Neurotrophin-3 rs1805149A>G variant in Hirschsprung disease: An investigative study

Abstract

BACKGROUND

Hirschsprung disease (HSCR) is a congenital disorder of the enteric nervous system (ENS) caused by defective migration of neural crest cells. Genetic factors, including neurotrophic genes such as neurotrophin-3 (NTF3), may contribute to its pathogenesis.

AIM

To investigate the association between the NTF3 rs1805149A>G variant and susceptibility to HSCR in a southern Chinese Han population. In addition, this study also aims to provide population-specific genetic data on HSCR and to explore whether this neurotrophin-related variant contributes to disease pathogenesis, potentially broadening the spectrum of candidate genes implicated in ENS development.

METHODS

A study was conducted involving 1470 HSCR patients and 1473 healthy controls. Genomic DNA was extracted and genotyping of the NTF3 rs1805149 variant was performed using a TaqMan real-time PCR system. Genotype and allele frequencies were analyzed using χ² tests.

RESULTS

The distribution of genotypes (AA, AG, GG) and allelic frequencies (A and G) showed no statistically significant differences between HSCR patients and controls. No association was found between the rs1805149 variant and specific clinical subtypes of HSCR.

CONCLUSION

The NTF3 rs1805149A>G variant does not appear to be associated with HSCR susceptibility in the studied southern Chinese cohort. Further studies with larger sample sizes and multi-gene analysis are warranted to better understand the genetic basis of HSCR.

Key Words: Hirschsprung disease; Neurotrophin-3; Single nucleotide polymorphism; Enteric nervous system; Genetic susceptibility

Core Tip: This case-control study explores the association between the neurotrophin-3 (NTF3) rs1805149A>G variant and Hirschsprung disease (HSCR) susceptibility in a southern Chinese Han population. Despite previous reports implicating NTF3 in HSCR pathogenesis, no significant association was found in this cohort. The results highlight the complexity of HSCR's genetic architecture and suggest that other factors, such as rare variants or multi-gene interactions, may play a more substantial role in its development. Further studies with larger sample sizes and diverse populations are needed to clarify the genetic basis of HSCR.

INTRODUCTION

Hirschsprung disease (HSCR) is a kind of intestinal dysfunction caused by the loss of ganglion cells in the distal bowels, which is characterized by continuous expansion and hypertrophy of the colorectal[1,2]. Globally, the incidence of HSCR is approximately 1.43:10000 with a 4:1 predominance in males[3]. The pathogenesis of HSCR is closely related to genetic abnormalities. Among HSCR patients, more than one-fifth of patients have a family history of the disease[4]. About 12% of patients present syndromes related to congenital abnormalities such as trisomy 21, Mowat-Wilson, Goldberg-Shprintzen, Shah-Waardenburg, and congenital central hypoventilation syndrome[5]. Genetic studies have identified key genes, including RET[6,7], EDNRB[8], EDN3[9], NRG1[10], PHOX2B[11], SOX10[12] that regulate neural crest cell migration, differentiation, and survival during enteric nervous system (ENS) development, with mutations contributing to HSCR’s polygenic etiology[2,7-13]. Notably, neurotrophin-3 (NTF3) and its receptor NTRK3 are critical for ENS neuron survival and differentiation, with specific variants like G76R and R645C implicated in HSCR pathogenesis[13,14].

NTF3 is a member of the neurotrophic factor family and plays an important role in the early differentiation and migration of neural crest cells[15]. NTF3 can bind to its tyrosine kinase receptors (TrkC) and activate PI3K/AKT and RAS/ERK cell signaling pathways, thereby regulating the growth and differentiation of neural cells[16,17]. During early embryonic development, enteric mesenchymal cells secrete NTF3 to act on TrkC-expressing neural crest-derived cells, facilitating their proper migration and integration into the developing ENS[18]. Moreover, immunohistochemical studies have shown the absence of NTF3 and its receptors in aganglionic intestinal segments from HSCR patients[19]. In another investigation, sequencing of two HSCR patients revealed missense mutations in NTF3[13]. Although limited by small sample size, these studies suggest that loss of NTF3 signaling may impair enteric neuronal survival, supporting its functional involvement in ENS development. NTF3 is also expressed in the ENS, where it supports the survival and differentiation of enteric neurons and glia, which are essential for intestinal motility and function[20,21]. Secreted by enteric mesenchyme, NTF3 acts on TrkC-expressing cells to promote ENS development[18,20]. Its absence in aganglionic segments of HSCR and reduced expression in experimental colitis suggest a role in ENS-related disorders and gut inflammation[22]. These findings highlight NTF3’s importance in maintaining intestinal function, and the association of NTF3 and HSCR still needs more in-depth research. These findings suggest that exploring NTF3 polymorphisms, such as rs1805149, could further elucidate the genetic basis of HSCR.

