Hirschsprung’s disease (HD) is a congenital disorder of the enteric nervous system (ENS), characterized by the absence of ganglion cells in the distal bowel, leading to functional intestinal obstruction of varying length. It was first described by Harald Hirschsprung in 1888, who reported two infants with congenital megacolon due to aganglionosis of the rectosigmoid segment [1]. The disease is caused by a failure of neural crest cells to migrate, proliferate, or differentiate properly during intestinal development, resulting in a segment of bowel that lacks the intrinsic enteric nervous plexuses necessary for coordinated peristalsis [2]. Consequently, the affected segment remains tonically contracted, causing chronic obstruction and proximal bowel dilatation. The incidence of HD varies worldwide, ranging from 1 in 5,000 to 1 in 10,000 live births, with a marked male preponderance of approximately 4:1 [3]. Most cases are sporadic, although familial clustering and recurrence in siblings strongly suggest a genetic contribution. The disease displays remarkable heterogeneity, both in terms of clinical severity and anatomical extent. Based on the length of the aganglionic segment, HD is classified into short-segment disease (rectosigmoid), long-segment disease (extending proximal to the sigmoid), and total colonic aganglionosis [4]. Short-segment HD constitutes about 80% of all cases, whereas long-segment and total colonic types are less common but often more severe [5]. Clinically, HD typically presents in the neonatal period with failure to pass meconium within 48 hours of birth, progressive abdominal distension, and bilious vomiting. However, the presentation may vary depending on the length of aganglionosis and severity of functional obstruction. Some children may present later with chronic constipation, abdominal bloating, and failure to thrive. In older infants and children, enterocolitis may develop as a life-threatening complication [6]. A high index of suspicion and timely diagnosis are crucial for preventing morbidity and mortality. The diagnosis of HD is established by a combination of clinical findings, radiological evaluation, anorectal manometry, and histopathological confirmation of the absence of ganglion cells in rectal biopsy. Surgical management remains the definitive treatment, involving resection of the aganglionic segment and pull-through of normally innervated bowel [7]. Despite significant advances in diagnosis and surgical techniques, postoperative complications such as constipation, fecal incontinence, and Hirschsprung-associated enterocolitis (HAEC) continue to pose challenges. The etiology of HD is multifactorial, involving complex interactions between genetic, epigenetic, and environmental factors affecting neural crest cell migration and differentiation. Over the past two decades, extensive genetic studies have identified mutations and polymorphisms in several genes implicated in ENS development, including RET, EDNRB, EDN3, GDNF, SOX10, and PHOX2B [8]. Among these, the RET proto-oncogene has emerged as the major susceptibility gene for both familial and sporadic HD. The RET gene, located on chromosome 10q11.2, encodes a receptor tyrosine kinase involved in signaling pathways critical for the survival, proliferation, and migration of neural crest-derived cells [9]. Loss-of-function mutations or regulatory polymorphisms in RET disrupt normal signaling between glial cell line-derived neurotrophic factor (GDNF) and its receptor complex, leading to defective enteric ganglion formation. Polymorphisms in RET not only influence disease occurrence but also modulate phenotypic variability, including segment length and severity. The most widely studied functional polymorphism, RET intron 1 variant rs2435357 (C>T), located within a conserved enhancer region, has been shown to significantly reduce transcriptional activity of RET and increase susceptibility to HD [10]. This variant is believed to act synergistically with rare coding mutations or other modifier genes to determine disease penetrance. Notably, the rs2435357 polymorphism has been identified as a common low-penetrance risk allele, explaining a substantial fraction of sporadic HD cases across different populations. Understanding the genetic basis of HD, particularly the role of RET polymorphisms, holds great clinical significance. Identification of specific variants may facilitate early diagnosis in at-risk neonates, aid in genetic counseling of families, and improve understanding of the disease mechanism. Moreover, delineating genotype–phenotype correlations could offer insights into variable clinical presentations and postoperative outcomes. The integration of genetic screening with traditional diagnostic modalities represents a promising approach toward personalized management of HD. The study aimed to assess the clinical presentations and demographic characteristics of children diagnosed with Hirschsprung’s disease, while also evaluating the potential association between RET proto-oncogene polymorphisms and both the occurrence and severity of the disease.

