Segregation of a missense variant in enteric smooth muscle actin γ-2 with autosomal dominant familial visceral myopathy.

BACKGROUND & AIMS: Familial visceral myopathy (FVM) is a rare inherited form of myopathic pseudo-obstruction; little is known about the genetic factors that cause this disorder. FVM is characterized by impaired functions of enteric smooth muscle cells, resulting in abnormal intestinal motility, severe abdominal pain, malnutrition, and even death. We searched for genetic factors that might cause this disorder. METHODS: We performed whole-exome sequence analysis of blood samples from 2 individuals in a family that had 7 members diagnosed with FVM. Sanger sequencing was used to analyze additional family members and 280 individuals without this disorder (controls). Intestinal tissue samples from 4 patients and 2 controls were analyzed by immunohistochemistry. Functional studies, including immunofluorescence, cell contractility, and actomyosin structure analyses, were performed using CRL-1976 and U2OS sarcoma cell lines. RESULTS: Whole-exome sequence analysis of DNA from 2 siblings identified 83 gene variants that were shared between the siblings and considered as possible disease-causing changes. A heterozygous variant, R148S in enteric smooth muscle actin γ-2 (ACTG2), segregated with disease phenotype. Intestinal smooth muscle (muscularis propria) from individuals with FVM had reduced levels of cytoplasmic ACTG2 and abnormal accumulation of the protein into intracellular inclusions compared with controls. Sarcoma cells that expressed exogenous ACTG2(R148S) incorporated reduced amounts of this protein into actin filaments compared with cells expressing ACTG2(wt) (P < .001). ACTG2(R148S) also interfered with actin cytoskeleton organization and the contractile activities of the cells, indicating a dominant-negative effect. These findings, along with the site of the variation in the protein, indicate that ACTG2 R148S interferes with actin filament assembly. CONCLUSIONS: We identified the R148S variant in ACTG2 as a cause of FVM in one family. The altered ACTG2 protein appears to aggregate, rather than form actin filaments, in intestinal smooth muscle tissue. This defect could impair contraction of the visceral smooth muscle cells and reduce bowel motility.

Abnormal development of intrinsic innervation in murine embryos with anorectal malformations.

INTRODUCTION: Constipation, soiling, and incontinence are common problems after definitive repair of anorectal malformations (ARMs) in children. We studied the expression of substance P (SP), vasoactive intestinal peptide (VIP), and c-kit in the rectum of murine embryos with or without ARMs at later developmental stages. METHODS: On the 9th embryonic day (E9), pregnant Institute of Cancer Research mice were fed etretinate, a synthetic vitamin A analogue (60 mg/kg), whereas controls were fed only with sesame oil. Embryos were excised between E14 and E18, and prepared for histological examination. The SP, VIP, and c-kit expressions were examined by immunohistochemical staining for the SP, VIP, and c-kit antigens, respectively. RESULTS: On E14 and E15, the expression levels of the anti-SP and anti-VIP antibodies in the rectum did not differ between the control and etretinate-treated group. However, as compared to the controls, a decreased SP and VIP immunoreactivity was observed in the circular muscle layer of the rectum between E16 and E18. On the other hand, on E14 and E15, the expression of anti-c-kit antibody in the rectum did not differ between the etretinate-treated and control group. However, c-kit immunoreactivity was slightly higher in the circular muscle layer of the rectum in the controls on E16 and E17, and considerably higher on E18 than that of the muscle layer in the etretinate-treated group. CONCLUSION: At later developmental stages, the expression levels of SP, VIP, and c-kit reduced in the circular muscle layer of the rectum in mice with etretinate-induced ARMs. This result indicates that reduced SP, VIP, and c-kit expression levels in the circular muscle layer may cause severe constipation in children who develop severe ARMs after definitive surgery.

Ascending contraction and descending relaxation in the distal colon of mice lacking interstitial cells of Cajal.

