Unraveling the association between COL4A2 polymorphisms and intracerebral hemorrhage

INTRODUCTION

Spontaneous intracerebral hemorrhage (ICH) is an emergency pathology associated with intracranial vessel rupture. ICH is the second most prevalent type of stroke, with over 3 million new cases registered worldwide in 2019.^[1,2] Despite the extensive history of research on this condition, to date, there has been no conclusive evidence to support the efficacy of surgical or medical treatments.

Due to the high levels of mortality and disability, it is a priority to prevent ICH. The established risk factors for ICH are arterial hypertension, obesity, diabetes mellitus, smoking, and alcohol abuse. There are also gender, racial, and ethnic characteristics.^[1-4] Due to the significant differences in the risk of occurrence and progression of ICH among individuals from European, Asian, and African populations, conducting genetic research is considered an upcoming trend. The greatest importance is given to specific single-nucleotide polymorphisms of the genes associated with the Renin-Angiotensin system, the integrity of cerebral vessels’ walls, lipid homeostasis, inflammatory processes in the endothelium, and vascular-platelet hemostasis.^[5-8]

COL4A1 (the protein accession ID P02462) and COL4A2 (the protein accession ID P08572) are inextricably bound. They encode the synthesis of Collagen Type IV Alpha 1 and Collagen Type IV Alpha 2. Forming heterotrimers, they are the main structural components of basement membranes. COL4A1 (NM_001845) and COL4A2 (NM_001846) have 52 and 48 exons, respectively, and are located on opposite strands on human chromosome 13, specifically at position 13q34. These two genes are separated by a 127-nucleotide region that contains a common bidirectional promoter. This promoter requires additional regulatory elements in order to control the tissue specificity, level, and ratio of gene expression.^[9]

Mutations in the COL4A1 and COL4A2 genes cause the development of highly penetrant multisystem anomalies, as well as damage to the central nervous system, vision, heart, kidneys, and musculoskeletal systems in both young and older individuals. The most pronounced brain damage associated with these mutations is seen in conditions such as Type I porencephaly, pre- and perinatal hemorrhages, and sporadic and recurrent ICHs in both young and aged patients. Subclinical manifestations of the mutation are also possible, where diffuse or periventricular leukoencephalopathy and calcification, intracranial aneurysms, and cerebral microbleeds are detected in the absence of an obvious neurological deficit. This suggests a potentially hidden and underestimated role in a wide range of cerebrovascular diseases.^[9-15]

Mutations in the COL4A2 gene can lead to disruption of the structure of Collagen Type IV, causing vessel fragility and susceptibility to rupture. Mutations in this gene are directly associated with an increased risk of ICH and other cerebrovascular diseases.^[9-15]

The investigation of polymorphisms in different ethnic groups is of critical importance due to population-specific patterns in the frequency and influence of COL4A2 alleles in both Asian and European populations.^[9-19] This implies that genetic risk factors may differ between ethnic groups, justifying the need for population-specific studies.^[20]

The aim of this study was to identify risk factors for the development of ICH in the Kazakh population.

MATERIALS AND METHODS

The main group included 162 patients diagnosed with ICH, who underwent inpatient treatment in the medical centers of the Karaganda region, Kazakhstan. Their diagnosis was verified using neuroimaging data. The control group consisted of 165 people, matched to the main group by sex and age, with no history of strokes and brain tumors, autoimmune diseases, and congenital malformations. All participants were ethnic Kazakhs, with at least two generations of their direct ancestors (parents and grandparents) being of Kazakh ethnicity. The recruitment period for this study was from October 01, 2020, to October 01, 2021. Informed consent to participate in the research was obtained from each patient. The DNA isolation process was conducted using the GeneJET Genomic DNA Purification Kit (Thermo Scientific^™), following the manufacturer’s protocol to ensure high-quality genomic DNA suitable for downstream applications. The isolated DNA was quantified using a spectrophotometer to assess its purity and concentration, with A260/A280 ratios maintained within the range of 1.8–2.0.

Genotyping was performed using real-time polymerase chain reaction (PCR) on the QuantStudio^™ 5 System, which ensures precise and reliable detection of single-nucleotide polymorphisms (SNPs). The analysis employed TaqMan^™ reagents, including the SNP Genotyping Assay and TaqMan^™ Genotyping Master Mix (Applied Biosystems^™), which were selected for their high sensitivity and specificity. The SNP Genotyping Assay was customized to target the polymorphisms of interest (rs9521732, rs9521733, and rs9515199 in the COL4A2 gene).

