Jagged1 (JAG1) mutations in Alagille syndrome: increasing the mutation detection rate.

Alagille syndrome (AGS) is caused by heterozygous mutations in JAG1, and mutations have been previously reported in about 70% of patients who meet clinical diagnostic criteria. We studied a cohort of 247 clinically well-defined patients, and using an aggressive and sequential screening approach we identified JAG1 mutations in 94% of individuals. Mutations were found in 232 out of 247 patients studied and 83 of the mutations were novel. This increase in the mutation rate was accomplished by combining rigorous clinical phenotyping, with a combination of mutation detection techniques, including fluorescence in situ hybridization (FISH), genomic and cDNA sequencing, and quantitative PCR. This higher rate of mutation identification has implications for clinical practice, facilitating genetic counseling, prenatal diagnosis, and evaluation of living-related liver transplant donors. Our results suggest that more aggressive screening may similarly increase the rate of mutation detection in other dominant and recessive disorders.

Consequences of JAG1 mutations.

BACKGROUND: Alagille syndrome (AGS) is a multi-system, autosomal dominant disorder with highly variable expressivity, caused by mutations within the Jagged1 (JAG1) gene. METHODS: We studied 53 mutation positive relatives of 34 AGS probands to ascertain the frequency of clinical findings in JAG1 mutation carriers. RESULTS: Eleven of 53 (21%) mutation positive relatives had clinical features that would have led to a diagnosis of AGS. Seventeen of the 53 (32%) relatives had mild features of AGS, revealed only after targeted evaluation following the diagnosis of a proband in their family. Twenty five of the 53 (47%) mutation positive relatives did not meet clinical criteria, and two of these individuals had no features consistent with AGS at all. The frequency of cardiac and liver disease was notably lower in the relatives than in the probands, characterising the milder end of the phenotypic spectrum. The characteristic facies of AGS was the feature with the highest penetrance, occurring almost universally in mutation positive probands and relatives. CONCLUSIONS: This study has implications for genetic counselling of families with AGS and JAG1 mutations.

Alagille syndrome and the Jagged1 gene.

Since the first descriptions of Alagille syndrome (syndromic bile duct paucity) 30 years ago, our appreciation of the clinical variability and complexity of this disorder has grown. In addition to the liver, Alagille syndrome is associated with abnormalities that involve the heart, eye, skeleton, kidneys, and the increasing importance of abnormalities of the central nervous system is being recognized. The developmental nature of the disorder has been proven with the identification of the disease-causing gene, Jagged1. Jagged1 is a cell surface protein that functions in an embryologically important signaling pathway, known as the Notch signaling pathway. Identification of the role of Jagged1 (JAG1) in the etiology of Alagille syndrome has improved diagnosis for this variably expressed disorder. In this review, we summarize information on the range of clinical abnormalities of the liver and other affected organs in affected individuals. Genetic studies have demonstrated the range of defects in JAG1 that cause Alagille syndrome. Mutations in JAG1 can be identified in 70% of Alagille syndrome patients, and they are inherited in 30-50%. These mutations include total gene deletions as well as mutations (frameshift, missense, and nonsense) in almost all regions of the 26 exons of the Jagged1 gene. This review focuses on clinical and genetic features of Alagille syndrome.

Failure of single-toxin assays to detect clostridium difficile infection in pediatric inflammatory bowel disease.

