NOTCH3 p.Arg1231Cys is Markedly Enriched in South Asians and Associated with Stroke

Background Genetic studies of stroke have focused predominantly on European ancestry populations. However, the genetic factors of stroke in South Asians are largely unexplored. Methods Exome sequencing was conducted in 75 thousand (K) Pakistanis, including a 31 K exome-wide association study (ExWAS) discovery cohort and a 44 K follow-up cohort that included 30 K with stroke case:control status for replication analysis. Further analysis was conducted in a 450 K United Kingdom Biobank (UKB) cohort, including 370 K with stroke case:control status and 33 K with brain MRIs. Findings ExWAS identified NOTCH3 p.Arg1231Cys enriched in South Asians and associated with subcortical hemorrhagic stroke [odds ratio (OR) = 3.39, 95% confidence interval (CI) = [2.26, 5.10], p value = 3.87×10-9, alternate allele frequency = 0.58% compared to 0.019% in UKB Europeans), which was replicated with similar effect size in the replication cohort. Meta analysis of 61 K PGR participants identified a significant association with all strokes (OR [CI] = 2.30 [3.01, 1.77], p value = 7.79×10-10). In UKB Europeans, p.Arg1231Cys was associated with white matter hyperintensity (WMH) (beta [CI] = 1.10 [0.61, 1.50], p value = 3.0×10-6). Interpretation Recurrent strokes and WMH loss are characteristics of Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), a rare disease caused by cysteine-altering variants in NOTCH3 . NOTCH3 p.Arg231Cys, present in approximately 1% of South Asians, incurs a 3-fold increased risk for hemorrhagic stroke, and is attributable for approximately 5.5% of hemorrhagic strokes and 1% of all strokes in South Asians. These results have major implications for genomic medicine and therapeutic development, especially for South Asian and West Asian populations, and highlight the value of diversity in genetic studies. Funding Fieldwork for this study was funded by the Center for Non-Communicable Diseases, Pakistan. DNA sequencing was funded by Regeneron Pharmaceuticals Inc. Evidence before this study Stroke is a multifactorial disease influenced by genetic and environmental factors. Family studies have identified mutations in several genes that cause rare monogenic forms of stroke, and large population genome-wide association studies have identified multiple common variants that modestly predict stroke risk. However, genetic studies of stroke have focused on predominantly European ancestry populations and ischemic stroke. Both the incidence and prevalence of hemorrhagic stroke is elevated in South Asians relative to Western Europeans. However, the genetic factors of stroke in South Asians are largely unexplored. Added value of this study Exome-wide association analysis identified a genome-wide significant association between p.Arg1231Cys in NOTCH3 and hemorrhagic stroke in Pakistani participants. Meta analysis of 61 K Pakistanis also observed a significant association with a general stroke phenotype for the same variant. While NOTCH3 p.Arg1231Cys is rare in European ancestry populations, it is present in approximately 1% of South Asians and associated with an approximately three-fold increased risk of hemorrhagic stroke. Brain magnetic resonance imaging of Europeans heterozygous for the risk allele had increased white matter hyperintensity, a pathology characteristic of CADASIL. Analysis of West Asian and North African populations (referred to as Greater Middle Eastern in population genetics) showed a broad range of variant enrichment beyond South Asians. The variant was estimated to be attributable for approximately 1% of all strokes in South Asians. This study demonstrates the value of conducting genetic analyses in understudied populations where disease prevalence and gene variant frequencies differ from European ancestry populations. In addition, this study expands the phenotypic spectrum of NOTCH3 mutations from classical rare autosomal dominant CADASIL to more common forms of stroke. Implications of all the available evidence CADASIL was previously thought of as a rare disease caused by Cys-altering variants in the first 6 epidermal growth factor-like repeat (EGFR) domains of the NOTCH3 extra-cellular domain (ECD). This study shows evidence of a milder form of CADASIL due to a Cys-altering variant affecting EGFR 31 (p.Arg1231Cys), shown here to be markedly enriched in both South Asian and West Asian populations. These findings have implications for therapeutic development priorities and stroke prevention in these populations. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Fieldwork for this study was funded by the Center for Non-Communicable Diseases, Pakistan. DNA sequencing was funded by Regeneron Pharmaceuticals Inc. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: PGR has received approval by the relevant research ethics committee of each of the institutions involved in participant recruitment, as well as centrally by the IRB board of the Center for Non-Communicable Diseases which is registered with the National Institutes of Health, USA. PGR has also been approved by the National Bioethics Committee, Islamabad Health Research Institute, National Institutes of Health of Pakistan. 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Yes All data produced in the present study are available upon reasonable request to the authors