Emerging Insights from the Genetics of Cerebral Small Vessel Disease

Abstract

Cerebral small vessel disease (cSVD) is a common cause of stroke, functional decline, vascular cognitive impairment, and dementia. Pathological processes in the brain’s microcirculation are tightly interwoven with pathology in the brain parenchyma and this interaction has been conceptualized as the neurovascular unit (NVU). Despite intensive research efforts to decipher the NVU’s structure and function to date, molecular mechanisms underlying cSVD remain poorly understood, which hampers the development of cSVD-specific therapies. Important steps forward in understanding the disease mechanisms underlying cSVD have been made using genetic approaches in studies of both monogenic and sporadic SVD. We provide an overview of the NVU’s structure and function, the implications for cSVD, and the underlying molecular mechanisms of dysfunction that have emerged from recent genetic studies of both monogenic and sporadic disease of the small cerebral vasculature.

Graphical abstract

A common cause of stroke is cerebral small vessel disease (cSVD), which results from pathological processes in the neurovascular unit (NVU). The review provides an overview of the structure and function of the NVU, the implications for cSVD, and the underlying molecular mechanisms of NVU dysfunction in cSVD that have emerged from recent genetic studies.

Background

Stroke is one of the leading causes of death and disability and its global burden is increasing.¹ Stroke is an acute manifestation of a chronic underlying disease process that can be caused by several different mechanisms, of which large artery atherosclerosis, cardioembolism, and cerebral small vessel disease (cSVD) are the most common. The latter one is the only etiological subtype of cerebrovascular disease that is considered intrinsic to the brain, and it is tightly interwoven with the pathological processes affecting the brain parenchyma since the cerebral microvasculature is the main component of the neurovascular unit (NVU).² cSVD is also an important risk factor for vascular cognitive impairment and dementia, as well as the overall functional decline in otherwise stroke-free aging adults.³ Neuro-pathological studies of cSVD patients show a number of abnormalities in the small perforating arteries, including both focal regions of atherosclerosis at the origin of, or in the proximal, perforating arteries, and more diffuse abnormalities affecting the small perforating vessels.⁴ These diffuse changes include thickening of the vessel wall due to the deposition of fibro-hyaline material, narrowing of the vessel lumen, and loss of smooth muscle cells in the tunica media with fibrinoid necrosis.⁵

Historically, cerebral blood vessels and brain cells have been considered two different entities, making a rigid distinction between cerebrovascular and neurodegenerative disease. However, the circulatory system is of utmost importance in brain health. The dependence of the brain on the circulatory system is demonstrated by the fact that the brain receives up to 20% of cardiac output, whereas it only comprises ~2% of total body mass.⁶ In addition, the brain consumes ~20% of the total body’s oxygen,⁶ and if brain regions are activated, the regional blood flow and oxygen delivery increases immediately. If blood flow is completely interrupted for more than a few minutes (for example, cerebral artery occlusion in stroke), significant brain damage or death is the consequence. Even if the blood flow is not completely interrupted but reduced or insufficient with respect to the energy demands of the tissue, more subtle brain alterations arise, leading to chronic brain injury.⁷

Despite the intensive research efforts to decipher the NVU’s structure and function to date, molecular mechanisms underlying cSVD remain poorly understood; this in turn serves as a major obstacle to the development of cSVD-specific therapies. Mechanistic studies to investigate the pathology underlying cSVD have been challenging because of the limited accessibility of the brain microvessels for biochemical or physiological studies, the difficulties to visualize these small vessels in vivo, and the lack of adequate animal models.⁸ However, important steps toward understanding the disease mechanisms underlying cSVD have been made using genetics; specifically, through the discovery of single genes responsible for numerous familial SVD syndromes (monogenic SVD). Also promising in this respect are recent large studies that have investigated the genetic basis of sporadic cSVD.

In this article, we provide a brief overview of the structure and function of the NVU (endothelium, smooth muscle cells, pericytes, glial cells, neurons, and extracellular matrix) (Fig. 1), the implications for cSVD, and the underlying molecular mechanisms of dysfunction that have emerged as a result of recent insights from genetic studies of both monogenic and sporadic disease of the small cerebral vasculature.