The missense variant rs1805149 (p.Gly76Arg) in NTF3 was selected for this study due to its potential functional impact on the neurotrophin protein and its prior implication in neurodevelopmental disorders[23]. This variant has been detected in several HSCR patients, suggesting a potential association with HSCR[13,24]. According to the gnomAD database, the frequency of this single nucleotide polymorphism (SNP) in East Asian populations is 0.173, compared with approximately 0.097 in European and 0.112 in African populations. This interethnic variation suggests that population-specific genetic backgrounds may influence the observed allele distribution. However, the association between rs1805149 and the risk of HSCR is still unclear, so we investigated its potential role as a risk factor for HSCR within the complex, multigenic architecture of this disease, aiming to enhance the understanding of HSCR from genetic and etiological perspectives and to contribute to broader efforts in multi-gene risk model development.

MATERIALS AND METHODS

Study subjects

This study was conducted at a single center (Guangzhou Women and Children's Medical Center). We consecutively recruited 1470 HSCR cases between October 24, 2017 and October 24, 2023. The diagnosis of HSCR was established based on clinical manifestations and histological examination after surgery or biopsy. The cases were divided into three subgroups according to the length of the affected bowel, as this classification reflects both the extent of aganglionosis and disease severity. Short-segment HSCR (S-HSCR) refers to aganglionosis confined to the rectosigmoid colon, long-segment HSCR (L-HSCR) involves aganglionosis extending proximal to the sigmoid colon, and total colonic aganglionosis (TCA) affects the entire colon[2,5]. This classification is clinically and biologically relevant, as the length of the aganglionic segment is associated with differences in embryonic neural crest cell migration, genetic background, and surgical prognosis. We simultaneously recruited 1473 healthy controls from community health screening programs in the same regions. Controls were selected based on the absence of any personal or family history of HSCR, other neurological disorders, or chronic gastrointestinal diseases. This sample size was determined based on a post-hoc power analysis, which indicated that this sample size provides > 80% power to detect an odds ratio (OR) of 1.5 at α = 0.05 for a variant with a minor allele frequency ≥ 0.17 in our population. Although initial recruitment aimed for age and sex matching, final demographic comparisons revealed considerable differences between cases and controls; therefore, all subsequent statistical analyses rigorously adjusted for both age and sex as covariates to control for potential confounding. Participants included in the study were all Han people from southern region of China. All procedures in studies involving human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study received ethical approval by the Research Ethics Committee of Guangzhou Women and Children Medical Center (Approval No. 2017102706).

SNP genotyping

We selected potential functional SNPs based on the candidate gene information and genome-wide association study using the SNPinfo Web Server (http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm). We analyzed linkage disequilibrium (LD) and functional characteristics of both coding and non-coding SNPs, allowing for an LD range of r² > 0.8 and using the Han Chinese in Beijing population for our analysis. Among the results, rs1805149A>G in the NTF3 gene emerged as the only coding SNP with a predicted regulatory function (regulome score of 2b), and no high-LD SNPs were found nearby, indicating its potential functional significance. To further study this SNP, we extracted whole genome DNA from the peripheral blood of participants using the TIANamp Blood DNA Kit (TianGen Biotech Co. Ltd., Beijing, China). We then performed SNP detection for rs1805149A>G using a TaqMan real-time PCR system following standard protocols. To ensure data reliability, we re-genotyped 10% of the samples, achieving a 100% concordance rate, which confirms the accuracy of our genotyping results.

