Background
Hirschsprung disease (HSCR) is a heterogeneous genetic disorder characterized by the absence of ganglion cells in the gastrointestinal tract, causing a functional obstruction. The most common classifications are short-segment HSCR, long-segment HSCR, and total colon aganglionosis [
1,
2]. The incidence of HSCR in Indonesia is higher (3.1 cases per 10,000 live births) [
3] than other populations (
vs. 1.5, 2.1, and 2.8 cases per 10,000 live births in European, African, and Asian ancestry cases, respectively) [
1,
2]. This difference might be caused by the genetic background of Indonesians, particularly the
RET rs2435357 and rs2506030 risk alleles [
4].
NRG1 has been successfully established as a gene candidate for HSCR disease [
5]. This genome-wide association study result can be confirmed with the
NRG1 variants in patients with HSCR from European and Chinese populations [
6,
7], where those mutations downregulate the protein level of NRG1 and cause HSCR disease. However, a current study showed that
NRG1 rare variant frequency in Indonesian patients with HSCR is only 0.9% [
8].
Moreover, expressions of genes involved in HSCR are influenced by epigenetic mechanisms, including methylation patterns [
9‐
11].
NRG1 hypermethylation has been associated with the downregulated
NRG1 expressions [
12]. In contrast, one study showed that
NRG1 expression was not affected by the methylation status [
13]. Moreover, while one study showed the aberrant
NRG1 expression in patients with HSCR compared to controls [
13], Tang et al
. [
14] revealed no differences in overall
NRG1 expressions between patients with HSCR and controls. These conflicting results concerning the role of NRG1 expressions and methylation level on HSCR pathogenesis emphasize the need for confirmation in other populations, mainly Indonesian.
Discussion
NRG1 variants have been associated with the development of HSCR across populations [
5‐
7]. However, the frequency of rare variants in our HSCR patients’ series is very low [
8]. Therefore, we looked for other factors that might have a role in the HSCR pathogenesis, including
NRG1 expressions and methylation patterns.
Here, we are able to show the aberrant
NRG1 expressions in patients with HSCR, both in the ganglionic and aganglionic colons. Our study demonstrates significantly upregulated
NRG1 expressions in patients with HSCR compared with control colons, indicating that the aberrant
NRG1 expression might impact HSCR pathogenesis. This finding further confirmed a previous study [
13]. However, our study has several novelties: 1) we tested the expressions of all isoforms of
NRG1, including types I, II, and III (
vs. only
NRG1 type I [
13]); 2) we quantitatively compared the
NRG1 expressions between patients with HSCR and control colons (
vs. only determined whether the
NRG1 was expressed in patients with HSCR and control colons [
6]), and 3) in the Indonesian population (
vs. Chinese population [
6,
13]). Interestingly, although from the same Chinese population, two studies revealed different findings: one study had the aberrant
NRG1 expression in patients with HSCR [
13], while another report [
14] showed no differences in
NRG1 expressions between patients with HSCR and controls. These findings together with our results highlight the differences in the epigenetic profile in HSCR patients among population.
In addition, a previous study showed the downregulated
NRG1 expressions in breast cancer cell lines compared to normal ones [
12]. In contrast, our results showed that
NRG1 expressions in Indonesian patients with HSCR are upregulated compared to controls. These differences might be due to 1) different diseases may have different impacts on
NRG1 expressions (developmental anomalies
vs. cancer), 2) variations in genetic backgrounds between populations (Indonesia
vs. Caucasian), and 3) different genetic resources (colon tissue
vs. cell lines).
It has been shown that some gene expressions involved in the HSCR pathogenesis or enteric nervous system development are affected by the methylation pattern, including
RET, GFRA4, EDNRB, and
SHH [
9]. Moreover, it has been hypothesized that
NRG1 expression was affected by its hypermethylation [
13]. However, they failed to prove the hypothesis and suggested a further study on different ethnic groups. Here, we successfully showed that the partially methylated
NRG1 was higher in patients with HSCR than controls. Hypermethylation has been shown to suppress gene expressions [
16]. Therefore, we suggest that the aberrant
NRG1 expressions in our patients might be due to the methylation status. The differences between our findings and a previous report [
13] might be due to the differences in genetic characteristics among populations within Asian people [
17].
Notably, limitations due to small sample size and single center study should be considered during interpretation of our findings. Moreover, it suggests a further multicenter studies with a larger sample size are necessary to identify other epigenetic factors that influence the NRG1 expression in patients with HSCR.
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