Subjects included 50 patients presenting neonatal cholestasis who had visited the Department of Pediatrics at the Kyungpook National University Children's Hospital between January 2013 and July 2016 (Table 1). Detailed clinical and family histories were obtained. Evaluations included laboratory examination, liver ultrasonography (USG), diisopropyl iminodiacetic acid (DISIDA) scan, percutaneous cholecystocholangiography (PCC), and liver biopsy. Genetic analysis was performed when a patient was suspected of having a syndromic disease. Two families also provided genetic material for analyses to confirm the diagnosis and were subjected to genetic counseling. Blood samples were collected and genomic DNA was extracted using a QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany). DNA quality and quantity were assessed using a Qubit Fluorometer (Invitrogen, Carlsbad, CA, USA) and a Quant-iT BR assay kit (Q32850; Invitrogen). The institutional review board of Kyungpook National University Hospital approved the protocol, and informed consent forms were obtained for genetic analysis and for utilization of the results for diagnosis and research purposes from the participants or from their legal guardian (IRB no. KNUH 2016-06-011).

Whole exome sequencing was performed on patients with ABCB11 spectrum liver disorders to analyze multiple candidate genes simultaneously. Targeted exonic regions were captured with the Agilent SureSelect XT Human All Exon v5 kit (Agilent Technologies, Santa Clara, CA, USA). Exome sequencing was performed on an Illumina HiSeq-2000 (Illumina, San Diego, CA, USA) platform with 100-bp paired-end runs at an average mean target depth of 100× coverage. Rare or novel mutations were prioritized.

The raw sequence data in fastq format were mapped to the reference human genome (UCSC hg 19, NCBI build 37.1) using the Burrows-Wheeler algorithm. Alignments for each sample were converted to binary alignment map (BAM) format, sorted, and indexed, and polymerase chain reaction duplicates were marked and then merged into one BAM file using Samtools. Alignments from the combined BAM file were then locally realigned around insertions/deletions, recalibrated, and variants were called for all samples together using the genome analysis toolkit. Mutations were further evaluated using bioinformatics software programs such as snpEff, SIFT score, polyphen-2 score, phyloP score, phastCons score, 1000 genomes, dbSNP (dbSNP137), and the Korean Personal Genome Project.

Sanger sequencing was performed on the patient and the family members to confirm suspected pathogenic mutations identified from whole exome sequencing and to determine whether other family members without obvious illness had inherited the same mutation. The target site of the variant and the flanking DNA sequences from each family member were amplified with forward and reverse primers. The amplified products were directly sequenced using an automated DNA sequencer (ABI3130; Applied Biosystems, Foster City, CA, USA) using a Big-Dye Terminator Cycle Sequencing kit version 3.1. The primer sequences are available upon request.

Fifty patients were divided based on the underlying cause of disease. Idiopathic or viral hepatitis was diagnosed in 17 (34%), metabolic disease in 10 (20%), total parenteral nutrition induced cholestasis in 8 (16%), extrahepatic biliary atresia in 7 (14%), genetic disease in 5 (10%) including PFIC2 and BRIC2, neonatal lupus in 1 (2%), congenital syphilis in 1 (2%), and choledochal cyst in 1 (2%) patient (Table 1).

Idiopathic hepatitis was confirmed as the sporadic form by clinical course, which presumably was undefined viral hepatitis. Of the 10 patients with metabolic disease, six were citrin deficient, two had hyperornithinemia-hyperammonemia-homocitrullinuria syndrome, and two had ornithine transcarbamylase deficiency. Of the five with genetic disease, two had siblings with PFIC2, one had BRIC2, one had Edward syndrome with heart failure, and the other had arthrogryposis-renal dysfunction-cholestasis syndrome.

Two families with proven ABCB11 spectrum liver disorders were subjected to genetic analyses (Fig. 1). Biochemical findings in the patients showed markedly elevated levels of aminotransferase with normal GGT levels.

In family A, with Korean ethnicity, genetic testing was performed on the second son (II-2), the third son (II-3), the parents (I-1 and I-2), and the maternal uncle (I-3). There was no access to the first son's (II-1) DNA because he had died 13 years before the study. The first and third sons had progressive cholestasis from the neonatal period. The parents of the affected siblings were non-consanguineous and healthy and there was no family history of liver disease.

The first son showed persistent jaundice from 7 days after birth and visited a hospital at 2 months of age. Diagnostic evaluations indicated intrahepatic cholestasis with normal GGT levels. As PFIC had not been reported in Korea at the time, genetic testing was not performed, and progressive cholestatic hepatitis did not lead to a specific clinical diagnosis. A liver biopsy was performed in the 1-year-old patient who showed significant pathological features of cholestasis. Liver transplantation was recommended but the parents deferred the decision. At the age of 27 months, the patient was referred to our hospital for urgent management of severe abdominal distension. A plain abdominal X-ray showed a markedly distended abdomen with no air-fluid levels or free gas under the diaphragm (Fig. 2A). Abdominal paracentesis with free drainage was performed and there was hemorrhagic ascitic fluid. Abdomen computed tomography revealed multinodular hepatocellular carcinoma and hemoperitoneum due to the ruptured tumor (Fig. 2B). He expired despite intensive care after moving to another tertiary hospital. The second son did not have any symptoms related to cholestatic liver disease.