OBJECTIVES: The aims of this retrospective study were 1) to determine the ability of single-toxin assays for Clostridium difficile to detect infection among pediatric patients with inflammatory bowel disease (IBD) and 2) to determine the toxin assays routinely used by pediatric tertiary care hospitals in the United States. METHODS: Stool specimens from patients with IBD (submitted from January, 1996, to August, 1999) were evaluated for the presence of C. difficile toxin A and toxin B. Toxin profile (toxin A alone, toxin B alone, toxin A and B together) was compared in positive specimens. A phone interview was conducted with representatives from laboratories in 22 pediatric hospitals to investigate which toxin assays were routinely used. RESULTS: A total of 697 specimens were submitted from 284 IBD patients. In all, 81 IBD patients (28.5%) had at least one documented infection. Toxin A assay failed to identify 41.5% of C. difficile infections. Toxin B assay failed to detect 34.9% of C. difficile infections. Toxin profile changed in 55% of patients with multiple infections. Of the hospitals surveyed, 59% did not test for both toxins. CONCLUSIONS: Single-toxin assays for C. difficile fail to detect a significant percentage of infections. The toxins identified during one infection are not predictive of the toxins identified in subsequent infections. Despite this, many pediatric hospitals do not routinely use both toxin assays to diagnose C. difficile infection. When infection is suspected, assays for C. difficile toxin A and toxin B should be requested.

Pediatric Crohn's disease: risk factors for postoperative recurrence.

OBJECTIVE: Postoperative recurrence of Crohn's disease in adults has been extensively studied; however, the course of Crohn's disease after surgery in children has not been well defined. The aim of this study was to examine the postoperative course of pediatric Crohn's disease and the factors that may predict early postoperative recurrence. METHODS: We identified 100 resective surgeries in 79 children with Crohn's disease seen at the Children's Hospital of Philadelphia between 1978 and 1996. A retrospective, multivariable analysis of factors potentially influencing postoperative clinical recurrence was performed. Preoperative and postoperative height measurements were compared, and z scores were computed for height-for-age. Two-tailed t test was used for the analysis. RESULTS: Clinical recurrence rates were 17% at 1 yr, 38% at 3 yr, and 60% at 5 yr. Patients with colonic Crohn's disease had a significantly shorter postoperative recurrence-free interval (median 1.2 yr) than patients with ileocecal (median 4.4 yr) or diffuse disease (median 3.0 yr) (p = 0.01). On multivariable analysis, a high Pediatric Crohn's Disease Activity Index at the time of surgery (p = 0.01) and preoperative use of 6-mercaptopurine (6-MP) (p < 0.005) were also independently associated with higher postoperative recurrence rates. There was a significant improvement in z scores for height (p = 0.04) after surgery. CONCLUSIONS: In children undergoing resective surgery for Crohn's disease, high rates of postoperative Crohn's disease recurrence are associated with severe disease at the time of surgery, colonic Crohn's disease, and the preoperative use of 6-MP. Patients who require preoperative use of 6-MP are likely to suffer from a more aggressive disease and would benefit from postoperative 6-MP prophylaxis. Height growth was improved after intestinal resection for Crohn's disease.

Mutation analysis of Jagged1 (JAG1) in Alagille syndrome patients.

Alagille syndrome (AGS) is an autosomal dominant disorder caused by mutations in Jagged1 (JAG1), a ligand in the evolutionarily conserved Notch signaling pathway. Previous studies have demonstrated that a wide spectrum of JAG1 mutations result in AGS. These include total gene deletions, protein truncating, splicing and missense mutations which are distributed across the coding region of the gene. Here we present results of JAG1 mutation screening by SSCP and FISH in 105 patients with AGS. For these studies, new primers were designed for 12 exons. Mutations were identified in 63/105 patients (60%). The spectrum of the JAG1 mutations presented here is consistent with previously reported results. Eighty three percent (52/63) of the mutations were protein truncating, 11% (7/63) were missense, 2% (1/63) were splice site, and 5% (3/63) were total gene deletions demonstrable by FISH. Six of the missense mutations are novel. As has been reported previously, there is no apparent relationship between genotype and clinical phenotype.

Growth, body composition, and nutritional status in children and adolescents with Crohn's disease.

