Intracranial aneurysms (IA) are cystic bulges caused by local defects or increased pressure in the intracranial artery wall (1). Unruptured intracranial aneurysms (UIA) occur in 3–5% of the global population and in 7% of Chinese persons aged 35–75 years (2–5). The annual risk of IA rupture is 0.5–1.8% (6, 7). The rupture of an IA can result in aneurysmal subarachnoid hemorrhage (aSAH), which can be fatal in 50% of patients and cause 33% of severe sequelae (8, 9). Costs associated with aSAH supervision exceed £510 million annually in the UK (10). Therefore, risk factors linked to IA should be determined as soon as possible to screen and treat IA more effectively.

In addition to the established acquired risk factors of cigarette smoking and hypertension, genetic factors also play significant roles in IA (3). Among patients with IA, 4% and 8% have first- and second-degree relatives with IA, respectively (11). The predicted probability of IA is higher among individuals with hereditary pathologies such as autosomal dominant polycystic kidney disease (ADPKD) and Marfan syndrome (12). The extracellular matrix (ECM) is an indispensable element for maintaining arterial wall stability. A disrupted balance between ECM synthesis and degradation may be the pathophysiological basis for IA formation (13, 14). For example, some single nucleotide polymorphisms (SNPs) of endothelin receptor type A (EDNRA), alpha 1 type III collagen (COL3A1), transcription factor SOX-17 (SOX17), and lysyl oxidase-like 2 (LOXL2), which are mainly involved in ECM reconstruction, are associated with IA (15–18). The LOXL2 gene belongs to the lysyl oxidase (LOX) family that comprises LOX and lysyl oxidase-like 1–4 (LOXL1, LOXL2, LOXL3, and LOXL4) (19). Lysyl oxidase can initiate covalent crosslinking between soluble collagen and elastin monomers that are converted into insoluble, stable fibers in the ECM, thereby ensuring the structural and functional stability of the arterial wall (20). Dysregulated LOX activity may play significant roles in cardiovascular diseases such as atherosclerosis and aneurysms (21), which are mediated by cross-linked structural ECM proteins. Thus, the LOX family of genes might be associated with IA.

So far, few population studies have explored associations between polymorphisms in LOX family genes and IA, and the results were inconsistent. For instance, associations between LOX gene polymorphisms and IA have not been found in Japanese, Central European, and South Indian populations (22–24). However, a recent population study in Korea found three SNPs of LOX (rs2303656, rs3900446, and rs763497) were significantly associated with IA (maximum OR, 20.15; P = 4.8 × 10^–5) (25). Akagawa et al. (18) examined associations between LOXL1–4 gene polymorphisms and susceptibility to familial intracranial aneurysms (FIA) in Japan and found that rs1010156 of LOXL2 was associated with FIA (OR, 1.49; P = 0.023). Using whole exome sequencing (WES), Wu et al. (26) also discovered that the rare variant of LOXL2, c.133C>T (rs142252012), is related to the FIA susceptibility in a Chinese population. Considering genetic heterogeneity among ethnic populations and that the genetic background of FIA might differ from that of sporadic intracranial aneurysms (SIA), we aimed to determine whether polymorphisms in LOX family genes are associated with susceptibility to IA in a Chinese population.

We aimed to determine associations between polymorphisms of LOX family genes and risk of IA in a Chinese population. We found that the LOX rs10519694 and LOXL2 rs1010156 polymorphism was associated with the risk of single and multiple IA respectively. As far as we can ascertain, this is the first systematic exploration of associations between LOX family genes polymorphisms and the risk of IA in a Chinese population, and the findings offer a new perspective for the prevention and treatment of IA.

The LOX family genes comprise five members which encode LOX and LOX–like isoenzymes (LOXL1, LOXL2, LOXL3, and LOXL4), which are copper-dependent oxidases with a conserved C-terminal region that corresponds to the catalytic domain and a different N-termini (29). Therefore, each isoform has similar amine oxidase activities in which the ϵ-amino group of peptidyl lysine is oxidized to generate peptidyl aldehyde (20, 30, 31). Collagen and elastin are the specific physiological substrates of LOX family oxidases. Cross-linked collagen and elastic fibers are essential for ECM stability and provide most of the tensile strength and structural integrity of connective tissue (32). The abnormal expression of LOX enzymes is related to changes in ECM composition characteristic of atherosclerosis and aortic aneurysms (21).