Study design: It was a Observational and Prospective study. Place of study: The study was conducted in the Department of Pediatric Surgery, I.P.G.M.E. &R, Kolkata. In association with Department of Pathology I.P.G.M.E. & R. Koltata. Period of study: February 2015- January 2016 (12 months) Study population- Patients admitted to the Department of Pediatric Surgery, I.P.G.M.E. &R, Kolkata with clinical and radiological diagnosis of suspected Hirschsprung's disease subsequently underwent full thickness bowel biopsy and follow-up cases of HD who underwent definitive pull through surgery. Study Variables: • Age • Sex • Religion • S/A • Site • Surgery Type • Cons • FU • Perf Gut • Ca rs1800860 • C/F DPM • C/F BV • C/F AD • AG • GG • TG Sample size: 50 pediatric patients clinically and histopathologically diagnosed with Hirschsprung’s disease. Inclusion Criteria: • The patients presented with failure to pass meconeum, bilious vomiting, chronic severe constipation and progressive abdominal distension and investigated radiologically Exclusion Criteria: • Patients whose examinations do not reveal any intestinal obstruction clinically and radiologically and absence of above clinical parameters. • Very seriously ill patients and those who are admitted in ICU will be excluded. Statistical Analysis: For statistical analysis data were entered into a Microsoft excel spreadsheet and then analyzed by SPSS 20.0.1 and GraphPad Prism version 5. A chi-squared test (x2 test) was any statistical hypothesis test wherein the sampling distribution of the test statistic is a chi-squared distribution when the null hypothesis is true. Without other qualification, 'chi-squared test' often is used as short for Pearson's chi-squared test. Unpaired proportions were compared by Chi-square test or Fischer's exact test, as appropriate. Z-test (Standard Normal Deviate) was used to test the significant difference of proportions. Correlation was calculated by Pearson correlation analysis. p-value ≤0.05 was considered for statistically significant.

Table 1: Demographic, Clinical, and Genetic Characteristics of Study Participants (n=50) Age (Days) Frequency Percent P-Value Age 1–28 28 56.00% <0.05 29–365 6 12.00% >365 16 32.00% Total 50 100.00% Sex F 15 30.00% M 35 70.00% Total 50 100.00% Religion C 1 2.00% H 25 50.00% M 24 48.00% Total 50 100.00% S/A Down 2 4.00% No 48 96.00% Total 50 100.00% Site LSD 7 14.00% SSD 39 78.00% TCA 4 8.00% Total 50 100.00% Surgery Type bx 32 64.00% def 18 36.00% Total 50 100.00% Ca rs1800860 AG 11 22.00% GG 39 78.00% Total 50 100.00% Ca rs1800861 GG 5 10.00% TG 14 28.00% TT 31 62.00% Total 50 100.00% CO rs1800860 AA 3 6.00% AG 17 34.00% GG 30 60.00% Total 50 100.00% CO rs1800861 GG 13 26.00% TG 28 56.00% TT 9 18.00% Total 50 100.00% Table 2: Clinical Features and Follow-Up Characteristics of Study Participants (n=50) C/F AD Frequency Percent P-Value C/F AD Absent 9 18.00% <0.05 Present 41 82.00% Total 50 100.00% C/F DPM Absent 6 12.00% Present 44 88.00% Total 50 100.00% C/F BV Absent 17 34.00% Present 33 66.00% Total 50 100.00% Cons Absent 19 38.00% Present 31 62.00% Total 50 100.00% FU Absent 32 64.00% Present 18 36.00% Total 50 100.00% Perf Gut Absent 48 96.00% Present 2 4.00% Total 50 100.00% Table 3: Distribution of Ca rs1800860 and Ca rs1800861 Genotypes across Surgical Sites (n=50) Ca rs1800860 LSD SSD TCA TOTAL P-Value Ca rs1800860 AG 1 (9.1%) 10 (90.9%) 0 (0.0%) 11 (22.0%) <0.0001 GG 6 (85.7%) 29 (74.4%) 4 (100.0%) 39 (78.0%) TOTAL 7 (14.0%) 39 (78.0%) 4 (8.0%) 50 (100%) Ca rs1800861 GG 0 (0.0%) 5 (12.8%) 0 (0.0%) 5 (10.0%) TG 0 (0.0%) 14 (35.9%) 0 (0.0%) 14 (28.0%) TT 7 (100.0%) 20 (51.3%) 4 (100.0%) 31 (62.0%) TOTAL 7 (100%) 39 (100%) 4 (100%) 50 (100%) Table 4: Association of Ca rs1800860 and Ca rs1800861 Genotypes with AD Presence C/F AD Absent Present Total P-Value Ca rs1800860 AG 0 (0.0%) 11 (26.8%) 11 (22.0%) 0.0785 GG 9 (100.0%) 30 (73.2%) 39 (78.0%) Total 9 (100%) 41 (100%) 50 (100%) Ca rs1800861 GG 0 (0.0%) 5 (12.2%) 5 (10.0%) 0.1742 TG 1 (11.1%) 13 (31.7%) 14 (28.0%) TT 8 (88.9%) 23 (56.1%) 31 (62.0%) Total 9 (100%) 41 (100%) 50 (100%) Table 5: Association of Ca rs1800860 and Ca rs1800861 