Statistical analysis

We used Pearson’s χ² test for consistency to assess the Hardy-Weinberg Equilibrium (HWE) test on NTF3 rs1805149A>G in the healthy controls. Pearson’s χ² test for independence was used to test the differences in genotype and allele frequency distributions between HSCR patients and healthy controls. Yates’s correction was not applied as all expected cell counts were greater than 5. A multivariate logistic regression analysis was performed to assess the association under additive, dominant, recessive, and genotypic genetic models. The genotype coding for each model was as follows: (1) Additive model: The number of effect alleles (A) was coded as 0, 1, or 2; (2) Dominant model: Genotypes AA and AG were combined and coded as 1, vs GG coded as 0; (3) Recessive model: Genotype AA was coded as 1, vs AG and GG combined coded as 0; and (4) Genotypic model: Two dummy variables were created to represent the three genotypes. No variable selection procedure (e.g., stepwise) was used. All models were adjusted for the covariates age and sex, which were included a priori based on their known clinical relevance. OR and 95% confidence interval (95%CI) were used to quantify such association. The assumptions of the multivariate logistic regression models were rigorously checked. The linearity of continuous predictors (age) with the logit of the outcome was assessed using the Box-Tidwell procedure, which confirmed no significant nonlinearity. Multicollinearity among explanatory variables was evaluated by calculating the variance inflation factor (VIF); all VIF values were below 2, indicating the absence of substantial multicollinearity. The presence of influential outliers was examined using Cook’s distance, and no observations with a Cook’s distance greater than 1 were identified, suggesting no single point exerted undue influence on the model estimates. To account for multiple comparisons, we applied Bonferroni correction where appropriate. The difference was considered to be statistically significant when two-side P < 0.05. We used SPSS V17.0 for statistical analysis.

RESULTS

Demographics and characteristics of the included participants

The detailed demographics of the included HSCR patients and healthy controls were shown in Table 1, which was also described in our previous study[25]. Notably, there were significant differences in gender and age distributions between cases and controls (P < 0.05). Specifically, males accounted for 83.67% of cases compared to 34.35% in controls, and the mean age of cases was 8.37 months vs 18.61 months in controls. Therefore, all subsequent analyses were adjusted for age and sex to control for potential confounding. Among the HSCR patients, 1033 (70.27%) were classified as S-HSCR, 294 (20.00%) as L-HSCR, and 82 (5.58%) as TCA.

Table 1 Demographics and characteristics of included patients and healthy control, n (%).

Variables	HSCR (n = 1470)	HC (n = 1473)	P value
Age (month)	8.37 ± 20.50	18.61 ± 19.75	< 0.05
< 2	745 (50.68)	1015 (68.91)
≥ 2	725 (49.32)	458 (31.09)
Sex			< 0.05
Male	1230 (83.67)	506 (34.35)
Female	240 (16.33)	967 (65.65)
HSCR subtype
S-HSCR	1033 (70.27)
L-HSCR	294 (20.00)
TCA	82 (5.58)
TIA	3 (0.2)

HC: Healthy control; HSCR: Hirschsprung disease; L-HSCR: Long segment Hirschsprung disease; S-HSCR: Short-segment Hirschsprung disease; TCA: Total colonic aganglionosis; TIA: Total intestine aganglionosis.

Association of SNP rs1805149A>G with HSCR risk

The results of the association between SNP rs1805149A>G and HSCR risk were shown in Table 2. The genotype distribution of rs1805149 was in HWE in the control group (P > 0.05). For thoroughness, we also assessed HWE in the case group and observed no significant deviation (P > 0.05). We analyzed the association between the NTF3 rs1805149A>G variant and HSCR risk under multiple genetic models. No significant association was observed in any model: Additive (adjusted P = 0.9942, OR = 1.00, 95%CI: 0.86-1.16), genotypic (adjusted P = 0.5586), dominant (adjusted P = 0.768, OR = 1.03, 95%CI: 0.87-1.21), or recessive (adjusted P = 0.3487, OR = 0.78, 95%CI: 0.46-1.31).

Table 2 Correlation between the NTF3 rs1805149A>G polymorphism and sensitivity of Hirschsprung disease.

