HBV pre-core mutant in genotype-D infected children is selected during HBeAg/anti-HBe seroconversion and leads to HBeAg negative chronic hepatitis B in adulthood.

Selection of HBeAg defective HBV mutants (mt) during childhood might influence infection outcome in adults. Aim of this study was to correlate the dynamics of pre-core HBV mutant (pre-C mt) selection with virological/clinical outcomes in children followed-up until adulthood. Eighty subjects (50-M/30-F), 70 HBeAg-positive (87.5%), and 10 (12.5%) HBeAg-negative/anti-HBe-positive at the admission, mostly genotype D infected (91.2%), with median age of 6.5 (range: 0.2-17) years, were followed-up for 14.3 years (range: 1.1-24.5); 46 (57.5%) received IFN treatment. HBV-DNA and q-HBsAg were tested by commercial assays, Pre-Core 1896 mt by direct-sequence, oligo-hybridization-assay, and allele-specific-PCR (sensitivity: 30%, 10%, and 0.1% of total viremia). HBeAg/anti-HBe seroconversion (SC) occurred in 55/70 (78.6%) children. After SC, 8 (14.6%) developed HBeAg-negative chronic hepatitis (CHB), 41 (74.5%) remain with HBeAg-negative chronic infection, and 6 (10.9%) lost HBsAg. Baseline HBV-DNA and HBsAg were lower in SC than in no-SC children (median: 7.35 vs 8.95 Log IU/mL, P = 0.005, and 4.72 vs 5.04 Log IU/mL, P = 0.015). The prevalence of pre-C mt increased rapidly (10-40%) around SC. Eventually, pre-C mt was detected in 100% of CHB, in 33% of chronic infections without disease, and in 16% of subjects who cleared HBsAg (P < 0.001). HBV-DNA levels remained slightly higher in carriers of HBeAg negative infection with dominant/mixed pre-C mt populations, than in those with dominant pre-C wt (mean Log IU/mL: 3.83 and 3.42 vs 2.67, P = 0.007). In conclusion, pre-C-mt is selected during HBeAg/anti-HBe SC in children with poor control of HBV replication, leading to HBeAg-negative chronic-active-hepatitis during adulthood.

Introduction of gluten, HLA status, and the risk of celiac disease in children.

BACKGROUND: The relationship between the risk of celiac disease and both the age at which gluten is introduced to a child's diet and a child's early dietary pattern is unclear. METHODS: We randomly assigned 832 newborns who had a first-degree relative with celiac disease to the introduction of dietary gluten at 6 months (group A) or 12 months (group B). The HLA genotype was determined at 15 months of age, and serologic screening for celiac disease was evaluated at 15, 24, and 36 months and at 5, 8, and 10 years. Patients with positive serologic findings underwent intestinal biopsies. The primary outcome was the prevalence of celiac disease autoimmunity and of overt celiac disease among the children at 5 years of age. RESULTS: Of the 707 participants who remained in the trial at 36 months, 553 had a standard-risk or high-risk HLA genotype and completed the study. At 2 years of age, significantly higher proportions of children in group A than in group B had celiac disease autoimmunity (16% vs. 7%, P=0.002) and overt celiac disease (12% vs. 5%, P=0.01). At 5 years of age, the between-group differences were no longer significant for autoimmunity (21% in group A and 20% in group B, P=0.59) or overt disease (16% and 16%, P=0.78 by the log-rank test). At 10 years, the risk of celiac disease autoimmunity was far higher among children with high-risk HLA than among those with standard-risk HLA (38% vs. 19%, P=0.001), as was the risk of overt celiac disease (26% vs. 16%, P=0.05). Other variables, including breast-feeding, were not associated with the development of celiac disease. CONCLUSIONS: Neither the delayed introduction of gluten nor breast-feeding modified the risk of celiac disease among at-risk infants, although the later introduction of gluten was associated with a delayed onset of disease. A high-risk HLA genotype was an important predictor of disease. (Funded by the Fondazione Celiachia of the Italian Society for Celiac Disease; CELIPREV ClinicalTrials.gov number, NCT00639444.).

