The impact of down-regulated SK3 expressions on Hirschsprung disease

Hirschsprung disease (HSCR: MIM# 142623) is a neurodevelopmental disorder characterized by the absence of ganglion cells, resulting in a functional intestinal obstruction in infants. According to the type of aganglionosis, HSCR is classified as follows: short-segment HSCR, long-segment HSCR, and total colon aganglionosis [1, 2]. Its incidence differs among race with 15, 21, and 28 cases per 100,000 live births in European, African and Asian ancestry cases, respectively [1, 2]. It might relate to the RET rs2435357 susceptibility allele frequency difference across populations [3, 4]. Our previous studies supported this hypothesis since the frequency of rs2435357 variant in Indonesian ancestry cases is higher than those of European ancestry cases (0.50 vs. 0.25) [5, 6].

The current treatment for Hirschsprung disease (HSCR) is surgical resection of the aganglionic segment of the intestines. Most HSCR patients have a satisfactory outcome after a properly performed pull-through operation, however some patients continue to have persistent bowel symptoms such as constipation, soiling and enterocolitis. The cause of persistent bowel symptoms might be the transition zone pull-through or residual aganglionosis, but many have no identifiable cause for their ongoing bowel dysfunction [7, 8]. The prevailing hypothesis is the down-regulated small-conductance calcium-activated potassium channel 3 (SK3) expression in ganglionic bowel in HSCR patients [9]. The SK3 channels have been shown to be involved in the outward currents activated by purines in the intact muscles in response to enteric inhibitory neurotransmission [10]. It is highly expressed in the platelet-derived growth factor receptor alpha-positive (PDGFRA+) cells [11], which together with interstitial cells of Cajal and smooth muscle cells regulate intestinal secretory activities and peristalsis [10]. In addition, a different genetic characteristic was previously revealed between the Indonesian and European populations with HSCR [12]. Therefore, we aimed to investigate the SK3 expression in HSCR patients after a properly performed definitive surgery in an Indonesian population, a genetically distinct group within Asia.

During a 1-year period of study, we recruited 16 HSCR patients and eight controls according to the inclusion criteria. We excluded two HSCR patients and two controls because of their low quality of total RNA, thus, we further analyzed 14 HSCR patients and six controls. Neither familial nor syndromic HSCR patients were involved in this study.

According to degree of aganglionosis, 86% of patients have a HSCR short-segment. The mean age at diagnosis and definitive surgery was 10.1 ± 31.6 months and 20.4 ± 35.9 months, respectively (Table 1). As for definitive surgery, 57% HSCR patients underwent the transanal endorectal pull-through, followed by the Duhamel and Soave pull-through procedures in 29% and 14% children, respectively (Table 1).

First, we genotyped HSCR patients for the RET rs2435357 variant since this variant has been strongly associated with HSCR in an Indonesian population [5, 6]. The genotype frequencies for RET rs2435357 variant among HSCR patients were as follows: TT (12/14, 86%), CT (0), and CC (2/14, 14%).

Quantitative RT-PCR showed that the SK3 expression was significantly lower (23-fold) in the ganglionic colon group compared to the control group (9.9 ± 4.6 vs. 5.4 ± 3.4; p = 0.044) (Fig. 1). The expression of SK3 in the aganglionic colon group was also significantly lower (43-fold) compared to the control group (10.8 ± 4.4 vs. 5.4 ± 3.4; p = 0.015) (Table 2).

We followed-up all patients for a mean of 10.8 ± 16.0 months following pull-through. Three individuals (patient 4, 6, and 11) developed enterocolitis in 5, 18, and 2 months, respectively, after pull-through, while only one subject (patient 6) suffered post-operative soiling (Table 1). All symptoms resolved with rectal irrigations and administration of oral metronidazole.

We describe new data on Indonesian HSCR patients which reveal a similar frequency of short-segment aganglionosis as reported in the literature [17, 18]. Our study clearly demonstrates that the SK3 expression was strongly reduced in the HSCR patient intestines, both in the ganglionic and aganglionic bowel, compared to the control intestines. These findings are consistent with previous observations [9, 19]. However, it should be noted that previous study [9] showed only half HSCR patients had reduced the SK3 expression. These differences might be attributed to the differences in the genetic background of analyzed population between previous report [9] versus our study.

Persistent bowel symptoms might occur in about 10% of children following pull-through procedure. They range from constipation to severe episodes of abdominal distension, vomiting, and enterocolitis [20]. We eliminated the possibility of any residual aganglionosis in our patients with persistent bowel symptoms since we have performed intraoperative histopathological evaluations. There was no stricture identified in these patients after evaluation by a contrast enema.

It has been proposed that the down-regulated SK3 in the HSCR colon results in unopposed cholinergic activity and a tonic hypercontraction, further causing a functional bowel obstruction in HSCR patients following pull-through [9]. Our study reveals a new evidence supporting this hypothesis by providing data from a genetically different population from previous reports [9, 19].

However, notably, the small sample size and limited power of the study, while a weakness of our report, implies that a significantly larger sample of patients needs to be involved to confirm our findings.

In this study, we involved the ARM patients as controls according to previous study [9]. It should be noted that most ARM patients suffer from a disturbance of the bowel motility [21]. Further study with more appropriate controls (e.g. trauma patients) is necessary to better clarify the impact of SK3 expression in the persistent bowel symptoms after a definitive surgery.

Furthermore, the molecular pathogenesis of HSCR still needs to be elucidated. It might involve the compromised condition of genes responsible for gangliogenesis of the enteric nervous system (ENS) [5, 6, 12, 22], the neurotransmitters expressed by the neurons of ENS [11, 23] and their interactions. Our results clarify the role of SK3 in the molecular pathogenesis of HSCR, particularly in Indonesia, a genetically distinct group within Asia [4‐6, 12]. It is important to note that the down-regulation in SK3 occurs in addition to other neurotransmitter signaling in HSCR. In addition, this study is the first report of SK3 expression in a sample population of Asian ancestry. Moreover, our recent study shows that epistasis between RET and NRG1 is important to ENS development [6]. It might be necessary to determine the PDGFRA expression level as a control and a marker for the PDGFRA+ cells that express SK3 for better understanding the persistence bowel symptoms’ pathogenesis in HSCR patients following pull-through procedure. Unfortunately, we do not have any data on immunostaining of the resection segment for SK3 due to limitation of resources in our institution. Furthermore, most patients (86%) in this cohort study carried the risk allele (T) for HSCR that are consistent with our previous findings [5, 6].

This study may contribute to extending the knowledge on mechanisms causing the persistence of bowel symptoms after an appropriately performed pull-through. The possibility of the continuing intestinal symptoms after an appropriately definitive surgery should be explained during surgery counseling to HSCR parents. Furthermore, the pediatric surgery involves more than conducting an operation but consists of total care of the patient and the family, of which proper diagnosis, prognosis and counseling are fundamental parts of the performance of procedure and the patient recovery process.

Our study shows that the down-regulated SK3 expression in ganglionic intestines might contribute to the persistent bowel symptoms following a properly performed pull-through surgery in Indonesian HSCR patients. Furthermore, this study is the first report of SK3 expression in a sample population of Asian ancestry.