SNP	A1/A2	Test	Affected (cases)	Unaffected (controls)	P value1	OR	95%CI	P value2
rs1805149	A/G	Additive	424/2434	428/2490	0.8569	1.00	0.86-1.16	0.9942
		Genotypic	27/370/1032	34/360/1065	0.564	NA	NA	0.5586
		Dominant	397/1032	394/1065	0.6397	1.03	0.87-1.21	0.768
		Recessive	27/1402	34/1425	0.4108	0.78	0.46-1.31	0.3487

¹χ² test of the distribution of genotype between patients with Hirschsprung disease and controls.

²Adjusted P value for bias factor including age and sex.

SNP: Single nucleotide polymorphism is located; OR: Odds ratio; 95%CI: 95% confidence interval.

Stratification analysis

The clinical manifestations of HSCR are diverse due to the heterogeneity of the disease. To further investigate whether rs1805149A>G is associated with different HSCR subtypes, we performed logistic regression analyses stratified by disease subtype (Table 3). No significant associations were found for S-HSCR (adjusted P = 0.5454, OR = 1.05, 95%CI: 0.89-1.24), L-HSCR (adjusted P = 0.2088, OR = 0.84, 95%CI: 0.64-1.10), or TCA (adjusted P = 0.8858, OR = 0.97, 95%CI: 0.62-1.52).

Table 3 Stratification analysis of the association between NTF3 rs1805149A>G polymorphism and sensitivity of Hirschsprung disease by subtypes.

CHR	SNP	Subtype	Affected (cases)	Unaffected (controls)	P value1	OR	95%CI	P value2
12	rs1805149	S-HSCR	311/1709	428/2490	0.4824	1.05	0.89-1.24	0.5454
		L-HSCR	72/494	428/2490	0.2273	0.84	0.64-1.10	0.2088
		TCA	23/137	428/2490	0.9192	0.97	0.62-1.52	0.8858

¹χ² test of the distribution of genotype between patients with Hirschsprung disease and controls.

²Adjusted P value for bias factor including age and sex.

CHR: Chromosome; SNP: Single nucleotide polymorphism is located; OR: Odds ratio; 95%CI: 95% confidence interval.

DISCUSSION

NTF3 plays a very important role in the development and maintenance of ENS and has long been considered to be associated with HSCR[13,20] Thus, exploring the relationship between NTF3 gene mutation and HSCR pathogenesis is of significant importance. This study focused on a functional SNP of the NTF3 gene (rs1805149>G) and investigated its association with HSCR risk using a large sample from the Chinese Han population in southern China.

In our study, we first investigated the relationship between NTF3 rs1805149A>G and HSCR susceptibility. Unexpectedly, we did not find a significant association between NTF3 rs1805149A>G and HSCR susceptibility. While our study found no association between the NTF3 rs1805149 variant and HSCR susceptibility, this does not preclude the importance of the NTF3/TrkC signaling pathway in ENS development, as demonstrated in animal and functional studies[13,20]. Previous research has shown that NTF3 and its receptor TrkC are critical for the survival and differentiation of late-developing enteric neurons, and their absence or dysfunction can lead to neuronal deficits in both myenteric and submucosal plexuses[20]. The lack of association observed in our cohort may be attributed to several factors, including the polygenic nature of HSCR, where the effect of a single common variant might be masked by stronger contributions from other genes such as RET, NRG1, or EDNRB[26], or the possibility that rare, rather than common, variants in NTF3 contribute to disease risk[13]. Indeed, a rare missense variant (G76R) in NTF3 has been identified in HSCR patients, suggesting that rare coding changes could play a role in a subset of cases[13]. Furthermore, the functional impact of the rs1805149 variant may be context-dependent, requiring interaction with other genetic or environmental factors not captured in this study[27,28]. Considering the complexity of HSCR, it may also be related to environmental factors and epigenetic abnormalities, which could obscure the effects of NTF3 mutations. Furthermore, since NTF3 plays an important role in promoting cell differentiation, maturation, and survival during the mid and late stages of ENS development[13], we reasonably hypothesized that NTF3 might be associated with different disease subtypes. Therefore, we conducted stratified analyses but did not find a significant association between rs1805149A>G and any specific subtype. This suggests that although NTF3 is crucial in ENS development, a single gene polymorphism may not significantly impact the overall risk of HSCR.