Management of chronic hepatitis C in childhood: the impact of therapy in the clinical practice during the first 2 decades.

BACKGROUND AND AIM: Treatment of chronic hepatitis C in children is controversial and its role in the clinical practice is unknown. We retrospectively investigated the impact of treatment in a large cohort of children with chronic hepatitis C over the past 20 years. METHODS: 376 hepatitis C virus RNA positive children were recruited consecutively in five Italian centres since 1990 and followed for 1-17 years. RESULTS: 86 (23%) subjects were treated: 73 with recombinant interferon alone and 13 with pegylated-interferon and ribavirin. Sustained clearance of hepatitis C virus RNA was observed in 25% of the former, in 92% of the latter and in 9% of untreated cases (p < 0.001). Loss of viraemia was recorded in all children with genotype 2-3 and in 6 of 7 with hepatitis C virus genotype 1 treated with combination therapy. At last evaluation 45% of patients were young adults and 15% had cleared viraemia. Overall, 152 (40%) were putative candidates to therapy. CONCLUSIONS: Few Italian children with chronic hepatitis C have been treated in the past 20 years. The poor propensity to spontaneous clearance of viraemia and the efficacy of combination therapy should encourage to consider treatment in attempt to shorten the duration of viral replication.

Variable phenotypic expression of chylomicron retention disease in a kindred carrying a mutation of the Sara2 gene.

Chylomicron retention disease is a recessive inherited disorder characterized by fat malabsorption and steatorrhea and is associated with failure to thrive in infancy. We describe a kindred carrying a mutation of Sara2 gene causing a chylomicron retention phenotype. The proband was a 5-month-old baby, born of consanguineous, apparently healthy parents from Morocco, with failure to thrive. There was a large quantity of fats in feces and malabsorption of fat-soluble vitamins. Intestinal biopsies showed a diffused enterocyte vacuolization with large cytosolic lipid droplets. Chylomicron retention disease or Anderson disease was hypothesized, and the Sara2 gene was analyzed by direct sequencing. Analysis of the Sara2 gene in the proband identified a 2-nucleotide homozygous deletion in exon 3 leading to a premature stop codon (c.75-76 del TG-L28fsX34). The father was heterozygous for the same mutation, whereas the proband's mother was homozygous, suggesting a variable phenotypic expression of the molecular defect. More studies are needed to understand the reasons of the phenotypic variability of the same molecular defect in the same family.

Thiopurine S-methyltransferase pharmacogenetics in a large-scale healthy Italian-Caucasian population: differences in enzyme activity.

AIMS: To investigate the influence of genotype, age and gender on the thiopurine S-methyltransferase (TPMT) phenotype in healthy Italian-Caucasian subjects. MATERIALS & METHODS: The study investigated the TPMT genotype and the TPMT phenotype of 943 healthy Italian-Caucasian subjects of different age and gender (age range: 0.08-68 years; 623 males 320 females). TPMT red blood cell activity was measured in all samples and genotype was determined for the TPMT alleles *2, *3A, *3B and *3C. RESULTS: TPMT activity levels in our whole population ranged from 1.6 up to 75.2 U/gHb. Significant TPMT activity differences between wild-type and heterozygous subjects were observed. We divided our TPMT activity into four categories according to our frequency distribution: low (0.1%), intermediate (32.9%), normal (60%) and high (7%), with arbitrary cut-off values of 8.0, 19.4 and 37.0 U/gHb, respectively. The whole population had a total of 94.5% of homozygous wild-type subjects, 5.4% heterozygous variants and one (0.1%) compound heterozygous variant TPMT*3B/*3C. The overall concordance rate between TPMT genotypes and phenotypes was 71.6%. The TPMT activity was significantly higher in wild-type children (0.08-17 years) than in wild-type adults (aged 18-68 years). Moreover, it was noted that wild-type infants from 0.08 to 5 years had a 9% higher average TPMT activity than the other wild-type groups, and only in children from 0.08 to 2 years was the TPMT activity higher in males than in females. CONCLUSION: The data obtained in this study show that genetic factors seem to be the major aspect in TPMT phenotype variability in adults, whilst, in children, other physiological factors should be taken into consideration when assessing the TPMT phenotype, such as age and gender.

