BACKGROUND & AIMS:Patients with severe bile salt export pump (BSEP) deficiency present as infants with progressive cholestatic liver disease. We characterized mutations of ABCB11 (encoding BSEP) in such patients and correlated genotypes with residual protein detection and risk of malignancy. METHODS: Patients with intrahepatic cholestasis suggestive of BSEP deficiency were investigated by single-strand conformation polymorphism analysis and sequencing of ABCB11. Genotypes sorted by likely phenotypic severity were correlated with data on BSEP immunohistochemistry and clinical outcome. RESULTS: Eighty-two different mutations (52 novel) were identified in 109 families (9 nonsense mutations, 10 small insertions and deletions, 15 splice-site changes, 3 whole-gene deletions, 45 missense changes). In 7 families, only a single heterozygous mutation was identified despite complete sequence analysis. Thirty-two percent of mutations occurred in >1 family, with E297G and/or D482G present in 58% of European families (52/89). On immunohistochemical analysis (88 patients), 93% had abnormal or absent BSEP staining. Expression varied most for E297G and D482G, with some BSEP detected in 45% of patients (19/42) with these mutations. Hepatocellular carcinoma or cholangiocarcinoma developed in 15% of patients (19/128). Two protein-truncating mutations conferred particular risk; 38% (8/21) of such patients developed malignancy versus 10% (11/107) with potentially less severe genotypes (relative risk, 3.7 [confidence limits, 1.7-8.1; P = .003]). CONCLUSIONS: With this study, >100 ABCB11 mutations are now identified. Immunohistochemically detectable BSEP is typically absent, or much reduced, in severe disease. BSEP deficiency confers risk of hepatobiliary malignancy. Close surveillance of BSEP-deficient patients retaining their native liver, particularly those carrying 2 null mutations, is essential. Abbreviations:ABC, ATP-binding cassette; AFP, α-fetoprotein; BSEP, bile salt export pump; CpG, cytosine-guanine; CC, cholangiocarcinoma; FIC1, familial intrahepatic cholestasis 1; γ-GT, γ-glutamyl transferase; HCC, hepatocellular carcinoma; IC, intracellular loop; MDR1, multidrug resistance protein 1; MDR3, multidrug resistance protein 3; MRP2, multidrug resistance-associated protein 2; NBF, nucleotide-binding fold; OLT, orthotopic liver transplantation; PEBD, partial external biliary diversion; PFIC, progressive familial intrahepatic cholestasis; PCR, polymerase chain reaction; RE, restriction endonuclease; SSCP, single-strand conformation polymorphism; TM, transmembrane domain; UDCA, ursodeoxycholic acid Acknowledgements We thank the families and the Children's Liver Disease Foundation for support and encouragement, and those who referred families for analysis, including Drs U Baumann, W Berquist, M de Vree, K Emerick, G Ferry, M Finegold, W Hardikar, S Horslen, R Houwen, R Jaffe, L Klomp, F Lacaille, K Mann, P McKiernan, H Sharp, R Sokol, E Sturm, L Szönyi, J Taminou, and J Watkins. We also thank Dr R Garcia-Kennedy for access...

Hepatocellular carcinoma (HCC) is rare in young children. We attempted to see if immunohistochemical and mutational-analysis studies could demonstrate that deficiency of the canalicular bile acid transporter bile salt export pump (BSEP) and mutation in ABCB11, encoding BSEP, underlay progressive familial intrahepatic cholestasis (PFIC)-or "neonatal hepatitis" suggesting PFIC-that was associated with HCC in young children. We studied 11 cases of pediatric HCC in the setting of PFIC or "neonatal hepatitis" suggesting PFIC. Archival liver were retrieved and immunostained for BSEP. Mutational analysis of ABCB11 was performed in leukocyte DNA from available patients and parents. Among the 11 nonrelated children studied aged 13-52 months at diagnosis of HCC, 9 (and a full sibling, with neonatal hepatitis suggesting PFIC, of a tenth from whom liver was not available) had immunohistochemical evidence of BSEP deficiency; the eleventh child did not. Mutations in ABCB11 were demonstrated in all patients with BSEP deficiency in whom leukocyte DNA could be studied (n ‫؍‬ 7). These mutations were confirmed in the parents (n ‫؍‬ 14). With respect to the other 3 children with BSEP deficiency, mutations in ABCB11 were demonstrated in all 5 parents in whom leukocyte DNA could be studied. Thirteen different mutations were found. In conclusion, PFIC associated with BSEP deficiency represents a previously unrecognized risk for HCC in young children. Immunohistochemical evidence of BSEP deficiency correlates well with demonstrable mutation in ABCB11.

CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and protein-losing enteropathy (the CHAPLE syndrome) is caused by abnormal complement activation due to biallelic loss-of-function mutations in CD55. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Background & AimsCongenital diarrheal disorders are rare inherited intestinal disorders characterized by intractable, sometimes life-threatening, diarrhea and nutrient malabsorption; some have been associated with mutations in diacylglycerol-acyltransferase 1 (DGAT1), which catalyzes formation of triacylglycerol from diacylglycerol and acyl-CoA. We investigated the mechanisms by which DGAT1 deficiency contributes to intestinal failure using patient-derived organoids.MethodsWe collected blood samples from 10 patients, from 6 unrelated pedigrees, who presented with early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure; we performed next-generation sequencing analysis of DNA from 8 patients. Organoids were generated from duodenal biopsies from 3 patients and 3 healthy individuals (controls). Caco-2 cells and patient-derived dermal fibroblasts were transfected or transduced with vectors that express full-length or mutant forms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption of DGAT1 in control intestinal organoids. Cells and organoids were analyzed by immunoblot, immunofluorescence, flow cytometry, chromatography, quantitative real-time polymerase chain reaction, and for the activity of caspases 3 and 7.ResultsIn the 10 patients, we identified 5 bi-allelic loss-of-function mutations in DGAT1. In patient-derived fibroblasts and organoids, the mutations reduced expression of DGAT1 protein and altered triacylglycerol metabolism, resulting in decreased lipid droplet formation after oleic acid addition. Expression of full-length DGAT2 in patient-derived fibroblasts restored formation of lipid droplets. Organoids derived from patients with DGAT1 mutations were more susceptible to lipid-induced cell death than control organoids.ConclusionsWe identified a large cohort of patients with congenital diarrheal disorders with mutations in DGAT1 that reduced expression of its product; dermal fibroblasts and intestinal organoids derived from these patients had altered lipid metabolism and were susceptible to lipid-induced cell death. Expression of full-length wildtype DGAT1 or DGAT2 restored normal lipid metabolism in these cells. These findings indicate the importance of DGAT1 in fat metabolism and lipotoxicity in the intestinal epithelium. A fat-free diet might serve as the first line of therapy for patients with reduced DGAT1 expression. It is important to identify genetic variants associated with congenital diarrheal disorders for proper diagnosis and selection of treatment strategies.

Neurologic complications (NCs) are a significant cause of morbidity and mortality in patients who undergo liver transplantation (LT). The aim of this study was to evaluate the incidence and type of NCs and associated factors in pediatric LT patients. We retrospectively reviewed NCs in the medical records of 40 consecutive infants, children, and adolescents who underwent LT at our institution. The subjects consisted of 23 boys and 17 girls (median age, 8.5 +/- 0.85 yr; range, 11 months to 17 yr). The indications for LT were Wilson's disease in 10 patients, fulminant hepatic failure (FHF) in nine, and other types of chronic liver disease in 21. NCs were found in 14 patients (35%). Those 14 individuals experienced a total of 16 episodes of NCs (two separate episodes in two of the patients). The most common NCs were seizure (seven episodes in six patients) and posterior leukoencephalopathy syndrome (PLES; five episodes in four patients). Seizure was the presenting symptom in three episodes of PLES. Two episodes of diffuse encephalopathy were observed in two patients, and two episodes of psychiatric symptoms occurred in two patients. We also noted one episode of tremor in one patient, one episode of acute dystonic reaction in one patient, and one episode of headache in one patient. Patients with Wilson's disease had a higher incidence of NCs (60%) than did patients without Wilson's disease (26.7%); however, this difference was not significant. The incidence of NCs was 44% in patients with FHF and 35% in those without FHF. That difference also was not significant. Immunosuppressive agents were the primary cause of 13 of the 16 episodes of NC. Uremia with hypertension, hypoxia, and hypomagnesemia caused one neurologic episode each. NCs, which are frequent in the first 30 days after pediatric LT, did not affect survival in this group. NCs were reversed by the discontinuation or reduction of immunosuppressive agents in 12 episodes, correction of hypomagnesemia and the reduction of immunosuppressive agents in one episode, and the correction of uremia and hypertension in one episode. Refractory epilepsy developed in one patient, and death unrelated to NCs occurred in one. The mortality rate was 7.1% (n = 1) in patients with NCs and 15.4% (n = 4) in those without NCs (p = 0.64). NCs are an important complication after LT. It is essential that each transplantation team collaborate with pediatric neurologists to ensure the rapid and accurate diagnosis of NCs in infants, children, and adolescents after LT and to prevent the delay of appropriate treatment.

Food allergy has been increasingly reported in children who had orthotopic liver transplantation (OLT). We aimed to conduct a prospective study to investigate the prevalence of sensitizations and food allergy in pediatric OLT recipients. We also aimed to identify potential risk factors. The study group consisted of 28 children (14 male, 14 female, mean age 4.96 +/- 0.76 yrs) who had OLT. Total eosinophil count (TEC), total IgE, and specific IgEs were studied before and 3, 6, 12 months after OLT. Six patients (21%) developed multiple food allergies. Mean age of six patients at OLT who developed food allergy was younger compared to the non-food allergy group (10.2 months vs. 68.9 months, p < 0.05). Food allergy has been developed within 1 yr in 5, and in 20 months in one patient after OLT. All six patients had cow's milk and egg allergy after OLT. Five children developed wheat, one children developed lentil and another one developed peach allergy in addition to cow's milk and egg allergy. Out of six food-allergic patients after OLT, four children developed Epstein-Barr virus (EBV) infection prior to food allergy. Before OLT, TECs and total IgE levels were not differed among food allergic and non-food allergic patients (p > 0.05). Mean of TECs were significantly higher in food allergic group compared to non-food allergic group at each time point after OLT (p < 0.05). Though statistically insignificant, mean of total IgE levels were also higher in the food allergic group (p > 0.05). These findings suggest that food allergy should be considered after OLT in patients who are younger than 1 yr of age, who developed hypereosinophilia, high total IgE levels or EBV viremia.

Symptomatic H. pylori infection in a child should always be treated. The urea breath test is an accurate and reliable way to identify H. pylori-positive patients and to determine the response to treatment. Triple-agent therapy is effective for eradicating H. pylori infection in children and usually helps reduce or eliminate dyspeptic symptoms. The level of H. pylori resistance to metronidazole is high in our region. The significant rate of resistance to clarithromycin (18.1%) may explain the treatment failure observed in this study.

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