Causal associations between human gut microbiota and hemorrhoidal disease: A two-sample Mendelian randomization study.

Hemorrhoidal disease (HEM) is a common condition affecting a significant proportion of the population. However, the causal relationship between the gut microbiota and hemorrhoids remains unclear. In this study, we employed a Mendelian randomization (MR) approach to investigate the potential associations between them. In this study, the exposure factor was determined by selecting summary statistics data from a large-scale gut microbiome whole-genome association study conducted by the MiBioGen Consortium, which involved a sample size of 18,340 individuals. The disease outcome data consisted of 218,920 cases of HEM and 725,213 controls of European ancestry obtained from the European Bioinformatics Institute dataset. Two-sample MR analyses were performed to assess the causalities between gut microbiota and hemorrhoids using various methods, including inverse-variance weighting, MR-Egger regression, MR Pleiotropy Residual Sum and Outlier (MR-PRESSO), simple mode, and weighted median. Reverse MR analyses were performed to examine reverse causal association. Our findings suggest phylum Cyanobacteria (OR = 0.947, 95% CI: 0.915-0.980, P = 2.10 × 10 - 3), genus Phascolarctobacterium (OR = 0.960, 95% CI: 0.924-0.997, P = .034) and family FamilyXI (OR = 0.974, 95% CI: 0.952-0.997, P = .027) have potentially protective causal effects on the risk of HEM, while genus Ruminococcaceae_UCG_002 (OR = 1.036, 95% CI: 1.001-1.071, P = .042), family Peptostreptococcaceae (OR = 1.042, 95% CI: 1.004-1.082, P = .029), genus Oscillospira (OR = 1.048, 95% CI: 1.005-1.091, P = .026), family Alcaligenaceae (OR = 1.048, 95% CI: 1.005-1.091, P = .036) and order Burkholderiales (OR = 1.074, 95% CI: 1.020-1.130, P = 6.50 × 10-3) have opposite effect. However, there was a reverse causal relationship between HEM and genus Oscillospira (OR = 1.140, 95% CI: 1.002-1.295, P = .046) This is the first MR study to explore the causalities between specific gut microbiota taxa and hemorrhoidal disease, which may offer valuable insights for future clinical interventions for hemorrhoidal disease.

CALM3 affects the prognosis of leukemia and hemorrhoids.

Leukemia is an abnormal proliferation of white blood cells in the bone marrow, resulting in a large accumulation of abnormal leukemia cells in the blood and bone marrow. Hemorrhoids are dilated and swollen veins in the rectum or anal area. However, the relationship between CALM3 and leukemia and hemorrhoids remains unclear. The hemorrhoids dataset GSE154650 and leukemia dataset GSE26294 were downloaded from GEO databases generated by GPL20301 and GPL571.The R package limma was used to screen differentially expressed genes (DEDs). Weighted gene co-expression network analysis (WGCNA) was performed. The construction and analysis of protein-protein interaction (PPI) network, functional enrichment analysis, Gene Set Enrichment Analysis (GSEA) and comparative toxicogenomics database (CTD) analysis were performed. TargetScan was used to screen miRNAs regulating central DEGs. It was verified by western blot basic cell assay. A total of 125 DEGs were co-identified. According to the GO analysis, they are mainly enriched in small molecule catabolic processes, skin development, and chemokine receptor binding. The KEGG analysis results show that the target cells are mainly enriched in the interaction of cytokines and cytokine receptors, as well as butyric acid metabolism. The GSEA analysis results indicate enrichment in small molecule catabolic processes, skin development, and chemokine receptor binding. Six core genes (CALM3, ACE2, PPARGC1A, XCR1, CFTR, PRKCA) were identified. We found that the core gene CALM3 is highly expressed in hemorrhoid samples, low in leukemia samples, and has low expression in normal samples, which may play a regulatory role in hemorrhoids and leukemia. Immunoinfiltration results showed a higher proportion of T_cells_CD4_memory_resting and a correlation with T_cells_CD8. WB experiment verified the result. CALM3 expression is low in leukemia, and the lower the expression is, the worse the prognosis is. CALM3 is highly expressed in hemorrhoids, and the higher the expression, the worse the prognosis.