Figure 1.

The neurovascular unit. Blood-brain barrier (BBB) and the neurovascular unit. Pial arteries branch out into smaller arteries called penetrating arteries, which eventually branch off into capillaries. Whereas the pial and penetrating arteries contain a smooth muscle cell layer and are separated from brain tissue by the parenchymal basement membrane (glia limitans), parenchymal arterioles and capillaries become associated with neurons and astrocytes. As vessels penetrate deeper into the brain, the smooth muscle cell layer and pia mater coverage disappears, while the neurovascular unit gains pericytes between the endothelial cell and astrocyte endfeet. Adapted from Ref. 9.

The neurovascular unit

Pial arteries that run on the brain surface in the subarachnoid space give rise to smaller arteries that penetrate into the brain tissue.⁷ These penetrating arteries consist of endothelial cells (ECs) and smooth muscle cells and are surrounded by a basement membrane, the perivascular (Virchow-Robin) space, pia mater, and astrocyte endfeet which collectively form the NVU (Fig. 1).⁹ As vessels penetrate deeper into the brain, the smooth muscle cell layer and pia mater coverage disappears, while the NVU gains pericytes between the EC and astrocyte endfeet.¹⁰ Neurons and astrocytes are in contact with other components of the NVU, where they can influence the function of the entire unit. The collective NVU enables tight regulation of blood flow through the vasculature, which has a unique structure in the brain.¹⁰

Brain endothelial cells

Endothelial cells lining the brain microvessels are the main component of the vascular blood-brain barrier (BBB), protecting the brain from factors present in the systemic circulation by limiting and regulating transport of blood-derived molecules into the brain.¹¹ Among the components of the BBB that contribute to its physical barrier function are tight junctions (TJs) and adherens junctions, which are formed between adjacent ECs.¹² TJ and adherens junction complexes restrict paracellular diffusion of ions, macromolecules, and other polar solutes (Fig. 2).⁹ TJs at the BBB are composed of a series of transmembrane proteins, including occludins, claudins, and junctional adhesion molecules.¹³ In addition to transport via TJ and adherens junctions, molecules such as nutrients, amino acids, nucleotides, and metabolic waste products are also selectively transported across EC membranes by specific influx and efflux transporters (Fig. 2).⁹

Figure 2.

Transport across the blood-brain barrier. Tight junctions (TJ) and adherens junction complexes restrict paracellular diffusion across the blood-brain barrier. In addition, molecules such as nutrients, amino acids, nucleotides, and metabolic waste products are also selectively transported across EC membranes by specific influx and efflux transporters. Abbreviations: ZO-1, zonula occludens-1; JAM, junctional adhesion molecule; VE-cadherin, vascular endothelial-cadherin, IgG, immunoglobulin G Adapted from Ref. 9.

Besides being a physical barrier, endothelial cells of the BBB have additional important functions, including regulation of vascular tone by producing vasoconstrictors and vasodilators, regulation of fibrinolysis and coagulation pathways, involvement in inflammatory responses, and blood vessel formation, repair and modeling.¹⁴ Endothelial dysfunction has been proposed as an important mechanism underlying cSVD, leading to impaired autoregulation, vessel reactivity, and BBB alterations.¹⁵ The latter has been shown in neuropathological studies by the presence of plasma proteins such as fibrinogen in the brain parenchyma and MRI studies demonstrating leakage of contrast agents such as gadolinium across the BBB, indicating that the BBB was open at some point.^16–18

Vascular smooth muscle cells

Smooth muscle cells (SMCs) of the cerebral microvasculature are a key component of the regulation of the overall as well as regional cerebral blood flow (CBF) in response to the systemic circulatory status or the organ’s metabolic demand (i.e., the brain CBF autoregulation process). Pial arterioles demonstrate multiple layers of SMCs separated from the endothelium by a prominent elastic lamina,¹⁹ whereas the SMC layer thins out along the gradient of the vasculature, from penetrating arterioles to intraparenchymal arterioles, where SMCs occur as a single or discontinuous layer, and where endothelial cells extend protrusions enriched with gap junctions through the basal lamina into SMCs (so-called myoendothelial projections).⁷