OBJECTIVE: To examine growth, body composition, and nutritional status in a large sample of children, adolescents, and young adults with Crohn's disease (CD). METHODS: One hundred thirty-two subjects (48 females) with CD, aged 5 to 25 years, and 66 healthy control subjects (37 females) of similar age. Growth, nutritional status, and body composition were measured by anthropometry and dual-energy x-ray absorptiometry. Genetic potential of linear growth was predicted using the adjusted heights of Himes et al. Pubertal status and skeletal age were assessed. Average Pediatric Crohn's Disease Activity Index (PCDAI) and lifetime steroid exposure (in milligrams per day) were obtained from medical charts. The variables were examined in relation to gender and measures of disease activity. RESULTS: Males and females with CD did not differ by age, disease duration, or PCDAI. Males with CD had significantly lower values for growth and nutritional status than control subjects (z-scores for weight: -0.66 +/- 1.18 vs. 0.26 +/- 0.95, P = 0.00002; height -0.81 +/- 1.14 vs. 0.28 +/- 0.93, P = 0.00001; adjusted height -1.05 +/- 1.03 vs. 0.40 +/- 1.03, P = 0.00001) and delayed skeletal age of 0.9 +/- 1.6 years. Impaired linear growth in the males was present regardless of pubertal stage. Associations between disease severity indicators and growth parameters were more consistent for females. CONCLUSIONS: Crohn's disease is associated with impaired growth. Despite similarities in disease duration, activity, and lifetime steroid exposure, growth in males was more impaired. Gender may confer risk for impaired growth in CD.

Azathioprine and 6-mercaptopurine for the treatment of perianal Crohn's disease in children.

Numerous adult studies show a 30-65% response rate to azathioprine (AZA) or 6-mercaptopurine (6-MP) for significant perianal Crohn's disease. The aim of this study was to evaluate whether these drugs healed pediatric perianal Crohn's disease. Records of pediatric Crohn's patients were retrospectively reviewed for significant perianal disease treated with AZA or 6-MP for > or =6 months. The patient's perianal disease was reviewed and evaluated for fistulas, drainage, induration, and tenderness. In addition, the patients were given a score using the Irvine Perianal Disease Activity Index (PDAI). Patients were retrospectively scored upon initiation of treatment and after six months of therapy. Possible scores ranged from 0-20. Twenty patients met the study criteria. Five patients were considered treatment failures. One patient required a colostomy after 1.5 months of therapy, one developed pancreatitis, and three were noncompliant with therapy. Of the remaining 15 patients who were treated for > or =6 months, 67% had an improvement in drainage, 73% in tenderness, 60% in induration, and 40% in fistula closure. The mean Irvine PDAI was 7.67 +/- 2.19 initially and 4.40 +/- 1.72 after six months of therapy. The improvement was statistically significant (p < 0.001). AZA and 6-MP are effective treatments for healing significant perianal Crohn's disease in pediatrics.

Clinical and molecular genetics of Alagille syndrome.

Alagille syndrome (AGS) is a dominantly inherited disorder characterized by bile duct paucity and resultant liver disease in combination with cardiac, skeletal, ocular, and facial abnormalities. Jagged1 (JAG1) has been identified as the AGS disease gene. It encodes a ligand in the Notch signaling pathway that is involved in cell fate determination. AGS is the first developmental disorder to be associated with this pathway. It shows highly variable expressivity, and diagnosis in mildly affected persons can be difficult without molecular analysis. Currently, JAG1 mutations are detected in about 70% of patients with AGS and include total gene deletions as well as protein truncating, splicing, and missense mutations. Mutations are located across the gene within the evolutionarily conserved motifs of the protein. There is no phenotypic difference between patients with deletion of the entire JAG1 gene and those with intragenic mutations. This suggests that haploinsufficiency for JAG1 is a mechanism causing AGS.

The expression of Jagged1 in the developing mammalian heart correlates with cardiovascular disease in Alagille syndrome.

