The present study was approved by the Institutional Review Board on Bioethics and Biosafety of The Third People's Hospital of Shenzhen (Shenzhen, China). Written informed consent was obtained from all participants. All of samples were obtained and analyzed according to appropriate ethical approvals.

Samples were obtained from two generations of a Chinese family. The proband (II:1; Fig. 1) was an 11 year-old boy diagnosed with chronic intrahepatic cholestasis resulting from AGS, who had a 9-year history of pruritus and a 7-year history of jaundice. He was the first full-term child of this family, and in 2005 presented with cutaneous pruritus with no clear cause, particularly on the lower limbs, accompanied with yellow urine. In 2007, the patient experienced loss of strength and appetite, jaundice, and occasional vomiting. In March 2015, liver function tests revealed that levels of total bilirubin (TBIL, 30.9 µmol/l), direct bilirubin (DBIL, 15.9 µmol/l), total bile acid (TBA, 49.6 µmol/l), alanine transaminase (ALT, 204 U/l), aspartate aminotransferase (AST, 128 U/l), γ-glutamyl transferase (γ-GGT, 517 U/l), total cholesterol (TC, 10.77 mmol/l) and free triiodothyronine (FT3, 7.04 pmol/l) in the patients were greater compared with healthy levels. Normal ranges are as follows: TBIL, 5.1–19.0 µmol/l; DBIL, 0.1–5.1 µmol/l; TBA 0–10 µmol/l; ALT, 0–40 U/l; AST, 0–40 U/l; γ-GGT, 7–32 U/l; TC, 3.35–6.45 mmol/l; FT3, 3.10–6.80 pmol/l. The right lobe of his liver was biopsied (length, 14 mm; diameter, 1 mm) and was observed to have minor lesions in the incomplete portal area analyzed (Fig. 2), suggesting that the proband had hepatobiliary hypoplasia. A B-scan ultrasound revealed that the liver, bladder, spleen and pancreas were healthy. The patient did not exhibit any other clear symptoms.

Peripheral blood was obtained from the proband and his healthy parents. Genomic DNA was extracted from the blood using a blood DNA extraction kit (QIAamp DNA Blood Midi kit; Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol.

To construct the capture library, DNA was sheared randomly by sonication using an LE220 Focused-Ultrasonicator (Covaris, Inc., Woburn, MA, USA); fragments of 200 to 250 bps were retained and subsequently purified by Ampure Beads (Beckman Coulter, Inc., Brea, CA, USA) according to the manufacturer's protocol. Following this, the two ends of the purified fragments were repaired, bound to A base and ligated with adapters. DNA fragments were subsequently amplified by ligation-mediated polymerase chain reaction (LM-PCR) using Platinum Pfx DNA polymerase (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Cycling conditions were as follows: An initial predenaturation step at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 sec, annealing 58°C for 30 sec, extension at 72°C for 30 sec, and a final extension step at 72°C for 7 min. PCR products were purified and hybridized to a customized gene-trapping chip (Roche Applied Science, Madison, WI, USA) for enrichment. Non-hybridized fragments were washed out using a NimbleGen Wash and Elution kit (Roche NimbleGen, Inc., Madison, WI, USA) as per the manufacture's protocol. A 2100 Bioanalyzer system (Agilent Technologies, Inc., Santa Clara, CA, USA) and an ABI StepOne Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) were used to estimate the magnitude of the enrichment, and the size and the concentration of the fragments in the two libraries. Subsequently, the library enriched for target regions was analyzed using a HiSeq 2500 Sequencing system (Illumina, Inc., San Diego, CA, USA) by paired-end sequencing to obtain raw reads, which were subsequently analyzed using Illumina Genome Analyzer Pipeline software version 1.3.4 (Illumina, Inc.).

The first step of the analysis was to assess the quality of the raw data and to remove contaminated or low quality reads. The reads were mapped to the reference hg19 (build 37.1) using Burrows Wheeler Aligner software version 0.7.12 (sourceforge.net/projects/bio-bwa/files) and estimated the effect capturing at the same time. Subsequently, Short Oligonucleotide Analysis Package single nucleotide polymorphism (SNP) software version 1.03 (soap.genomics.org.cn/soapsnp.html) and SAMtools software version 1.2 (samtools.sourceforge.net) were used to detect single nucleotide variants, and insertions and deletions (indels), respectively. The last step was to annotate and screen the suspected mutations. The coverage of targeted region and sequencing depth calculation were based on all mapped reads. All alterations were compared and filtered against exome data from the SNP database (dbSNP build 137; www.ncbi.nlm.nih.gov/snp), the 1000 human genome dataset (http://www.internationalgenome.org/), HapMap (http://www.ncbi.nlm.nih.gov/probe/docs/projhapmap/) and an in-house database of 100 Chinese healthy adults. Sorting Intolerant From Tolerant software version 1.03 (sift.jcvi.org) was used to predict if amino acid substitutions, insertions or deletions affected protein function (30).

The samples of the three individuals in this pedigree were used for validation. The primers were designed using Primer 3 software version 4.0.0 (primer3.ut.ee). PCR amplification and Sanger sequencing were conducted to validate the candidate mutation (human JAG1; locus, NM_000214; c.3254_3255insT) using standard protocols on an ABI 3730XL sequencer (Applied Biosystems). The forward primer for JAG1 was 5′-TTGGTGGTGTTGTCCTCAGA-3′ and the reverse primer was 5′-AGGGATAAAGGGCAGGAGAA-3′ (product size, 244 bp). PCR amplification was performed using Ex Taq^™ DNA polymerase (Takara Bio, Inc., Otsu, Japan) with an initial predenaturation step at 95°C for 5 min, followed by 33 cycles of denaturation at 95°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 30 sec and a final extension at 72°C for 7 min. PCR products were pooled and subsequently purified with an AxyPrep DNA Gel Extraction kit (Axygen Biosciences, Inc., Union City, CA, USA) according to the manufacturer's protocol.

Targeted region capture sequencing of the proband in a Chinese family with chronic intrahepatic cholestasis linked with JAG1, NOTCH2, UGT1A1, ATP8B1, ABCB11 and ABCB4 genes was performed. The reads were aligned with the human genome reference. The average depth of the target region was 186.55-fold and the coverage of the target region was 99.67%. A total of 10,277 genetic variants were revealed, including 10,132 SNPs and 1435 indels. Of the variants, there were 758 missense, 3 nonsense, 1087 synonymous, 248 splice site, 7047 intron and 2188 3′ untranslated region (UTR) mutations, 311 5′UTR and 8 frame-shift coding indels that were more likely to be pathogenic compared with the other variants. Priority was given to frame-shift, non-synonymous and splice site mutations. Using a preliminary screening process, ten candidate sites were revealed (Table I). Considering the frequency in the public and in-house databases, nine other candidate sites were excluded. A novel insertion mutation, c.3254_3255insT (p.Leu1085PhefsX24), was identified in exon 26 of the JAG1 gene in the proband. The mutation in the JAG1 gene was selected for further validation, as it is known to be the disease-causing gene.

The mutation was subsequently sequenced in three members of the family via Sanger sequencing. The results of Sanger sequencing demonstrated that the mutation was present in the proband but absent in unaffected members (Fig. 3). The mutation results in a premature stop codon and a truncated protein, which affects the Notch signaling pathway, influencing multi-system development in the embryonic period.