Antal Nemeth

A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis.

The progressive familial intrahepatic cholestases (PFIC) are a group of inherited disorders with severe cholestatic liver disease from early infancy. A subgroup characterized by normal serum cholesterol and γ-glutamyltranspeptidase (γGT) levels is genetically heterogeneous with loci on chromosomes 2q (PFIC2) and 18q. The phenotype of the PFIC2-linked group is consistent with defective bile acid transport at the hepatocyte canalicular membrane. The PFIC2 gene has now been identified by mutations in a positional candidate, BSEP, which encodes a liver-specific ATP-binding cassette (ABC) transporter, sister of p-glycoprotein (SPGP). The product of the orthologous rat gene has been shown to be an effective bile acid transporter in vitro. These data provide evidence that SPGP is the human bile salt export pump (BSEP).

Hepatocellular carcinoma in ten children under five years of age with bile salt export pump deficiency

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. (HEPATOLOGY 2006;44:478–486.)

Severe bile salt export pump deficiency : 82 different ABCB11 mutations in 109 families

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.

The viral association of neonatal cholestasis in Sweden : A possible link between Cytomegalovirus infection and extrahepatic biliary atresia

BACKGROUND In addition to earlier reports on the association between viral infections and intrahepatic neonatal cholestasis, in recent studies, investigators have suggested a similar link to extrahepatic biliary atresia. METHODS Fifty-nine cholestatic infants (mean age 8 weeks) were investigated for signs of infection with a large spectrum of viruses. Twenty-one infants had extrahepatic biliary atresia, 38 had intrahepatic cholestasis. The virologic methods included serologic investigation in 59 infants and 54 mothers, virus isolation from stools (49 infants), urine (58 infants) and liver biopsies (40 infants). Polymerase chain reaction was used to detect cytomegalovirus DNA in 25 of the liver biopsy specimens. Two control groups, one with 35 noncholestatic infants and one with 111 healthy, pregnant women were checked for serologic signs of cytomegalovirus. RESULTS Nineteen of 59 (32%) cholestatic infants, including 8 of 21 (38%) with extrahepatic biliary atresia, compared with 2 of 35 (6%) control infants had cytomegalovirus-immunoglobulin (Ig) M detected in serum (p < 0.01). Fifty-one of 54 (94%) tested mothers of cholestatic infants were seropositive for cytomegalovirus, compared with 83 of 111 (75%) control mothers (p < 0.01). Cytomegalovirus DNA in liver specimens was detected by polymerase chain reaction in 9 of 18 (50%) analyzed patients with biliary atresia and in specimens from 3 of 7 patients with intrahepatic cholestasis. CONCLUSIONS Cytomegalovirus infection may play a role, not only in intrahepatic neonatal cholestasis, as was suggested earlier, but also in extrahepatic biliary atresia. The pathogenetic mechanism for this link remains to be established.

Hepatocyte transplantation for inherited metabolic diseases of the liver.

Inherited metabolic diseases of the liver are characterized by deficiency of a hepatic enzyme or protein often resulting in life‐threatening disease. The remaining liver function is usually normal. For most patients, treatment consists of supportive therapy, and the only curative option is liver transplantation. Hepatocyte transplantation is a promising therapy for patients with inherited metabolic liver diseases, which offers a less invasive and fully reversible approach. Procedure‐related complications are rare. Here, we review the experience of hepatocyte transplantation for metabolic liver diseases and discuss the major obstacles that need to be overcome to establish hepatocyte transplantation as a reliable treatment option in the clinic.

Renal function before and long after liver transplantation in children

Background. Renal dysfunction occurs in children with liver diseases and renal function is often further impaired after orthotopic liver transplantation (OLT). Inaccurate methods of determining renal function are used in many cases. We studied renal function with accurate methods before and repeatedly after OLT to analyze the effect of the underlying diseases, hypertension, and the immunosuppressive agents. Methods. A total of 46 children were studied both before and annually after OLT with clearances of inulin and paraaminohippuric acid to determine the glomerular filtration rate (GFR) and effective renal plasma flow (ERPF). The clearance of inulin was also compared with the formula creatinine clearance. Results. GFR and ERPF decreased from before to after OLT and decreased further during the first years after OLT. Patients with extrahepatic biliary atresia and with tumours showed higher GFR 1 year after OLT than those with metabolic and miscellaneous disorders. No significant change in GFR of individual patients occurred from the first to the last values determined at around 1 and 6 years after OLT. No difference in renal function was seen during the first years between patients treated with cyclosporine as compared to those treated with tacrolimus, but 4 years after OLT, the GFR was higher in the tacrolimus-treated patients. Patients on antihypertensive agents had lower GFR than the normotensive ones. There was no agreement between GFR, determined by clearance of inulin, and that calculated on the basis of serum creatinine and the height of the patients. Conclusions. Renal function is reduced by OLT and decreases further during the first years after OLT. Patients with metabolic disorders and those on antihypertensive treatment have the lowest GFR. Determination of GFR by the formula creatinine clearance is inaccurate in children after liver transplantation.

