Short-segment Hirschsprung Disease Research Articles

Is It Safe to Operate without Frozen Section Biopsies in Short-Segment Hirschsprung's Disease? An Overview of 60 Cases.

Background: Advancements in surgical management in a single-stage procedure made intraoperative frozen section biopsies critical for determining of level of resection to avoid the potential risk of leaving a retained aganglionic segment. However, in most low-income countries, due to the lack of this facility, the surgeon's intraoperative judgment is used for the determination of the resection level. Objective: This study aims to evaluate the accuracy of determining the level of bowel resection in short-segment Hirschsprung's disease based on macroscopic changes. Materials and methods: Intraoperative macroscopic evaluations were assessed using postoperative microscopic findings to determine whether the surgeons' intraoperative judgments were accurate in determining the level of bowel resection in 60 cases of operated short-segment Hirschsprung's disease. In addition, Pearson's correlation coefficient was used to determine whether the sensitivity and specificity of both methods were significantly correlated. Results: The microscopic results showed that the level of resection based on the macroscopic evaluation was performed in normally ganglionated segment in cases of short-segment Hirschsprung's disease. Conclusions: Macroscopic intraoperative assessment by an experienced surgeon is highly accurate method of determining the level of bowel resection in short-segment HSCR.

Outcome of Primary Transanal pull through for Short segment Hirschsprung's disease in children without frozen section

BackgroundHirschsprung's disease is a congenital disorder of the enteric nervous system. There are many type of pull through surgeries (e.g.) Duhamel, Soave, Swenson etc. and are done openly as well as laparoscopically. This can be performed in multiple stages or in a single stage (primary). MethodologyThis cross sectional study was conducted at Pediatric Surgery Department, Lady Reading Hospital, Peshawar from January 2022 till December 2022, after approval from ethical committee (IRB # 489/LRH/MTI). All Children up to 16 year age presented with short segment Hirschsprung's disease on contrast enema and with confirmed absence of ganglion cells on rectal biopsy were included in the study. Patients with syndromic Hirschsprung's disease, those with stoma, previous surgery, not responding/compliant to rectal washout and parents unwilling for the procedure were excluded. All patient underwent Primary Transanal Pull Through (PTAPT) without frozen section. All patients were followed up to 6 months post-op. ResultsOut of 88 patients, 18(20.4%) had complications. Anastomotic stricture was found to be the most common. Complications were found to be more in male patients who were less than 6 months old. None of the patient had aganglionic or transitional zone pull through. ConclusionPTAPT can be done safely in Short segment Hirschsprung's disease in centers without frozen section facility with comparable results. At least 5 cm resection should be done proximal to the transition zone to avoid aganglionic bowel or transition zone pull through. The incidence of complications is more in males and in patients aged less than 6 months. Level of evidenceLevel IV

Comparison of clinical features and prognosis between ultrashort-segment and short-segment hirschsprung disease.

To compare the differences in clinical features, postoperative complications, and long-term bowel function outcomes of ultrashort-segment Hirschsprung disease (USHD) and short-segment Hirschsprung disease (SHD). A retrospective study was conducted to compare patients with USHD or SHD who underwent transanal endorectal pull-through (TEPT) at Beijing Children's Hospital between January 2014 and June 2021. Clinical details were collected from medical records. A long-term bowel function questionnaire (age > 4 years old) was completed by the patients' parents. A total of 84 patients (USHD = 15, SHD = 69) were included. Age at diagnosis and radical surgery in the USHD group were significantly older than the SHD group (46 [38, 66] vs. 34 [6, 55] months, p = 0.002; 51 [39, 68] vs. 37 [10, 68] months, p = 0.001, respectively). Compared with the SHD group, patients with USHD are more likely to suffer anastomosis leakage and postoperative enterocolitis after TEPT ([3/15, 33.3%] vs. [1/69, 1.4%], p = 0.017; [5/15, 33.3%] vs. [6/69, 8.7%], p = 0.023). In addition, patients in the USHD group are inclined to suffer lower bowel function scores (12.0 [7.5, 18.3] vs. 17 [15, 19], p = 0.018).Patients in the USHD group were more likely to suffer poorer ability to hold back defecation (p = 0.023), soiling (p = 0.011), fecal accidents (p = 0.004), and social problems (p = 0.004). Compared with patients with SHD, patients with USHD are diagnosed and performed TEPT at an older age. and they are inclined to suffer postoperative enterocolitis, anastomosis leakage, and poorer long-term bowel function following TEPT.