The LOX located at 5q23.1, which was the first identified subtype and represent of the LOX family genes.LOX catalyzes the translation of peptidyl lysine and oxidizes it to peptidyl aldehyde and α–aminohexyl–δ semialdehyde. The collagen and elastin crosslinking initiated by this chemical reaction assures deposition of these fiber proteins in the ECM (33). Changes in the ECM disrupt the vascular wall, as evidenced in atherosclerosis, a cardiovascular disease that features vigorous destruction of the ECM (34, 35). Some morphological changes caused by atherosclerosis can result in the deposition of fibrous tissues that ultimately develop into IA (13, 36). Thus, we hypothesis that the disrupted LOX expression might be associated with IA that is characterized by ECM destruction. The present study found that LOX rs10519694 was associated with the increased risk of single IA in recessive and additive models and the association was verified in the additional population control. Furthermore, similar to the findings of a case-control study by Hong et al., who associated the A allele of another polymorphism of LOX (rs2303656) with decreased risk of IA (P = 8.2 × 10^-4) in Korean population, we also identified that this variant was associated with the decreased risk of single IA in Chinese, even a definite conclusion couldn’t be drawn considering the fact of that the statistical power was relatively low and the association was not replicated in the additional control. Hong et al. also found that the C allele of LOX rs3900446 and the G allele of rs763497 with increased risk of IA (OR, 20.15; P = 4.8 × 10^-5 and OR, 2.26; P = 4.8 × 10^-5) in Korean population, respectively (25). However, the polymorphisms of LOX gene is not associated with IA in Japanese and Dutch (23), central European (22) and South Indian (24) populations. Lan Ma et al. found that another SNP of LOX (rs1800449) was associated with the susceptibility to coronary artery diseases in Chinese population (37) and a recent mouse model also suggested that the missense mutation (p.G473A) caused by rs1800449 could lead to loss tumor suppressor function of the LOX propeptide and thus accelerate carcinogen-induced tumor formation (38). However, this SNP was not found to be associated with IA in our study. We speculate that these results are associated with the population genetic heterogeneity or limited statistical power of previous studies. These lines of evidence illustrated that the polymorphisms of LOX may perform roles in IA susceptibility in particular populations. The detail functions of the identified variants were not clear, considering the fact of that rs10519694 and rs2303656 are both intronic variants, as a tagSNP, the associations between them and IA might exist different possibilities: the polymorphism has a causal role or the polymorphism has no causal role but is associated with a nearby causal variant. The detailed mechanism of LOX polymorphisms on IA should be further explored.

LOXL1–4 are located at 15q24.1, 8p21.3, 2p13.1, and 10q24.2, respectively. Similar with LOX, LOXL1 also contains an N-terminal propeptide, while the N-terminal of LOXL2-4 consists of four scavenger receptor cysteine-rich (SRCR) domains (29). The biological effects of LOXL1 are similar to those of LOX, and are significant not only for elastogenesis but also for supporting the deposition of elastin (39). For example, a large-scale GWAS study found that LOXL1 variants can increase the deposition of elastin and fibrillin-1 that stabilizes the ECM, thereby protecting against exfoliation syndrome (40). Since LOXL2 affects the pathophysiological processes of cancer, such as cell adhesion and invasion (30) and promotes endothelial tube formation by cross–linking collagen IV in the vascular system, its expression may be related to tumor angiogenesis and progression (21). The glycosylation and proteolytic processing of extracellular LOXL2 are essential for cross–linking basement membrane type IV collagen in the ECM (41, 42). A recent crystal structure study found that a point mutation of LOXL2 (N455Q) could affect the activity of the catalytic domain by eliminating the glycosylation in the fourth SRCR domains of LOXL2, and another mutation (R257G) could prevent LOXL2 from proteolytic cleavage by serine protease, resulting in LOXL2 couldn’t remove the first two SRCR domains and subsequently can’t bind to type IV collagen, finally disturb the structural and functional stability of ECM (29, 41). LOXL3 is expressed abundantly in the retina and central nervous system; abnormal LOXL3 expression is associated with cleft palate and spinal deformities (43). These anomalies may be associated with the inability of LOXL3 to effectively crosslink collagen (30). The LOXL4 is the latest discovery in the LOX family and limited information is available regarding this isoform. However, LOXL4 plays specific roles in the proliferation and metastasis of cells in certain malignancies, such as gastric and liver cancer (44, 45). Considering the role of LOXL4 in tumorigenesis, it may serve as a potential independent prognostic marker and therapeutic target for these cancers. Further detailed investigations are necessary to fully understand the biological functions of LOX family gene members.