Identification of the microRNA alterations in extracellular vesicles derived from human haemorrhoids.

NEW FINDINGS: What is the central question of this study? What are the morphological features and microRNA (miRNA) expression features of extracellular vesicles (EVs) from haemorrhoids (Hae-EVs) and normal tissues? What are the potential functions of the differentially expressed (DE) miRNAs in Hae-EVs? What is the main finding and its importance? We present, for the first time, the morphological features and miRNA profile of human Hae-EVs. Four hundred and forty-seven significant DE-miRNAs were identified. Gene ontology and pathway analysis of the DE-miRNAs indicated diverse roles of the Hae-EVs through different pathways. Our findings provide EV-based pathological features and the underlying mechanism of haemorrhoids. ABSTRACT: Extracellular vesicles (EVs) play important roles in many pathophysiologies as cell-to-cell communication vehicles. However, the features and potential functions of the EVs in haemorrhoids remain unclear. Therefore, we performed microRNA (miRNA) microarray analysis in EVs derived from haemorrhoid tissue to identify the profile of miRNAs in these EVs and predict their potential functions. We obtained typical EVs from both haemorrhoid and control tissues. Microarray analysis identified 447 miRNAs with significant differential expresssion (DE): 245 upregulated and 202 downregulated. The top three upregulated miRNAs in haemorrhoid EVs (Hae-EVs), namely miR-6741-3p, miR-6834-3p and miR-4254, were detected by RT-qPCR in both Hae-EVs and haemorrhoid tissues. Interestingly, we found a different expression pattern in the haemorrhoid tissues from that in Hae-EVs. The potential target genes of these DE-miRNAs were predicted by the miRWalk and miRDB databases. Gene ontology (GO) analysis of the target genes showed that the DE-miRNAs contributed mainly to protein kinase activity, transcriptional activity and ubiquitin-protein function. KEGG search found that the DE-miRNAs might regulate the MAPK and Ras signalling pathways. These findings revealed, for the first time, the miRNA profiles in Hae-EVs and provided potential targets and pathways involved in the pathological process.

miR-412-5p targets Xpo1 to regulate angiogenesis in hemorrhoid tissue.

Hemorrhoid is a common and recurrent proctological disease, which is often accompanied by angiogenesis and edema. MicroRNAs in the DLK1-DIO3 imprinted clusters are involved in the development and pathogenesis of mammalian hemorrhoids. Results of the present study indicated multiple, differential expression of DLK1-DIO3 imprinted cluster microRNA between hemorrhoid and normal tissues, where miR-412-5p expression in hemorrhoid tissue was significantly decreased. Fluorescein reporter assays showed that miR-412-5p silenced Xpo1 mRNA expression by targeting its 3'-UTR. Overexpression of miR-412-5p in human umbilical vein endothelial cells (HUVECs) indicated that proliferation, migration and formation of vascular structures in HUVECs were inhibited in vitro. In addition, overexpression of miR-412-5p significantly inhibited Xpo1 expression and promoted upregulation of the p53 protein and its retention in the nucleus. Simultaneously, expression of p66SHC and p16 proteins was activated. In summary, downregulation of endogenous miR-412-5p expression in hemorrhoid vascular endothelial cells leads to high expression of the target gene Xpo1 and translocation of the p53 protein out of the nucleus, rendering it unable to activate p66SHC and p16. This ultimately weakens regulation of the vascular endothelial cell cycle, thereby accelerating the division of hemorrhoid vascular endothelial cells, leading to angiogenesis.

Effect of Genetic Factors on the Etiopathogenesis of Thrombosed Hemorrhoidal Disease.

