A Specially Designed Multi-Gene Panel Facilitates Genetic Diagnosis in Children with Intrahepatic Cholestasis: Simultaneous Test of Known Large Insertions/Deletions

Abstract

Background and aims: Large indels are commonly identified in patients but are not detectable by routine Sanger sequencing and panel sequencing. We specially designed a multi-gene panel that could simultaneously test known large indels in addition to ordinary variants, and reported the diagnostic yield in patients with intrahepatic cholestasis.

Methods: The panel contains 61 genes associated with cholestasis and 25 known recurrent large indels. The amplicon library was sequenced on Ion PGM system. Sequencing data were analyzed using a routine data analysis protocol and an internal program encoded for large indels test simultaneously. The validation phase was performed using 54 patients with known genetic diagnosis, including 5 with large insertions. At implement phase, 141 patients with intrahepatic cholestasis were evaluated.

Results: At validation phase, 99.6% of the variations identified by Sanger sequencing could be detected by panel sequencing. Following the routine protocol, 99.8% of false positives could be filtered and 98.8% of retained variations were true positives. Large insertions in the 5 patients with known genetic diagnosis could be correctly detected using the internal program. At implementation phase, 96.9% of the retained variations, following the routine protocol, were confirmed to be true. Twenty-nine patients received a potential genetic diagnosis when panel sequencing data were analyzed using the routine protocol. Two additional patients, who were found to harbor large insertions in SLC25A13, obtained a potential genetic diagnosis when sequencing data were further analyzed using the internal program. A total of 31 (22.0%) patients obtained a potential genetic diagnosis. Nine different genetic disorders were diagnosed, and citrin deficiency was the commonest.

Conclusion: Specially designed multi-gene panel can correctly detect large indels simultaneously. By using it, we assigned a potential genetic diagnosis to 22.0% of patients with intrahepatic cholestasis.

Fig 1. Schematic diagram of primer design for large indels.

①, ②, ③ were three pair primers designed for each large indel, and yield three amplicons AP1, AP2, AP3 respectively. BC was the inserted or deleted sequence.

Fig 2. The optimized protocol for panel sequencing data analysis.

The data from 195 patients were presented as median [P25, P75]. MAF, minor allele frequency; TGP, 1000 Genomes Project; ExAC, Exome Aggregation Consortium; dbSNP, Single Nucleotide Polymorphism database; HGMD, Human Gene Mutation Database.