The third son displayed similar symptoms and visited our hospital presenting with jaundice from 7 days after birth. Physical examination showed mild hepatomegaly and initial biochemical analysis indicated conjugated hyperbilirubinemia (6.8 mg/dL) and elevated levels of aspartate aminotransferase (AST), 100 U/L; alanine aminotransferase (ALT), 53 U/L; serum bile acid, 124 µmol/L; with normal ranges of ammonia, 40 µmol/L; and GGT, 31 U/L. Liver USG revealed normal hepatic parenchymal echogenicity with a visible common bile duct, and triangular cord sign was not identified. A DISIDA scan, obtained 6 hours after Tc^99m injection, did not reveal passage to the small bowel (Fig. 2C), but PCC demonstrated a contrast-filled gall bladder and excreted contrast material in the small bowel loops, which could exclude extrahepatic biliary atresia (Fig. 2D). A liver biopsy was performed at 3 weeks after birth and indicated mild portal inflammation and lobular disarray (Fig. 2E), mild portal and focal periportal fibrosis by Masson's trichrome stain (Fig. 2F), mild bile canalicular and lobular cholestasis with mild ballooning change, focal lobular inflammation, and apoptosis (Fig. 2G). Electron microscopy revealed distended canaliculi with loss of microvilli (Fig. 2H) and filamentous bile, which is a distinctive finding of PFIC2 (Fig. 2I). Cholestatic hepatitis progressed drastically and maximum conjugated bilirubin, AST, and ALT were 11.4 mg/dL, 8,000 U/L, and 3,740 U/L, respectively. After the genetic confirmation of PFIC2, treatment with 4-phenylbutyric acid (4-PBA) was started but showed no effectiveness. The patient underwent liver transplantation from a living donor at the age of 6 months, and is currently recovering and showing normal liver function.

In family B with Korean ethnicity, genetic testing was performed on the parents (I-1 and I-2), daughter (II-1), and son (II-2). The son presented with increasing jaundice since birth. He was born as a full-term baby following a normal pregnancy and delivery. He was the second child of healthy unrelated parents with no family history of liver disease. Upon physical examination, he was normal except for mild hepatomegaly. Initial liver function tests showed conjugated hyperbilirubinemia with a level of 6.2 mg/dL, high alkaline phosphatase (ALP) of 875 U/L, AST of 464 U/L, and ALT of 357 U/L. GGT were within normal limits. Liver USG, a DISIDA scan, and PCC showed unremarkable results and a liver biopsy demonstrated similar findings to that of the PFIC2 patient, distended canaliculi with loss of microvilli (Fig. 2J) and filamentous bile (Fig. 2K).

He was positive for IgM antibodies to herpes simplex simplex virus (HSV), but negative for other viral work ups. Acute cholestatic attack lasted for more than 10 months, and maximum ALP, conjugated bilirubin, AST, and ALT were 884 U/L, 8.2 mg/dL, 1,538 U/L, and 1,141 U/L, respectively. There was the possibility of PFIC2 because the onset was neonatal, the biochemical manifestations were severe, and the acute phase was relatively long. Genetic analysis demonstrated no pathogenic mutation for PFIC2. After 4-PBA was initiated, biochemical improvement occurred dramatically and serum bilirubin and liver enzymes normalized completely within several months. This benign evolution was consistent with a diagnosis of BRIC2.

Whole exome sequencing was performed on the third son of family A and the son of family B, which generated over 50 million reads. Common variants were filtered and the candidate genes associated with inherited cholestatic disease were analyzed. Forty-one variants of ATP8B1, ABCB11, and ABCB4 were identified in the third son of family A. Among those 41, only two novel mutations in ABCB11 (NM_003742.2), c.11C>G (p.Ser4^*) and c.1543A>G (p.Asn515Asp), were suspected to be causative mutations. The nonsense and the missense mutations were shown to be conserved in orthologs of mammalian ABCB11, and in silico analyses also demonstrated high probabilities of pathogenicity. These mutations have never been reported in PFIC2 patients or in the general population.

In the son of family B, 13 variants of ATP8B1 and ABCB11 were detected. Among those, ABCB11 c.1331T>C, p.Val444Ala (rs2287622), c.3084A>G, and p.Ala1028Ala (rs497692) had the potential to be causative mutations. The allele frequencies of Val444Ala and Ala1028Ala are 74.5% and 67.2%, respectively [8]. Although both variants are commonly found in the general population, Val444Ala has previously been reported in ICP and drug induced cholestasis with a higher allele frequency than in normal controls [910]. Val444Ala has also been reported in BRIC2 and ICP when present in combination with other ABCB11 or ABCB4 mutations [1112]. Ala1028Ala promotes exon skipping in vitro and is possibly associated with primary biliary cirrhosis [1314].

The mutations were validated by Sanger sequencing in the patient and family members. In family A, the patient had combined heterozygous mutations of Ser4^* and Asn515Asp. The patient inherited Ser4^* from the mother and Asn515Asp from the father. The mutations were not identified in the other family members (Fig. 3). In family B, Val444Ala was homozygous in the patient and sister, and heterozygous in the parents. Ala1028Ala was heterozygous in the patient, sister, and father, but homozygous in the mother.