Lack of association between HOTTIP rs3807598 C>G and venous malformation risk in Chinese children

Introduction

Venous malformations (VMs), classified as congenital low-flow vascular anomalies, arise from dysregulated vascular development during embryogenesis (1). They represent the most prevalent congenital vascular developmental anomalies, with a prevalence ranging from 1 to 5 per 10,000 births and an overall incidence of 1% (2). These malformations can occur in the superficial skin and mucous membranes of the body or in deeper tissues, and the extent of the lesions can gradually and proportionally increase with age (3). Its pathology is characterized by flattened endothelial cells and scattered smooth muscle cells (3). More than 90% of clinical VMs are sporadic, with a small percentage having a familial predisposition (3). Recent genetic studies have been successful in identifying the underlying etiology of familial cases of VMs and have found that over 50% of sporadic VMs are associated with specific genetic factors (4). VM pathogenesis is usually associated with the phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin (PI3K/AKT/mTOR) and Ras/MEK/ERK pathways, which play a role in most cases (5). Although genetic factors have been identified in most VMs, the pathogenesis of some subtypes has not been clarified. Further in-depth studies are required to elucidate the molecular mechanisms involved in these cases.

HOTTIP functions as a scaffolding long non-coding RNA (lncRNA) that recruits the WDR5/MLL complex to chromatin, specifically regulating the transcriptional activation of 5' HOXA genes including HOXA1, HOXA4, HOXA6, and HOXA7 (6). Crucially, HOX genes exhibit precise spatiotemporal expression patterns that establish positional identity along the body axis during embryogenesis. The upper and lower limbs develop under distinct HOX regulatory programs, with HOXA genes showing differential expression patterns between forelimb and hindlimb buds during critical developmental windows (7). Moreover, HOX genes regulate vascular development and remodeling, participating in endothelial differentiation and angiogenesis, such as HOXA3, HOXA5, HOXA9, HOXD3, and HOXD10 (8). This developmental context-dependency suggests that genetic variants affecting HOX regulatory elements could plausibly exhibit anatomically restricted effects on vascular morphogenesis. Furthermore, the upper extremity vasculature develops under unique hemodynamic conditions and mechanical constraints compared to other body regions, potentially creating a developmental milieu where subtle HOXA gene perturbations become phenotypically manifest (9).

The regulatory roles of lncRNAs in critical signaling pathways, such as the PI3K/AKT pathway implicated in osteosarcoma pathogenesis (10), underscore their importance in diseases of cellular growth and development. Given that VMs often arise from genetic predispositions, lncRNAs are plausible candidates for involvement through epigenetic modulation of gene expression patterns that govern vascular development (11). Within this context, we focused on the lncRNA HOTTIP, and specifically its C>G polymorphism at rs3807598. The rationale for this targeted selection is grounded in compelling prior evidence from oncology: this specific variant has been robustly associated with an increased risk for malignancies like gastric and colorectal cancer (12-14), pathologies where aberrant vascularization is a defining hallmark. This convergence of evidence from both oncology, where the single nucleotide polymorphism (SNP) is linked to vascular dysregulation, and from fundamental vascular biology, where the proposed epigenetic mechanism is operative (11), establishes a strong pathophysiological parallel to the developmental errors underlying VMs.

Although the potential role of the HOTTIP rs3807598 C>G polymorphism in VMs has not been fully investigated, this SNP may have a significant impact on the pathogenesis of VMs, given its known impact on other diseases (15-18). Therefore, we aimed to investigate the association between the HOTTIP rs3807598 C>G SNP and risk of VM in children through a case-control study. We present this article in accordance with the STROBE reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-320/rc).

Methods

Study subjects

This case-control study was conducted at Guangzhou Women and Children’s Medical Center, Guangzhou Medical University from 2018 to 2021. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by ethics board of Guangzhou Women and Children’s Medical Center, Guangzhou Medical University (No. 2018061106) and informed consent was obtained from the parents or legal guardians of all patients. A total of 1,113 patients with a clinical and pathologically confirmed diagnosis of VM were recruited. VM was diagnosed based on characteristic clinical features (blue, soft, compressible lesions), imaging studies [such as magnetic resonance imaging (MRI) or ultrasound] showing typical dilated vascular channels), and pathological confirmation (biopsy demonstrating dilated venous channels with flattened endothelium) (19). Healthy controls were matched to cases for age (±2 years) and sex, and they were recruited during routine physical examinations at the same hospital and were excluded if they had a history of autoimmune disease, cancer, and any vascular abnormalities. Blood samples were collected from both groups.