Pericytes

Pericytes are mural cells embedded in the basement membrane of blood vessels. Their thin processes are extended around and along the brain microvessels (pre-capillary arterioles, capillaries, and post-capillary venules) (Fig. 1).¹¹ Studies in mice have shown that several pericyte subpopulations exist, depending on the position along the vascular bed.²⁰ Pericytes have diverse functions in BBB permeability, angiogenesis, clearance of cellular debris, immune cell entry, and CBF regulation.^{11, 20} Because of the basement membrane, most of pericyte bodies processes do not attach to EC’s. However, through gap junctions and with other pericytes via peg- and-socket contacts, pericytes are able to communicate directly with cerebral ECs.^{21, 22}

Astrocytes

Astrocytes are the main class of glial cells and the classic view of their function is to provide metabolic, biochemical, and physical support to other cells of the central nervous system.

Astrocytes are distributed throughout the brain and surround the microvessels and interact with EC’s through their endfeet (Fig. 1).²³ Individual astrocytes can contact through their processes up to 140,000 synapses, as well as capillaries.²⁴ As part of the NVU, astrocytes are centrally positioned between neurons and ECs. This position of cellular linkage between neurons and ECs allows astrocytes to help regulate CBF by responding dynamically to synaptic activity and neuronal metabolism.²⁵ Another feature of the astrocyte endfeet is the expression of high levels of aquaporin-4 water channel proteins, which are thought to play an important role in perivascular clearance mechanisms.²⁶

Neurons

Neurons are able to regulate regional CBF in response to increased neuronal metabolic demand by generating signals that act on local blood vessels to initiate a vascular response.⁷ These signals can act either directly or via cells in between the neuron and the blood vessel. Vasoactive substances released by neurons include COX-2–derived prostanoids, nitric oxide, vasoactive intestinal polypeptide, acetylcholine, corticotropin-releasing factor, neuropeptide Y, and somatostatin.¹⁰

Matrisome

The matrisome of cerebral blood vessels is an often overlooked but key component of the neurovascular unit.²⁷ It comprises the extracellular matrix (ECM) and the matrisome-associated proteins. The ECM is the core matrisome and consists of collagens, proteoglycans, and glycoproteins. The basement membrane (BM) is a specialized form of ECM that covers the basal side of endothelial cells, facing the elastic lamina, at the interface between pericytes and astrocyte endfeet.²⁷ The matrisome-associated proteins comprise several hundred of proteins, which can be grouped into secreted factors, ECM regulators, and ECM-associated proteins. The matrisome is critical to the proper functioning of the NVU as it regulates the activation state of numerous receptors on the cells of the NVU.¹⁰

Pathological, clinical, and neuroimaging manifestations of common cSVD subtypes

Cerebral small vessel disease (cSVD) is a broad term describing a wide range of clinical pathological and neuroimaging features that reflect acute and chronic injury to the NVU throughout the brain.²⁸ Clinically, cSVD can be diagnosed in the setting of an acute small-vessel stroke, insidious onset of cognitive impairment or dementia, gait dysfunction, incontinence, late-life depression, or based on only neuroimaging findings without any clinical symptoms at all.

Acute ischemic small-vessel stroke (SVS) is one of the clinically overt manifestations of cSVD and comprises one quarter of all ischemic strokes. SVS is often referred to as lacunar stroke,²⁹ and it occurs as a result of occlusion of the small perforating arteries of the brain. Pathological studies of SVS patients have described diffuse arteriopathy of the small arterioles (40–200 μm diameter), with infiltration of plasma components and inflammatory cells into the vessel wall and perivascular tissue, resulting in vessel wall damage.^{4, 30}

