Hirschsprung disease (HD) is a congenital disorder of enteric nervous system characterized by the absence of enteric ganglia in the distal part of the colon, leading to abnormal contractions (1). Until now, the only efficient treatment for HD is surgery to remove the aganglionic segment of the colon and reconstruct the healthy, innervated intestine to the anus (2). However, approximately one-third of HD patients still experience complications after surgery, including fecal soiling, obstructive symptoms, and Hirschsprung-associated enterocolitis (HAEC) (3). The cause of these complications is not well understood. Various studies have suggested possible mechanisms such as genetic mutation (4), dysfunction of the immune system in the gut (5), and an imbalance of the gut microbiome (6).

Gut microbiome (GM) is a distinct microbial ecosystem within the digestive system that plays a critical role in regulating human health and disease (7). The imbalance and dysfunction of microbial composition in GM, resulting from a metabolic disorder, can lead to several diseases (8). GM is deemed an extra “organ” with more genes than the host and is influenced by genetics and the environment (9). Several studies have shown that GM is associated with a range of illnesses, such as diabetes (10), cardiovascular disorders (11), neurological diseases (12), and immune diseases (13). Genome-wide association studies (GWAS) have challenged the view that the gut microbiota is solely an environmental factor (14). However, the extent of its genetic influence remains a topic of debate.

In order to investigate the potential mediation effect that could bridge the GM to the incidence of HD, metabolites are considered. Metabolites refer to tiny molecules produced either as a result or at the end of various metabolic reactions (15). Multiple factors, such as genetics, lifestyle, disease, and GM, can impact metabolite levels in the body (16, 17). The changes in metabolites can impact the incidence of disease. The high heritability of metabolites makes them convenient to study and potential therapeutic targets for disease (18). Based on these findings, we hypothesize that metabolites mediate the effect of GM on HD. However, it is difficult to establish the causal relationship and avoid confounding bias in an observational study. Therefore, an innovative methodology should be adopted to investigate the causal relationship.

Mendelian randomization (MR) is an effective method that can avoid the confounding bias and evaluate the effect of exposures on outcomes (19). MR analysis assumes that during conception, random assignment of alleles to offspring follows Mendel's law of inheritance, mirroring the concept of a randomized controlled trial (20). Using single nucleotide polymorphisms (SNPs) associated with specific IVs as proxies for exposures, MR analysis can assess the causal effects between exposures and outcomes (21). When the effect between exposures and outcomes is confusing, a mediation MR analysis can help to understand the cause better and identify intermediate variables that could be potential targets for intervention (22).

Our study aimed to investigate the relationship between GM and HD and explore the potential role of metabolites as mediators by conducting a bidirectional two-step MR analysis.

This study explored the causal relationship between GM and HD, and assessed the mediating effect of serum metabolite traits between GM and HD in a comprehensive bidirectional two-step MR analysis. Our study revealed that genetically variants in Ruminococcus2, ErysipelotrichaceaeUCG003, Roseburia, Paraprevotella, Peptococcus, RuminococcaceaeNK4A214group, and Eggerthella were significantly associated with the incidence of HD. The results did not indicate bidirectional causal relationship. Our two-step MR analyses revealed that Roseburia contributed to an increased risk of HD, which was partially mediated by lower levels of X-21733. As the original paper stated that X-21733 is an unknown molecule in serum (15), we have excluded it from further discussion. On the other hand, Peptococcus was found to be associated with a reduced risk of developing HD, partially mediated by levels of stearoyl sphingomyelin (d18:1/18:0) and lysine.

Studies conducted on animals and humans have confirmed a connection between the diversity of GM and HD. The human gastrointestinal tract commonly acts as a natural ecosystem and supplier of nutrients for microbiota. In return, microbiota aids in gut development, strengthen the immunity, and improve the defense mechanisms by generating different metabolites (44). Cheng et al. found decreased fecal microbiota alpha diversity in HD mice after microsurgical pull-through surgery on Ednrb knock-out mice (45). A study on children with HD showed reduced gut microbiota richness after surgery compared to control group (46). HD may require different lengths of surgical therapy depending on the degree of aganglionic involvement, which in turn may affect gut microbiota homeostasis (47). However, studies on the mechanism of GM underlying the development of HD were limited. Through our MR analyses, we found lines linking GM and HD by the mediation of metabolites.

