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Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis
Published on: August 26, 2020
Diagnosis of ABCB11 gene mutations in children with intrahepatic cholestasis using high resolution melting analysis
Guorui Hu1, Ping He1, Zhifeng Liu1
1Medical College of Nanjing University, Nanjing, Jiangsu 210093, P.R. China.
Insights
High-resolution melting (HRM) analysis combined with direct sequencing offers an effective, sensitive, and economical method for detecting mutations in disease-causing genes. This approach aids in diagnosing progressive familial intrahepatic cholestasis type 2 (PFIC2) by analyzing ABCB11 gene mutations.
Area of Science:
- Genetics
- Molecular Biology
- Pediatric Gastroenterology
Background:
- Intrahepatic cholestasis disorders present significant challenges in childhood, often leading to chronic liver dysfunction.
- Inherited factors are increasingly recognized as key contributors to pediatric cholestasis, necessitating advanced diagnostic tools.
- Existing molecular diagnostic methods for inherited cholestasis vary in efficiency, cost, and complexity.
Purpose of the Study:
- To introduce and validate a cost-effective, sensitive, and efficient method for detecting mutations in cholestasis-associated genes.
- To investigate the spectrum of mutations and single nucleotide polymorphisms (SNPs) in the ABCB11 gene in patients with progressive familial intrahepatic cholestasis type 2 (PFIC2).
- To enhance the clinical diagnosis of PFIC2 by integrating genetic findings with clinical features.
Main Methods:
- Utilized high-resolution melting (HRM) analysis and direct sequencing, based on standard polymerase chain reaction (PCR).
- Applied HRM analysis to screen for mutations in the ABCB11 gene, the causative gene for PFIC2.
- Conducted comprehensive analysis of genetic findings alongside clinical features for accurate PFIC2 diagnosis.
Main Results:
- Identified 14 types of mutations/polymorphisms in the ABCB11 gene among 20 patients from mainland China.
- Discovered six missense mutations, one nonsense mutation, and seven SNPs, including five novel mutations.
- Diagnosed PFIC2 in 4 out of 20 patients by correlating genetic results with clinical presentations. Highly prevalent polymorphisms (V444A and A1028A) were noted in the Chinese population.
Conclusions:
- High-resolution melting (HRM) analysis is an efficient and effective method for detecting ABCB11 gene mutations, expanding the known mutation spectrum.
- The study highlights the utility of integrated genetic and clinical analysis for diagnosing PFIC2.
- The findings provide valuable insights into the genetic landscape of PFIC2 in the mainland Chinese population.
Abstract:
Intrahepatic cholestasis represents a heterogeneous group of disorders that begin during childhood, most commonly manifesting as neonatal cholestasis, and lead to ongoing liver dysfunction in children and adults. For children, inherited pathogenic factors of cholestasis have gained increasing attention owing to the rapid development of molecular biology technology. However, these methods have their advantages and disadvantages in terms of simplicity, sensitivity, specificity, time required and expense. In the present study, an effective, sensitive and economical method is recommended, termed high-resolution melting (HRM) analysis and direct sequencing, based on general polymerase chain reaction, to detect mutations in disease‑causing genes. As one type of inherited intrahepatic cholestasis, progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by pathogenic mutations in the ABCB11 gene, HRM was used to detect mutations in the ABCB11 gene in the present study, and the diagnosis for PFIC2 was made by comprehensive analysis of genetic findings and clinical features. Furthermore, the characteristics of mutations and single nucleotide polymorphisms (SNPs) in the ABCB11 gene were elucidated. A total of 14 types of mutations/polymorphisms were identified in 20 patients from mainland China, including six missense mutations (p.Y337H, p.Y472C, p.R696W, p.Q931P, p.D1131V and p.H1198R), one nonsense mutation (p.R928X) and seven SNPs (p.D36D/rs3815675, p.F90F/rs4148777, p.Y269Y/rs2287616, p.I416I/rs183390670, p.V444A/rs2287622, p.A865V/rs118109635 and p.A1028A/rs497692). Five mutations were novel. The majority of the mutations were different from those detected in other population groups. A total of 4/20 patients (1/5) were diagnosed to be PFIC2 by combining genetic findings with the clinical features. Polymorphisms V444A and A1028A, with an allele frequency of 74.5 and 67.2%, respectively, were highly prevalent in the mainland Chinese subjects. No differences were found between the patients with cholestasis and the control subjects. Efficient genetic screening facilitates the clinical diagnosis of genetic disorders. The present study demonstrated that HRM analysis was efficient and effective in detecting mutations and expanded the known spectrum of ABCB11 gene mutations.
