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 [1,2,3,4]. 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 [1,2,3,4,5]. 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.

Materials and methods

Patients

Twenty patients under 18 years old presenting with non-syndromic HSCR whose ganglionic and aganglionic colons were collected during pull-through surgery are included in this study. We did not have any genetic data for those 20 HSCR patients. Sixteen control colon samples were collected during stoma closure for anorectal malformation patients. One HSCR patient was excluded due to low-quality RNA; thus, 19 of the total RNA of HSCR patients were further analyzed.

Each patient’s parents completed a written informed consent form before participating in this study. This study was approved by the Institutional Review Board of the Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada/Dr. Sardjito Hospital (KE/FK/0442/EC/2022 and KE/FK/0758/EC/2022). All experiments were conducted in conformity with the necessary legislation and guidelines.

Total RNA isolation and quantitative real-time polymerase chain reaction

Total RNA extraction of the HSCR patient’s colon (aganglionic and ganglionic) and the control colon was done by using a total RNA Mini Kit (Tissue) (Geneaid Biotech Ltd., New Taipei City, Taiwan). The samples were stored at − 80 °C for future use.

One-step quantitative real-time polymerase chain reaction (qPCR) was performed using Kapa SBYR Fast qRT-PCR One Step Kit Universal (Kapa Biosystems, Massachusetts, USA) and BioRad CFX Real-Time PCR System (California, USA). The primers used for qPCR were as follows: SOX10 5’-ATGAACGCCTTCATGGTGTGGG-3’ (forward) and 5’-CGCTTGTCACTTTCGTTCAGCAG-3’ (reverse) [9], and RET 5′-CTGCCAAGTCCCGATG-3′ (forward) and 5′-TGGAGTACGCCAAATACG-3′ (reverse) [10]. The GAPDH gene was used as an internal control with the following primers: 5′-GCACCGTCAAGGCTGAGAAC-3′ (forward) and 5′-TGGTGAAGACGCCAGTGGA-3′ (reverse). As a reference gene, GAPDH has been validated in a diverse set of several tissues representing different organs, including the liver of biliary atresia and other diseases (gallbladder hydrops, omphalocele, internal bleeding, liver abscess, and choledochal cyst) and ganglionic and aganglionic colon of HSCR and anorectal malformation colon patients in our previous studies [11, 12]. The GAPDH showed a constant expression in a significant variation of human tissue samples [11, 12].

Statistical analysis

The expression level of SOX10 and RET was measured using the Livak method (2− ΔΔCT). The data is presented as mean ± SD and analyzed using an independent t-test; p < 0.05 was considered significant. All statistical analyses were performed using the IBM SPSS version 23 (Chicago, USA).

Results

Patient characteristics

Nineteen subjects were involved in this study. Most patients were males (68.4%) and short aganglionosis segment (78.9%) (Table 1).

Table 1 Characteristics of HSCR patients in this study
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SOX10 expressions in HSCR patients

There were significant upregulated SOX10 expressions in both ganglionic (2.84-fold) and aganglionic (3.72-fold) colon of HSCR patients compared to controls’ colon (p = 0.032 and 0.003, respectively) (Table 2; Fig. 1).

Table 2 SOX10 expressions in HSCR patients and controls
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Fig. 1
figure 1

Box-plot graph of ΔCT value of the SOX10 expressions in HSCR ganglionic colon (ΔCT 5.21 ± 2.04), HSCR aganglionic colon (ΔCT 4.82 ± 1.59), and control colon (ΔCT 6.71 ± 1.90). Box-plot graph of ΔCT value reveals the median values as lines across the box. Lower and upper boxes represent the 25th percentile to the 75th percentile, while whiskers indicate the maximum and minimum values. *p < 0.05

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RET expressions in HSCR patients

Subsequently, we determined the RET expressions in HSCR patients and controls. Interestingly, significant downregulated RET expressions were noted in both ganglionic (11.71-fold) and aganglionic (29.96-fold) colon of HSCR patients compared to controls’ colon (p = 0.0004 and 0.0001, respectively) (Table 3; Fig. 2).

Table 3 RET expressions in HSCR patients and controls
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Fig. 2
figure 2

Box-plot graph of ΔCT value of the RET expressions in HSCR ganglionic colon (ΔCT 12.54 ± 2.21), HSCR aganglionic colon (ΔCT 13.90 ± 2.64), and control colon (ΔCT 8.99 ± 3.13). Box-plot graph of ΔCT value reveals the median values as lines across the box. Lower and upper boxes represent the 25th percentile to the 75th percentile, while whiskers indicate the maximum and minimum values. *p < 0.05

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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 [14,15,16]. 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.

Conclusions

Our study shows aberrant SOX10 and RET expressions in HSCR patients, implying the critical role of SOX10 and RET in the pathogenesis of HSCR, particularly in the Indonesian population. Our study further confirms the involvement of SOX10-RET within the GNR during the ENS development.