Cerebral SVD can also result in acute intracerebral hemorrhage (ICH)., ICH can be classified into “deep”, “nonlobar” (thalamus, basal ganglia, brainstem, cerebellum), or “lobar” (junction cortical gray matter/subcortical white matter), according to its location. A main cause of deep ICH is chronic hypertension, whereas cerebral amyloid angiopathy (CAA) is a leading cause of lobar ICH.³¹ A discussion of lobar ICH is however beyond the scope of this review. Cognitive impairment, dementia, depression, and functional disability are the more insidious consequences of chronic cSVD, which become overt as the previously silent burden of cerebral microvascular disease accumulates.³ Chronic cSVD can be detected using T2 magnetic resonance imaging (MRI) of the brain, and its common features include white matter hyperintensities (WMH), lacunes, enlarged perivascular spaces (ePVSs), cortical microinfarcts, and cerebral microbleeds (CMBs).³⁰ Of these, WMH is the most commonly studied chronic cSVD marker that is detected as an increased signal of the cerebral white matter on T2-weighted or fluid-attenuated inversion recovery (FLAIR) MRI.²⁸ A frequent radiographic finding, WMH varies significantly in severity in association with age and common vascular risk factors, especially hypertension.^{32, 33} Furthermore, despite its reputation as an incidental, or age-related non-specific finding, WMH carries a significant burden of mortality and morbidity,³⁴ including risk of incidental and recurrent stroke. In patients with clinical stroke, a greater burden of WMH is associated with higher rates of acute treatment complications and poor post-stroke outcomes.^{35, 36} WMH are more common and more extensive in patients with acute small-vessel stroke than in patients with other stroke subtypes,³⁷ and are associated with lacunes,³⁸ ePVSs and CMBs.^{39, 40}

Genetics of cerebral small vessel disease: monogenic disorders

Support for a genetic basis of cSVD is mainly derived from family studies, but increasingly also from genome-wide association (GWA) studies (Fig. 3). WMH heritability has been estimated to be >60% in a broad range of studies.^41–44 The estimated heritability of SVD traits in GWA studies is generally lower, in the range of 15–30%.⁴⁵ This discrepancy can be at least partly explained by the contribution of rare variants with high impact in families, or monogenic SVD.

Figure 3.

Risk loci for monogenic and sporadic cSVD subtypes. Shown are the genomic locations of genes underlying monogenic cSVD and published risk loci reaching genome-wide significance in previous GWA studies for sporadic cSVD.

The most prevalent monogenic SVD is cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), with a minimum prevalence between 2 and 5 in 100,000.⁴⁶ The most common symptoms include early-onset subcortical lacunar stroke, migraine with aura, mood disturbances, and vascular dementia. Brain imaging generally shows extensive WMH changes, lacunar strokes, and less frequently, CMBs. CADASIL is caused by mutations in the NOTCH3 gene on chromosome 19q1, which encodes the Notch3 transmembrane receptor, which is involved in arterial differentiation and vascular SMC remodeling.⁴⁷ Pathological studies on a macroscopic level showed typical chronic SVD features, including lacunes, dilated PVSs, and rarefaction of the hemispheric white matter.⁴⁸ Microscopically, the small penetrating cerebral and leptomeningeal arteries show arterial wall thickening, leading to stenosis of the lumen, the presence of granular osmiophilic material within the media, and SMC alterations.⁴⁸

Other genes involved in monogenic cSVD include HTRA1, COL4A1, COL4A2, TREX1, FOXC1, PITX2, CECR1 and CTSA. A brief overview of the associated pathology and clinical symptoms for these genes is provided in Table 1. Many manifest with common phenotypes, such as WMH or CMBs, prompting a hypothesis of shared underlying disease biology. Recently Joutel et al. proposed the concept that perturbations of the cerebrovascular matrisome is a convergent pathological pathway in these monogenic forms of SVD.²⁷ Furthermore, albeit genetically heterogenous and rare, the monogenic cSVDs share clinical and radiological features with sporadic cSVD and, therefore, provide important insights into the mechanisms underlying the common and highly prevalent cerebrovascular forms of disease.

Table 1.

