Intracranial aneurysm (IA) is a major public health burden that affects 3.2%–7.0% of the adult population.1 Subarachnoid haemorrhages resulting from a ruptured IA are associated with a 35% mortality within 3 months and 50% of survivors suffer from serious neurological dysfunction.2 The high morbidity and mortality of this disease are partly attributable to a lack of effective preventive approaches. IA rupture often occurs suddenly and most patients are not aware of the presence of an aneurysm.3 4 Previous studies have aimed to identify the risk factors for the formation, growth and rupture of IA, but these investigations have been limited to unmodifiable risk factors such as gender, age, menopause, family history, etc.5 The identification of novel biologic pathways underlying IA development is important to informing the development of new preventive and therapeutic strategies.

There are few IA biomarkers; these include plasma total homocysteine (tHcy),6 which remains controversial.7 8 tHcy is an amino acid generated by the metabolism of dietary methionine and is sensitive to vitamin B12, folate and various lifestyle factors.9 Multiple studies demonstrate a role for tHcy in endothelial injury,10 11 the mechanisms of which overlap with the pathogenesis of IA.12 13 Some clinical studies have questioned the relationship between tHcy and IAs; however, their findings are limited by the utilisation of small study cohorts and the conventional observational design of the research.7 8 14 It remains unclear whether the association between circulating tHcy levels and IA is causal.

Mendelian randomisation (MR) analysis, thus, offers the most appropriate approach for evaluating the causal association between tHcy and IA formation from a genetic perspective. In this approach, gene data are used to test causal relationships between genetic variants and outcomes while avoiding confounding biases.15 Several published MR studies report negative results but have not rigorously excluded potential modifiers and mediators; the reported single nucleotide polymorphisms (SNPs) have included rs1801133, rs154657 and rs7422339.16 17 However, these findings violate MR criteria because these SNPs are strongly associated with hypertension (p<5×10−8) according to the results of genome-wide association studies (GWAS) from the UK biobank,18 complicating the exact causal pathophysiological pathway.19 In short, further convincing evidence is required to properly test what role tHcy plays in the occurrence of IAs.

In this study, we used a two-sample MR analysis and a large-sample, multicentre observational analysis to explore the effect of tHcy on IA formation from the perspective of genetic variation data and real-world data. These two research methods complement each other as MR analysis provides causal evaluations that redress the limitations of conventional observational studies, while the latter can assess the real effect size of tHcy on the risk of IA formation, which verifies the results of MR and remedy the deficiency of the MR evidence in extrapolation.

In this study, we use a two-sample MR analysis of genetic variations and found evidence of a genetic-based increase in tHcy that is causally associated with the risk of IAs. This was followed up with a 1:1 propensity score-matched cohort consisting of 9902 patients, which verified that tHcy is a risk for IAs; the relationship was positively correlated with a linear trend. We found that the effects of tHcy on IA formation are dose dependent but independent of hypertension and modified by hypertension, age, and sex.

To our knowledge, this is the first MR analysis testing the association between tHcy and IAs using SNPs under the criteria of instrumental variables’ assumption in MR studies,19 and the largest multicentre observational study was conducted on this topic. Two published MR studies on tHcy and IA used three gene variants, rs1801133, rs7422339 and rs154657,16 17 which are associated with hypertension; this approach violates the principle of MR and, thus, leads us to question the negative findings. After removing these SNPs associated with confounders, we reassess the causal relationship between tHcy and IAs by a strict two-sample MR analysis following the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)-MR.19 Further sensitivity analyses indicated no evidence of horizontal pleiotropy confounding the causal inference, confirmed by the MR-Egger regression (p=0.85) and the MR-PRESSO test (p=0.60). However, the deficiency of MR evidence was that the estimation of the effect size of tHcy on IA risk was based on exposure to abnormal tHcy for a life long time, while being independent of other risk factors such as hypertension and age. This was not correspondent with clinical practice, and, therefore, we further verified the genetic effect size in a real-world cohort using multicentre data. By observing the real effect size and heterogeneity of tHcy for risk of IA formation among different subgroups, it makes such association embodied concretely in clinical settings and reveals the potential value of the application of tHcy in identifying specific population at risk of IA. This approach provides high-confidence findings that could only be surpassed by a large prospective cohort study or randomised clinical trial which, for ethical reasons, cannot be conducted. What is more, our results differed from the previously negative conclusions. 7 The large-sample, multicentre cohort could guarantee the reliability of our results, with strict inclusion and exclusion of patients and rigorous statistical methods mitigating potential distortion and bias.

