In these patients four clinical features: (i) age at onset of the biliary symptoms; (ii) occurrence of acute complications, e.g., recurrent cholangitis or pancreatitis; (iii) occurrence of chronic complications, e.g., secondary sclerosing cholangitis, segmental MLN8237 datasheet dilatations of the intrahepatic biliary tract filled with gallstones; (iv) occurrence of ICP with or without severe complications (spontaneous premature delivery, fetal distress, stillborn fetus), were studied according

to the presence or not of ABCB4 variant, and the type of variation if present. All patients were identified by the clinicians responsible for their care. Informed consent was obtained from all subjects and the study was approved by the local Ethical Committee. Genomic DNA was obtained from peripheral white blood cells using standard procedures. To check selleck kinase inhibitor for the presence of sequence variants of the ABCB4 gene, 27 pairs of exon-specific primers were used to amplify the 27 coding exons of the ABCB4 gene together with their respective exon/intron boundaries. After purification, the polymerase chain reaction (PCR) products were sequenced using amplification

primers and the Big Dye Terminator Chemistry. Sequencing products were run after purification on an ABI 3130 Genetic Analyser (Applied Biosystems). Identification and localization of ABCB4 gene sequence variations were assessed by sequence comparisons with the SeqScape Software (v. 2.5; Applied Biosystems). Quantitative variables are expressed as means ± SD. Continuous variables were compared using the Wilcoxon rank sign test or the Kruskal-Wallis test when more than two groups were compared. Quantitative variables were compared using the chi-squared test. A difference was considered statistically significant when the P < 0.05. The R software was used

for all comparisons. A variant was detected in 79 (61 missense and 18 truncating sequence variants) of the 156 patients. The lists providing the sequence variations (nature, location, status) are provided in Tables 1 and 2. Among the 61 patients with missense variants, three were homozygotes and nine were compound heterozygotes. All the patients with a truncating variant were heterozygotes and four were compound heterozygotes. As shown in Table 3, age at onset of symptoms, sex ratio, frequency of either acute (cholangitis PTK6 or pancreatitis), or chronic complications (cholangitis with or without segmental dilatations of the intrahepatic biliary tree), ICP with or without fetal complications (spontaneous prematurity, fetal distress, stillborn fetus) did not differ significantly between the patients with or without ABCB4 variation. Overall, 70% of the patients with an altered genotype were women (P < 0.001); the mean age at the onset of symptoms was 38.7 years for men and 29.1 years for women (P < 0.003). Age at the onset of symptoms differed also according to the type of variant and gender.