Prevalence and natural history of gastroesophageal reflux: pediatric prospective survey.

OBJECTIVE: The prevalence and natural history of gastroesophageal reflux in infants have been poorly documented. The aim of this study was to evaluate the prevalence and natural history of infant regurgitation in Italian children during the first 2 years of life. METHODS: A detailed questionnaire, according to the Rome II criteria, was completed by 59 primary care pediatricians to assess infant regurgitation in consecutive patients seen in their office over a 3-month period. A total of 2642 patients aged 0 to 12 months were prospectively enrolled during this 3-month period. Follow-up was performed at 6, 12, 18, and 24 months of age. RESULTS: A total of 313 children (12%; 147 girls) received the diagnosis of infant regurgitation. Vomiting was present in 34 of 313 patients. Their score for the Infant Gastroesophageal Reflux Questionnaire was 8.51 +/- 4.75 (mean +/- SD). Follow-up visits were conducted to the end in 210 of 313 subjects. Regurgitation had disappeared in 56 of 210 infants by the first 6 months of age, in 128 by the first 12 months, in 23 at 18 months, and in 3 patients by the first 24 months. At follow-up, 1 of 210 patients had developed a gastroesophageal reflux disease with esophagitis, proven endoscopically and histologically; another patient received a diagnosis of cow milk protein intolerance. The Infant Gastroesophageal Reflux Questionnaire score reached 0 after 8.2 +/- 3.9 months in breastfed infants (89 of 210) and after 9.6 +/- 4.1 months in artificially fed infants. CONCLUSIONS: We found that 12% of Italian infants satisfied the Rome II criteria for infant regurgitation. Eighty-eight percent of 210 infants who had completed a 24-month follow-up period had improved at the age of 12 months. Only 1 patient later turned out to have gastroesophageal reflux disease. Use of breast milk was associated with a shorter time necessary to reach an Infant Gastroesophageal Reflux Questionnaire score of 0.

Genotype-phenotype correlation in Italian children with Wilson's disease.

BACKGROUND/AIMS: Wilson's disease phenotype is very variable for clinical and laboratory features. Our aim was to assess the role of the type of ATP7B disease causing mutations on Wilson's disease phenotype. METHODS: We retrospectively evaluated the data of children with Wilson's disease from eight pediatric departments. RESULT: Fifty-eight patients (34 male, median age at diagnosis 7.4 years) from 47 unrelated families were studied, carrying 34 different mutations. The most common mutations were the missense p.H1069Q and p.M769V, the nonsense p.R1319X, the frameshift c.2299delC, c.2298_2299insC and c.2530delA, and the splice site mutation c.2447+5G>A. Serum ceruloplasmin and copper were lower among the patients' homozygotes for nonsense and frameshift mutations than in patients with missense mutations. A normalization of serum alanine aminotransferase after therapy was not achieved in 23.6% of patients with missense mutations versus 45.5% of patients with nonsense/frameshift mutations. A direct linear correlation was found between age at diagnosis and urinary copper excretion at diagnosis. CONCLUSIONS: The type of mutation explains at least a part of Wilson's disease phenotype, and mutation analysis should be considered as an integrative tool for such a challenging diagnosis. Urinary copper excretion appears to be correlated to the age at diagnosis rather than genotype.

Duodenal bulb biopsies in celiac disease: a multicenter study.

OBJECTIVES: Celiac disease (CD)-related lesions have been reported in duodenal bulb biopsies, sometimes the bulb mucosa being the only one affected. The aim was to verify in a significant series whether histological lesions are always present in the bulb of celiac patients, what is the prevalence of lesions when isolated to the bulb, and if similar lesions are present in nonceliac subjects. METHODS: We studied 665 children with CD (241 males, range 9 months-15 years, 8 months), at diagnosis on a gluten-containing diet, and 348 age- and sex-matched gastroenterological controls submitted to upper endoscopy for gastroenterological complaints. During endoscopy, multiple biopsies (1 bulb and 4 distal duodenum samples) were taken. Anti-endomysium antibodies were evaluated by immunofluorescence method, anti-human tissue-transglutaminase antibodies by an enzyme-linked immunosorbent assay or radioimmunoassay. Human leukocyte antigen-DRB1, -DQA1, and -DQB1 genes were typed by polymerase chain reaction sequence-specific primers repeat method. RESULTS: In all of the patients with CD, histological lesions were present in the bulb sample; in 16 of them, the lesions were present only in the bulb. Patchy villous atrophy was found in 20 children. All of the patients with CD were anti-endomysium and/or antitransglutaminase positive. The controls showed neither autoantibody positivity nor mucosal changes compatible with CD. CONCLUSIONS: This study demonstrated that CD-related histological lesions are always present in the bulb; sometimes this specific site is the only one affected. Therefore, we suggest taking 2 biopsies from the bulb and 2 from the distal duodenum for CD diagnosis.