OBJECTIVE: The aim of this study is to investigate whether genetic factors known to increase thrombosis risk play a role in the etiopathogenesis of thrombosed hemorrhoidal disease. Methods: Genomic DNA from patients with thrombosed hemorrhoidal disease was analyzed for the presence of factor V Leiden, prothrombin G20210A, methylenetetrahydrofolate reductase C677T, and methylenetetrahydrofolate reductase A1298C mutations. Results: No significant differences were found in the allele frequencies of factor V Leiden, prothrombin G20210A, methylenetetrahydrofolate reductase C677T, and methylenetetrahydrofolate reductase A1298C mutations between patients with thrombosed hemorrhoidal disease and controls (p 0.05). Moreover, there were no significant differences in the genotype (heterozygous and homozygous mutations) of factor V Leiden, prothrombin G20210A, methylenetetrahydrofolate reductase C677T and A1298C mutations between patients with thrombosed hemorrhoidal disease and controls (p 0.05). Conclusions: Our findings indicate that mutations associated with venous thromboembolism do not play a role in the etiopathogenesis of thrombosed hemorrhoidal disease; however, several environmental, mechanical, and hemodynamic factors may contribute to the etiopathogenesis of hemorrhoidal disease.

Exhaustive Genome-Wide Search for SNP-SNP Interactions Across 10 Human Diseases.

The identification of statistical SNP-SNP interactions may help explain the genetic etiology of many human diseases, but exhaustive genome-wide searches for these interactions have been difficult, due to a lack of power in most datasets. We aimed to use data from the Resource for Genetic Epidemiology Research on Adult Health and Aging (GERA) study to search for SNP-SNP interactions associated with 10 common diseases. FastEpistasis and BOOST were used to evaluate all pairwise interactions among approximately N = 300,000 single nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) ≥ 0.15, for the dichotomous outcomes of allergic rhinitis, asthma, cardiac disease, depression, dermatophytosis, type 2 diabetes, dyslipidemia, hemorrhoids, hypertensive disease, and osteoarthritis. A total of N = 45,171 subjects were included after quality control steps were applied. These data were divided into discovery and replication subsets; the discovery subset had > 80% power, under selected models, to detect genome-wide significant interactions (P < 10(-12)). Interactions were also evaluated for enrichment in particular SNP features, including functionality, prior disease relevancy, and marginal effects. No interaction in any disease was significant in both the discovery and replication subsets. Enrichment analysis suggested that, for some outcomes, interactions involving SNPs with marginal effects were more likely to be nominally replicated, compared to interactions without marginal effects. If SNP-SNP interactions play a role in the etiology of the studied conditions, they likely have weak effect sizes, involve lower-frequency variants, and/or involve complex models of interaction that are not captured well by the methods that were utilized.

Biological findings from the PheWAS catalog: focus on connective tissue-related disorders (pelvic floor dysfunction, abdominal hernia, varicose veins and hemorrhoids).

Pelvic floor dysfunction, specifically genital prolapse (GP) and stress urinary inconsistency (SUI) presumably co-occur with other connective tissue disorders such as hernia, hemorrhoids, and varicose veins. Observations on non-random coexistence of these disorders have never been summarized in a meta-analysis. The performed meta-analysis demonstrated that varicose veins and hernia are associated with GP. Disease connections on the molecular level may be partially based on shared genetic susceptibility. A unique opportunity to estimate shared genetic susceptibility to disorders is provided by a PheWAS (phenome-wide association study) designed to utilize GWAS data concurrently to many phenotypes. We searched the PheWAS Catalog, which includes the results of the PheWAS study with P value < 0.05, for genes associated with GP, SUI, abdominal hernia, varicose veins and hemorrhoids. We found pronounced signals for the associations of the SLC2A9 gene with SUI (P = 6.0e-05) and the MYH9 gene with varicose veins of lower extremity (P = 0.0001) and hemorrhoids (P = 0.0007). The comparison of the PheWAS Catalog and the NHGRI Catalog data revealed enrichment of genes associated with bone mineral density in GP and with activated partial thromboplastin time in varicose veins of lower extremity. In cross-phenotype associations, genes responsible for peripheral nerve functions seem to predominate. This study not only established novel biologically plausible associations that may warrant further studies but also exemplified an effective use of the PheWAS Catalog data.

MicroRNA let-7b-regulated epidermal stem cell proliferation in hypertrophied anal papillae.