Recently an essential role of interstitial cells of Cajal (ICC) within myenteric plexus (ICC-MY) was suggested in ascending contraction and descending relaxation in the mouse ileum. The role of ICC in these neural reflexes was examined in the distal colonic segments prepared from the wild type and c-kit mutant, W/W(V) mice, in the present study. Localized distension of the segments from the wild type mice by using a small balloon resulted in ascending contraction and descending relaxation. In the segments from the mutant mice, localized distension also induced these neural reflexes similar to those observed in the wild type mice. Immunohistochemical examination demonstrated that ICC-MY and ICC present in muscle layers (ICC-IM) were severely disrupted in the mutant mouse, but only ICC, present within submucosal plexus (ICC-SMP), remained unchanged. In the small strips with ICC-SMP absent prepared from the mutant mouse, electrical field stimulation induced contraction or relaxation in the absence or presence of atropine, respectively. It was suggested that ICC have no important role in the ascending and descending neural reflexes in the mouse distal colon, this is in direct contrast to the role of ICC-MY in the ileum.

Absence of peristalsis in the ileum of W/W(V) mutant mice that are selectively deficient in myenteric interstitial cells of Cajal.

It is well known that the enteric nervous system plays a key role in the generation of gastrointestinal peristaltic movements. Recently, the networks of interstitial cells of Cajal (ICC) have been found to be essential in the generation of spontaneous gastrointestinal movements. However, the role of ICC in the mechanisms involved in the generation of peristaltic movements is still controversial. The aim of the present study was to reveal how pacemaker myenteric ICC (ICC-MY) and the enteric nervous system contribute to the mechanisms involved in the generation of intestinal peristalsis. We compared spontaneous peristaltic movements of the ileum in wild type (WT) mice with those in W/W(V) mutant mice which are selectively deficient in ICC-MY. Simultaneous recordings were made from both the circular and longitudinal muscle of a 4-cm long segment of ileum under hydrostatic pressure of 0--0.5 cm H(2)O. Mechanical activity and continuous video-images of the ileum were compared between WT and W/W(V) mutant mice under control conditions, in the presence of N-nitro-L-arginine methyl ester (L-NAME) and after tetrodotoxin (TTX). In the WT mouse ileum, peristaltic waves to propagate from the oral to the anal end were frequently observed. The frequency of these peristaltic waves and their associated synchronous longitudinal and circular muscle contractions was increased by L-NAME. The peristaltic waves were abolished by TTX. In the W/W(V) mutant mouse ileum, no peristaltic waves to propagate from the oral to the anal end were observed in control and even after L-NAME, although the local spontaneously generated longitudinal and circular muscle contractions were enhanced by L-NAME. These local contractions were not abolished by TTX. The results presented here suggested that ICC-MY are essential for the generation of spontaneous intestinal peristaltic movements. It is conceivable that ICC-MY may determine the polarity of the excitation of the intestine such that longitudinal and circular muscle contractions propagate from the oral to the anal end of the intestinal segments, although the question of why ICC-MY are necessary for the neural pathways remains unresolved.

Congenital interstitial cell of cajal hyperplasia with neuronal intestinal dysplasia.

Interstitial cells of Cajal (ICCs) are intestinal pacemaker cells that initiate peristalsis in the stomach and intestine, and are considered to be precursors of gastrointestinal stromal tumors (GISTs). We report a 2-year-old girl who suffered from scanty stool passage since birth. On barium enema, the distal colon was rigid with narrow lumen, whereas the proximal colon was dilated and atonic. She received right hemicolectomy and ileostomy. Histopathologically, there was continuous proliferation of spindle cells located between the layers of the muscularis propria throughout the right colon. These spindle cells were positive for c-kit and CD34 but negative for myogenic or neurogenic markers, indicating they are ICCs. No germline or somatic mutation of the juxtamembrane domain of c-kit gene was detected. In addition, the changes of the submucosal plexus fulfilled the histologic criteria of neuronal intestinal dysplasia type B. To our knowledge, this is the first reported case of congenital ICC hyperplasia. Further studies of ICC development may contribute to better understanding of the pathogenesis of this congenital malformation and the tumorigenesis of GIST.