The transition zone in Hirschsprung's bowel contains abnormal hybrid ganglia with characteristics of extrinsic nerves

The aganglionic bowel in short-segment Hirschsprung's disease is characterized both by the absence of enteric ganglia and the presence of extrinsic thickened nerve bundles (TNBs). The relationship between the TNBs and the loss of enteric ganglia is unknown. Previous studies have described decreasing numbers of ganglia with increasing density of TNBs within the transition zone (TZ) between ganglionic and aganglionic gut, and there is some evidence of spatial contact between them in this region. To determine the cellular interactions involved, we have analysed the expression of perineurial markers of TNBs and enteric ganglionic markers for both neural cells and their ensheathing telocytes across four cranio-caudal segments consisting of most proximal ganglionic to most distal aganglionic from pull-through resected colon. We show that in the TZ, enteric ganglia are abnormal, being surrounded by perineurium cells characteristic of TNBs. Furthermore, short processes of ganglionic neurons extend caudally towards the aganglionic region, where telocytes in the TNB are located between the perineurium and nerve fibres into which they project telopodes. Thus, enteric ganglia within the TZ have abnormal structural characteristics, the cellular relationships of which are shared by the TNBs. These findings will help towards elucidation of the cellular mechanisms involved in the aetiology of Hirschsprung's disease.

Is the appendix a good organ to diagnose total colonic aganglionosis?

The use of the appendix for diagnosis of Total Colonic Aganglionosis (TCA) remains controversial. This study aimed to categorize the presence of ganglion cells in the appendix and determine its reliability as a histological specimen for the diagnosis of TCA. This was a combined retrospective and prospective study. Permanent histological specimens of normal appendices removed during appendectomy for malrotation or falsely presumed appendicitis, and from patients with short segment Hirschsprung's disease (HD) or TCA were included. Permanent specimens of the appendix tip from Malone procedures were prospectively collected. All specimens were independently reviewed by two experienced pathologists in a standardized manner to assess for the presence of ganglion cells. A total of 112 appendices were evaluated. Nine came from patients with a diagnosis of TCA, and five from patients with HD. Ganglion cells were present in all specimens from patients with diagnoses other than TCA and were absent in all specimens from patients with TCA. In the correct clinical setting, the absence of ganglion cells in the appendix can allow for a reliable diagnosis of TCA.

Optimal timing for Soave primary pull-through in short-segment Hirschsprung disease: A meta-analysis

BackgroundThe optimal age for endorectal pull-through (ERPT) surgery in infants with short-segment Hirschsprung disease varies, with a trend toward earlier surgery. However, it is unclear if the timing of surgery impacts functional outcomes. We undertook the present study to determine the optimal timing of ERPT in infants with short-segment Hirschsprung disease. MethodsThe NCBI PubMed database was searched for English-language manuscripts published between 2000 and 2019 analyzing functional outcomes for patient following the initial Soave ERPT for short-segment Hirschsprung disease. Raw data from these studies was obtained from the corresponding author for each manuscript. We combined data from these papers with our own institutional data and performed a meta-analysis. ResultsA total of 780 infants were included in our meta-analysis. Constipation occurred in 1.0–31.7%, soiling 1.3–26.0%, anastomotic stricture 0.0–14.6%, and anastomotic leak 0.0–3.4%. Regarding age at ERPT, younger infants at the time of initial corrective surgery had higher rates of soiling, stricture, and leak. On sub-group analysis, patients <2.5 months at their initial corrective surgery had higher rates of soiling (25.9% vs. 11.4%, p<0.01), as well as stricture (10.0% vs 1.7%, p<0.01) and leak (5.5% vs 1.3%, p<0.01). ConclusionWhile age at Soave endorectal pull-through for short-segment Hirschsprung disease has decreased over time, functional outcomes associated with this trend have only recently been examined. Our findings suggest that patients <2.5 months old at the time of endorectal pull-through may have worse functional outcomes, emphasizing the need to consider further study of the timing of surgery in this population.

