Background
Hirschsprung disease (HSCR) is a complex congenital disorder characterized by the absence of intrinsic ganglion cells in the intestinal tract, starting distally and extending proximally to variable lengths [
1‐
4]. Its incidence is higher in Indonesia (3.1:10,000) than in other populations, including Asians (2.8:10,000) and Caucasians (1.5:10,000) [
1,
5]. These facts might be due to the greater risk allele frequency of
REarranged during Transfection (RET) rs2506030 in Indonesia compared to other populations [
6].
HSCR has been associated with more than 35 genes, including
SRY-box transcription factor 10 (SOX10) and
RET [
1‐
5]. However, the thirty-five genes only provide 62% of the frequency of HSCR. Therefore, the pathogenesis of HSCR in the remaining patients remains unclear [
2,
5]. HSCR can be associated with the alteration of gene expressions [
7,
8].
SOX10 is a transcription factor that affects
RET expression by binding to one of its cis-regulatory elements (CREs) located in intron 1 of
RET: RET-7,
RET-5.5, and
RET + 3. Therefore, in the presence of variants affecting CREs,
SOX10 is not able to bind anymore, leading to decreased
RET expression [
7]. Here, we aimed to evaluate the expressions of
SOX10 and
RET in HSCR patients and compare them with the controls.
Discussion
Our study is able to show the aberrant
SOX10 and
RET expressions in HSCR patients. A previous study showed lower
SOX10 expressions were associated with hypertrophic nerve trunks in HSCR patients [
8]. They suggested that the aberrant
SOX10 expressions might involve HSCR pathogenesis via interaction with other neurotrophic factors in non-syndromic patients without any pathogenic variants in the
SOX10 gene. Our study provides new evidence of the aberrant
SOX10 expressions in HSCR patients from a different population from a previous study [
9]. In addition, most
SOX10 pathogenic variants were found in syndromic HSCR patients [
13]. Interestingly, a recent study revealed that three common variants within CREs of
RET decreased the binding of transcription factors, including
SOX10, to those three CREs. These interactions caused the decrease of
RET expressions and disruption of other HSCR and enteric nervous system (ENS) genes within the
RET–EDNRB GRN [
7].
SOX10 is a gene encoding a member of the SRY-related HMG-box (SOX) family of transcription factors that regulate embryonic development and determine cell fate. The SOX10 protein acts as a nucleocytoplasmic shuttle protein, essential for neurogenesis and neural crest cells (NCCs) development [
13].
SOX10 has significant roles in the development of NCCs, one of which is regulating the migration of NCCs, which form the ganglionic plexus of the ENS [
14].
SOX10 helps ensure the survival and pluripotency of NCCs during and after migration and contributes to determining their fates and differentiation [
14‐
16]. It is also known that direct
SOX10 interaction with
Cadherin-19 (
Cdh19) mediates early sacral NCCs migration by forming cadherin-catenin complexes. These complexes interact with the cytoskeleton filamentous actin in the migration of NCCs [
17].
SOX10 expression is regulated by several transcription factors, such as
SOX9, Olig2, WNT, FoxD3, and Snail [
16,
18]. Overexpression of
SOX10 has been shown to inhibit the differentiation of NCCs [
14,
18]. After the differentiation of the NCCs,
SOX10 expression is maintained in enteric glial cells while downregulated in neurons and smooth muscle cells [
14,
16].
Several weaknesses of our study were noted, including a small sample size, and our findings did not consider other ENS and HSCR gene expressions involved within the
RET–EDNRB gene regulatory network (GRN). Two housekeeping genes should always be included to account for technical variations during qPCR. Our study only used
GAPDH as an internal control. It is essential to conduct a further study to determine whether the increased
SOX10 expressions are due to the increased glial cells or enhancement of
SOX10 expressions in ENS cells. Moreover, it is also interesting to determine whether the increased
SOX10 expressions due to the cell numbers of
SOX10-expressing cells are changed or the
SOX10 promoter activity is enhanced. In the postnatal period,
SOX10 is mainly expressed in glial cells. The quantification of the glial cell population in the colon from patients and control is crucial. Checking aberrant expression of
SOX10 in other cell types is also needed. Alternatively, an isolated explant or cell culture experiment is required. Using this system, it is possible to estimate promoter or enhancer activities of
SOX10 and high levels of
SOX10 in ENS cells. In addition, we do not validate the protein levels of SOX10, including immunohistochemistry, in HSCR patients due to limited resources. Pathogenic variants in
GLI, resulting in upregulated
Sox10 expression in vitro, have been detected in patients with non-syndromic HSCR [
19]. Therefore, screening pathogenic variants in transcription factors regulating
SOX10 in patients or estimating promoter or enhancer activities of
SOX10 in isolated human ENS cells is essential.
SOX10 is required for
RET expression, and decreased
RET expression causes HSCR [
7,
14,
20]. During the development of ENS,
SOX10 controls specific genes, including
RET, EDNRB, and
SOX10 itself [
14]. The decrease of
RET expressions disrupts the other HSCR and enteric nervous system (ENS) genes within the
RET–EDNRB GRN, including
GATA2, SOX10, RARB, and
NKX2.5 [
21]. However, no direct evidence indicates that increased
SOX10 leads to aganglionosis, i.e., HSCR. Interestingly, upregulated
SOX10 expression promotes the migration of neural crest-like cells of the neural tube; however, it hampers their differentiation [
22]. We further determined the
RET expressions in our HSCR patients. Intriguingly, the expressions of
RET were significantly downregulated in patients compared to controls. These decreased
RET expressions might lead to HSCR. It is important and interesting to conduct a further study on how upregulated
SOX10 causes decreased
RET expression, resulting in HSCR.
Moreover, our findings might be beneficial during the surgical counseling to the parents that in a polygenic disorder, such as HSCR, a complex interaction between genes might result in different disease phenotypes. This evidence further confirms the complexity of the pathogenesis of HSCR, including the disruption of the GNR during the ENS development.
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