Screening Mutation Of ABCB11 Gene In Children With Cholestasis Using High Resolution Melting Analysis

Background and Objective: Cholestasis is one of the most manifestations in infantile hepatitis syndrome. Genetic factors resulted from mutations of relevant genes are the second etiologies after biliary atresia. The ABCB11 gene codes one membrane protein called bile salt export pump (BSEP) whose function is secretion of bile salt from hepatocyte to bile capillary, and BSEP is the greatest factor for ductal bile secretion. The ABCB11 mutations resulting to abnormal expression or function of BSEP have been related to many diseases such as progressive intrahepatic cholestasis type 2 (PFIC2) in pediatrics. A lot of technique platforms for detecting mutation have been applied to perform clinically genetic diagnosis of inherited diseases. As a result, progressive intrahepatic cholestasis can be discriminated from infantile hepatitis syndrome to make a definite diagnosis. Current methods for genetic diagnosis are mostly based on polymerase chain reaction and direct sequencing of coding exons and promote using Sanger’s method. Direct sequencing is the current gold standard. However, there are many shortcomings such as high costs and complex operations, especially when the gene has so many exons that those defects are extremely obvious. To solve the problem, we need to search a method which has some advantages such as low costs, high throughput, easy operations, accuracy and good reliability. High resolution melting (HRM) analysis is considered. In this study, we aimed to establish a method based on the combination between HRM and direct sequencing to detect mutations of the ABCB11 gene in children who suffered from intrahepatic cholestasis providing an inexpensive method for clinical gene mutation screening.Materials and Methods:DNA samples were obtained from 20 children who were suspicious of suffering from PFIC2. Genetic variations in the 27 coding exons together with at least 10 bp of adjacent intronic sequences of ABCB11 gene were prescreened using HRM analysis and confirmed by direct sequencing. Variations were submitted to Mutation Taster to relevant literatures. SIFT, PolyPhen-2 and SNPs&GO were used to predict the functional consequences of novel missense mutations. Moreover, amino acid changes of BSEP were classified as evolutionarily conserved (EC) or non-conserved (EN) based on sequence alignments with six mammalian orthologs (bonobo, mouse, rat, rabbit, dog and cattle). Single nucleotide polymorphisms V444A and A1028A were analyzed in patients (n=20) and control subjects (n=200).Results:Fourteen types of mutations/polymorphisms were identified, including seven SNPs (p.D36D/rs3815675, p.F90F/rs4148777, p.Y269Y/rs2287616, p.I416I/rs183390670, p.V444A/rs2287622, p.A865V/rs118109635 and p.A1028A/rs497692), six missense mutations (p.Y337H, p.Y472C, p.R696W, p.Q931P, p.D1131V and p.H1198R), and one nonsense mutation (p.R928X). Five mutations were novel. Four patients could be diagnosed with PFIC2 based on the comprehensive analysis of clinical features and genetic findings. The allele frequency of V444A and A1028A in controls were 74.5% and 67.2%, respectively. As to cholestasis patients, they were 80% and 75%, respectively. No differences were found between patients with cholestasis and control subjects.Conclusion: It is essential to make genetic diagnosis for patients who are characterized by recurrent cholestasis and persistent jaundice. The combination between HRM and direct sequencing is an efficient and effective method which could be used to detect ABCB11 gene mutations to make a genetic diagnosis for children who is clinically diagnosed with PFIC2. The allele frequency of V444A and A1028A in patients and controls show no statistically significant difference.