Supporting
chr5:75594657	ACT/A	p.Ser515CysfsTer3	Frameshift Deletion	NA	HC	NA	NA	0	0%	1	0.018%	LD4	-	-
chr5:75594708	A/G	p.Tyr531Cys	Missense	D	NA	0.0002	NA	1	0.023%	2	0.036%	LD4	-	BP4: Supporting
chr5:75596620	G/A	p.Gly568Glu	Missense	D	NA	NA	NA	1	0.023%	0	0%	LD4	-	BP4: Supporting
chr5:75596742	C/T	p.Arg609Cys	Missense	D	NA	5.56E-05	0	1	0.023%	0	0%	CD4	Uncertain significance	BP4: Supporting
chr5:75621215	C/T	p.Ala676Val	Missense	D	NA	NA	NA	1	0.023%	0	0%	TM11	Uncertain significance	BP4: Supporting
chr5:75621313	G/A	p.Val709Met	Missense	D	NA	0	0	1	0.023%	0	0%	TM12	Uncertain significance	BP4: Benign Moderate

^a: D: probably damaging; P: possibly damaging.

^b: HC: high confidence.

^c: CD: cytoplasmic domain; TM: transmembrane domain; LD: luminal/extracellular domain.

Among the common variants, only p.Asp543Asn (rs31244) showed significant association with PD (Supplemental Table 2) (allele frequencies: 10.72% cases, 9.14% controls, OR = 1.156, 95% CI = 1.049–1.273, p = 0.003). The other two common variants (Supplemental Table 2) showed no significant association or nominal association with PD (p.Arg120Arg [rs10070440]: allele frequencies 33.79% cases, 32.27% controls, OR = 1.028, 95% CI = 0.967–1.094, p = 0.372; p.Thr482Ser [rs2270927]: allele frequencies 19.69% cases, 17.63% controls, OR = 1.079, 95% CI = 1.002–1.163, p = 0.045) and were not in high linkage disequilibrium with the lead GWAS SNP rs246814 (r²< 0.5) in East Asians. We did not observe significant gene burden of rare pathogenic SV2C variants (Table 1) in PD cases (0.512% carriers) compared to controls (0.408% carriers) using stratified CMH (OR = 1.213, 95% CI = 0.799–1.844, p = 0.356; Supplemental Table 3). Testing with sequence kernel association test also showed a lack of association between SV2C rare pathogenic variants gene burden with PD (SKAT-O p = 0.483; SKAT p = 0.458; Burden p = 0.319; Table 1). Similar results were obtained when Varsome was used to classify rare pathogenic variants (CMH: OR = 1.451, 95% CI = 0.869–2.423, p = 0.145; SKAT-O p = 0.271; SKAT p = 0.204, Burden p = 0.174). The 4 variants predicted to cause loss-of-function at high confidence were found in 1 PD case and 5 controls (CMH: OR = 0.287, 95% CI = 0.033–2.454, p = 0.224).

Discussion

Overall, we observed no evidence for association of rare missense variants in SV2C with PD in a sequencing study of 9810 East Asian samples. SV2C knockout mice were shown to exhibit mild motor deficits and impaired dopamine release. ⁴ However, we identified only 1 patient with a high confidence heterozygous loss-of-function variant in SV2C, suggesting that loss-of-function mutations in SV2C do not play a major role in PD risk in the East Asian population. Consistent with our observations, rare pathogenic (MAF < 0.1%, LoF High Confidence + CADD > 20) SV2C variants were not enriched (p > 0.05) in PD cases (0.226%) compared to controls (0.567%) in European-ancestry populations (UK Biobank).^17,18

The common missense SNP p.Asp543Asn does not appear to cause loss-of-function of the SV2C protein. The mechanisms through which it contributes to PD susceptibility remain to be investigated, although it is predicted to introduce a new glycosylation site in the extracellular/luminal domain of SV2C. None of the other rare missense variants we identified in SV2C are likely to influence glycosylation of this domain. Overall, our results show that rare variants in SV2C do not play an important role in PD susceptibility in the East Asian population. Further work is needed to understand the effects of the GWAS-associated SNP on SV2C gene function and PD susceptibility.