The relationship between tHcy and IA occurrence was positively correlated with a linear trend. Taking low tHcy as a reference, the ORs increased with increasing tHcy (moderate tHcy, OR 2.13 (95% CI 1.93 to 2.36) and high tHcy, OR 3.66 (95% CI 2.71 to 4.95), p trend<0.001). Similar tHcy risks exist in CHD and stroke, where increases of 3–5 µmol/L markedly increase the risk of disease.24 25 In these conditions, it is widely recommended that tHcy ≥15 µmol/L is classified as hyperhomocysteinemia (16 µmol/L in the American Heart Association/American Stroke Association (AHA/ASA) Guideline) to assist therapeutic decision-making.23 26 Nevertheless, the OR of group tHcy (>13.2 µmol/L) increasing risk of IAs was also observed with a smaller CI when subdividing tHcy into three groups by tertiles (OR 3.12, 95% CI 2.82 to 3.45, taking tHcy <10 µmol/L as the reference). It is suggested that further prospective studies are warranted to establish a better cut-off value of tHcy for stratifying IA risk.

Further subgroup analysis stratified by age demonstrated that higher ORs were observed in IA patients aged ≥60 years, suggesting that the detrimental effects of tHcy may develop over the lifetime. Multiple experimental studies have shown that tHcy contributes to endothelial dysfunction by inducing the inflammatory response, oxidative stress, DNA damage and cell apoptosis.27–29 We theorise that chronically abnormal tHcy impairs the endothelial cell and smooth muscle cell remodelling function, weakening the arterial wall and thereby permitting IA formation.13 Based on these considerations, we hypothesise that tHcy influences the formation of IAs by weakening the arterial wall over time, in a concentration-dependent manner. Further experimental validation in cell and animal models is required to confirm this hypothesis. These findings agree with the higher population-attributable risk of tHcy for stroke in the population of older than 60, which was estimated from he NHANES Epidemiologic Follow-up Study III.26 Based on our findings and that of others, tHcy may be a potential biomarker, and individuals with higher tHcy levels should be assessed for IA risk, including early screening using MRA/CTA of older patients with a presumably prolonged exposure to higher tHcy.

Hyperhomocysteine correlates with hypertension and human and animal studies have shown that attenuation of hypertension can prevent IA rupture,30 but no evidence directly addresses whether tHcy causes IA formation by mediating hypertension. Our MR analyses support a causal relationship between tHcy and IA risk independent of hypertension and any other known confounders such as smoking. Further verified by observational analysis, the OR of high tHcy was 8.05 (95% CI, 4.65 to 13.90) among patients without hypertension; this risk was even higher than in those with hypertension. It needs cautious interpretation for the reason that the effect of tHcy on the risk of IA formation may be masked by hypertension to a certain degree, which is a well-established traditional risk factor for IA.1 In general, the simultaneous presence of hyperhomocysteine and hypertension also produce a positive interaction for IA formation (RERI31=1.65, 95% CI 1.29 to 2.01; AP=0.66, 95% CI 0.58 to 0.74, online supplemental table S4). These findings are clinically relevant, as tHcy screening for hyperhomocysteinemia (tHcy ≥15 µmol/L) is prioritised in patients at risk of CHD and stroke widely for those with hypertension,22 23 whereas our findings suggest that such screening should be broadened. In particular, patients with tHcy >30 µmol/L need to be identified and promptly treated.

The association between tHcy and IA formation is independent of sex. A significant interaction between sex and tHcy was tested, demonstrating that men with high tHcy were associated with greater odds of IA formation than women. For one thing, it is well established that men have a higher mean concentration of tHcy than women throughout all ages.23 For another, men exposed to high tHcy have a higher rate of hypertension and unhealthy lifestyles such as smoking.23 32 33 Therefore, men with high tHcy potentially suffer more from additive adverse effects, contributing to higher risks for IA formation than women.

tHcy is also a treatable risk factor as low-cost interventions are available; these include dietary changes, and supplementation with folate or vitamin B.23 Folate or vitamin B therapy treatment of patients with increased tHcy can effectively reduce the incidence of ischaemic stroke and CHD.34 35 Further MR and clinical evidence is warranted to test whether folate or vitamin B supplementation can decrease the risk of IA occurrence.

There are some limitations to our work. (1) The two-sample MR study used GWAS data from a European population and the real effects of tHcy were explored in a Chinese population. Despite the genetic and non-genetic population differences, the direction of elevated tHcy contributing to IAs risk remains consistent among these two populations. (2) We used a retrospective case–control study to verify tHcy effects on IA formation; this limits the level of evidence but a prospective longitudinal cohort study would be too costly to implement and a randomised control study is not feasible. (3) Balanced baseline characteristics achieved by PSM were limited to observed confounders because analyses do not address unobserved confounders associated with the formation of IAs. (4) Limited by the retrospective nature of the available data, we did not take folate and B vitamin therapy into consideration; this might influence the estimated effect size of tHcy. (5) Although the adjustment for dyslipidaemia and diabetes did not change the results in general, further studies are warranted to investigate whether different classifications of dyslipidaemia or diabetes are associated with the risk of IA formation. (6) Despite the causal role of tHcy on IA formation evaluated by MR, patients with ruptured IA might have acute changes in tHcy, confounding the estimation of ORs in the observational analyses. We are conducting a further study focusing on the association of tHcy with the development (growth/rupture), quantity and size of IA to elaborate on this topic.