Machine learning-based quantitative analysis of barium enema and clinical features for early diagnosis of short-segment Hirschsprung disease in neonate

ObjectiveTo develop a mathematical model based on a combination of clinical and radiologic features (barium enema) for early diagnosis of short-segment Hirschsprung disease (SHSCR) in neonate. MethodsThe analysis included 54 neonates with biopsy-confirmed SHSCR (the cases) and 59 neonates undergoing barium enema for abdominal symptoms but no Hirschsprung disease (the control). Colon shape features extracted from barium enema images and clinical features were used to develop diagnostic models using support vector machine (SVM) and L2-regularized logistic regression (LR). The training cohort included 32 cases and 37 controls; testing cohort consisted 22 cases and 22 controls. Results were compared to interpretation by 2 radiologists. ResultsIn the analysis by radiologists, 87 out of 113 cases were correctly classified. Six SHSCR cases were mis-classified into the non-HSCR group. In the remaining 20 cases, radiologists were unable to make a decision. Both the SVM and LR classifiers contained five clinical features and four shape features. The performance of the two classifiers was similar. The best model had 86.36% accuracy, 81.82% sensitivity, and 90.91% specificity. The AUC was 0.9132 for the best-performing SVM classifier and 0.9318 for the best-performing LR classifier. ConclusionA combination of clinical features and colon shape features extracted from barium enemas can be used to improve early diagnosis of SHSCR in neonate.

Functional outcomes of patients with short-segment Hirschsprung disease after transanal endorectal pull-through

BackgroundTransanal endorectal pull-through (TEPT) is considered the most preferable treatment method for Hirschsprung disease (HSCR) since it is less invasive and has fewer morbidities than transabdominal pull-through. Here, functional outcomes in short-segment HSCR patients after TEPT were assessed and associated with the prognostic factors.MethodsKrickenbeck classification was used to assess the functional outcomes in patients with HSCR after TEPT surgery at our institution from 2012 to 2020.ResultsFifty patients were involved in this study. Voluntary bowel movement (VBM) was achieved in 82% of subjects. Nine (18%) subjects had soiling grade 1, while two (4%) and two (4%) patients suffered constipation that was manageable with diet and laxative agents, respectively. Patients who underwent TEPT at ≥ 4 years old tended to have soiling more than patients who underwent TEPT at < 4 years old (OR = 16.47 [95% CI 0.9–301.61]; p = 0.06), whereas patients with post-operative complications had 10.5-fold higher risk for constipation than patients without post-operative complications (p = 0.037; 95% CI 1.15–95.92). Multivariate analysis showed male sex was significantly associated with VBM (OR = 9.25 [95% CI 1.34–63.77]; p = 0.024), while post-operative complications were strongly correlated with constipation (OR = 10 [95% CI 1.09–91.44]; p = 0.04).ConclusionsThe functional outcomes of HSCR patients after TEPT in our institution are considered relatively good. Moreover, the VBM, soiling, and constipation risk after TEPT might be affected by sex, age at TEPT performed, and post-operative complications, respectively, while the age at TEPT performed might not be associated with functional outcomes. Further multicenter studies with a larger sample size are necessary to clarify and confirm our findings.

Increasing Trend of Calretinin-Positive Mucosal Innervation from Aganglionic Zone toward Transition Zone in Hirschsprung's Disease.

Diagnosis of very short-segment Hirschsprung's disease (vsHD) by rectal suction biopsy is challenging as its aganglionic zone (AZ) overlaps with physiologic hypoganglionic zone and calretinin-positive mucosal nerves may extend from the transition zone (TZ) into AZ. We studied whether an increasing trend/gradient of calretinin-positive mucosal nerves along the distance from AZ toward TZ aids in diagnosis of HD. In this study, 46 rectal suction biopsies from non-HD and HD, and 15 pull-through specimens from short-segment HD were evaluated by mucosal calretinin immunostain (CI) and image processing and analysis (IPA) to measure pixel count (PC, the percentage of calretinin stained pixels in the mucosa). Consecutive longitudinal sections of proximal AZ toward distal TZ in HD pull-through specimens were utilized as a vsHD surrogate model. First, we studied variability of mucosal CI in non-HD biopsies along the distance from dentate line. Second, we determined a cutoff point of mucosal CI by IPA that separated non-HD versus HD and applied this cutoff to longitudinal sections from proximal AZ to distal TZ segments in HD pull-through specimens. Third, we studied whether an increasing trend of mucosal CI was universally observed in HD pull-through. Our findings included a significant variability in PC along the biopsy distance in non-HD cases. Positive mucosal CI was found in proximal AZ in 6 (43%) of 14 HD pull-through, among which 1 case lacked submucosal nerve hypertrophy in the proximal AZ. All 14 HD pull-through cases showed an increasing trend/gradient of PC from AZ toward TZ. Based on our findings, the presence or absence of mucosal CI positivity and submucosal nerve hypertrophy may not reliably diagnose vsHD in rectal suction biopsy. While we acknowledge that the density of mucosal innervation in variable contexts and anatomical locations is unknown and yet to be explored, our study suggests that an increasing trend of positive mucosal CI from AZ toward TZ by IPA might prove to be a useful tool for the diagnosis of vsHD in the future.

