Gene Variants and Altered Blood mRNA Levels as Risk Factors for Post-Stroke Cognitive Impairment in Indians

Research Background

Cognitive impairment is a common clinical outcome after stroke, occurring in about 30% of stroke survivors. Research has shown that Brain-Derived Neurotrophic Factor (BDNF) plays an important role in this process, and BDNF is regulated by circadian rhythms. At the molecular level, circadian clock genes are closely related to stroke timing, but research on the susceptibility of these genes to post-stroke cognitive impairment (PSCI) is relatively limited. This paper aims to explore: 1) genetic risk variants of circadian clock genes and BDNF; 2) potential mechanisms by which dysregulation of circadian clock genes CLOCK, BMAL1, and BDNF expression levels may be associated with PSCI.

Paper Source

This research paper was written by Dipanwita Sadhukhan and her team, with authors mainly from institutions such as the University of Calcutta and the National Neurosciences Centre Calcutta. The paper was published online in the journal “Neuromolecular Medicine” on October 9, 2023.

Research Process

Research Subjects and Methods

The study included 119 stroke survivors and 292 controls, all from the eastern region of India with the same ethnic and linguistic background. All stroke survivors underwent detailed cognitive assessments to determine whether they had cognitive impairment, thus dividing them into PSCI and PSCN groups. Additionally, 15 PSCI cases and 12 controls were selected from stroke patients for gene expression analysis in peripheral blood mononuclear cells (PBMCs), and plasma BDNF levels were measured by ELISA to validate mRNA data.

Specific Steps

1. Selection and Processing: Blood samples were collected, genomic DNA was extracted for genotyping of single nucleotide variants (SNVs). PCR products were digested with corresponding restriction enzymes and detected using polyacrylamide gel electrophoresis.
2. Gene Expression Analysis: PBMCs were isolated from fresh blood, total RNA was extracted using Trizol reagent, and mRNA expression levels of CLOCK, BMAL1, and BDNF were measured by real-time quantitative PCR (qPCR).
3. Plasma BDNF Level Measurement: Fasting blood samples were collected, and mature BDNF (mBDNF) and precursor BDNF (pro-BDNF) levels were detected by ELISA.

Research Results

Association of Gene Polymorphisms with PSCI

Among the studied gene variants, the ‘C’ allele of CLOCK gene rs4580704 was found to be significantly associated with PSCI (p = 0.001), and its ‘C’ allele was also associated with attention deficits. Additionally, the distribution of variants in BDNF rs6265 and CRY2 rs2292912 showed associations with specific cognitive domains (such as language and memory).

Gene Expression Analysis

Results showed that CLOCK and BDNF gene expression levels in PBMCs were significantly reduced in PSCI patients compared to the control group. This finding supports the notion that transcriptional dysregulation of these genes may be a potential mechanism for post-stroke cognitive impairment. Furthermore, no significant difference was found in BMAL1 gene expression levels between the two groups.

Comparison of Plasma BDNF

Biochemical analysis of plasma levels showed that mature BDNF levels were significantly lower in the PSCI group compared to the PSCN group, but there was no significant difference in pro-BDNF levels. Plasma mBDNF levels showed a significant positive correlation with lower memory function scores.

Research Conclusions

The study demonstrates for the first time that variants in CLOCK and CRY2 genes are susceptibility factors for PSCI. Furthermore, gene expression analysis suggests that changes in CLOCK and BDNF gene expression may be associated with post-stroke cognitive impairment. Future research should explore the role of overall circadian rhythms and other clock-controlled genes in PSCI, emphasizing their importance as potential therapeutic targets.

Research Highlights

1. Gene Variant Associations: Discovered associations between specific variants of CLOCK and CRY2 genes and PSCI, which is a first globally.
2. Gene Expression Differences: Revealed significant differences in CLOCK and BDNF gene expression between PSCI patients and the control group, supporting the view of transcriptional dysregulation as a potential mechanism for post-stroke cognitive impairment.
3. Plasma BDNF Levels: Decreased plasma mBDNF levels were associated with PSCI and showed a significant positive correlation with memory function scores.

Research Value

This study provides new insights into the molecular mechanisms of post-stroke cognitive impairment, particularly the role of circadian clock genes and BDNF. Furthermore, the results highlight the significance of CLOCK and CRY2 gene variants as potential genetic risk factors for PSCI, providing targets for future therapeutic interventions.