Intractable Infant Diarrhea with Epithelial Dysplasia Associated with Polymalformation

Abstract

Intractable diarrhea of infancy (IDI) is defined as persistent and severe diarrhea despite protracted bowel rest that requires long-term parenteral nutrition in children who have no specific disease that can be effectively treated (1). It is a clinical syndrome shared by a heterogeneous group of diseases, which have been recently classified as: IDI associated with microvillus inclusion disease, IDI from immune or autoimmune origin, and nonimmune IDI (2). Two distinct entities have been described within non-immune IDI. Syndromatic IDI is characterized by a typical, abnormal phenotype including facial dysmorphism and hair abnormalities with trichorrhexis nodosa, associated with functional immunodeficiency (1). Intestinal biopsy specimens from children with syndromatic IDI show villous atrophy with no striking epithelial abnormalities. Intractable diarrhea of infancy associated with intestinal epithelial dysplasia (IED) is defined by changes in the intestinal epithelium, including disorganization of surface enterocytes with focal tufting and abnormal regenerative crypts(3,4). Basement membrane abnormalities with reduced laminin staining along the crypts have also been described recently in cases of IED (4). No phenotypic abnormalities have been reported in cases of IED to date. This report describes two children who had IDI associated intestinal epithelial changes similar to those described as IED by Reifen et al.(3) and Goulet et al. (4) Both patients had polymalformation syndrome. CASE REPORTS CASE 1 This 2-year-old boy was born to nonconsanguineous parents at 40 weeks of gestational age with a birth weight of 2.45 kg. The pregnancy was marked by metrorrhagia and intrauterine growth retardation. Both the father's and mother's family histories included transient neonatal diarrhea that was slowly resolved with a diet free of cow's milk proteins. The boy was brought to our institution at 2 weeks of age with severe, watery diarrhea, vomiting, and impaired growth velocity. He failed to tolerate a diet free of cow's milk and semielemental enteral feeding (Alfare [Nestlí, Marne-la-Vallíe, France]; Pepti 2000 Nutritia, Rueil-Malmaison, France]) and parenteral nutrition were initiated. Examination of the stools for pathogens and studies of gastrointestinal motility (esophageal and rectal manometry, surgical rectal biopsy) showed no usual cause of diarrhea. Immunodeficiency was eliminated by normal immunologic assessments including serum immunoglobulin (IgA, IgM, IgG, IgE) and complement levels, levels of circulating autoantibodies, counts of lymphocyte subsets, determination of mitogen-induced lymphocyte transformation, and positive antibody responses to vaccinal antigens. Results of a radio-allergosorbent test and blast transformation of lymphocytes with cow's milk proteins were negative. Secretory diarrhea with high stool output (up to 60 ml/kg per day) and vomiting persisted despite long-term parenteral nutrition and bowel rest. Jejunal and colonic biopsy specimens were taken when the patient was 3, 5, 7, and 16 months of age. Clinical examination showed phenotypic abnormalities including primordial short stature, microcephaly and unusual facial appearance associated with psychomotor retardation, hyperactivity, and delayed onset of eczematous skin lesions. Facial dysmorphism included large long nose, ocular hypertelorism, blepharophimosis, ear dysplasia (big and sloping backward), micrognathia, flat supraorbital ridges, and high sloping forehead. The overall pattern of clinical manifestations was characteristic of Dubowitz syndrome(5). Hair was woolly and fair but not easily removed. Microscopic examination showed no features of trichorrhexis nodosa. Skeletal radiographs showed sacral malformation, abdominal echography was normal, and magnetic resonance imaging demonstrated mild left ventricular dilation. Findings in high-resolution karyotype were normal. CASE 2 This 2.5-year-old girl was born to unrelated parents at 35 weeks of gestational age with a birth weight of 2.2 kg. Pregnancy was obtained after insemination from an anonymous sperm donor. No particular familial history was noted. In the first weeks of life, the child had severe, watery diarrhea, associated with vomiting and abdominal distension. A diet free of cow's milk and semielemental enteral feeding were unsuccessful. Results of extensive investigations, including stool examination for pathogens and immunologic assessments identical to those described in patient 1, were normal. Diarrhea persisted (stool output up to 80 ml/kg per day) despite bowel rest and long-term parenteral nutrition. Small intestinal biopsies were performed when the patient was 3, 8, 13, and 24 months of age, and a surgical specimen of Meckel diverticulum was obtained at 24 months of age (when continuity was re-established after surgical treatment for anal imperforation). Gastrointestinal symptoms were unchanged at 30 months of age, and the child was still dependent on total parenteral nutrition. Clinical examination showed polymalformation syndrome that involved facial dysmorphism, anal imperforation with a vaginal fistula (treated at 2 weeks of age), lacrimal duct and choanal imperforation, coloboma of the right optic nerve, and hypoplasia of the right fourth finger. Facial phenotypic abnormalities included hypoplasia of the middle face, small palbebral clefts, ocular hypertelorism, Gothic palate, and a dimple on the upper part of the filtrum. Neurologic examination revealed hypotony of the lower limbs and trunk, but psychomotor development was normal. Skeletal radiographs were normal, except for hypoplasia of the middle face. Cerebral echography demonstrated moderate bilateral ventricular dilation. High-resolution karyotype showed no abnormalities. CONTROL PATIENTS Duodenal biopsy specimens from four children served as normal control specimens. The biopsies were performed for failure to thrive, but none of the children had diarrhea, and gastrointestinal disease was subsequently eliminated. Control subjects also included two children with IDI of neonatal onset, who had IED with no polymalformation syndrome. These cases were identical to those described by Reifen et al. (3) and Goulet et al.