Monogenic cSVDs

Disease	Gene	Gene function	Vascular pathology	Clinical symptoms	Refs.
CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) (OMIM: 125310)	NOTCH3	Encodes NOTCH3 transmembrane receptor that is involved in arterial differentiation and vascular smooth muscle cell remodeling.	NOTCH3 ectodomain aggregation and accumulation in the extracellular space of small vessels, intimal thickening, degeneration of SMCs.	Subcortical lacunar infarcts, vascular dementia, migraine with aura, psychiatric disturbances.	48, 78–80
CARASIL (cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy) (OMIM: 600142)	HTRA1	Encodes High temperature requirement protein A1, a serine protease. Loss of HTRA1 function is associated with dysregulation of the TGF-β pathway. Emerging evidence suggest that heterozygous mutations also can cause early onset cSVD.	TGF-β signaling has a key role in vessel development and maintenance, acting in both endothelial and SMCs. Pathological studies showed extensive loss of SMC and deposition of fibro-hyaline material in the media.	Subcortical lacunar infarcts, vascular dementia, alopecia, spondylosis.	81–83
COL4-related angiopathies (OMIM: 607595; 614519)	COL4A1, COL4A2	Encodes alpha1 and alpha2 collagen chains, which are important components of the extracellular matrix.	Basement membrane abnormalities.	ICH, infantile hemiparesis, Axenfeld-Rieger anomaly, nephropathy, porencephalopathy.	27, 84
RVCL-S (retinal vasculopathy with cerebral leukodystrophy and systemic manifestations) (OMIM: 192315)	TREX1	Encodes DNase III, which plays a role in DNA repair.	Basement membrane defects in capillaries.	Retinal vasculopathy, subcortical lacunar infarcts, white matter hyperintensities, pseudotumors, migraine, cognitive impairment, psychiatric disturbances.	85, 86
FOXC1/PITX2-related SVD	FOXC1, PITX2	FOXC1 encodes forkhead box transcription factor C1, which is involved in blood vessel development. PITX2 encodes paired-like homeodomain transcription factor 2, which is involved in left-right asymmetry of internal organs. FOXC1 interacts with PITX2.	Changed endothelial and pericyte proliferation and impaired blood-brain barrier integrity in animal models.	Stroke, WMH, Axenfield-Rieger anomaly.	87–89
Deficiency of ADA2 (DADA2)	CECR1	Encodes adenosine deaminase 2 (ADA2). ADA2 plays a role in downregulation of extracellular adenosine, and cellular proliferation and differentiation.	Neutrophils and macrophages in interstitium with perivascular T-lymphocytes.	Small subcortical ischemic and hemorrhagic strokes, intermittent fevers, raised acute phase proteins, livedoid rash, hepatosplenomegaly	90, 91
CARASAL (cathepsin A related arteriopathy with strokes and leukoencephalopathy)	CTSA	Encodes cathepsin A, which is involved in the lysosomal transport, activation, and stabilization of β-galactosidase and neuraminidase-1. Cathepsin A inactivates selected neuropeptides and regulates a lysosomal pathway of protein degradation.	Fibrous thickening of the small vessels.	Subcortical ischemic and hemorrhagic strokes, cognitive impairment, swallowing difficulties, dry eyes and mouth, muscle cramps, treatment-resistant hypertension	92, 93

Genetics of cerebral small vessel disease: sporadic WMH

Due to its high heritability estimates, WMH has been the model sporadic cSVD trait investigated extensively using the latest generation GWA approach. Genetic loci that showed robust associations with sporadic cSVD in GWA studies are listed in Table 2. A meta-analysis of GWA studies in more than 10,000 stroke-free individuals from the population-based CHARGE consortium identified the first genome-wide significant risk locus on chromosome 17q25.⁴⁹ This locus contains six genes (TRIM65, TRIM47, WBP2, MRPL38, FBF1, and ACOX1). A larger follow-up meta-analysis in the same CHARGE population identified four additional genome-wide significant loci (chr10q24 (SH3PXD2A), chr2p21 (HAAO), chr1q22 (PMF1-BGLAP), and chr2p16 (EFEMP1)).⁵⁰ HAAO encodes 3-hydroxyanthranilate 3,4-dioxygenase, which catalyzes the synthesis of quinolinic acid (QUIN) from 3-hydroxyanthranilic acid. QUIN has been implicated in other neurodegenerative diseases, including Alzheimer’s disease and Huntington’s disease.⁵¹ PMF1-BGLAP encodes polyamine-modulated factor 1, a nuclear protein regulated by polyamines that is required for normal chromosome alignment and segregation during mitosis. EFEMP1 encodes the extracellular matrix glycoprotein fibulin-3.