Genotyping

In this study, we first extracted DNA from the peripheral blood of participants using the TIANamp kit to ensure high quality of the samples. The extracted DNA was diluted to 10 ng/µL and stored at −20 ℃. TaqMan polymerase chain reaction (PCR) premix was used for real-time quantitative polymerase chain reaction (qPCR) detection in 384-well plates (20-22) for genotyping. This method can efficiently and precisely detect polymorphisms. All samples were processed using the same equipment and protocol to ensure comparability. To ensure reliability of the results, we randomly selected 10% of the samples for repeat genotyping.

Statistical analysis

We first analyzed the differences in demographic characteristics and genotype frequency distributions between patients with VM and healthy controls using two-sided χ² tests to ensure the reliability and validity of the study. The study population included 1,113 VM patients and 1,158 healthy controls. Subsequently, a goodness-of-fit χ² test was performed on healthy controls using Hardy-Weinberg equilibrium (HWE) to test whether the selected SNPs deviated from HWE, which is an important step in assessing the genetic balance of genetic markers. To adjust for the effects of potential confounders, such as age and sex, we used multivariate logistic regression analysis to assess the association between the HOTTIP rs3807598 C>G polymorphism and VM susceptibility by calculating the odds ratio (OR) and corresponding 95% confidence intervals (CIs). Based on the regulatory role of HOTTIP on the HOXA gene cluster and the region-specific function of Hox genes in axial pattern formation, we designed a comprehensive anatomical location stratification analysis. All statistical analyses and tests were performed using the statistical analysis system (SAS) statistical package (version 9.4; SAS Institute, Cary, NC, USA), which ensured the accuracy and reproducibility of statistical analyses. In the interpretation of the results, P values <0.05 were considered significant. The sample size was calculated to detect an OR of 1.5 with 80% power and α =0.05, assuming a minor allele frequency of 20%.

Results

Association of gene HOTTIP rs3807598 C>G polymorphism with VM risk

A total of 1,113 patients with VM and 1,158 healthy controls were included in this study, and their demographic characteristics were comprehensively analyzed (Tables S1,S2). The mean age was 68.99 months for patients and 22.22 months for controls, with no significant difference (t-test, P>0.05). Gender distribution showed no significant difference between cases and controls (χ² test, P>0.05).

Genotyping of the HOTTIP rs3807598 C>G polymorphism was successfully completed in most participants. Further validation showed that the genotype frequency distribution of this polymorphism in the healthy control group was in accordance with the HWE principle of genetics (P=0.12), indicating a representative control cohort. Analyses showed no significant difference in the genotype frequency distribution of the HOTTIP rs3807598 C>G polymorphism between the VM and healthy control groups (Table 1). Based on this finding, we hypothesized that the HOTTIP rs3807598 C>G polymorphism is not directly associated with the risk of VM development.

Stratification analysis

We performed a stratified analysis of VM subtypes for different anatomical sites (the head, neck, trunk, upper extremity, lower extremity, and multiple sites) (Table 2). After correcting for age and sex, we found that the HOTTIP rs3807598 C>G polymorphism was associated with an increased risk of upper-extremity VMs (adjusted OR =1.55; 95% CI: 1.002–2.39; P=0.049). Given the proximity of the P value to the conventional threshold, this finding should be interpreted with caution and requires validation in larger, independent cohorts.

Discussion

In this case-control study, we investigated the potential association between HOTTIP rs3807598 C>G polymorphism and susceptibility to VM in children. To the best of our knowledge, this specific genetic variant has not previously been examined in the context of VM. Our study suggests that the HOTTIP rs3807598 C>G polymorphism has a limited effect on the likelihood of VM development. However, additional and more extensive research is necessary to substantiate these findings, including the analysis of TEK mutations and a broader examination of genetic and environmental factors.

HOTTIP, a long intergenic non-coding RNA (lincRNA) transcribed from the 5' end of the HOXA gene cluster, orchestrates the activation of multiple HOXA genes at their 5' ends (6). This lincRNA modulates gene transcription by directly interacting with WDR5 protein, a component of the WDR5/MLL complex (6). HOTTIP binds to WDR5 and redirects the complex to the HOXA locus, where it catalyzes the trimethylation of histone H3 at lysine 4 (H3K4me3) (6). This specific histone modification is a well-established epigenetic signal for transcriptional activation, thereby enhancing the expression of the target HOXA genes. HOTTIP functions underscore the intricate interplay between noncoding RNAs and chromatin-modifying complexes in the regulation of gene expression, with profound implications for developmental processes and cellular differentiation. HOTTIP has been found to play an important role in several diseases, particularly cancer (23). For example, HOTTIP lncRNA promotes hematopoietic stem cell self-renewal and triggers acute myeloid leukemia-like (AML)-like disease in mice (24). Studies have reported that Fn infection-induced exosomal HOTTIP promotes gastric cancer progression through the miR-885-3p/EphB2/PI3K/AKT axis (25). Tang et al. (26) revealed that solamine inhibits hepatocellular carcinoma (HCC) cell growth and enhances the anticancer effect of sorafenib through the HOTTIP-TUG1/miR-4726-5p/MUC1 signal pathway. Similarly, Feng et al. (15) found that lncRNA HOTTIP promotes tumorigenesis carcinoma cells by regulating HOXA13 expression. Recently, it has been shown that HOTTIP regulates cellular autophagy in renal cell carcinoma through the PI3K/AKT/ATG13 signaling pathway, and that HOTTIP silencing decreases the expression of p-PI3K and p-AKT, and increases the expression of ATG13; whereas HOTTIP overexpression shows the opposite trend (27). In addition, HOTTIP has been found to promote inflammation and angiogenesis through multiple mechanisms, in particular through activation of the p38/MAPK signaling pathway (28), and through interactions with WDR5 and MLL, which orchestrate the transcription of intercellular adhesion molecule 1 (ICAM-1) and vascular endothelial growth factor (VEGFR), respectively (29).