We found that one SNP of LOXL2 rs1010156 associated with the risk of multiple IA after adjusting for potential confounders (OR, 1.92; 95% CI, 1.02–3.62, P = 0.044). Even the result was not verified in the additional control, we thought that the previous analysis was more reasonable due to that we just performed univariate logistic regression analysis in the additional controls and potential confounders were not adjusted due to these informations was not available. Our findings were similar to the results obtained by Akagawa et al. (18) who systematically screened LOXL family genes and reported that the LOXL2 polymorphism was associated with susceptibility to FIA. However, a comparison of SIA and FIA between patients and controls did not reveal any associations. In addition, using WES, Wu et al. found that a rare variant of LOXL2 c.133C>T (rs142252012) can increase susceptibility to FIA (26), however, we could not verify the association between this variant and susceptibility to IA in Chinese patients with SIA. We found an association between LOXL2 and multiple IA but not single IA, which may be attributed to the involvement of different pathophysiological pathways in the formation of multiple and single IA. Unlike single IA, multiple IA were not distributed randomly in the Circle of Willis, but were rather arranged in clusters near the index aneurysm, which may predict the formation of mirror aneurysms. Such aneurysmal clusters may be affected by genetic and hemodynamic variables (46). Multiple IA that develop more frequently in patients with familial IA can be larger and rupture at a higher frequency in younger individuals (14), which further suggests a genetic predisposition. This study did not find an association between LOXL1, LOXL3, and LOXL4 polymorphisms and susceptibility to IA, which was consistent with the results of Akagawa et al., who found no associations between LOXL1, LOXL3, and LOXL4 gene polymorphisms and IA susceptibility in a Japanese population (18). Considering the limited power of the statistics, larger sample size studies are needed in the future.

This study has several limitations. First, the controls comprised individuals who had not been diagnosed with IA or other cerebrovascular diseases. As some participants were assessed by imaging modalities, classification might have been biased. However, this influence was probably small considering that few people had IA in China. Second, we explored associations between polymorphisms in LOX family genes and risk of single and multiple IAs. Due to the sample size was relatively small and the statistical power was limited, the results may be not stable, further studies with larger sample size are needed to verify our findings. Third, considering the fact that the identified associated variants were tagSNP of target genes, we didn’t know whether these variants were the real causal ones or just the surrogate of nearby causal variants, the potential functions and the detail pathological mechanisms of polymorphisms of LOX family genes on IA are needed to further explored. Furthermore, this study has been performed in a central south Chinese population and the results may can’t directly extrapolate to other ethnicities considering the fact that there may be population genetic heterogeneity. Our results need to be validated in further studies. Nevertheless, this study is the first to reveal associations between polymorphisms of the LOX family genes and susceptibility to IA in the Chinese population and offers a new perspective for the early diagnosis of IA.

Our findings showed that LOX and LOXL2 polymorphisms were associated with risk of single IA and multiple IA in a Chinese population, suggesting potential roles of these genes in IA. Further investigations are imperative to elucidate the effects of these genes on IA.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by The Ethics Committee at Central South University (Permit No: CTXY–150002–1). The patients/participants provided their written informed consent to participate in this study.

Conceptualization: CL, CH, and JY. Data curation: CL, BL, and JL. Formal analysis: CL and BL. Funding acquisition: JY. Investigation: CL, CH, BL, JL, LH, RD, XL, JZ, LX, SL, YL, DY, and WJ. Methodology: CH, CL, and JY. Project administration: CL, BL, and JY. Resources: CH, JL, WJ, and JY. Supervision: CL, CH, and JY. Writing—original draft preparation: CL. Writing—review and editing: JY. All authors contributed to the article and approved the submitted version.

This work was supported by the National Nature Science Foundation, China [grant numbers: 81502881], and the Graduate Student Innovative Scientific Research Project of Central South University, China [grant numbers:1053320192459].

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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