The role of vascular dementia associated genes in patients with Alzheimer's disease: A large case–control study in the Chinese population

Abstract Aim The role of vascular dementia (VaD)‐associated genes in Alzheimer's disease (AD) remains elusive despite similar clinical and pathological features. We aimed to explore the relationship between these genes and AD in the Chinese population. Methods Eight VaD‐associated genes were screened by a targeted sequencing panel in a sample of 3604 individuals comprising 1192 AD patients and 2412 cognitively normal controls. Variants were categorized into common variants and rare variants according to minor allele frequency (MAF). Common variant (MAF ≥ 0.01)‐based association analysis was conducted by PLINK 1.9. Rare variant (MAF < 0.01) association study and gene‐based aggregation testing of rare variants were performed by PLINK 1.9 and Sequence Kernel Association Test‐Optimal (SKAT‐O test), respectively. Age at onset (AAO) and Mini‐Mental State Examination (MMSE) association studies were performed with PLINK 1.9. Analyses were adjusted for age, gender, and APOE ε4 status. Results Four common COL4A1 variants, including rs874203, rs874204, rs16975492, and rs1373744, exhibited suggestive associations with AD. Five rare variants, NOTCH3 rs201436750, COL4A1 rs747972545, COL4A1 rs201481886, CST3 rs765692764, and CST3 rs140837441, showed nominal association with AD risk. Gene‐based aggregation testing revealed that HTRA1 was nominally associated with AD. In the AAO and MMSE association studies, variants in GSN, ITM2B, and COL4A1 reached suggestive significance. Conclusion Common variants in COL4A1 and rare variants in HTRA1, NOTCH3, COL4A1, and CST3 may be implicated in AD pathogenesis. Besides, GSN, ITM2B, and COL4A1 are probably involved in the development of AD endophenotypes.


| INTRODUC TI ON
Dementia is characterized by progressive cognitive impairment and ultimately impaired independent living. The number of dementia cases was estimated at 50 million in 2018 and is expected to triple by 2050. In China, about 15 million individuals aged 60 years or older have dementia, imposing a huge burden on society and family. 1 Alzheimer's disease (AD) is the most prevalent dementia type worldwide, accounting for approximately 60%-80% of all dementia cases. 2 The etiology and pathology of AD are complex and remain elusive. In addition to AD, vascular dementia (VaD) is another major subtype of dementia and accounts for 15%-20% of dementia patients in Western countries. VaD refers to dementia caused mainly by vascular pathology. Typically, patients with VaD present with memory problems and executive dysfunction. 3 It is estimated that around 30% of dementia cases are diagnosed as VaD in Asia. 4 Vascular pathology coexists in a large proportion of AD cases and reduces the threshold for dementia. 5 Emerging evidence implicates cerebral microbleeds, white matter lesions, increased blood-brain barrier permeability, attenuated cerebral blood flow, and diminished neurovascular coupling in the development of AD. [6][7][8] Microvascular alterations, such as increased capillary tortuosity and capillary rarefaction, also exist in AD brains. 9 Additionally, epidemiological studies suggested that AD and VaD share similar risk factors, including hypertension, obesity, and diabetes. 10 Vascular risk factors contribute to increased amyloid precursor protein processing and reduce amyloid beta (Aβ) clearance. 11 Given that AD and VaD exhibit overlapping pathological changes and share similar clinical features, genetic studies may provide the underlying biological links between them. Previous studies have demonstrated that VaD-associated genes were implicated in AD risk. For example, the NOTCH3 gene was associated with AD using the c-alpha test in the United Kingdom and North America. 12 NOTCH3 rs149307620, a missense variant, was enriched in AD patients compared to controls in individuals of European ancestry. 13 However, NOTCH3 was not associated with AD risk in the Chinese population. 14,15 To illustrate the role of VaD-associated genes in the pathogenesis of AD, we comprehensively investigated the associations between these genes and AD risk in a large Chinese cohort via a targeted sequencing panel.

| Genomic DNA isolation
Using phenol-chloroform extraction and ethanol precipitation, 17 genomic DNA was extracted from the peripheral blood leukocytes of each individual. The quality and quantity of DNA were assessed with a NanoDrop spectrophotometer (Thermo Scientific). All DNA samples were diluted to 50-100 ng/μl.

| Gene selection
A detailed literature search in PubMed was manually conducted to select genes associated with VaD. The candidate genes were selected with more than one of the following features: (1)

| Statistical analysis
With the use of PLINK 1.9, 28 we filtered out the following variants with genotyping rate <95%, Hardy-Weinberg equilibrium p-value <1 × 10 −6 in controls, genotype quality (GQ) ≤20, allelic balance departing from 25%/75% ratio of referent and alternate allele reads in the heterozygote, and allelic balance departing from 95% ratio of the homozygote. We performed the common variant-based association analysis between 1192 AD patients and 2412 cognitively normal controls using PLINK 1.9. Age, gender, and APOE ε4 status (APOE ε4+, APOE ε4−) were adjusted for each common variant. Furthermore, we also performed age at onset (AAO) and Mini-Mental State Examination (MMSE) association studies in AD patients using the linear regression models in PLINK 1.9.
Additionally, using the Sequence Kernel Association Test-Optimal 29 gene-based association tests were conducted by combining rare variants between AD patients and cognitively normal controls. Rare variants were further categorized as followings: rare damaging variants (MAF <0.01, LoF or ReVe >0.7), rare damaging missense variants (MAF <0.01, ReVe >0.7), rare LoF variants (MAF <0.01, LoF), and rare missense variants (MAF <0.01, missense). Age, gender, and APOE ε4 status were also adjusted by SKAT-O. Besides, the rare variants association studies were also conducted using PLINK 1.9. A cutoff p-value * n < 0.05 was considered statistically significant based on Bonferroni correction (n is defined by the number of common variants or genes). Variants or genes not surviving the Bonferroni correction, but with uncorrected p-values less than 0.05, were considered "suggestive."