Intestinal neuronal dysplasia.

Intestinal neuronal dysplasia (IND) is a clinical condition that resembles Hirschsprung's disease. In the past many years investigators have raised doubts about the existence of IND as a distinct histopathologic entity. One strong piece of evidence that IND is a real entity stems from animal models. Recently, two different HOX11L1 knockout mouse models and a heterozygous endothelin B receptor-deficient rat demonstrated abnormalities of the submucous plexus similar to that observed in human IND. This review describes in detail the diagnostic criteria of IND, staining techniques, correlation between histological findings and clinical symptoms, and management of IND.

The development of colon innervation in trisomy 16 mice and Hirschsprung's disease.

AIM: To study the colon innervation of trisomy 16 mouse, an animal model for Down's syndrome, and the expression of protein gene product 9.5 (PGP 9.5) in the stenosed segment of colon in Hirschsprung's disease (HD). METHODS: Trisomy 16 mouse breeding;cytogenetic analysis of trisomy 16 mice; and PG P9.5 immunohistochemistry of colons of trisomy 16 mice and HD were carried out. RESULTS: Compared with their normal littermates, the nervous system of colon in trisomy 16 mice was abnormally developed. There existed developmental delay of muscular plexuses of colon, no submucosal plexus was found in the colon, and there was 5mm aganglionic bowel aparting from the anus in trisomy 16 mice. The mesentery nerve fibers were as well developed as shown in their normal littermates. Abundant proliferation of PGP 9.5 positive nerve fibers was revealed in the stenosed segment of HD colon. CONCLUSION: Trisomy 16 mice could serve as an animal model for Hirschsprung's disease for aganglionic bowel in the distal part of colon. Abundant proliferation of PGP 9.5 positive fibers resulted from extrinsic nerve compensation, since no ganglionic cells were observed in the stenosed segment of the colon in HD. HD has a genetic tendency.

Congenital aganglionosis of the entire colon in neonates.

A report of six cases and their radiological diagnosis in neonates. Total colonic aganglionosis (TCA) is a variety of what is commonly known as long segment Hirschsprung's disease. This is not a rare condition, but is often unrecognized. It has a high mortality, frequently with a complicating enterocolitis. Aganglionosis of some part of the colon should be suspected in all babies who show obstructive plain film changes. In TCA the barium enema changes may easily be passed as unremarkable. However, free ileal reflux during the examination, with a transition point in the ileum, and retention of barium in the entire colon after the examination, are diagnostic.

Alternative operative procedure for total colonic aganglionosis.

The authors have utilized a technique recently described by Boley as the corrective procedure in two patients with complex long segment aganglionosis. The one-stage operation combines a right colon onlay patch for enhanced absorptive and reservoir purposes with an ileoendorectal pull-through. Both patients had only 3 to 4 stools per day by the end of the first postoperative month. The obligatory period of intestinal adaptation needed to achieve an acceptable stooling pattern is significantly reduced in comparison with results obtained with other commonly used procedures.

Extensive aganglionosis: further experience with the colonic patch graft procedure and long-term results.

During the period from 1979 to 1986, seven patients have been treated for extensive aganglionosis involving the colon and distal ileum (5 to 40 cm) employing the colonic patch graft (CPG) procedure. This consists of (1) ileostomy; (2) creation of a longitudinal side-to-side ileocolostomy between normal ileum and aganglionic ascending colon, forming a CPG; and (3) the definitive pull-through procedure. In all patients, the initial course after ileostomy was complicated by severe diarrhea, which was significantly improved by creation of the CPG. At the definitive operation several months later, the mesocolon attached to the CPG was severed to mobilize the ileocolostomy segment to the distal pelvis. Four patients in this series have been followed for 5 to 8 years. No patient has developed enterocolitis requiring hospitalization. Body weight became normal for age in all patients within 4 years of the definitive operation.