Predictive factors for the development of postoperative Hirschsprung-associated enterocolitis in children operated during infancy

The risk factors for postoperative Hirschsprung-associated enterocolitis (HAEC) are still incompletely understood, especially age at which surgery is performed. Therefore, the aim of this study was to identify the risk factors for the development of postoperative HAEC in children operated during infancy. Thirty-five children who had undergone radical surgery for Hirschsprung disease (HD) during infancy were included in the study. They were divided into two groups; those who developed postoperative HAEC (HAEC, 14 patients) and those who did not (no HAEC, 21 patients). Their medical records were retrospectively reviewed for clinical details. Developing postoperative HAEC was significantly associated with long-segment HD (p = 0.020) and the age at radical surgery (p = 0.0241). No other factors had a significant association with postoperative HAEC. In the patients who developed postoperative HAEC (n = 14), those with Trisomy 21 had significantly longer hospitalizations than those without. Patients with long-segment HD had a higher hospitalization rate than those with short-segment HD. This study clearly showed that long-segment HD and older age at radical surgery are risk factors for developing postoperative HAEC.

Whole-genome analysis of noncoding genetic variations identifies multiscale regulatory element perturbations associated with Hirschsprung disease.

It is widely recognized that noncoding genetic variants play important roles in many human diseases, but there are multiple challenges that hinder the identification of functional disease-associated noncoding variants. The number of noncoding variants can be many times that of coding variants; many of them are not functional but in linkage disequilibrium with the functional ones; different variants can have epistatic effects; different variants can affect the same genes or pathways in different individuals; and some variants are related to each other not by affecting the same gene but by affecting the binding of the same upstream regulator. To overcome these difficulties, we propose a novel analysis framework that considers convergent impacts of different genetic variants on protein binding, which provides multiscale information about disease-associated perturbations of regulatory elements, genes, and pathways. Applying it to our whole-genome sequencing data of 918 short-segment Hirschsprung disease patients and matched controls, we identify various novel genes not detected by standard single-variant and region-based tests, functionally centering on neural crest migration and development. Our framework also identifies upstream regulators whose binding is influenced by the noncoding variants. Using human neural crest cells, we confirm cell stage–specific regulatory roles of three top novel regulatory elements on our list, respectively in the RET, RASGEF1A, and PIK3C2B loci. In the PIK3C2B regulatory element, we further show that a noncoding variant found only in the patients affects the binding of the gliogenesis regulator NFIA, with a corresponding up-regulation of multiple genes in the same topologically associating domain.

Contemporary management of Hirschsprung disease in New Zealand.

The aim of this study was to report the contemporary management of Hirschsprung disease (HD) in New Zealand. We undertook a national multi-centre retrospective review of all newly diagnosed cases of HD during a 16-year period (2000-2015). Demographics, genetic and syndromic associations, family history, radiology and histology results and surgical interventions were analysed. A total of 246 cases (males:females 4:1) were identified, an incidence of 1:3870 live births. Short-segment disease was present in 81.7%, long-segment disease in 8.5%, total colonic aganglionosis in 6.5% and unknown in 3.3%. HD was diagnosed by 4 weeks' corrected gestational age in 67%. Thirty cases (12%) also had Trisomy 21. Fifty-three (21.5%) patients required a repeat rectal biopsy for definitive diagnosis. A contrast enema was performed in 55% and identified the transition zone with 69% accuracy. Primary pull-through surgery was undertaken in 59% (65% of short-segment cases) at a median age of 27 days; others were initially managed by a defunctioning stoma. The commonest definitive procedure was a Soave-Boley endorectal pull-through (79%) (or similar variant). During a median follow-up of 7.4 years, six (2.5%) survivors underwent a redo pull-through, 13 (5.5%) an appendicostomy, 16 (6.8%) a defunctioning stoma and 10 never had a definitive procedure. Total colonic aganglionosis was significantly more likely to be fatal (12.5% versus 0.5%, P < 0.0005) or associated with a permanent end stoma (27.5% versus 4.5%, P < 0.0005). Most New Zealand born infants with short-segment HD are currently managed by primary pull-through, usually in the first months of life.