In this study, we explored the association between the rs3807598 C>G polymorphism and the risk of VMs in HOTTIP. This study found no significant association between the HOTTIP rs3807598 C>G polymorphism and overall VM risk. However, subgroup analysis of upper limb VM suggested a potential protective effect of the GG genotype (OR =1.55; P=0.049), warranting further investigation. While scientific prudence dictates caution in interpreting any signal that does not withstand rigorous correction for multiple testing, to dismiss this as a mere statistical anomaly may be to overlook a deeper biological narrative. This observation may instead exemplify a foundational principle of complex trait genetics: the action of a given variant is not absolute but is profoundly shaped by a constellation of modifier genes that dictate its ultimate penetrance and expressivity (30). The upper extremity-specific signal may reflect the unique developmental program governing forelimb vascular morphogenesis, where HOXA gene expression patterns differ markedly from those in the hindlimb or axial structures. Our finding could be interpreted not as evidence for rs3807598 being a primary causative factor, but rather as a risk modifier whose subtle influence on angiogenic regulation is only unmasked in the specific developmental milieu of the upper limbs (30).

In the current study, we focused on an SNP in one gene, HOTTIP, which may not account for the full range of genetic variants that may affect VM risk. Although our study adjusted for some potential confounders (e.g., age and sex), it is important to consider potential confounders, such as alcohol consumption, obesity, and hormone use, which may affect the risk of VM. Study limitations include the sample size possibly being insufficient to detect small effects, lack of functional validation (e.g., gene expression analysis), and not accounting for environmental factors or comorbidities. Since participants were all from southern China, caution should be taken in extrapolating results to other ethnicities or regions. Selection bias may exist as controls were hospital-based; however, frequency matching by age/sex and HWE validation in controls mitigated this risk. Residual confounding by unmeasured factors (e.g., maternal exposures) cannot be excluded future studies should include larger cohorts and mechanistic experiments to confirm these findings.

Future studies should collect and adjust these data for their analyses. There are several limitations in this study. First, we focused on a single SNP in HOTTIP, and other polymorphisms or genetic variations within this gene or related genes may also contribute to VM risk. Second, functional studies are needed to understand how the rs3807598 C>G polymorphism affects HOTTIP expression or function and its downstream effects on vascular development. Third, we did not account for potential environmental factors or other genetic confounders that might influence VM risk. Fourth, the study did not perform a power analysis to determine the adequacy of the sample size for detecting small effect sizes. Fifth, our relatively small sample size precludes detailed subtype analysis within each anatomical region.

It is important to acknowledge limitations. This study provides an initial exploration of the relationship between HOTTIP and VMs. Although no significant association was observed in the overall sample, it provides clues for further validation in specific subgroups or larger studies. In conclusion, our study provides initial evidence that the HOTTIP rs3807598 C>G polymorphism is not significantly associated with overall VM risk in children. However, the potential association with upper-extremity VM warrants further investigation. Comprehensive studies encompassing multiple genetic variants, functional analyses, anatomical stratification, and larger cohorts are necessary to fully understand the role of HOTTIP in VM pathogenesis.

Conclusions

Our findings suggest that the HOTTIP rs3807598 C>G polymorphism is not significantly associated with overall VM risk, though further studies are needed to validate its potential role in upper-extremity VMs. Future research should focus on functional mechanisms with larger sample sizes.

Acknowledgments

We thank the Clinical Biological Resource Bank of Guangzhou Women and Children’s Medical Center, Guangzhou Medical University for providing clinical samples.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-320/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-320/dss

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-320/prf

Funding: This study was funded by the grant from the Guangzhou Medical University Research Enhancement Program (No. 02-410-2302148XM).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-320/coif). Z.L. received research funding from Guangzhou Medical University for this study. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by ethics board of Guangzhou Women and Children’s Medical Center, Guangzhou Medical University (No. 2018061106) and informed consent was obtained from the parents or legal guardians of all patients.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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