The establishment of the cardiovascular system represents an early, critical event essential for normal embryonic development, and defects in cardiovascular development are a frequent cause of both in utero and neonatal demise. Congenital cardio-vascular malformations, the most frequent birth defect, can occur as isolated events, but are frequently presented clinically within the context of a constellation of defects that involve multiple organs and that define a specific syndrome. In addition, defects can be a primary effect of gene mutations or result from secondary effects of altered cardiac physiology. Alagille syndrome (AGS) is an autosomal dominant disorder characterized by developmental abnormalities of the heart, liver, eye, skeleton and kidney. Congenital heart defects, the majority of which affect the right-sided or pulmonary circulation, contribute significantly to mortality in AGS patients. Recently, mutations in Jagged1 ( JAG1 ), a conserved gene of the Notch intercellular signaling pathway, have been found to cause AGS. In order to begin to delineate the role of JAG1 in normal heart development we have studied the expression pattern of JAG1 in both the murine and human embryonic heart and vascular system. Here, we demonstrate that JAG1 is expressed in the developing heart and multiple associated vascular structures in a pattern that correlates with the congenital cardiovascular defects observed in AGS. These data are consistent with an important role for JAG1 and Notch signaling in early mammalian cardiac development.

Risk factors for low bone mineral density in children and young adults with Crohn's disease.

OBJECTIVE: Low bone mineral density (BMD) is a recognized complication of Crohn's disease (CD). The aim of this study was to identify the risk factors for low BMD in pediatric patients with CD. STUDY DESIGN: One hundred nineteen subjects with CD ranging in age from 5 to 25 years were enrolled. BMD of the lumbar spine was measured by dual-energy x-ray absorptiometry. Growth parameters were assessed by examination. Disease-specific variables and use of selected medications were determined by chart review. RESULTS: Powerful risk factors for low BMD z-score included hypoalbuminemia, exposure to nasogastric tube feeds, total parenteral nutrition, 6-mercaptopurine, and corticosteroids. Corticosteroid dosing at a level >7.5 mg/d, 5000 mg lifetime cumulative dose, or >12 months of lifetime exposure were significant risk factors for low BMD z-score. Weaker but significant associations with low BMD z-scores included measures of disease severity such as pediatric Crohn's disease activity index, hospital admissions, and length of hospital stay. Site and duration of disease were not predictive. CONCLUSIONS: The presence of several clinically available factors was predictive of poor bone mineral status in this sample of subjects with CD. Hypoalbuminemia, corticosteroid exposure, nasogastric tube feeds, total parenteral nutrition, and 6-mercaptopurine were the most powerful risk factors for low bone mineral status.

Bone mineral density in children and young adults with Crohn's disease.

Reduced bone mineral density (BMD) has been reported in adults with Crohn's disease (CD). Less is known about abnormal BMD in children and young adults with CD. The aims of this study are to determine the prevalence of low BMD and to evaluate the effect of growth and pubertal development on BMD in children and young adults with CD. One hundred-nineteen patients with CD underwent dual-energy X-ray absorptiometry (DXA) to determine BMD. Anthropometry and pubertal development were measured. Bone age was measured only in patients older than 8 years of age and who had not grown in height during the last year. One hundred-nineteen patients (72 male, 47 female) were evaluated. Seventy percent of patients had BMD z-scores < or = -1.0 and 32% had z-scores < or = -2.0. Weight and height z-scores were significantly associated with BMD z-scores. BMD z-scores based on bone age and on chronological age were highly correlated, except when the chronological age BMD z-score was < or = -2.0. BMD z-score was significantly different between males and females for the group (-1.75 +/- 1.06 vs. -1.08 +/- 1.00), respectively. Children and young adults with CD have a high prevalence of low BMD and routine evaluation by DXA is indicated. In patients with a chronological age-based BMD z-score < or = -2.0, a bone age-based BMD should be considered.

Inflammatory bowel disease in pediatric and adolescent patients.

IBD is a chronic pediatric disease that needs to be treated by a team of experts consisting of pediatricians, pediatric gastroenterologists, psychologists, nutritionists, social workers, and nurses. A critical factor in successful management of this disease is the willingness of the patient to participate and cooperate with the team. Parents and patients must be educated and supported to treat these disorders effectively. Much further research is necessary to understand the specific causative and therapeutic issues unique to young patients with IBD.

