Single variant association analyses identified a significant association of an intronic variant in HTRA1 (rs2293871-T, frequency = 0.19) with extreme SVD (Table 2). This association was significant after correcting for multiple testing (permutation derived 95% empirical significance threshold P < 2.89 × 10⁻⁴, Supplementary material, part A) and remained significant after additionally adjusting for hypertension status (Table 2). Moreover the top-SNP locus-based test implemented in the VEGAS2 software (Mishra and Macgregor, 2015) confirmed that the association of rs2293871 with extreme SVD is independent of the regional properties of the HTRA1 locus: the linkage disequilibrium structure or number of variants in the region (Table 2). The effect estimates of rs2293871-T appeared larger when comparing the 58 extensive SVD participants with lacunes to minimal SVD participants [OR (95%CI) = 3.04 (1.67–5.50), P = 2.56 × 10⁻⁴] than when comparing the 203 participants with extensive SVD without lacunes to minimal SVD participants [OR (95%CI) = 1.80 (1.27–2.56), P = 9.60 × 10⁻⁴]. We replicated the association of rs2293871 in independent cohorts of European ancestry (n extreme SVD = 3308) using genome-wide genotype data for this common intronic variant (Table 2). The association of rs2293871 was not significant in the only African ancestry sample (rs2293871-T frequency = 0.14, Supplementary Table 3). The inverse variance weighted meta-analysis of discovery and replication cohorts of European ancestry showed an association of rs2293871-T with extensive SVD at an OR (95%CI) of 1.29 (1.14–1.46), P = 4.72 × 10⁻⁵ (Table 2). The same allele at rs2293871 was also associated with increased risk of small vessel ischaemic stroke defined using the CCS system in 16 851 cases and 31 259 controls in the NINDS-SiGN study [NINDS Stroke Genetics Network (SiGN) and International Stroke Genetics Consortium (ISGC), 2016]: OR (95%CI) = 1.12 (1.03–1.22), P = 6.14 × 10⁻³ for causative CCS and OR (95%CI) = 1.12 (1.04–1.22), P = 4.68 × 10⁻³ for phenotypic CCS. The rs2293871 variant showed nominal association with continuous WMH burden (n = 17 936, P-value = 0.03) in a previously reported GWAS meta-analysis (Verhaaren et al., 2015). Functional explorations using HaploReg (Ward and Kellis, 2012) suggest that rs2293871 lies in the H3K9ac promoter and H3K4me1, H3K4me3 and H3K27ac enhancer histone marks (Supplementary Table 4). Two proxies of rs2293871 (rs876790 and rs2736928, r² = 0.75 with rs2293871) are eQTL for HTRA1 in blood (Westra et al., 2013), with C alleles at rs876790 and rs2736928 (in phase with rs2293871-T) showing significant association with lower HTRA1 transcript levels (false discovery rate-corrected P-value = 0.03 and 0.04, respectively).