Pan-enteric neuropathy and dysmotility are present in a mouse model of short-segment Hirschsprung disease and may contribute to post-pullthrough morbidity

PurposeHirschsprung disease (HSCR) is characterized by distal intestinal aganglionosis. While surgery is lifesaving, gastrointestinal (GI) motility disorders persist in many patients. Our objective was to determine whether enteric nervous system (ENS) abnormalities exist in the ganglionated portions of the GI tract far proximal to the aganglionic region and whether these are associated with GI dysmotility. MethodsUsing Ednrb-null mice, a model of HSCR, immunohistochemical analysis was performed to evaluate quantitatively ENS structure in proximal colon, small intestine, and stomach. Gastric emptying and intestinal transit were measured in vivo and small and large bowel contractility was assessed by spatiotemporal mapping ex vivo. ResultsProximal colon of HSCR mice had smaller ganglia and decreased neuronal fiber density, along with a marked reduction in migrating motor complexes. The distal small intestine exhibited significantly fewer ganglia and decreased neuronal fiber density, and this was associated with delayed small intestinal transit time. Finally, in the stomach of HSCR mice, enteric neuronal packing density was increased and gastric emptying was faster. ConclusionsENS abnormalities and motility defects are present throughout the ganglionated portions of the GI tract in Ednrb-deficient mice. This may explain the GI morbidity that often occurs following pull-through surgery for HSCR.

Is Single-stage Primary Transanal Endorectal Pull through (TEP) feasible for Short Segment Hirschsprung's Disease in Neonates?- Our Experience in a Tertiary Hospital

Background: The surgical management of Hirschsprung's Disease (HD) includes so many procedures. TransanalEndorectal Pull through (TEP) represents the latest development in the concept of the minimally invasive surgery for HD. The purpose of this study was to evaluate the out come of the procedure in neonates.&#x0D; Methods: This retrospective study was carried out in a tertiary pediatric hospital during the period from January 2007 to December 2012 (5 years). The study included neonates of both sex, weight more than 2 kg, who were clinically suspected HD, radiologically transition zone at rectosigmoid and midsigmoid region and rectal biopsy proven HD and no evidence of sepsis or entrocolitis. Short segment HD with associated anomalies, and operated cases with less than 6 months' or irregular follow up were excluded.&#x0D; Results: During study period, single-stage transanalendorectal pull through (TEP) operation was done for short segment HD in 63 neonates, 9 patients were excluded from the study for irregular follow up. The mean operative time, mean blood loss, postoperative hospital stay, follow up period were 113 minutes, 20ml, 6.8 days and 19.6 months respectively. Transverse colostomy was needed in 6 patients for anastomotic leakage, thereafter developed anastomotic stricture, managed with regular anastomotic dilatation.&#x0D; Conclusion: Advancement in pediatric anesthesia, improvement of pediatric surgical expertise, perioperative management and nursing care has made single-stage primary transanalendorectal pull-through a feasible and safe surgical procedure for the treatment of short segment Hirschsprung's disease in neonate.&#x0D; Journal of Surgical Sciences (2014) Vol. 18 (2) : 45-50

Risk factors for the development of post-operative enterocolitis in short segment Hirschsprung's disease.

