Hirschsprung disease (HSCR: MIM# 142623) is a complex genetic disorder, characterized by the lack of ganglion cells in the bowel, resulting in a functional obstruction during infancy (1). HSCR is categorized into the following types: short-segment, long-segment, and total colonic aganglionosis (1, 2).

HSCR incidence differs among ethnic groups, with 1.5, 2.1, and 2.8 cases per 10,000 live births in European, African, and Asian ancestry cases, respectively (1, 2). There are at least 17 genes responsible for the development of HSCR, with most of them being members of the RET and EDNRB signaling pathways (1, 2). Two genetic risk factors are the RET rs2435357 and rs2506030 variants (3, 4). Our recent studies showed that the RET rs2435357 and rs2506030 risk alleles have higher frequency in Indonesian ancestry cases as compared with European ancestry cases (5, 6), which might relate to the higher incidence of HSCR in Indonesia (3.1 cases per 10,000 live births) than other populations (7).

The third signaling pathway of HSCR pathogenesis includes class 3 semaphorins (SEMA3s), involving SEMA3D (4, 8, 9) SEMA3D has been implicated in the development of HSCR and contributes to risk through both common and rare variants in European ancestries (4, 8, 9), as evidenced by (1) the detection of both common and rare SEMA3D variants in HSCR patients; (2) the expression of SEMA3D in the human, mouse, and zebrafish intestines and, particularly, the enteric nervous system (ENS); and (3) the joint effect of Ret and Sema3d loss of function in an aganglionosis animal model. However, our recent study showed that the effect of SEMA3 rs11766001 common variant on HSCR depends on the ethnic background (10). In addition, the allele frequencies of common variants might differ among Asians, since the North Asians, Han Chinese, Japanese, and Southeast Asians can be distinguished based on their Y chromosome variants (11). Moreover, alterations in the expression of specific genes have been implicated in the development of HSCR (12–15). Therefore, we wished to investigate the role of SEMA3D variants, both rare and common variants, as well as its mRNA expression in Indonesian HSCR patients.

Most of our HSCR patients were male (70%) and short-segment aganglionosis type (98%), with the mean age at diagnosis being 34.6 ± 44.5 months (Table 1). Among the 54 HSCR patients, 49, 3, and 2 children underwent a definitive surgery, a colostomy, and a full-thickness rectal biopsy waiting a pull-through procedure, respectively, with the most common definitive surgery conducted being transanal endorectal pull-through (43%) (Table 1); and our controls were six males and seven females, with a mean age during stoma closure of 47 ± 45.1 months.

We could not identify any rare variant in all 17 exons of SEMA3D gene in 54 HSCR patients, but encountered one common variant in exon 17: p.Lys701Gln (rs7800072) (Figure 1). The genotype frequencies for rs7800072 variant among HSCR patients were as follows: AA (32/54, 60%), AC (19/54, 35%), and CC (3/54, 5%). Subsequently, we compared the risk allele (C) frequency of rs7800072 in 54 Indonesian HSCR cases and the 1000 Genomes and ExAC East Asian ancestry controls (Table 3). The risk allele (C) frequency was similar in HSCR cases (23%) and the 1000 Genomes (27%) and ExAC (28%) East Asian ancestry controls, with p-values of 0.49 and 0.41, respectively (Table 3). According to the conservation score prediction using PhyloP, the p.Lys701Gln variant did not reach a deleterious threshold of 0.84, while the predicted deleterious effect (Condel) showed the p.Lys701Gln variant as being neutral (4). Furthermore, the SIFT and PolyPhen-2 analysis of p.Lys701Gln showed the variant as being tolerated and benign, respectively (18).

Next, we compared SEMA3D expression in 18 aganglionic and ganglionic colons of HSCR patients and 13 control colons. The distribution of SEMA3D expression data were normal for ganglionic, aganglionic, and control colons (p = 0.2, 0.17, and 0.2, respectively). A significant different of SEMA3D expression was observed between groups (p = 0.04). Furthermore, qPCR showed that the SEMA3D expression was strongly up-regulated (5.5-fold) (Figure 2) in the ganglionic colon of HSCR patients compared to control colon (ΔC_T 10.8 ± 2.1 vs. 13.3 ± 3.9; p = 0.025) (Table 4, Figure 3), while the SEMA3D expression was not significantly different between the aganglionic colon of HSCR patients and the control colon (ΔC_T 13.1 ± 3.0 vs. 13.3 ± 3.9; p = 0.89) (Table 4, Figure 3).