Multifocal ganglioneuroblastoma coexistent with total colonic aganglionosis.

Neuroblastoma and Hirschsprung's disease are considered aberrations of neural crest cell growth, migration, or differentiation. Their coexistence, however, is rare. We present the case of an only child with total colon Hirschsprung's disease diagnosed shortly after birth, who was found to have noncontiguous ganglioneuroblastomas without metastases at age 16 months. The spectrum of neural crest anomalies, long segment Hirschsprung's disease and multifocal neuroblastoma, in this child is unique and previously unreported.

Quantitative assessment of the stage of neuronal maturation in the developing human fetal gut--a new dimension in the pathogenesis of developmental anomalies of the myenteric plexus.

There are two main theories of the normal development of the enteric nervous system: the classical theory suggests that the enteric neuroblasts migrate along the alimentary tract in a single cranio-caudal direction. However, the second theory postulates a dual gradient of neuronal development. The present study aims to support the second theory and contribute to the understanding of the pathogenesis of Hirschsprung's disease and its allied gut motility disorders. Using a histomorphometric method, quantitative estimation of the myenteric intraganglionic neuronal development was made in tissues from various selected levels of the gut in 32 normal human fetuses of 11 to 24 weeks ovulation age. A parallel study with special chromatin staining was carried out from these materials to investigate different neurons in the myenteric plexus. Development of intraganglionic neurons was shown to be more advanced in the esophagus, less so in the rectosigmoid and least so in the ileocecal region: the mean values of neuronal and nuclear volume were found in the esophagus greater than in rectosigmoid and ileocecal region as ovulation age progressed. Intraganglionic mitotic figures were detected at all the selected levels of the gut. A decrease of the mitosis index as age progressed was observed. We showed that myenteric neurons followed a dual gradient of development proceeding from both ends to the middle of the gut in mid-trimester human fetuses. Our findings are compatible with the suggestion that any alterations in the fetal gut microenvironment may affect seriously the normal development of a multipotential precursor cell population resulting in various congenital anomalies of the myenteric plexus.

Clinicopathologic relationship of hypoganglionosis.

BACKGROUND/PURPOSE: Congenital motor dysfunction of the intestine associated with a morphologically abnormal myenteric nervous plexus (MP) is known as Hirschsprung's disease allied disorder (HAD). However, the clinicopathologic features of HAD are not well understood, partially because a standardized method of histologic evaluation of MP has not been established. To elucidate the clinicopathologic relationship of HAD the authors reviewed 6 cases of HAD using a newly devised histologic evaluation method. METHODS: Flat-mounted frozen sections of the ileum were stained for S-100 protein by fluorescent immunohistochemistry. Quantitative evaluation of MP was performed by measuring the fluorescence-positive area (MP ratio), and the results were compared with those of age-matched normal controls. RESULTS: All of 6 patients required laparotomy within 1 month after birth and enterostomy between 23 days and 10 months. Three died of intractable enteritis by the age of 2.2 years and were totally dependent on parenteral nutrition (PN) throughout their lives. The other 3 have survived for 6 to 10 years but have required PN occasionally. MP ratio in controls was more than 0.34 at all ages, whereas that in HAD was significantly lower than that in controls according to the clinical severity. CONCLUSION: MP size measured on 2-dimensional demonstration is suggested to be an indicator of clinical severity of HAD. J Pediatr Surg 36:898-900.

Abnormal Auerbach plexus in the esophagus and stomach of patients with esophageal atresia and tracheoesophageal fistula.

The Auerbach plexus of the upper gastrointestinal tract of five patients with esophageal atresia and tracheoesophageal fistula (EA-TEF) who had not had surgical reconstruction of the esophagus was studied by a microdissection technique to investigate the etiology of dysphagia, gastroesophageal reflux, and delayed gastric emptying; common findings in patients with repaired EA-TEF. All five patients showed a looser than normal Auerbach plexus in the distal esophagus, and to a lesser degree in the proximal esophagus, confirmed by point-count morphometric studies. The Auerbach plexus of the gastric fundus of all the patients was also abnormal. These findings suggest the existence of congenital functional impairment of the upper gastrointestinal tract in patients with EA-TEF, due to abnormal development of the myenteric plexus.

