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Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease
Published on: April 4, 2018
Most common SLC25A13 mutation in 400 Chinese infants with intrahepatic cholestasis
Hai-Yan Fu1, Shao-Ren Zhang, Hui Yu
1Center for Pediatric Liver Diseases, Children's Hospital of Fudan University, 399 Wanyuan Road, Minhang District, Shanghai 201102, China.
Insights
This study developed a rapid real-time PCR method to detect the SLC25A13 gene mutation 851del4 in infants with intrahepatic cholestasis. The new RT-PCR technique achieved 100% accuracy, offering a fast diagnostic tool.
Area of Science:
- Molecular Biology
- Genetic Diagnostics
- Pediatric Diseases
Background:
- Intrahepatic cholestasis in infants can be caused by genetic mutations.
- The SLC25A13 gene mutation 851del4 is a significant cause of this condition.
- Accurate and rapid diagnostic methods are crucial for timely intervention.
Purpose of the Study:
- To establish a real-time fluorescence polymerase chain reaction (RT-PCR) assay.
- To enable fast detection of the SLC25A13 gene mutation 851del4.
- To validate the assay's accuracy in a large infant cohort.
Main Methods:
- Designed specific primers and dual-labeled fluorescence probes for SLC25A13 mutation 851del4 detection.
- Analyzed take-off curves from a single RT-PCR run for rapid results.
- Enrolled 400 infants (<1 year) with unexplained intrahepatic cholestasis from China.
- Confirmed results of 24 positive and 14 negative samples using direct sequencing.
Main Results:
- Identified 46 mutant alleles, with a mutation rate of 5.8% (46/800).
- Detected 8 homozygous and 30 heterozygous mutations.
- Observed higher mutation prevalence in intermediate and southern China compared to northern China.
- RT-PCR demonstrated 100% accuracy when validated by direct sequencing.
Conclusions:
- RT-PCR with dual-labeled probes is a highly accurate method for detecting the SLC25A13 851del4 mutation.
- This assay provides a fast and reliable diagnostic tool for infants with intrahepatic cholestasis.
- The findings support the use of this RT-PCR method in clinical settings for genetic screening.
Aim:
To establish the real time fluorescence polymerase chain reaction (RT-PCR) with dual labeled probes for fast detection of SLC25A13 gene mutation 851del4.
Methods:
Four hundred infants (< 1 year of age) with unexplained intrahepatic cholestasis from 18 provinces or municipalities in China were enrolled in this study for detecting their SLC25A13 gene mutation 851del4. Suitable primers and fluorescence-labeled probes for detecting SLC25A13 gene mutation 841del4 were designed. Normal and mutant sequences were detected by PCR with two fluorescence-labeled probes. After a single RT-PCR, results were obtained by analyzing the take-off curves. Twenty-four positive and 14 negative samples were retested by direct sequencing.
Results:
Eight homozygous and 30 heterozygous mutations were detected in 46 mutant alleles with a 851del4 mutation rate of 5.8% (46/800). Twenty-six and 20 mutant alleles were observed respectively, in 474 and 242 alleles from the intermediate and southern areas of China. No mutant allele was detected in 84 alleles from northern China. Twenty-four positive samples including 4 homozygous and 20 heterozygous mutations, and 14 negative samples were retested by direct sequencing, which confirmed that the accuracy of RT-PCR was 100%.
Conclusion:
RT-PCR can detect the mutation 851del4 in infants with intrahepatic cholestasis with an accuracy of 100%.
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