The present study investigated the role of epidermal stem cell-expressed microRNA let-7b in the pathogenesis of hypertrophied anal papillae. Hypertrophied anal papillae were examined for the presence of epidermal stem cells. Epidermal stem cells were identified using flow cytometry and immunofluorescent staining for the cell surface markers, integrin α6 and integrin ß1 subunits. Expression levels of microRNA let­7b in α6+/ß1+and α6­/ß1­cells were compared using reverse transcription­quantitative polymerase chain reaction and northern blotting. Lentivirus­mediated expression of microRNA let­7b in epidermal stem cells was utilized in order to study the effects of this microRNA on the cell cycle proteins, cyclin D1 (CCND1) and cyclin­dependent kinase 4 (CDK4). MicroRNA let­7b­overexpressing cells were examined using flow cytometry, in order to determine the effects of the microRNA on cell cycle progression. α6+/ß1+epidermal stem cells were identified in hypertrophic anal papillae. Following isolation and enrichment of the α6+/ß1+population, these cells were found to have a rapid rate of proliferation in vitro. The expression of cell cycle­related proteins was elevated in this population, compared with that in α6­/ß1­cells. The expression of microRNA let­7b in α6+/ß1+epidermal stem cells was significantly lower than that in α6­/ß1­cells. Two microRNA let­7b target genes, CCND1 and CDK4, were found to be upregulated in α6+/ß1+cells. When the exogenous precursor, microRNA let­7, was overexpressed in α6+/ß1+ epidermal stem cells, the cell proliferation rate was significantly lower than that in cells expressing microRNA let­7 containing a mutated seed sequence. The addition of exogenous microRNA let­7 resulted in an increased expression level of mature microRNA let­7b, while the expression of CCND1 and CDK4 was reduced. Epidermal stem cells transfected with microRNA let­7b were arrested in the G2/M phase and the percentage of cells in S­phase was significantly reduced. In conclusion, let­7b expression results in upregulation of the cell cycle-related proteins, CCND1 and CDK4, resulting in the excessive proliferation that leads to the formation of hypertrophic anal papillae.

Expression pattern, ethanol-metabolizing activities, and cellular localization of alcohol and aldehyde dehydrogenases in human large bowel: association of the functional polymorphisms of ADH and ALDH genes with hemorrhoids and colorectal cancer.

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are principal enzymes responsible for metabolism of ethanol. Functional polymorphisms of ADH1B, ADH1C, and ALDH2 genes occur among racial populations. The goal of this study was to systematically determine the functional expressions and cellular localization of ADHs and ALDHs in human rectal mucosa, the lesions of adenocarcinoma and hemorrhoid, and the genetic association of allelic variations of ADH and ALDH with large bowel disorders. Twenty-one surgical specimens of rectal adenocarcinoma and the adjacent normal mucosa, including 16 paired tissues of rectal tumor, normal mucosae of rectum and sigmoid colon from the same individuals, and 18 surgical mixed hemorrhoid specimens and leukocyte DNA samples from 103 colorectal cancer patients, 67 hemorrhoid patients, and 545 control subjects recruited in previous study, were investigated. The isozyme/allozyme expression patterns of ADH and ALDH were identified by isoelectric focusing and the activities were assayed spectrophotometrically. The protein contents of ADH/ALDH isozymes were determined by immunoblotting using the corresponding purified class-specific antibodies; the cellular activity and protein localizations were detected by immunohistochemistry and histochemistry, respectively. Genotypes of ADH1B, ADH1C, and ALDH2 were determined by polymerase chain reaction-restriction fragment length polymorphisms. At 33mM ethanol, pH 7.5, the activity of ADH1C*1/1 phenotypes exhibited 87% higher than that of the ADH1C*1/*2 phenotypes in normal rectal mucosa. The activity of ALDH2-active phenotypes of rectal mucosa was 33% greater than ALDH2-inactive phenotypes at 200µM acetaldehyde. The protein contents in normal rectal mucosa were in the following order: ADH1>ALDH2>ADH3≈ALDH1A1, whereas those of ADH2, ADH4, and ALDH3A1 were fairly low. Both activity and content of ADH1 were significantly decreased in rectal tumors, whereas the ALDH activity remained unchanged. The ADH activity was also significantly reduced in hemorrhoids. ADH4 and ALDH3A1 were uniquely expressed in the squamous epithelium of anus at anorectal junctions. The allele frequencies of ADH1C*1 and ALDH2*2 were significantly higher in colorectal cancer and that of ALDH2*2 also significantly greater in hemorrhoids. In conclusion, ADH and ALDH isozymes are differentially expressed in mucosal cells of rectum and anus. The results suggest that acetaldehyde, an immediate metabolite of ethanol, may play an etiological role in pathogenesis of large bowel diseases.