Long-term course of chronic hepatitis C in children: from viral clearance to end-stage liver disease.

BACKGROUND & AIMS: The natural course of chronic hepatitis C (CHC) in children is not well understood. The aim of this study was to assess the long-term course of CHC in a large sample of otherwise healthy children. METHODS: From 1990 to 2005, 504 consecutive antihepatitis C virus (HCV)-positive children were enrolled at 12 centers of a national observatory and were followed up retrospectively/prospectively. RESULTS: Putative exposure was perinatal in 283 (56.2%) cases, parenteral in 158 (31.3%), and unknown in 63 (12.5%). At baseline, 477 (94.6%) cases were HCV RNA seropositive, 118 (24.7%) of which were treated with standard interferon alpha. Ten years after putative exposure, the outcome in 359 HCV RNA-positive, untreated patients was (1) undetectable viremia in 27 (7.5%) (by Cox regression analysis, spontaneous viral clearance was independently predicted by genotype 3 [hazard ratio 6.44; 95% confidence interval: 2.7-15.5]) and (2) persistent viremia in 332 (92%) cases. Six of these 332 cases (1.8%) progressed to decompensated cirrhosis (mean age, 9.6 years). This latter group included 5 Italian children perinatally infected with genotype 1a (4 of the mothers were drug users). Thirty-three (27.9%) treated patients achieved a sustained virologic response. CONCLUSIONS: Over the course of a decade, few children with chronic HCV infection cleared viremia spontaneously, and those who did were more likely to have genotype 3. Persistent viral replication led to end-stage liver disease in a small subgroup characterized by perinatal exposure, maternal drug use, and infection with HCV genotype 1a. Children with such features should be considered for early treatment.

Twenty-four novel mutations in Wilson disease patients of predominantly Italian origin.

Herein we report the results of mutation analysis of the ATP7B gene in a group of 134 Wilson disease (WD) families (268 chromosomes) prevalently of Italian origin. Using the SSCP and sequencing methods we identified 71 disease-causing mutations. Twenty-four were novel, while 19 more mutations already described, were identified in new populations in this study. A known mutation G591D showed a regional distribution, since it was only detected in 38.5% of the analyzed chromosomes in WD patients originating from Apulia, a region of South Italy. Detection of new mutations in the ATP7B gene increases our capability of molecular analysis that is essential for early diagnosis and treatment of WD.

Epidemiological profile of 806 Italian children with hepatitis C virus infection over a 15-year period.

BACKGROUND/AIMS: To evaluate the epidemiological profile of Italian children with hepatitis C virus (HCV) infection over a 15-year period. METHODS: Fifteen tertiary care centers, belonging to a national Observatory established in 1998, retrospectively/prospectively recruited 806 consecutive HCV-infected, otherwise healthy, children seen from 1990 to 2004. RESULTS: Seven hundred and sixty four were Italian and 42 from foreign countries. Newly-diagnosed cases declined from 332 in 1995-1999 to 196 in 2000-2004, while the proportion of foreign children rose from 3% to 13%. Transfusion-transmitted infection disappeared after 1992. Maternal infection (with drug abuse in 63% of cases in the North) has become the most important mode of HCV diffusion throughout Italy and the exclusive source for all children infected in 2000-2004. The prevalence of HCV genotypes 3 and 4 increased and that of genotype 1b decreased significantly (p<0.02). Male/female ratio was significantly (p<0.001) lower among vertically infected (0.6) than in transfused children (1.3). CONCLUSIONS: The number of children with newly-diagnosed HCV infection is declining in Italy and most post-transfusion cases are now young adults. Thus foreign children could significantly contribute to the reservoir of pediatric infection in years to come. New infections result from maternal transmission and seem to privilege females and genotypes 3 and 4.