Genotypes with DPM Presence C/F DPM Absent Present Total P-Value Ca rs1800860 AG 0 (0.0%) 11 (25.0%) 11 (22.0%) 0.1655 GG 6 (100.0%) 33 (75.0%) 39 (78.0%) Total 6 (100%) 44 (100%) 50 (100%) Ca rs1800861 GG 0 (0.0%) 5 (11.4%) 5 (10.0%) 0.1238 TG 0 (0.0%) 14 (31.8%) 14 (28.0%) TT 6 (100.0%) 25 (56.8%) 31 (62.0%) Total 6 (100%) 44 (100%) 50 (100%) Table 6: Association of Ca rs1800860 and Ca rs1800861 Genotypes with CONS Presence CONS Absent Present Total P-Value Ca rs1800860 AG 2 (10.5%) 9 (29.0%) 11 (22.0%) 0.1252 GG 17 (89.5%) 22 (71.0%) 39 (78.0%) TOTAL 19 (100%) 31 (100%) 50 (100%) Ca rs1800861 GG 0 (0.0%) 5 (16.1%) 5 (10.0%) 0.0282 TG 3 (15.8%) 11 (35.5%) 14 (28.0%) TT 16 (84.2%) 15 (48.4%) 31 (62.0%) TOTAL 19 (100%) 31 (100%) 50 (100%) Table 7: Association of Ca rs1800860 and Ca rs1800861 Genotypes with PERF GUT Presence PERF GUT Absent Present Total P-Value Ca rs1800860 AG 10 (20.8%) 1 (9.1%) 11 (22.0%) 0.3292 GG 38 (79.2%) 1 (2.6%) 39 (78.0%) TOTAL 48 (96.0%) 2 (4.0%) 50 (100%) Ca rs1800861 GG 5 (10.4%) 0 (0.0%) 5 (10.0%) 0.5281 TG 14 (29.2%) 0 (0.0%) 14 (28.0%) TT 29 (60.4%) 2 (100.0%) 31 (62.0%) TOTAL 48 (100%) 2 (100%) 50 (100%) Among the 50 participants, the majority were aged 1–28 days (56%), followed by >365 days (32%) and 29–365 days (12%), with a statistically significant age distribution (p < 0.05). Males predominated (70%) compared to females (30%). The religious distribution was nearly equal between Hindus (50%) and Muslims (48%), with only one Christian participant (2%). Most participants did not have Down syndrome (96%). The most common surgical site was SSD (78%), followed by LSD (14%) and TCA (8%), while biopsy (bx) was the predominant surgery type (64%) over debridement (def) (36%). Genetic analysis showed Ca rs1800860 primarily as GG (78%) and AG (22%), while Ca rs1800861 was mainly TT (62%), followed by TG (28%) and GG (10%). For CO rs1800860, GG was most frequent (60%), followed by AG (34%) and AA (6%), whereas CO rs1800861 was predominantly TG (56%), followed by GG (26%) and TT (18%). Among the 50 participants, 82% had a history of C/F AD, which was statistically significant (p < 0.05), while 18% did not. Most participants also had C/F DPM (88%) and C/F BV (66%), with the remaining being absent in 12% and 34%, respectively. Consistency (Cons) was present in 62% of cases. Follow-up (FU) was noted in 36% of participants, whereas 64% did not have follow-up. Gastrointestinal perforation (Perf Gut) was rare, occurring in only 4% of participants, with the majority (96%) unaffected. The distribution of Ca rs1800860 genotypes differed significantly across surgical sites (p < 0.0001). Among participants with the AG genotype, 90.9% underwent SSD, while only 9.1% had LSD and none had TCA. For the GG genotype, the majority were also in SSD (74.4%), followed by LSD (85.7%) and TCA (100%). Overall, AG was present in 22% and GG in 78% of participants. For Ca rs1800861, TT was the predominant genotype (62%), with all LSD and TCA cases carrying TT, while in SSD, 51.3% were TT, 35.9% TG, and 12.8% GG. TG accounted for 28% overall, and GG was least frequent (10%). In the study population, for Ca rs1800860, the AG genotype was observed in 26.8% of participants with AD and absent in those without AD, while the GG genotype was present in 73.2% of participants with AD and 100% of those without AD. The association was not statistically significant (P = 0.0785). For Ca rs1800861, the TG and TT genotypes were more frequent among participants with AD (31.7% and 56.1%, respectively) compared to those without AD, whereas the GG genotype was absent in participants without AD and present in 12.2% of those with AD. This association also did not reach statistical significance (P = 0.1742). Among participants, for Ca rs1800860, the AG genotype was observed in 25.0% of those with DPM and absent in participants without DPM, whereas the GG genotype was present in 75.0% of participants with DPM and 100% of those without DPM. This association was not statistically significant (P = 0.1655). For Ca rs1800861, the TG and TT genotypes were observed in 31.8% and 56.8% of participants with DPM, respectively, while the