(4). Intestinal epithelial dysplasia was diagnosed from examination of duodenal or jejunal biopsy specimens obtained at several times during the course of the disease. Growth and psychomotor development were normal. Extensive laboratory investigations, including examination of the stools for pathogens and immunologic assessments, were negative. The gastrointestinal symptoms persisted in these children at the ages of 4 and 13 years, with a stool output of 70 ml/kg and 60 ml/kg per day, respectively, and both children were still dependent on total parenteral nutrition. METHODS Small intestinal and colonic (case 1) biopsy specimens obtained during endoscopy were fixed in formalin, and paraffin sections were stained with hematoxylin-eosin and periodic acid Schiff (PAS). Some specimens were frozen for immunohistochemical investigations. Small intestinal samples were collected from both children for electron microscopy, fixed in phosphate-buffered 3% glutaraldehyde, and embedded in epoxy resin. Immunohistochemical staining was performed on frozen intestinal sections with the following antibodies: CD3, CD4, CD8, CD25 (IL2R), and HLA DR(Dakopatts Glostrup, Denmark). The distribution of the basement membrane component laminin in the intestinal mucosa was also studied using the monoclonal antibodies LN26-7 and LN41-11 (dilution 1:200; TaKaRa Biomed, Shiga, Japan). A three-stage immunoperoxidase procedure was used with a biotinylated second antibody and peroxidase-labeled streptavidin. Staining was visualized using diaminobenzidine. RESULTS Histopathologic Findings The specimens obtained in repeated small intestinal biopsies performed in both patients all showed moderate villous atrophy with elongation and hyperplasia of the crypts. The lamina propria had a normal to slightly elevated number of mononuclear cells. The number of intraepithelial lymphocytes was normal (fewer than 20 per 100 epithelial cells). The biopsy and surgical specimens from patient 2 showed surface epithelium "tufts," with focal crowding of closely packed enterocytes (Fig. 1). The enterocytes had a rounded apical plasma membrane at the surface of the tufts, giving them a teardrop appearance. No brush border abnormalities were detected after PAS staining. Transmission electron microscopy revealed normal brush border microvilli on enterocytes. There were none of the inclusion bodies or secretory granules described in microvillus inclusion disease, and the intracytoplasmic organelles appeared normal.FIG. 1: Duodenal biopsy specimen from patient 2 showing villous atrophy and disorganization of the surface epithelium with focal tufting (arrows; hematoxylin-eosin; magnification,× 250. Inset: hematoxylin-eosin; magnification, ×1000).Two sets of colon biopsy specimens obtained in patient 1 at 3 and 16 months of age showed surface epithelium tufts identical to those described in the small intestine. The mucosal architecture was normal, and there was no evidence of inflammatory changes in the lamina propria (Fig. 2).FIG. 2: Colonic biopsy specimen from patient 1 showing surface epithelium tufts (arrow) (hematoxylin-eosin, magnification, ×400).The intestinal specimens obtained from the four normal control patients without gastrointestinal disease had normal morphology with no tufting features. Specimens obtained in repeated duodenal or jejunal biopsies in the two control patients with isolated IED showed villous atrophy without mucosal inflammatory changes. All had focal tufting of the surface epithelium, identical to those just described. No brush border abnormalities were detected by PAS staining or by electron microscopy. Colon specimens from both patients also showed superficial epithelial tufts with no other morphologic changes. Immunohistochemical Findings All the small intestinal specimens from both patients showed normal epithelial expression of HLA DR, moderate in the superficial epithelium and negative in the crypts. The distribution of mucosal T-cell subpopulations was normal, mainly CD3+ CD8+ within the epithelium, and mainly CD3+ CD4+ in the lamina propria. When compared with CD25+ cells in the normal control subjects, no increase was detected. Immunohistochemical staining for laminin was identical with both antibodies. The distribution and intensity of laminin staining were all similar in the normal control biopsy specimens, the specimens from control children with IED, and all specimens from patients 1 and 2. All the samples had continuous, linear, and thin deposits of laminin on the basement membranes lining the surface epithelium and the crypts (Fig. 3). There was also positive staining on the basement membranes of the blood vessels.FIG. 3: Duodenal biopsy specimen from patient 2. Immunostaining with monoclonal antibody antilaminin LN26-7 showing a continuous and linear positivity on the basement membranes of both the surface epithelium and the crypts (magnification, ×400).DISCUSSION The definition and classification of intractable diarrhea of infancy (IDI) has been modified since its first descriptions by Avery et al.