Table 2.

Genes identified in genome-wide association studies as being associated with sporadic cSVD

SNP	CHR	Nearest Gene	Phenotype	Population	References
rs7214628	17	TRIM65*	WMH	Community, clinical stroke	50, 52
rs72848980	10	NEURL1	WMH	Community	50, 52
rs7894407	10	PDCD11	WMH	Community	50, 52
rs12357919	10	SH3PXD2A	WMH, SVS	Community	50, 52
rs7909791	10	SH3PXD2A	WMH	Community	50, 52
rs78857879	2	EFEMP1*	WMH	Community, clinical stroke	50, 52
rs2984613	1	PMF1-BGLAP	WMH, ICH, SVS	Community, clinical stroke	50, 52, 65
rs11679640	2	HAAO	WMH	Community	50, 52
rs72934505	2	NBEAL1	WMH	Community, clinical stroke	50, 52
rs941898	14	EVL	WMH	Community, clinical stroke	50, 52
rs962888	17	C1QL1	WMH	Community, clinical stroke	50, 52
rs9515201	13	COL4A2*	WMH	Community, clinical stroke	50, 52
rs12445022	16	ZCCHC14	WMH, SVS	Stroke	70
rs12204590	6	FOXF2*	WMH, SVS	Community, clinical stroke	72, 74
rs275350	6	PLEKHG1*	WMH	Community, clinical stroke	56
rs13164785, rs67827860	5	VCAN*	MD, FA, WMH	Community	58

Abbreviations: SNP, single nucleotide polymorphism; CHR, chromosome.

^*Genetic loci that have been putatively linked to the various NVU domains and associated SVD dysfunction

Exploring the hypothesis of shared genetic contribution to WMH between different populations, a meta-analysis of WMH in stroke-free individuals and 3670 patients with ischemic stroke identified 4 additional genome-wide significant loci (chr2q33 (NBEAL1), chr14q32 (EVL), chr17q21 (C1QL1), and chr13q34 (COL4A2)).⁵² NBEAL1 encodes neurobeachin like 1. A variant of this locus, in high linkage disequilibrium (R² = 0.87) with the WMH locus, has been associated with coronary artery disease.⁵³ The COL4A2 gene encodes one of the six subunits of type IV collagen, the major structural component of the basement membrane. Rare mutations in this gene are known to cause a monogenic form of SVD (see above). Common variants in this gene have also been associated with ICH and ischemic SVS.^{54, 55}

The most recent and even larger meta-analysis of stroke and population-based studies additionally identified chr6q25 (PLEKHG1) as genome-wide significant locus for WMH.⁵⁶ Pleckstrin homology and RhoGEF domain containing G1 (PLEKHG1) belongs to a family of Rho guanine nucleotide exchange factors (Rho-GEFs). In the vascular endothelium, Rho-GEFs—including PLEKHG1—are involved in cyclic stretch–induced cell reorientation.⁵⁷

Further exploring the role of common genetic contributions to cerebral microvascular health, the largest GWA study to date, including 8448 population-based individuals from the UK Biobank, studied fractional anisotropy and mean diffusivity derived from diffusion tensor imaging as measures of microstructural integrity of the white matter.⁵⁸ This analysis identified a genome-wide significant locus for both mean diffusivity and fractional anisotropy at chr5q14, which maps to an intron of VCAN. In this same study, this locus was nominally associated with WMH (P = 3 × 10⁻⁶). VCAN encodes the extracellular matrix proteoglycan versican, a versatile protein that plays a role in intercellular signaling and in connecting cells with the extracellular matrix.⁵⁹ Furthermore, versican may play a role in the regulation of cell motility, growth, and differentiation.