The canine copper toxicosis gene MURR1 is not implicated in the pathogenesis of Wilson disease.

BACKGROUND: It has recently been demonstrated that the Wilson disease (WD) protein directly interacts with the human homolog of the MURR1 protein in vitro and in vivo, and that this interaction is specific for the copper transporter. The aim of the present study was to clarify the role of MURR1 in the pathogenesis of WD as well as in other WD-like disorders of hepatic copper metabolism of unknown origin. METHODS: Using the single-strand conformation polymorphism (SSCP) method followed by sequencing, we analyzed the 5' untranslated region (UTR) and three exons of the MURR1 gene in three groups of patients: 19 WD: patients in whom no mutations were detected in the ATP7B gene, 53 WD: patients in whom only one mutation in the ATP7B gene was found, and 34 patients in whom clinical and laboratory data suggested a WD-like disorder of hepatic copper metabolism of unknown origin. RESULTS: We detected in these patients six rare nucleotide substitutions, namely one splice-site consensus sequence and one missense and four silent nucleotide substitutions. All substitutions except one were found in the heterozygous state. No difference in the frequencies of the various substitutions was observed between patients and controls. CONCLUSIONS: These data suggest that the MURR1 gene and its protein product are unlikely to play a primary role in the pathogenesis of Wilson disease. More extensive studies with larger numbers of clinically homogeneous patients should be carried out to establish whether nucleotide alterations in the MURR1 gene may have a role in causing WD or WD-like disorders or act as modifying factors in the phenotype variability in WD.

Serum transaminases in children with Wilson's disease.

OBJECTIVES: The response of serum transaminase levels to penicillamine and zinc treatment in Wilson's disease is poorly understood. The aim of this multicenter retrospective study was to evaluate transaminase levels after penicillamine and zinc treatment in children with Wilson's disease. PATIENTS AND METHODS: One hundred and nine patients with Wilson's disease (median age at diagnosis, 7.2 years; range, 1 to 18 years), treated for at least 12 months and observed in the last 20 years at 11 Paediatric Departments were studied. Clinical, laboratory and histologic features at diagnosis and initial treatment were recorded. Efficacy parameters were normalization of serum transaminase level and improved clinical and/or laboratory signs. One hundred and two patients had clinical or laboratory signs of liver disease. RESULTS: Fifty-six of 87 patients (64%) given penicillamine normalized serum alanine aminotransferase (ALT) levels within a median of 17 months (range, 2 to 96 months). Of the 29 patients with persistent hyper-ALT, 17 (59%) switched to zinc; only four of these normalized ALT on zinc within a median period of 38 months (range, 7 to 48 months). Eleven (50%) of the 22 patients given zinc alone normalized ALT within a median period of 6 months (range, 1 to 36 months). Of the 11 patients with persistent hyper-ALT, five switched to penicillamine. Three of the five normalized ALT within a median period of 6 months (range, 6 to 9 months). Overall, in penicillamine-treated and zinc-treated patients with persistent hypertransaminasemia, ALT decreased from a basal median of 236 IU/L (range, 54 to 640 IU/L) to a median of 78 (range, 46 to 960 IU/L) at the end of follow-up (P = 0.0245). Poor compliance was suspected in only 10% of cases. No predictive factor of persistent hypertransaminasemia was identified. Liver disease did not worsen in any patient during the study. CONCLUSIONS: Although the efficacy of penicillamine and zinc is well documented, it is notable that a subset of children with Wilson's disease-related liver disease (36%) had hypertransaminasemia despite appropriate treatment with penicillamine or zinc.

Fibrosis in chronic hepatitis C acquired in infancy: is it only a matter of time?