Jagged1 mutations in patients ascertained with isolated congenital heart defects.

Mutations in Jagged1 cause Alagille syndrome (AGS), a pleiotropic disorder with involvement of the liver, heart, skeleton, eyes, and facial structures. Cardiac defects are seen in more than 95% of AGS patients. Most commonly these are right-sided defects ranging from mild peripheral pulmonic stenosis to severe forms of tetralogy of Fallot. AGS demonstrates highly variable expressivity with respect to all of the involved systems. This leads us to hypothesize that defects in Jagged1 can be found in patients with presumably isolated heart defects, such as tetralogy of Fallot or pulmonic stenosis. Two patients with heart defects of the type seen in AGS and their relatives were investigated for alterations in the Jagged1 gene. Jagged1 was screened by a combination of cytogenetic and molecular techniques. Patient 1 was studied because of a four-generation history of pulmonic stenosis. Molecular analysis showed a point mutation in Jagged1 in the patient and her mother. Patient 2 was investigated owing to the finding of tetralogy of Fallot and a "butterfly" vertebra on chest radiograph first noted at age 5 years. She was found to have a deletion of chromosome region 20p12 that encompassed the entire Jagged1 gene. The identification of these two patients suggests that other patients with right-sided heart defects may have subtle findings of AGS and Jagged1 mutations.

Features of Alagille syndrome in 92 patients: frequency and relation to prognosis.

We have studied 92 patients with Alagille syndrome (AGS) to determine the frequency of clinical manifestations and to correlate the clinical findings with outcome. Liver biopsy specimens showed paucity of the interlobular ducts in 85% of patients. Cholestasis was seen in 96%, cardiac murmur in 97%, butterfly vertebrae in 51%, posterior embryotoxon in 78%, and characteristic facies in 96% of patients. Renal disease was present in 40% and intracranial bleeding or stroke occurred in 14% of patients. The presence of intracardiac congenital heart disease was the only clinical feature statistically associated with increased mortality (P <.001). Initial measures of hepatic function in infancy including absence of scintiscan excretion were not predictive of risk for transplantation or increased mortality. The hepatic histology of these AGS patients showed a significant increase in the prevalence of bile duct paucity (P =.002) and fibrosis (P <.001) with increasing age. Liver transplantation for hepatic decompensation was necessary in 21% (19 of 92) of patients with 79% survival 1-year posttransplantation. Current mortality is 17% (16 of 92). The factors that contributed significantly to mortality were complex congenital heart disease (15%), intracranial bleeding (25%), and hepatic disease or hepatic transplantation (25%). The 20-year predicted life expectancy is 75% for all patients, 80% for those not requiring liver transplantation, and 60% for those who required liver transplantation.

Growth, nutritional status, body composition, and energy expenditure in prepubertal children with Alagille syndrome.

OBJECTIVES: To describe the patterns of growth, nutritional status, body composition, and resting energy expenditure (REE) in prepubertal children with Alagille syndrome (AGS) before the onset of end-stage liver disease. STUDY DESIGN: Thirteen prepubertal subjects with AGS (8 male; mean age, 6.8 2.8 years) were evaluated for growth parameters, body composition by skinfolds and by dual-energy x-ray absorptiometry, and REE by indirect calorimetry. The children with AGS were compared with a healthy, age-matched reference group of 37 prepubertal children. RESULTS: Compared with healthy children, children with AGS had significantly reduced (P <. 05) growth (weight, weight z score, height, height z score), nutritional status (midarm circumference, triceps skinfold, and midarm muscle area), and body composition (fat mass and fat-free mass). Subscapular thickness, percent body fat, and REE were not different. The AGS subgroup (n = 4) with REE greater than 110% predicted value had a reduced percent body fat (P <.02). CONCLUSIONS: Growth and body composition abnormalities are common in prepubertal children with AGS.

Experiences with 6-mercaptopurine and azathioprine therapy in pediatric patients with severe ulcerative colitis.