| VaD genes in the Chinese and European populations
To further investigate the role of VaD genes in the Chinese and European populations, we searched them in AD patients from the webserver AlzData, 30,31 a freely accessible database in the Chinese population (http://www.alzda ta.org/). Meanwhile, the suggestive common variants between AD and controls were also searched in a recent large meta-genome-wide association study (GWAS) in the European population. 32

TA B L E 1 Demographic and clinical information of AD patients and controls
was no significant age difference between AD patients and controls (p = 0.06). The MMSE scores of AD patients were statistically higher than those of controls (p = 4.84×10 −6 ). All participants were of southern Han Chinese ancestry (Table 1).

| Common variant association analysis
Forty common variants remained after quality control, including 22 The LD patterns of the COL4A1 variants (rs874203-rs874204-rs16975492-rs1373744) were similar between AD patients and controls ( Figure 1). No nominally significant associations were found between the four COL4A1 variants and AD in a large meta-GWAS study in the European population (p > 0.05). 32

| Rare variant aggregation testing
Gene-based aggregation testing was performed by combining the rare variants within genes. In the rare missense variants group, although the association was nonsignificant after the Bonferroni correction, we observed a suggestive association of HTRA1 with AD. Specifically, 0.50% of the AD cases and only 0.16% of the controls carried HTRA1 missense variants (p = 4.64 × 10 -2 ) ( Table 3).
In the remaining three groups, including rare damaging variants, rare damaging missense variants, and rare LoF variants, none of the VaD-associated genes were correlated with AD risk (p > 0.05).

| AAO and MMSE association studies
We performed AAO and MMSE association studies to elucidate the relationships between VaD-associated genes and AD endophenotypes. In the AAO association study, although no variants TA B L E 2 The nominal significant common variants between AD patients and controls

| VaD genes in the AlzData database
The association results for rare coding and damaging variants were available in the AlzData database from the whole-exome sequencing data of Chinese AD patients. Eight VaD-associated genes were searched in AlzData. In total, we identified 13 variants in NOTCH3, one variant in HTRA1, three variants in TREX1, three variants in GSN, one variant in ITM2B in the AlzData. None of these variants exhibited associations with AD (p > 0.05).

| DISCUSS ION
In our study, to explore the role of VaD-associated genes in AD, VaD refers to a variety of cerebrovascular diseases resulting in cognitive impairment. 33 Multiple genes were associated with VaD, such as NOTCH3, 34 HTRA1, 35 GLA, 36 and COL4A1. 37 The widely recognized pathological changes include hemorrhages, infarcts, white matter injury, and ischemic brain injury, which were not specific to VaD but also seen in AD. 38 Accumulating evidence demonstrated that alterations in small or large cerebral vessels were implicated These studies suggested that AD and VaD overlap pathologically and genetically.
Our study found that four COL4A1 common variants were nominally associated with AD risk. COL4A1, located on chromosome 13q34, encodes the α1 chain of type IV collagen. In 2005, COL4A1 mutations were identified to segregate with human familial porencephaly. 42 Later, it was recognized that these mutations cause a spectrum of cerebrovascular diseases, ranging from smallvessel disease to intraparenchymal hemorrhage. 43 in an autosomal recessive form. 19 Most of these mutations potentially lead to increased IGFβ signaling activity and a reduced level of protease activity, resulting in the degeneration of smooth muscle cells. 48 Moreover, a significant association was observed between HTRA1 rs2293871 and cerebral small vessel disease in the elderly. 49 In 5xFAD mouse analysis and human brain mass spectrometry, HTRA1 was correlated with Aβ levels. 50 Specifically, HTRA1 is in- Abbreviations: adjusted p, adjusted by age, gender, and APOE ε4 status; CI, confidence interval; OR, odds ratio; UTR, untranslated region. and stroke. 53 In a South East Asian cohort, four rare missense variants in NOTCH3 were marginally associated with AD susceptibility. 15 We identified that a rare variant in NOTCH3 achieved suggestive evidence of association with AD, suggesting that NOTCH3 may confer genetic susceptibility to AD in the Chinese population. Mutations in CST3 can cause amyloidosis characterized by deposition of abnormal protein fibrils. 54,55 A meta-analysis showed that the G73A variant of CST3 was associated with AD risk in Caucasian populations but not in Asians. 55 Our study revealed that two rare variants in CST3 were suggestively associated with AD, indicating that CST3 may also be a risk gene for AD in the Chinese population.
Substantial evidence indicates that genetic risk factors are involved in AD endophenotypes. 56 in AD endophenotypes.

ACK N OWLED G M ENTS
The authors are grateful to all subjects for participation in our study.
This study was supported the National Key R&D Program of China

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest associated with the contents of this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.