The objective of this study is to identify risk factors associated with the development of post-operative enterocolitis (HAEC), in short segment Hirschsprung's disease (HSCR-S). A retrospective study was carried out for post-operative patients with HSCR-S from 1997 to 2017. HSCR-S was defined as the most proximal extension of aganglionosis limited to the sigmoid colon. An episode of HAEC was defined as the presence of (1) vomiting or explosive diarrhea; (2) abdominal distension; (3) fever and (4) leukocytosis. Risk factors for the development of HACE were determined using multivariate logistic regression. The medical records of 96 patients were reviewed. The overall incidence of HAEC was 20.8% (n = 20) and 65.0% (n = 13) of HAEC occurred within the first year of operation. After a univariate logistic regression analysis, three risk factors for HAEC were identified: (1) presence of other major anomalies [OR: 1.43 (1.12-2.32), p = 0.041]; (2) creation of pre-operative defunctioning stoma [OR: 2.28 (1.47-3.23), p = 0.035]; (3) extension of aganglionosis to the sigmoid colon [OR: 1.89 (1.05-3.19), p = 0.049]. After multivariate logistic regression analysis, a significant association was demonstrated for creation of pre-operative defunctioning stoma [OR: 1.81 (1.08-3.22), p = 0.045] and extension of aganglionosis to the sigmoid colon [OR: 1.91 (1.37-2.98), p = 0.038]. The requirement of pre-operative defunctioning stoma and a more proximal extension of aganglionosis are risk factors for the development of post-operative HAEC in HSCR-S. Patients with these risk factors should be closely followed up especially during the first year after the operation.

Optimal time for single-stage pull-through colectomy in infants with short-segment Hirschsprung disease.

Short-segment Hirschsprung disease (HSCR) is the predominant type of HSCR that affects approximately 75% of patients. Whether single-stage endorectal pull-through (ERPT) surgery is appropriate for neonatal patients with HSCR has not been definitively determined. This retrospective cohort study concerning infants with short-segment HSCR investigated the optimal age for single-stage ERPT surgery, regardless of the operative approach. The 198 patients were stratified by operative age ≤ 3 or > 3months (groups A or B, respectively, n = 62 and 136, respectively). Diagnoses of short-segment HSCR were conducted by preoperative contrast enema and rectal suction biopsy with acetylcholinesterase immunohistochemical staining. The perioperative clinical course for all patients was reviewed and the accuracy rate of the preoperative diagnoses and postoperative short- and midterm outcomes were assessed. The rates of diagnostic accuracy, according to the results of the preoperative contrast enema or rectal suction biopsy, were lower in group A (67.2 and 93.5%, respectively) than in group B (81.4 and 94.9%, respectively). In groups A and B, 49 (79.1%) and 108 (79.4%) infants, respectively, completed follow-up examinations. The short-term outcomes were postoperative HSCR-associated enterocolitis, adhesive bowel obstruction, anastomosis leakage, and anal stenosis during the first 12months after surgery. The midterm outcomes were incontinence and constipation at ~24months after surgery. Compared with group B, group A experienced more incidences of anastomotic leakage in the short-term and more soiling in the midterm. In groups A and B, the rates of constipation recurrence were nil and 1.9%, respectively. Infants with HSCR ≤3months old at the time of single-stage ERPT surgery showed lower rates of accurate and conclusive diagnostic results and poorer postoperative outcomes. Waiting to perform this surgery until infants are older might be more beneficial.

Utility of Image Processing and Analysis (IPA) for Diagnosis of Very Short Segment Hirschsprung Disease (vsHD)

Utility of Image Processing and Analysis (IPA) for Diagnosis of Very Short Segment Hirschsprung Disease (vsHD)

Are all patients with short segment Hirschsprung's disease equal? A retrospective multicenter study.

Short segment Hirschsprung's disease (HSCR) carries a better prognosis than long segment disease, but the definition of short is controversial. The objective of this study is to determine anatomically the extent of disease involvement that would be associated with a better functional outcome. This is a retrospective multicenter (n = 3) study with patients (≥ 3years) who had transanal pullthrough operation done for aganglionosis limited to the recto-sigmoid colon were reviewed. The extent of disease involvement and bowel resection was retrieved by reviewing the operative records as well as histopathological reports of the resected specimens. Clinical assessment was performed according to the criteria of a seven-itemed bowel function score (BFS) (maximum score = 20). Manometric assessment was performed with anorectal manometry. The study period started from 2003 to 45 patients were studied with median age at assessment = 52.0 months and operation = 3.0months. The disease involvement was categorized into upper sigmoid-descending colon (DC) (n = 8), sigmoid colon (SC) (n = 12), upper rectum (UR) (n = 14) and lower rectum (LR) (n = 11) according to the level of normal biopsy result. There was no significant difference in the age of assessment between the four groups. The median BFSs in the DC, SC, UR and LR were 13, 15, 17 and 17, respectively (p = 0.01). Nine patients from the DC and SC groups reported soiling for more than twice per week. Sub-group analysis comparing patients with and without the entire sigmoid colon resected revealed worse functional outcomes in terms of the incidence of soiling (40.7 vs 22.2%, p = 0.05) and the BFS (14 vs 18, p = 0.04) in the former group. Anorectal manometry did not reveal any significant difference between the four groups, but a higher proportion of patients in the UR and LR groups appeared to have a normal sphincter resting pressure (DC vs SC vs UR vs LR = 62.5 vs 75.0 vs 85.7 vs 80.0%, p = 0.10). Patients with short segment HSCR are not equal at all. HSCR patients with aganglionosis limited to the rectum without the need of removing the entire sigmoid colon have a better bowel control and overall functional score. Less bowel loss and colonic dissection maybe the underlying reasons. Although future studies with a larger sample size and a longer follow-up period are required to validate the results of this study, it has provided a new insight to the current understanding of short segment disease in HSCR.