BACKGROUND: The effectiveness of 6-mercaptopurine combined with azathioprine in treating severe ulcerative colitis has been shown in several adult studies. Reported pediatric experiences are rare. The purpose of this study was to investigate the safety and the potential efficacy of 6-mercaptopurine and azathioprine in the treatment of active ulcerative colitis in a pediatric population. METHODS: The medical records of patients with active ulcerative colitis who were under observation at The Children's Hospital of Philadelphia and its satellite clinics from January 1984 through December 1997 were retrospectively reviewed. Patients were included who had received a diagnosis of ulcerative colitis, who met no criteria for Crohn's colitis, and who had received treatment with 6-mercaptopurine and azathioprine. They were then analyzed for the development of side effects, the indication to use 6-mercaptopurine and azathioprine, and the ability to discontinue corticosteroid use in those patients taking 5-acetylsalicylic acid products who were corticosteroid-dependent or whose disease was refractory to treatment. Excluded from the corticosteroid analyses were patients who underwent surgery for their disease and patients treated with 5-acetylsalicylic acid only. Statistical analysis was performed by the Kaplan-Meier survival curve and paired Student's t-test. RESULTS: In a review of 200 medical records of patients with active ulcerative colitis, 20 patients met the criteria. The patients' average age at the initiation of treatment with 6-mercaptopurine and azathioprine was 13.8 years. Sixteen patients (80%) were corticosteroid dependent and 3 (15%) had ulcerative colitis refractory to corticosteroid treatment. One patient had severe colitis treated with 5-acetylsalicylic acid only. Discontinuation of corticosteroid was accomplished in 12 (75%) of 16 patients. The median time to discontinuation of corticosteroid after initiation of 6-mercaptopurine and azathioprine therapy was 8.4 months. Eight patients (67%), observed from 3 months to 65 months, have continued without corticosteroid therapy. Side effects included pancreatitis and shingles that resulted in discontinuation of 5-acetylsalicylic acid, leukopenia corrected by withholding 6-mercaptopurine, and self-resolved hepatitis. CONCLUSIONS: The data support the safety of 6-mercaptopurine and azathioprine use in the treatment of pediatric patients with ulcerative colitis; side effects were minimal and reversible. Eighteen (90%) of 20 patients tolerated the therapy well. The results also show that 12 (75%) of 16 pediatric patients with ulcerative colitis will benefit from the use of 6-mercaptopurine and azathioprine after initial discontinuation of corticosteroid therapy. Although 6-mercaptopurine and azathioprine may not prevent further relapses, medical management of these flares may be less intense and may not require long-term corticosteroid use. Prospective clinical trials in pediatric patients are necessary to delineate further the role of 6-mercaptopurine and azathioprine in pediatric ulcerative colitis.

Single toxin detection is inadequate to diagnose Clostridium difficile diarrhea in pediatric patients.

BACKGROUND & AIMS: Clostridium difficile is an important cause of symptomatic diarrhea in pediatric patients. The bacterium produces two toxins, although many laboratories assay for only one. We questioned this diagnostic approach when patients had positive results for C. difficile at our institution, but initially had tested negative at outside laboratories. METHODS: We retrospectively analyzed relative frequencies of C. difficile toxin A alone, toxin B alone, and toxins A and B from pediatric patients with diarrhea. Results were stratified according to toxin detection and patient age. RESULTS: Of 1061 specimens, 276 (26.8%) were positive for C. difficile toxin(s). Fifty-one (18.5%) were positive for toxin A alone, 133 (48.2%) for toxin B alone, and 92 (33.3%) for both toxins. Assaying for toxin B identified C. difficile infection more frequently than did assaying for toxin A (P < 0.0001). The frequency of toxin B detection was significantly higher for older children but not for infants. CONCLUSIONS: Testing for C. difficile toxin A or toxin B alone will result in more frequent misdiagnosis than testing for both toxins. This practice may lead to inappropriate further invasive investigations in children, although this finding may not be applicable to adults.