Genetics of cerebral small vessel disease: other cSVD markers

Genetic studies of MRI-detected cSVD markers other than WMH have been limited to date due to challenges related to phenotype ascertainment and lack of power to uncover associations that are smaller in effect size and driven, generally, by lower estimates of heritability.⁶⁰ A meta-analysis of GWA studies of silent brain infarcts in 9401 participants from the CHARGE consortium did not identify any genome-wide significant locus. Recently a larger meta-analysis of studies of the CHARGE consortium identified 3 genome-wide significant loci (FBN2, GRK6P1/ZDHHC20, and SV2B), but these could not be replicated in an independent, albeit smaller, sample.⁶¹

Enlarged PVS have been rarely investigated in genetic studies. So far, no GWA study has been reported for this trait. A recent meta-analysis of studies from the CHARGE consortium including 1597 participants investigated the heritability of ePVS and its shared heritability with other MRI markers of cSVD.⁶² They demonstrated an estimated heritability of 0.59 for ePVS burden. Furthermore, they showed differential heritability patterns for ePVS in white matter and basal ganglia, which may be due to partly distinct underlying biological processes.

To date, only candidate gene studies have been reported on genetic associations with CMBs. A review of candidate gene studies of CMBs found that only the APOE gene had been investigated in more than 100 persons.⁶³ In this study, compared with people with the ε3/ε3 genotype, carriers of the ε4 allele (ε4+) were more likely to have CMBs.

ICH

Achieving sufficient sample sizes for GWA studies of ICH is challenging because of its low incidence (15% of all strokes) and heterogeneous biology. ⁶⁴ The International Stroke Genetics Consortium published the first ICH GWA study, including 1545 cases, and identified 1q22 (PMF1-BGLAP) as the first nonfamilial genetic locus for nonlobar ICH, the subtype thought to be mainly driven by cSVD mechanisms.⁶⁵ As described above, this locus has been subsequently also identified as a genome-wide significant locus for WMH, supporting the at least partly shared mechanisms between otherwise different (both clinically and radiographically) cSVD phenotypes. Significant association with ICH has also been demonstrated for the APOE locus in a large-scale genetic association study of 2189 ICH cases and 4041 controls from 7 cohorts within the International Stroke Genetics Consortium.⁶⁶ As described above, candidate-gene associations for the APOE locus have also been demonstrated for other cSVD traits, including CMBs, WMH, and silent brain infarcts.⁶⁷ Similarly, another candidate study suggested a role for common variants in COL4A2, a gene involved in monogenic cSVD^{54, 55} in sporadic clinical forms of ICH.

Genetics of cerebral small vessel disease: ischemic small-vessel stroke

To date, GWA studies in sporadic, clinically overt SVS have not yet been as successful in identifying common variants as compared to other vascular diseases or main stroke subtypes (large-artery and cardioembolic stroke), which is likely due to a combination of relatively small sample sizes and issues related to phenotyping and the heterogeneity of cSVD.^{68, 69} An age-at-onset–informed GWAS approach for the first time identified an SVS locus at 16q24 (ZCCHC14).⁷⁰ Recently, the largest meta-analysis of stroke and its subtypes (MEGASTROKE), published by the International Stroke Genetics Consortium, replicated the association of 16q24 with small-vessel stroke.⁷¹ ZCCHC14 is highly expressed in arterial and brain tissues, but its function has not yet been well defined.