Correction of Hirschsprung-Associated Mutations in Human Induced Pluripotent Stem Cells Via Clustered Regularly Interspaced Short Palindromic Repeats/Cas9, Restores Neural Crest Cell Function

Hirschsprung disease is caused by failure of enteric neural crest cells (ENCCs) to fully colonize the bowel, leading to bowel obstruction and megacolon. Heterozygous mutations in the coding region of the RET gene cause a severe form of Hirschsprung disease (total colonic aganglionosis). However, 80% of HSCR patients have short-segment Hirschsprung disease (S-HSCR), which has not been associated with genetic factors. We sought to identify mutations associated with S-HSCR, and used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system to determine how mutations affect ENCC function. We created induced pluripotent stem cell (iPSC) lines from 1 patient with total colonic aganglionosis (with the G731del mutation in RET) and from 2 patients with S-HSCR (without a RET mutation), as well as RET+/- and RET-/- iPSCs. IMR90-iPSC cells were used as the control cell line. Migration and differentiation capacities of iPSC-derived ENCCs were analyzed in differentiation and migration assays. We searched for mutation(s) associated with S-HSCR by combining genetic and transcriptome data from patient blood- and iPSC-derived ENCCs, respectively. Mutations in the iPSCs were corrected using the CRISPR/Cas9 system. ENCCs derived from all iPSC lines, but not control iPSCs, had defects in migration and neuronal lineage differentiation. RET mutations were associated with differentiation and migration defects of ENCCs invitro. Genetic and transcriptome analyses associated a mutation in the vinculin gene (VCL M209L) with S-HSCR. CRISPR/Cas9 correction of the RET G731del and VCL M209L mutations in iPSCs restored the differentiation and migration capacities of ENCCs. We identified mutations in VCL associated with S-HSCR. Correction of this mutation in iPSC using CRISPR/Cas9 editing, as well as the RET G731del mutation that causes Hirschsprung disease with total colonic aganglionosis, restored ENCC function. Our study demonstrates how human iPSCs can be used to identify disease-associated mutations and determine how they affect cell functions and contribute to pathogenesis.

Calretinin Variant in Hirschsprung Disease: Pretransitional Sign and Surgical Planning.

Objectives Calretinin immunohistochemistry (IHC) with slight submucosal positivity (variant) has been reported in short forms of Hirschsprung disease (HD). The first aim of our study was to confirm this variant being a pretransitional sign. The second was to evaluate if its presence on suction rectal biopsy was reliable enough for surgical planning. Methods We performed a monocentric retrospective study of all rectosigmoid HD between 2009 and 2014. Suction rectal biopsy defined two groups of calretinin results: P- group with no staining and P+ group with slight positivity. P- group final resection specimen was retrospectively reanalyzed looking for the variant to appear within the aganglionic bowel. P+ group surgical management was analyzed in terms of initial symptoms, radiological results, type of surgery and outcome, before and after formal identification of the variant by our pathologists (2011). Results Overall, 54 patients with rectosigmoid HD were included from 2009 to 2014. In the P- group (48.2%), the variant pattern finally appeared on all surgical resection specimens, 1 cm before the transitional zone (minimum-maximum: 0-5 cm). In the P+ group, 46% of these patients had peroperative biopsies before the transanal pull through before 2011 against 10% after 2011, with 100% of ganglionic coloanal anastomosis and similar outcome. Conclusion We confirmed our hypothesis of this varied pattern of calretinin being a pretransitional sign only visible on rectal suction biopsies in short segment HD and reliable enough to plan direct transanal pull through.