Several human genetic studies have previously investigated the association between NTF3 variants, including rs1805149, and susceptibility to HSCR, but the findings remain inconsistent across populations. In 2008, Ruiz-Ferrer et al[13] first reported two rare missense mutations (G76R and R81C) in NTF3 among European HSCR patients, suggesting a possible pathogenic role of NTF3 variants in ENS development[13]. Luzón-Toro et al[24] later performed exome sequencing in familial HSCR cases and identified high genetic heterogeneity involving NTF3 and other neurodevelopmental genes. However, these studies were based on small sample sizes and were limited to European populations. To date, no large-scale Asian study has confirmed the association between the NTF3 rs1805149 polymorphism and HSCR susceptibility. Our present work, based on 1470 HSCR cases and 1473 controls from southern China, provides the largest dataset in an East Asian population and did not replicate the previously suggested association. This discrepancy may reflect population-specific allele frequencies, genetic background differences, or distinct environmental influences.

Although our study found no association between the NTF3 rs1805149A>G variant and HSCR susceptibility, previous research has implicated NTF3 and its receptor NTRK3 in HSCR pathogenesis through disruptions in ENS development[27]. Mutations in NTF3, such as the novel G76R variant identified in HSCR patients, may alter NTF3/TrkC signaling, leading to impaired neural crest cell differentiation and survival essential for ENS formation[13]. Similarly, a point variant R645C in NTRK3 has been linked to HSCR via haploinsufficiency, potentially acting in concert with RET mutations[14]. Other studies report absent neurotrophin and TrkC localization in aganglionic HSCR segments, with decreased TrkC expression reflecting developmental abnormalities in neural survival[19,21,29]. These observations suggest that while rs1805149 is not a risk factor, other NTF3/NTRK3 variants contribute to HSCR's polygenic etiology.

Previous studies have shown that other genes such as RET, GDNF, EDNRB, PHOX2B, and SOX10 also play critical roles in the early development and migration of the ENS[30,31]. Mutations in these genes have been proven to be closely related to the occurrence of HSCR. Large-scale genetic studies have demonstrated that HSCR risk is influenced by complex interactions between common and rare variants in multiple genes[26,32,33]. For instance, recent work identified PLK5 as a novel susceptibility gene and showed that a genetic risk score combining variants in RET and PLK5 can dramatically increase HSCR risk in Chinese populations[32]. Furthermore, interactions between variants in genes such as NRG1 and RET have been shown to synergistically disrupt ENS development through pathways like PI3K/Akt/SOX10[28]. Therefore, while NTF3 is involved in neural crest development, its contribution to HSCR may be modulated by multi-gene interactions, environmental factors, or epigenetic mechanisms. Future studies should prioritize multi-gene analyses and incorporate functional validation to fully elucidate the complex pathogenesis of HSCR.

Limitations of the study

This study has several limitations that should be considered. First, despite the large sample size, the participants were exclusively of Chinese Han descent from southern China, which may limit the generalizability of the findings to other populations. Second, significant differences in age and sex distribution existed between cases and controls. Although statistical adjustments were applied, residual confounding cannot be entirely ruled out. Most importantly, this study focused solely on the rs1805149 variant in NTF3. HSCR is a complex polygenic disorder, and analyzing a single SNP does not capture the potential cumulative effects or interactions with other genetic and epigenetic factors known to contribute to disease pathogenesis. Future studies should adopt multi-gene or genome-wide approaches in diverse, multi-ancestry cohorts to better elucidate the genetic architecture of HSCR.

CONCLUSION

In summary, this large case-control study found no significant association between the NTF3 rs1805149 variant and HSCR susceptibility or its major subtypes in a southern Chinese Han population. While NTF3 remains biologically relevant to ENS development, our results suggest that this specific polymorphism may not be a major risk factor for HSCR in this cohort. Future research should focus on larger-scale, multi-gene studies incorporating rare NTF3 variants and other genetic or environmental factors to elucidate HSCR’s complex etiology. Additionally, exploring the clinical implications of NTF3/TrkC signaling could inform targeted therapeutic strategies.