(6) and Ricour et al. (7), and new entities have been described. The term IDI is restricted to symptoms of severe diarrhea with no specific cause, persistence despite protracted bowel rest, and requirement for long-term parenteral nutrition(1). A classification has been proposed, based on clinical, biologic, and histologic criteria (2). The two patients described in this report had IDI of neonatal onset, with persistent severe diarrhea and histologic abnormalities, despite long-term parenteral nutrition. No known enteric pathogens were identified, and food intolerance was excluded, particularly in patient 1, who had a family history of intolerance of cow's milk proteins. The absence of abnormalities of the enterocyte brush border detected by PAS staining or transmission electron microscopy ruled out the diagnosis of microvillus inclusion disease and related disorders in both patients(8,9). The histopathologic changes in these children also differed from those reported for IDI of immune origin, where there is severe villous atrophy, crypt necrosis and heavy infiltration of the lamina propria by mononuclear cells, together with features of local immune activation (10). The present patients had no mucosal inflammation (or mild and focal), no crypt necrosis, and no signs of immune activation including CD25+ cells and HLA DR expression by crypt enterocytes. The absence of extraintestinal symptoms and signs of autoimmunity also excluded the diagnosis of IDI caused by autoimmune disease(10). A group of patients with IDI of nonimmune origin has recently been described. These patients had moderate villous atrophy with no evident local immune activation (2). Nonimmune IDI covers two distinct entities, syndromatic IDI and IDI associated with intestinal epithelial dysplasia (IED) (1,3,4). The clinical, immunologic, and histologic features of these two entities are summarized inTable 1.TABLE 1: Comparative features of nonimmune intractable diarrhea of infancyAlthough the two cases reported herein had some clinical features in common with the group of patients with syndromatic IDI, such as low birth weight and mild mental retardation (in patient 1), the phenotypic abnormalities were different. Children with syndromatic IDI have a typical malformation syndrome with recognizable facial deformities and woolly, easily removable hair with trichorrhexis nodosa. In contrast, our first patient showed clinical manifestations pointing to Dubowitz syndrome, without trichorrhexis nodosa. The second child had polymalformation syndrome including anal and choanal atresia, which showed no similarities to syndromatic IDI, although we had no specific overall diagnosis. Whereas functional immune deficiency is found in all the reported cases of syndromatic IDI, the antibody responses to antigens and the mitogen-induced lymphocyte response were normal in our patients. Lastly, the intestinal epithelial changes that occurred in both children have never been described in association with syndromatic IDI. The epithelial abnormalities found in the repeated small intestinal biopsy specimens are identical to those described by Reifen et al.(3) and Goulet et al. (4) as tufting enteropathy, or intestinal epithelial dysplasia (IED), with a total of nine reported cases. However, none of the previously reported cases of IDI with IED was associated with a malformation syndrome. We included as control cases two new patients with IED-associated IDI with similar histopathologic changes in the small intestine. These two children and the child with Dubowitz syndrome all showed focal features of epithelial dysplasia in their colonic specimens. Colonic epithelial changes have not been previously described in IED, except in one recent case reported in abstract form(11). This suggests that the epithelial abnormalities associated with IDI are not limited to the small intestine, but can involve other parts of the digestive tract. Features of epithelial dysplasia seem milder in the colon, however, than in the small bowel. This observation could be explained in part by differences in the kinetics of epithelial cell renewal between the colon and the small bowel. A recent study supports the hypothesis of a cell turnover dysfunction in the pathogenesis of IED(12). The association of phenotypic abnormalities with IDI and features of epithelial dysplasia is unlikely to be incidental, but the link between the clinical and histologic manifestations is unclear. Although diarrhea and vomiting have been described in Dubowitz syndrome, the symptoms are mild, and patients never require parenteral nutrition (13). An association between congenital IDI and choanal atresia was recently reported in four children (14) who had clinical similarities to our second patient. These cases were reported in abstract form, and no information regarding the presence or absence of epithelial tufts was provided in the histopathologic description. Basement membrane abnormalities with decreased laminin expression along the crypts have been described in cases of IED (4). The investigators suggested that the abnormal matrix deposit could be responsible for constitutive alteration in the proliferation and maturation of the small intestinal epithelium, which could explain the early onset and severity of diarrhea. However, we detected no changes in laminin expression in isolated IED or in IED associated with polymalformation syndrome. Although it cannot be excluded that these conflicting results may be related to the use of different methods of assessment, these data suggest that the basement membrane abnormalities observed in other cases of IED could be the result of an epithelial alteration rather than a primary defect. A recent study of IED, increased epithelial desmoglein expression and ultrastructural changes of desmosomes were reported, strongly suggesting that alterations of cell-cell interactions may be involved in the pathogenesis of the disease(12). Previous reports have suggested that IED may be a congenital inherited autosomal recessive disease, because of the family histories of four of the children. Despite the absence of family history and parental consanguinity, this hypothesis is supported in our first patient because of the association with Dubowitz syndrome, which is believed to represent the homozygous state of an autosomal recessive mutation (5). In conclusion, we report two cases of IDI with IED that fall into no previously defined category of IDI. The association of IED with malformation syndrome of autosomal recessive inheritance could help to identify the genetic abnormalities that are possibly involved in IED.