OBJECTIVE: The natural history of chronic hepatitis C acquired in infancy is not well understood. The progression of fibrosis was analyzed in untreated children with chronic hepatitis C virus infection and no other hepatotoxic cofactors. METHODS: A total of 112 pediatric patients (13 with paired liver biopsies) were considered. Fibrosis was assessed by METAVIR score (i.e., stage F1 to F4). The ratio between the stage of fibrosis (METAVIR units) and the presumed duration of infection represented the "estimated" rate of fibrosis progression per year. In patients with paired biopsies, the "observed" rate of fibrosis progression was defined as the difference between the stage of fibrosis in the two biopsies divided by the time interval between them. RESULTS: Both age of patients at biopsy and duration of infection correlated with stage of fibrosis (p < 0.002 and p < 0.0005, respectively). Stage of fibrosis differed significantly between patients with infection lasting less or more than 10 yr (p < 0.0006). Sex, hepatitis C virus genotype, and route of infection did not correlate with stage of fibrosis. Among the 13 patients with paired biopsies, stage of fibrosis increased in seven and did not change in six; the median rate of estimated fibrosis progression per year was 0.142. The difference between estimated and observed fibrosis progression rates was significant (coefficient of determination, r(2) = 0.031), which demonstrated that the prediction of the fibrosis progression was unreliable in 97% of patients. CONCLUSIONS: Chronic hepatitis C acquired in childhood is a progressive, slow-moving, fibrotic disease. Fibrosis progression inferred on the basis of linear mathematical models should be critically evaluated in the clinical practice.

Hepatitis C virus infection associated with liver-kidney microsomal antibody type 1 (LKM1) autoantibodies in children.

OBJECTIVE: To evaluate the clinical pattern and evolution of chronic hepatitis C in children with liver/kidney microsomal antibody type 1 autoantibodies (LKM1). STUDY DESIGN: A multicenter, retrospective study, including the following groups of children with hepatitis C virus infection: (1). 21 consecutive LKM1-positive patients, (2). 42 age- and sex- matched LKM1-negative patients, and (3). 4 interferon-induced LKM1-positive cases. LKM1 reactivity to human microsomes and recombinant cytochrome P450IID6 (CYP2D6) was assayed by immunoblotting. RESULTS: Clinical and biochemical features overlapped in LKM1-positive and LKM1-negative children, but a fibrosis score >3 (range 0-6) was significantly more frequent (P =.04) in the former. Reactivity to microsomal protein and CYP2D6 was significantly (P =.02) associated with LKM1 titers >or=1:320 and was found in 39% of patients, including severe cases and both children (of 4 treated) who achieved a sustained alanine aminotransferase (ALT) normalization after steroid treatment. Five of 7 LKM1-positive children treated with interferon had an ALT exacerbation. CONCLUSIONS: LKM1-positive hepatitis C in children is characterized by a wide spectrum of biochemical, serologic, and histologic features. Whether autoimmunity may contribute to liver damage in a subgroup of patients with more severe liver disease, high LKM1 titers, and reactivity to CYP2D6 is a question deserving further investigation.

Chronic hepatitis C virus infection in childhood: clinical patterns and evolution in 224 white children.

The characteristics and evolution of hepatitis C virus (HCV) infection were retrospectively investigated in a study of 224 HCV RNA-seropositive white children who were consecutively recruited at 7 European centers in 1980-1998. At presentation, all patients were positive for antibodies to hepatitis C virus, 87% were asymptomatic, and 48% had alanine aminotransferase (ALT) levels that were < or =2 times the upper limit of the range considered to be normal. Of 200 children followed for 1-17.5 years (mean follow-up +/- standard deviation [SD], 6.2+/-4.7 years), only 12 (6%) achieved sustained viremia clearance and normalization of the ALT level. In 92 revised liver biopsy specimen analyses, the mean fibrosis score (+/-SD) was 1.5+/-1.3 for children <15 years of age and 2.3+/-1.2 for children > or =15 years of age (range, 0-6 years; P<.01). Pediatric HCV infection is usually mild, but few patients, especially those who are perinatally infected, clear viremia in the medium-term follow-up. Conversely, the higher rates of fibrosis observed in older patients suggest the possibility of an insidious progression of HCV-associated liver disease.