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

Abstract

Background and AimsLarge 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.MethodsThe 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.ResultsAt 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.ConclusionSpecially 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.

Defects in myosin VB are associated with a spectrum of previously undiagnosed low γ‐glutamyltransferase cholestasis

“…In this study, approximately one fifth (7 of 31) of the first two cohorts of undiagnosed low‐GGT cholestasis patients carried biallelic mutations in MYO5B —mutations that either had been reported as pathogenic in other patients or are predicted in silico to be pathogenic (Table )—but not in genes known to be mutated in cholestasis (http://onlinelibrary.wiley.com/doi/10.1002/hep.29020/suppinfo). Histologically, we observed coarse granular marking for MYO5B (Fig. ) and disruption of canalicular distribution of BSEP (Fig.…”

“…Study subjects were Han Chinese enrolled from 2011 to 2016, with informed consent, under a clinical‐diagnosis protocol approved by Children's Hospital and Jinshan Hospital of Fudan University (Shanghai, China) and according to the ethical guidelines of the 1975 Declaration of Helsinki. The following enrollment criteria were used: elevated serum total bilirubin (TB) and direct bilirubin (DB); GGT <100 IU/L; failure to ascertain an etiology of disease through testing listed in http://onlinelibrary.wiley.com/doi/10.1002/hep.29020/suppinfo; and parental DNA available. All patients were analyzed either by WES or targeted sequencing.…”

TJP2 hepatobiliary disorders: Novel variants and clinical diversity

“…Other biomarkers (serum alkaline phosphatase, γ‐glutamyl transpeptidase [GGT], and 5′‐ribonucleotidase activity values) were not used as selection criteria to cast our net as widely as possible. We carried out multiple‐gene panel sequencing (at least ABCB11 , ATP8B1 , TJP2 , BAAT , UGT1A1, CYP27A1 , CYP7B1 , HSD3B7 , VPS33B , and VIPAS39 ; Wang et al, ) in 522 patients and whole‐exome sequencing (WES) in the 153 remaining patients, as clinically requested. When biallelic predictedly pathogenic variants in TJP2 were detected, we verified the variants by Sanger sequencing and confirmed their presence in the parents.…”

“…Genome DNA manipulation and analysis in WES were as described (Qiu et al, ), as was panel sequencing (Wang et al, ). Briefly, WES was done on HiSeq 2500 sequencers with 2 × 150 paired‐end modules (Illumina, San Diego, CA).…”

“…The sequencing depth of 98% targeted regions is above 20×. Filtering procedure for both initially removes sequencing errors and variants with a frequency >1% in public and in‐house databases; secondly, identify suspected genes with pathogenic/predictedly pathogenic variants; finally, designate the primary disease‐causing candidate gene, taking into account autosomal recessive inheritance pattern and recurrence rate in families and/or individual samples (Qiu et al, ; Wang et al, ). Criteria for designating variants as pathogenic/predictedly pathogenic were (a) allele frequency <0.01 in both the 1000 Genomes Project (1000G, http://www.internationalgenome.org/data) and the Exome Aggregation Consortium Browser (ExAC, http://exac.broadinstitute.org); (b) pathogenicity predicted by at least one of the three programs MutationTaster (Schwarz, Cooper, Schuelke, & Seelow, ), Polymorphism Phenotyping v2 (Polyphen‐2; Adzhubei, Jordan, & Sunyaev, ), and Sorting Tolerant From Intolerant (SIFT; Kumar, Henikoff, & Ng, ).…”