The PCR protocol included an initial denaturation step, followed by 40 amplification cycles consisting of denaturation, annealing, and extension. After PCR, the genotyping data were analyzed using QuantStudio^™ Design and Analysis Software, ensuring robust allele discrimination and compliance with Hardy–Weinberg equilibrium. These methodological steps were optimized to minimize variability and ensure accurate identification of genotypes.

RESULTS

DISCUSSION

Our study identified a significant association between the rs9515199 polymorphism in the COL4A2 gene and a reduced risk of ICH within the Kazakh population, suggesting a protective effect of the C allele. This finding stands in direct contrast to prior meta-analyses conducted exclusively in European cohorts, which reported the same allele to be a risk factor for deep ICH (rs9515199: OR = 1.28, 95% CI = 1.14–1.44;^[11] OR = 1.24, 95% CI = 1.11–1.4^[16]). This discrepancy highlights the critical influence of ethnic background on genetic risk assessments for ICH.

The observed protective effect in our cohort is consistent with the higher frequency of the protective C allele in Asian populations (e.g., 0.47 in East Asians) compared to European populations (0.4), as recorded in the ALFA database. The Kazakh population, with its unique admixed ancestry combining East and West Eurasian heritage,^[17] provides a valuable model for exploring these ethnic disparities. Our data show that the allele frequencies of rs9515199 and rs9521732 align with East Asian patterns, while rs9521733 aligns with European population, further support this admixed status, and suggest that population-specific genetic architecture can profoundly modify the effect of a variant.

The complex role of COL4A2 across different ethnicities is further illustrated by conflicting data from other East Asian studies. A large exome-wide study in Japan found no association between COL4A2 mutations and ICH,^[18] while research in the Chinese Han population implicated different polymorphisms within the same gene (rs1049931 and rs1049906) in increasing risk.^[19] This suggests that the genetic etiology of ICH is not only population-specific but may also involve different causal variants within the same gene or interactions with differing genetic backgrounds and environmental factors.

Our study has several limitations. The sample size, while sufficient to detect an association, is relatively modest compared to large international consortia. This limits the power for more sophisticated subgroup analyses (e.g., by ICH location) or for investigating rare variants. The findings require validation in an independent, larger Kazakh cohort to confirm the effect size and direction.

Future research directions are promising. The planned multicenter ERICH-GENE study,^[20] which will perform whole-genome sequencing on over 10,000 patients from diverse ethnicities, is poised to definitively address these questions of ethnic heterogeneity and discover novel variants. In conclusion, our investigation identifies rs9515199 as a potential protective genetic marker for ICH in the Kazakh population, underscoring the necessity of including diverse ethnic groups in genetic research. While this marker is not yet ready for clinical application, it represents a step toward more precise risk stratification and underscores the need for developing personalized medicine approaches that account for genetic ancestry.

CONCLUSION

Our study identified several risk factors for ICH in the Kazakh population. A history of arterial hypertension, diabetes mellitus (OR = 3.11; 95% CI: 1.19–8.09), and moderate-to-heavy alcohol consumption (OR = 4.43; 95% CI: 2.12–9.27) were significant medical and social predictors. Genetic analysis revealed a strong association between the rs9515199 polymorphism in the COL4A2 gene and a reduced risk of ICH. A protective effect was observed under the co-dominant (CT genotype: OR = 0.60; 95% CI: 0.36–0.99; p = 1e-04; CC genotype: OR = 0.22; 95% CI: 0.11–0.46; p = 1e-04), dominant (CT/CC vs. TT: OR = 0.45; 95% CI: 0.28–0.73; p = 9e-04), and recessive (CC vs. CT/TT: OR = 0.29; 95% CI: 0.15–0.56; p = 1e-04) models. The identified protective effect of the C allele contrasts with previously reported associations in European populations, where the same allele was linked to an increased risk. This discrepancy underscores the critical role of ethnic genetic background in modifying disease risk and highlights the importance of conducting genetic association studies in diverse populations. Further validation in larger, independent cohorts is essential to confirm these associations. Ultimately, integrating genetic markers like rs9515199 with established clinical risk factors could improve risk stratification and pave the way for personalized prevention strategies for ICH.