Linkage to the FOXC2 region of chromosome 16 for varicose veins in otherwise healthy, unselected sibling pairs.

BACKGROUND: The FOXC2 gene on 16q24 is mutated in lymphoedema distichiasis (LD), in which varicose veins (VV) are a common feature. We hypothesised that this gene might be implicated in the development of VV in the normal population, therefore, after performing a classical twin study, we tested for linkage and association in white women. We also tested for linkage with haemorrhoids (H), as a separate venous anomaly at the same locus. METHODS: A total of 2060 complete female twin pairs aged 18-80 years from the St Thomas' Adult UK Twin registry replied to questions on VV and H as part of a broader postal survey of 6600 twins (62% response rate). Dizygotic female twin pairs were tested for linkage and association to the candidate marker D16S520 (1903 individuals genotyped), which is located about 80 kb from FOXC2. RESULTS: Casewise concordance rates were significantly higher for monozygotic than dizygotic twins for both phenotypes (VV 67% v 45%; p = 2.2x10(-6); H 68% v 59%; p = 0.01; H including during pregnancy 73% v 64%; p = 2.1x10(-4)), corresponding to additive genetic heritabilities in liability of 86% (95% confidence interval (CI) 73% to 99%) for VV and 56-61% for H (95% CI 43% to 73%). The presence of VV and H were significantly correlated. We found significant evidence of linkage to the marker for VV (MLS(ASP) = 1.37, p = 0.01; GLM(ASP/DSP) Z = 3.17 p = 0.002), but no association. Both linkage and association tests were negative for H. The combined phenotype of having VV and H did not show any evidence of linkage or association. CONCLUSION: These results demonstrate VV and H to be heritable, related conditions, and the data strongly suggest FOXC2 to be implicated in the development of VV in the general population.

Anal human papillomavirus infection: a comparative study of cytology, colposcopy and DNA hybridisation as methods of detection.

OBJECTIVE: To compare anal cytology, colposcopy and DNA hybridisation as methods of detecting anal HPV infection. SUBJECTS AND DESIGN: Patients attending: (1) a genitourinary medicine (GUM) clinic with ano-genital warts; (2) a surgical out-patient department with anal fissure or haemorrhoids were examined for evidence of anal HPV infection. RESULTS: Considering GUM clinic attenders, 17% (38/225) and 40% (90/225) had perianal or anal canal warts respectively. Colposcopic examination revealed anal acetowhite lesions without warts in 28% (63/225). Cytological evidence of HPV infection was found in 98%, 83%, and 90% of patients with anal canal warts, perianal warts and acetowhite lesions respectively. Anal intraepithelial neoplasia (AIN) was documented in 22% of patients with anal canal warts compared with 6% with perianal warts (p less than 0.01). HPV DNA was detected from the anal brushings of 71%, 50%, 32%, and 29% of patients with anal canal warts, perianal warts, acetowhite lesions and a normal anal examination respectively. HPV type 6/11 was detected in the majority of HPV positive samples. Considering surgical out-patient attenders with no history or signs of anal warts, 25% showed cytological evidence of anal HPV infection and HPV DNA was detected from anal brushings in 3% (2/71). CONCLUSION: Anal examination with the colposcope is a useful method for detecting subclinical HPV infection. Anal cytology may prove helpful for detecting AIN, however, since koilocytosis was rarely seen, the specificity of the cytological criteria for anal HPV infection in the absence of AIN is uncertain. DNA analysis of anal brushings proved only moderately sensitive.