Familial aspects of Hirschsprung's disease.

Twenty-eight of 370 patients (14 families) treated for Hirschsprung's disease (HD) over a 34 year period had a family member with histologically proven HD. These 14 represented 4% of the 351 families: more than one affected child per family in 10 (2.8%) and both parent and child in 2 families. Neuronal intestinal dysplasia (NID) in a parent was associated with total colonic aganglionosis in two siblings of one family which suggests a similar genetic inheritance pattern to HD. Aganglionosis extended beyond the rectosigmoid in 61% of the familial group as opposed to 27% of the non-familial group. A significantly higher number of total colonic aganglionosis (TCA) was noted in those with a family history; 11 out of 28 (39%) as opposed to 19 out of 342 (5.6%) without a family history (p less than 0.001). Fifty percent of males with TCA had a family history but in only 2 cases was this transmitted through a female sibling. Although no significant difference was noted between male and female probands, a three times higher incidence of familial occurrence was noted in females with rectosigmoid disease than in males. Progression of length of segment in succeeding generations was noted in two families. Associated anomalies occurred in 16% without familial occurrence and 11% of the familial group.

Ileocolonic aganglionosis in white progeny of overo spotted horses.

The congenital absence of myenteric ganglia in the terminal portion of the ileum, cecum, and entire colon of white foals with overo spotted parents was reported. Males as well as females were affected. The foals were generally normal at birth but did not defecate. Signs of colic were noticed between 5 and 24 hours after birth, with death occurring at 23 to 132 hours.

Constipation and congenital disorders of the myenteric plexus.

Full-thickness muscle biopsies have been taken from patients with severe disabling chronic constipation that has not responded to conservative measures. Assessment by neurohistochemical techniques has revealed that a range of neuronal dysplasias of the myenteric plexus are responsible in many cases; these include aganglionosis (Hirschsprung's disease), hypoganglionosis and hyperganglionosis. In cases considered unlikely to be Hirschsprung's disease on clinical grounds, the procedure used has often been anorectal myectomy; this has not only provided tissue for diagnosis but has also been of therapeutic value in most cases of hypoganglionosis and some cases reported as 'normal'.

Does maternal drug ingestion cause megacystis microcolon intestinal hypoperistalsis syndrome? I. Clomiphene trial.

PURPOSE: Megacystis Microcolon Intestinal Hypoperistalsis Syndrome (MMIHS) is a congenital and lethal disease, and the etiology of the disease is not clear. It is speculated that maternal ingestion of some drugs during pregnancy may be an etiologic factor. In this study we aimed to investigate the effect of maternal ingestion of clomiphene on fetal bladder and colon in pregnant rats. METHODS: We separated animals into a control group including 14 rats and a clomiphene group with 30 rats. Nothing was given to the control group during pregnancy. Two mg/kg/day clomiphene intraperitoneally was given to the study group from the 6th to 12th day of pregnancy. All of them were sacrificed on the 20th day of pregnancy. Histopathological examination of the fetal colon and bladder was performed. RESULTS: In the clomiphene group a significant decrease in the thickness of the bladder wall, an increase in bladder epithelium, an increase in muscle atrophy of the colon and bladder wall, an increase in vacuoler degeneration of the muscles of the bladder and colon wall, a decrease in ganglion cell numbers in the myenteric plexus of the bladder and a decrease in the thickness of the bladder tunica muscularis were determined. CONCLUSION: In our rat model we found histological structural changes in the rats' colon and bladder walls as a result of using clomiphene on days 6-12 of pregnancy; a similar pathological finding to those found in some of the MMIHS patients' colons and bladders.