The genus Roseburia belongs to the phylum Firmicutes, class Clostridia, order Clostridiales, and family Lachnospiraceae (48). There are five well-characterized Roseburia species: Roseburia intestinalis, Roseburia hominis, Roseburia inulinivorans, Roseburia faecis, and Roseburia cecicola (49). Roseburia intestinalis is known to protect against several inflammatory diseases by minimizing intestinal inflammatory reaction (48). The expression of colonic mucosal melatonin is positively related to Roseburia hominis (50). Roseburia inulinivorans, a newly discovered motile member of the Firmicutes, helps form butyrate from different dietary polysaccharide substrates present in the human large intestine (51). Roseburia faecis, a Gram-positive anaerobic bacterium that produces butyrate, has been tested for its usefulness in treating irritable bowel syndrome induced by repeated water avoidance stress in rat models (52). To date, no research has been carried out to explore the presence of the genus Roseburia in individuals with HD. Our MR analysis found that Roseburia may be involved in HD as a risk factor due to the mediation effect of serum X-21733. However, the information regarding serum X-21733 is unknown, and further molecular experiments are required to elucidate this mystery.

There has been debate regarding the precise role of Peptococcus in the human gut. Bourgault et al. conducted a study revealing Peptococcus as the most frequently occurring species of anaerobic Gram-positive cocci in significant infections (53). Moreover, it appears to be particularly pathogenic in infections of bones and joints or when present with foreign bodies (53). However, Gu et al. conducted research that demonstrated the protective effect of Peptococcus on inflammatory disorders of the breast (54). According to their study, there is a strong positive correlation between Peptococcus and the presence of valeric acid and butyrate, which regulate the inflammatory response and participate in the energy supply of tight junction proteins (54). Our MR analyses supported the protective effect of Peptococcus on HD, which may be mediated by the levels of stearoyl sphingomyelin (d18:1/18:0) and lysine. In the future, success may come from therapeutics targeting Roseburia or Peptococcus, including probiotics, fecal microbiota transplantation, prebiotics, and synbiotics.

Other bacteria were not associated with the development of HD. Ruminococcus gnavus is a Gram-positive strict anaerobe bacterium that forms chains. The bacteremia has been associated with an acute flare of ulcerative colitis (55). Erysipelotrichaceae has effect on the metabolism of lipid, warranting additional investigation into the metabolic profiles of these organisms. Erysipelotrichaceae may be correlated to inflammation and auto-immunogenic procedure (56).

Metabolomics is an advanced technology with great promise for uncovering the underlying mechanisms linked to various disease processes (57). Yang et al. identified 21 HD biomarkers in serum samples using metabolomic analysis (58). Sphingomyelin (d18:1/18:0) is a sphingolipid commonly found in cell membranes, particularly in the membranous myelin sheath surrounding nerve cell axons (59). Plekhova et al. discovered that lysine catabolism was increased in HD patients. This has been associated with inadequate bacterial butyrate production in the large bowel (60). Our study has found that serum levels of sphingomyelin (d18:1/18:0), lysine, and X-21733 were involved in mediating the GM-HD effect. However, the structure and function of X-21733 is still unknown. Our MR analyses have confirmed that metabolites are involved in the mechanism of HD development. To fully understand the underlying pathologies, further experiments are necessary.

This study has several advantages. Firstly, we used summary data from the GWAS consortium in our MR analyses, which increases statistical power since genetic variation is unaffected by confounding factors. Secondly, we combined bidirectional and two-step MR analyses to explore the potential mediation effects of metabolites, producing more precise and reliable results. This innovative approach provides a comprehensive understanding of the causal relationship between GM and HD.

There are a few limitations to consider in this study. Firstly, even if all three assumptions are fulfilled, it is impossible to avoid weak instrumental bias. However, GWAS data with larger sample sizes can help reduce this bias. Secondly, since the GWAS participants were only of European descent, it may not be appropriate to generalize the findings. Therefore, similar studies need to be conducted across multi-ethnic groups. Thirdly, MR analysis typically reveals exposure over a lifetime, and the presence of canalization may lead to an overestimation of effect size. Therefore, it is recommended that further randomized controlled trials be conducted to examine the effect.

In conclusion, our study indicated a causal relationship between Peptococcus and the incidence of HD that is partially mediated by serum levels of stearoyl sphingomyelin (d18:1/18:0) and lysine. Levels of Peptococcus in feces and stearoyl sphingomyelin (d18:1/18:0) and lysine levels in serum could serve as novel biomarkers and therapeutic targets for HD.