Different expression of HLA-DR and ICAM-1 in livers from patients with biliary atresia and Byler's disease

BACKGROUND/AIMS Extrahepatic biliary atresia (EHBA) and chronic progressive intra-hepatic cholestasis (Bylers disease) are two distinct disorders of unknown etiology in the neonate, characterized by profound cholestasis. The aim of the present study was to investigate the inflammatory reaction in liver tissue from patients with EHBA and Bylers disease. METHODS The expression of HLA-DR and ICAM-1 and the number of CD4+, CD8+ and gamma/delta+ T-cells were investigated with immunoperoxidase technique on cryostat sections of liver tissue from patients with EHBA (n=11), Bylers disease (n=7) and (healthy controls (n=5). RESULTS Only mild inflammation was seen, mainly with CD4+ cells, predominantly in the portal tracts in EHBA and throughout the lobuli in Bylers disease. Neither ICAM-1 nor HLA-DR was present on the bile duct cells or hepatocytes in the control patients. The bile duct cells in all EHBA patients expressed both ICAM-1 and HLA-DR. HLA-DR was expressed on the hepatocytes in five EHBA patients, but ICAM-1 was only found on the hepatocytes in two EHBA patients. In Bylers disease the bile duct cells did not express ICAM-1 or HLA-DR, but the hepatocytes from all Byler patients expressed both ICAM-1 and HLA-DR. CONCLUSIONS Although neither EHBA nor Bylers disease is characterized by intense inflammatory cell infiltration, an aberrant expression of ICAM-1 and HLA-DR was found in the liver in both patients with EHBA and patients with Bylers disease. The expression of HLA-DR and ICAM-1, was clearly distinct between the two disorders, indicating that it is not merely an unspecific event secondary to cholestasis.

Cytomegalovirus DNA detection on Guthrie cards in patients with neonatal cholestasis

AIM To time the onset of cytomegalovirus (CMV) infection in patients (n=39) with CMV associated neonatal cholestasis by analysing CMV DNA on Guthrie cards sampled at 3 days of age. METHODS CMV infection was diagnosed by serology/urine isolation or by CMV DNA detection (polymerase chain reaction) in liver biopsy specimens. In order to time the infection dry blood filter paper discs were punched out from stored Guthrie cards. After phenol–chloroform extraction CMV DNA was detected by nested polymerase chain reaction. RESULTS All cards from control children (n=8) with congenital CMV tested positive; none of the negative controls (n=4) did so. Two of 39 cholestatic infants were CMV DNA positive; their mothers had serological signs compatible with infection during the second half of the pregnancy. All other cholestatic infants tested negative. CONCLUSIONS CMV DNA was not detected in most of the children using Guthrie cards, suggesting that infection developed at or soon after birth.

Budesonide versus Prednisone with Azathioprine for the Treatment of Autoimmune Hepatitis in Children and Adolescents

OBJECTIVE To compare the effect of budesonide vs prednisone therapy both in combination with azathioprine in pediatric patients with autoimmune hepatitis (AIH). STUDY DESIGN Forty-six patients with AIH (11 males and 35 females) aged 9-17 years were enrolled in a 6-month, prospective, double-blind, randomized, active-controlled, multicenter phase IIb study evaluating budesonide (n = 19; 3 mg twice or 3 times daily) vs prednisone (n = 27; 40 mg/day tapered to 10 mg/day), both with azathioprine (1-2 mg/kg/day), followed by a further 6 months of open-label budesonide therapy. The primary efficacy endpoint was complete biochemical remission (normal serum alanine aminotransferase and aspartate aminotransferase levels) without predefined steroid-specific side effects. RESULTS We observed no statistically significant difference in the percentage of patients who met the primary endpoint between the budesonide (3 of 19; 16%) and prednisone groups (4 of 27; 15%) after 6 months, nor in the percentage of patients who experienced biochemical remission (budesonide, 6 of 19 [32%]; prednisone, 9 of 27 [33%]), lack of steroid-specific side effects (budesonide, 10 of 19 [53%]; prednisone, 10 of 27 [37%]). The mean weight gain was 1.2 ± 3.5 kg in the budesonide group and 5.1 ± 4.9 kg in the prednisone group (P = .006). A total of 42 patients received open-label budesonide treatment for another 6 months. After 12 months, 46% of these patients achieved complete remission. CONCLUSION Oral budesonide with azathioprine can induce and maintain remission in pediatric patients with AIH and may be considered an alternative therapy to prednisone. The treatment causes fewer side effects and does not lead to weight gain; however, it may be less effective than prednisone in inducing remission.

Cognitive and emotional outcome after pediatric liver transplantation

Abstract: The aim of the study was to evaluate the cognitive and emotional development after pediatric liver transplantation. A total of 21 patients, aged 4–16.9 yr (median 9.6 yr) were tested 1–9 yr (median 4.2 yr) after the transplantation. The pretransplant diagnoses included biliary atresia (eight patients), various metabolic diseases (n = 6), acute liver failure (n = 3), and miscellaneous (n = 4). The cognitive functions were tested with Wechsler preschool and primary scale of intelligence (WPPSI)‐R or Wechsler intelligence scale for children (WISC)‐III according to age. The Piers–Harris self‐concept scale and the evaluation of human figure drawings according to Koppitz were used to detect emotional problems. All tests in all patients were performed by the same psychologist. A significantly lower result on cognitive tests was seen when compared with the expected normal values (p < 0.01). The number of patients with results within or under the lower normal range was higher than expected. Although the mean value of the Piers–Harris self‐concept scale was normal, there was a large spread within the group. Indicators of emotional problems were found in the human figure drawings of 50% of the patients. To some extent, low cognitive scores coincided with low scores on self‐concept scale and indicators of emotional difficulties. We conclude that the high degree of cognitive and emotional problems after liver transplantation is an important argument for routine psychologic follow‐up and support in these patients.