In this study, we have performed an in-depth genetic and gene expression study of the SEMA3D in Indonesian HSCR patients. We did not detect any rare variants in this gene, although previous studies in European ancestry cases have identified rare coding variants in SEMA3D associated with HSCR. Our results indicate that the association of such variants to the disease may be restricted to specific ethnic groups. It may still be the case that other semaphorin 3 genes could play a role in HSCR pathogenesis (4, 9). A previous study showed the existence of other genetic factors conferring risk to HSCR in specific ethnic populations (3). For example, there were two different RET haplotypes involving the enhancer mutation that were over-transmitted to the HSCR offspring in the European sample, while in the Chinese sample, only one of those haplotypes was present (3). It might be speculated that the enhancer mutation arose in one haplotype, which after the Asian–Caucasian split, rearranged to also give the other haplotype, but exclusively in the Caucasian (22). Another example is the NRG1 rs7835688 genetic marker, which has been originally discovered in Chinese HSCR patients (23) and has been shown in other Asian ancestry cases (5, 24), but is rare and shows no effect in the Caucasian population (8, 22). Makhmudi et al. (25) also demonstrated that the MTHFR c.677C>T is a genetic risk factor for Indonesian gastroschisis, but not seen in Caucasians (26, 27). A recent study showed that loss of Sema3d in null homozygotes mice had no impact on the intestinal transcriptome (28). The authors were unable to find evidence for Ret and Sema3d interaction affecting survival, presence of myenteric plexus, or intestine transcriptome (28). Furthermore, Tang et al. revealed that the effects of RET and NRG1 variants are universal across Caucasian and Asian ancestries, but the impact of SEMA3 variant was restricted to Caucasian ancestries (29). It should be noted that our screening method did not cover the promoter and enrich regions of CpG of SEMA3D.

In addition, the observed SEMA3D rs7800072 variant frequency in Indonesian HSCR patients is similar to those reported for the 1,000 Genomes Project and ExAC East Asian ancestry controls (17, 18). Therefore, we might conclude that this common variant does not have a role on the development of HSCR in Indonesia.

ENS development is a complex process, regulated by a large range of molecules and signaling pathways. This strictly controlled process needs the correct regulation of ENS-specific gene expression. The expressions of several genes implicated in HSCR development have been shown to be regulated by epigenetic mechanisms. RET expression was regulated by retinoic acid through DNA methylation on this 5′-CG-3′-rich enhancer region (30), while a significantly lower level of EDNRB methylation was also detected in HSCR patients (31). Interestingly, our study clearly demonstrates that SEMA3D gene expression was strongly up-regulated in the ganglionic intestines of HSCR patients as compared to controls. To the best of our knowledge, our study is the first report of the aberrant expressions of SEMA3D in HSCR patients. Another novelty in our findings is we conducted the study on patients with Indonesian ancestry [vs. European (4) ancestry cases]. It has been shown that reduced sema3d expression by morpholino resulted in severe effects on the intestine and its innervation of zebrafish (4). In addition, sema3d bound to nrp1a to facilitate the axonal guidance and contributed to peripheral axon outgrowth interdependently with dpysl3 (32, 33) Binding of Sema3d to neuropilin and plexin receptors introduced biochemical responses in specific neurons and stimulated the neuron migration (34, 35). Therefore, we might hypothesize that the aberrant expressions of SEMA3D will have an impact in our HSCR patients by affecting the neuronal guidance during ENS development.

It has been shown that some HSCR patients have persistent bowel symptoms, such as constipation, soiling, and enterocolitis, after an appropriate pull-through procedure. Most HSCR patients with persistent bowel symptoms do not have any identifiable etiology for their ongoing bowel dysmotility (36). The current hypothesis is that the aberrant expression of some genes in the ganglionic colon of HSCR patients includes SK3, Cx26, ChAT, and nNOS (37–40). Our study presented the altered SEMA3D expressions in the ganglionic colon of HSCR patients. Therefore, we might hypothesize that the aberrant SEMA3D expressions in the ganglionic colon involve in the pathogenesis of persistent bowel symptoms in HSCR patients following a properly performed pull-through surgery.

Our results should be interpreted with some caution, however, because they are based on overall means without accounting for other factors generating variation in the data such as gender, age, and degree of aganglionosis. Further studies of the methylation pattern of the SEMA3D gene are required to investigate whether the aberrant expression of SEMA3D in HSCR patients is due to abnormal DNA methylation. It is also necessary to compare the SEMA3D protein expression level between HSCR patients and controls and to identify its location in the colon tissue to prove the increased SEMA3D expressions. Unfortunately, we do not have any data on the methylation pattern, protein expression, and immunohistochemistry of SEMA3D due to resource limitations in our institution. Furthermore, our study utilized ARM patients' colon as controls. It has been shown that most ARM patients have abnormal colonic motility (41) and low expression of interstitial cells of Cajal marker (42). Therefore, further research with more proper control colon (e.g., autopsy bowel material from healthy infants or trauma patients) is necessary to better confirm the role of SEMA3D expression in the pathogenesis of HSCR. Noteworthy, however, our small sample size is a limitation of our study and suggests that a multicenter larger sample population needs to be studied to clarify our findings.

In conclusion, we report the first study of aberrant SEMA3D expressions in HSCR patients and suggest further understanding into the contribution of aberrant SEMA3D expression in the development of HSCR. In addition, this study is the first comprehensive analysis of SEMA3D variants in the Asian ancestry.

All data generated or analyzed during this study are included in the submission. The raw data are available from the corresponding author on reasonable request.

The Institutional Review Board of the Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/Dr. Sardjito Hospital gave approval for this study (KE/FK/1356/EC/2015). All parents signed a written informed consent form before participating in this study.

G and KI conceived the study. G drafted the manuscript. KI and JV critically revised the manuscript for important intellectual content. AK, NB, and FR performed total RNA extraction and qPCR. HH, AM, MF, and DY conducted the experimental PCR-based work for Sanger sequencing. All authors have read and approved the manuscript and agreed to be 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.