Although the MEGASTROKE study did not identify any additional loci with genome-wide significance, secondary analysis provided further support for the roles of some loci in SVS. In this analysis, 12q24 (SH2B3), 16q24 (ZCCHC14), 1q22 (PMF1-BGLAP), 6q25 (FOXF2), and 10q24 (SH3PXD2A) were identified as associated predominantly with SVS.⁷² Among these, only the 12q24 locus demonstrated evidence of involvement with all stroke subtypes. Furthermore, this locus also showed associations with coronary artery disease, blood pressure, and HDL levels.⁷¹ The gene mapping to this locus, SH2B3, is a gene encoding for SH2B adapter protein 3 (also known as lymphocyte adapter protein), which promotes a proinflammatory state in human vascular endothelial cells.⁷³ As described above, the 1q22 (PMF1-BGLAP) locus has also been linked with WMH and ICH, and the 10q24 (SH3PXD2A) locus with WMH. An association of the 6q25 (FOXF2) locus with SVS and the presence of WMH was already suggested in an earlier GWA study.⁷⁴ Zebrafish knockout models for FOXF2 orthologs showed signs of defects in pericyte maturation and smooth muscle, suggesting involvement of FOXF2 in the differentiation of cerebral vascular mural cells.⁷⁴ Suggested endothelial genes have been investigated as several candidate genes in SVS, and cSVD in general. The investigated genes included ACE, AGT, eNOS, and MTHFR, but the results have been inconsistent.^{75, 76}

Genetic insights into structural and functional integrity of the NVU

Mounting evidence to date points to the critical role of the structural and functional integrity of NVU in overall brain health. Disorders compromising any of the previously discussed NVU elements may lead to the indolent and chronic, yet relentless, progression of the burden of diffuse small cerebral vasculature pathology, which in turn inevitably results in (1) functional deterioration of otherwise healthy, aging adults; (2) vascular cognitive impairment and dementia; (3) increased risk of stroke; and(4) poor post-stroke outcomes.32, 34–36 As presented above, recent genetic studies of the known monogenic and sporadic cSVD phenotypes with high heritability estimates provide important insights regarding the susceptibility of the NVU elements to pathological processes ultimately leading to clinical manifestations (such as SVS or ICH) or radiographic detection (WMH, ePVSs, CMBs) of diffuse cerebral microangiopathy. For example, resilience of the matrisome to injury could be compromised in those carrying pathogenic variants in EFEMP1, which encodes the extracellular matrix glycoprotein fibulin-3; COL4A2, which encodes one of the six subunits of type IV collagen, the major structural component of the basement membrane; or VCAN, which encodes the extracellular matrix proteoglycan versican, an integral part of extracellular matrix signaling.52, 56, 58 Endothelial dysfunction is a key mechanism of cSVD, and based on the new GWA studies, several putative pathways linked to the role of endothelial cells within the NVU have been proposed. These include pathways linked to the activity of TRIM65 and TRIM47, a superfamily of ring-finger B-box coiled-coil (RBCC) proteins involved in the mechanisms of innate immunity.77 In addition, PLEKHG1, which belongs to the family of Rho guanine nucleotide exchange factors, has been identified and thought to be involved in cyclic stretch–induced cell reorientation in the vascular endothelium.56 These findings are complementary to the well-known paradigm of monogenic influence of NOTCH3 on endothelial cell function, leading to the broad spectrum of clinical CADASIL syndrome.46 Another locus linked to a rare segmental deletion syndrome at 6q25 (FOXF2) in humans with extensive WMH showed decreased SMC and pericyte coverage in an zebrafish ortholog model.74 Most of the results discussed in this review are derived from GWA studies focusing on neuroimaging phenotypes or clinical endpoints. The next steps to further interpret the functional consequences of identified genetic variants for the NVU could include in vitro and in vivo experimental models, as in the example of the FOXF2 zebrafish model discussed above.74

Whereas the roles of most of the common genetic variants (e.g., TRIM65, EFEMP1, COL4A2, FOXF2, PLEKHG1, and VCAN) emerging in association with sporadic forms of cSVD have yet to be elucidated, it has become clear that each of these variants can potentially disrupt specific components of the NVU’s structure and function. A diseased NVU state leads to diffuse cerebral microvascular injury and loss of structural and functional integrity of the white matter, with associated cognitive impairment, functional decline, and increased risk of clinical stroke. Detailed characterization of these pathways may lead to discovery of novel molecular targets for future therapies.