GG genotype was present in 11.4% of participants with DPM and absent in those without DPM. This association was also not statistically significant (P = 0.1238). In participants with CONS, the AG genotype of Ca rs1800860 was observed in 29.0% and the GG genotype in 71.0%, compared to 10.5% and 89.5%, respectively, in those without CONS; this association was not statistically significant (P = 0.1252). For Ca rs1800861, the GG genotype was present in 16.1% of participants with CONS and absent in those without CONS, while the TG and TT genotypes were observed in 35.5% and 48.4% with CONS, respectively. This association was statistically significant (P = 0.0282), suggesting a potential link between Ca rs1800861 genotypes and CONS presence. For Ca rs1800860, the AG genotype was observed in 9.1% of participants with PERF GUT and 20.8% of those without, while the GG genotype was present in 2.6% with PERF GUT and 79.2% without. This difference was not statistically significant (P = 0.3292). For Ca rs1800861, the TT genotype was observed in all participants with PERF GUT (100%) and 60.4% without, whereas the GG and TG genotypes were present only in participants without PERF GUT. The association between Ca rs1800861 genotypes and PERF GUT was not statistically significant (P = 0.5281).

In this study of 50 participants, early-onset presentations were predominant, with the majority aged 1–28 days (56%), followed by >365 days (32%) and 29–365 days (12%) [11]. This age distribution aligns with Singh et al., who reported a similar neonatal predominance in congenital and early pediatric disorders [12]. The significant p-value (<0.05) in age distribution suggests a relationship between age and disease onset or surgical timing, consistent with Chaudhary et al., who observed early surgical interventions are more common in neonates and infants due to symptomatic presentations [13]. Males constituted 70% of the study population, indicating male predominance, in agreement with Kumar et al., who reported higher male representation in congenital and pediatric surgical conditions [14]. Religious distribution was nearly equal between Hindus (50%) and Muslims (48%) and was not statistically significant, consistent with Ali et al., who found no religion-based differences in pediatric surgical presentations [15]. Clinically, 82% had a history of C/F AD, 88% had C/F DPM, and 66% had C/F BV, reflecting frequent multi-feature involvement, as observed by Ramaswamy et al. [16]. Consistency (Cons) was noted in 62% of participants, while only 36% had follow-up (FU), highlighting heterogeneity and challenges in post-operative monitoring. Gastrointestinal perforation (Perf Gut) was rare (4%), consistent with complication rates reported in similar pediatric studies [17]. Regarding surgical characteristics, SSD was the most common site (78%), followed by LSD (14%) and TCA (8%). Biopsy (bx) was the predominant procedure (64%) compared to debridement (36%), similar to findings by Bose et al., who reported preference for biopsy in early or uncertain diagnostic stages [18]. Genetic analysis revealed Ca rs1800860 as predominantly GG (78%) over AG (22%), and Ca rs1800861 as TT (62%), TG (28%), and GG (10%). This distribution aligns with Chen et al., who reported predominance of homozygous variants (GG or TT) in Asian populations [19]. The significant distribution difference of Ca rs1800860 across surgical sites (p < 0.0001) suggests genetic variation may influence disease localization or tissue response. Participants with AG genotype predominantly underwent SSD (90.9%), indicating genotype-linked predilection, consistent with Lee et al., who demonstrated that calcium channel gene polymorphisms can modulate local tissue remodeling [20].

Fractionated intrathecal injection provides a more controlled onset, greater hemodynamic stability, and fewer adverse effects compared to bolus injection, while maintaining comparable anesthetic efficacy and duration. Therefore, it represents a safer and more reliable technique for lower abdominal, lower limb, and perineal surgeries under spinal anesthesia.

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