Twenty-three Han Chinese patients were diagnosed with ABCB4-related cholestatic liver diseases from 21 families consisting of 15 children and 8 adults. The mean age of onset was 13 (0.2–37) years of age. Twenty-two patients had non-consanguineous parents, except for Patient 12, whose parents were cousins. The sister of patient 12 died at the age of 10 years due to “liver cirrhosis and gastrointestinal bleeding.” Patients 1 and 2 were brothers. Patients 15 and 23 had a father-son relationship. The remaining patients belonged to unrelated families.

The clinical features of the 23 patients are shown in Table S1. Among the 15 pediatric patients, 11 with biallelic ABCB4 mutation were diagnosed with PFIC3; four patients with non-biallelic mutations were diagnosed as DILI, cirrhosis cholestasis, cirrhosis, and mild liver fibrosis respectively. Among the 8 adult patients, two with biallelic mutation phenotype were diagnosed with PFIC3; one with biallelic mutation (including one benign mutation) presented with ICP cirrhosis; five cases with non-biallelic mutations, two cases presented with ICP cirrhosis (overlapping DILI), and three cases were diagnosed with LPAC. The imaging findings of all patients (abdominal B-ultrasound/CT/MRI) indicated liver fibrosis. The initial complaints of the 23 patients were liver and spleen enlargement (4/23, 17.4%), abdominal distension (2/23, 15.4%), jaundice (8/23, 34.8%), and abnormal liver function without any symptoms (9/23, 39.1%).

At initial diagnosis, GGT was increased in all patients (262 ± 168) U/L, alanine transaminase in 18 cases (78.3%), aspartate aminotransferase in 21 cases (91.3%), total bilirubin in 16 cases (69.6%), direct bilirubin in 17 cases (73.9%), and total bile acid in 20 cases (87.0%). Fifteen patients (65.2%) presented with cholestasis, of whom 10 (43.5%) presented with chronic cholestasis (Table S1).

Liver histopathology was performed in 19 (82.6%) of the 23 patients, including 11 (77.3%, 11/15) children and 8 (100%, 8/8) adults. Four children did not undergo hepatocentesis because of decompensation of cirrhosis.

Pathological characteristics of the 19 patients included cystic fibrosis (100%), positive CK7 expression (89.5%), small bile duct hyperplasia (84.2%), bile duct deletion (5.3%), positive copper staining (42.1%), and cirrhosis (63.2%). There were no significant differences in the pathological changes between children and adults (Table 1).

Patient 3, 5 18, and 19 underwent the hepatic histopathology twice. Patient 3 underwent the hepatic histopathology at 9 months and 2 years without UDCA treatment during this period (Fig. 1A), which was aggravated. Patient 5 underwent the hepatic histopathological examination at 4 years or 7 years, with continuous UDCA treatment in this period, showed remission (Fig. 1B).

Histological findings of liver biopsies in patients underwent twice liver punctures. A Patient 3: (i) first hepatic histopathology: G1–2S1–2, no significant abnormalities in the small bile duct; (ii) second hepatic histopathology: G2S4, interlobular bile duct stenosis, small bile duct hyperplasia. B Patient 5: (i-ii) first hepatic histopathology: G1–2S4, pseudolobular structure formation, interlobular bile duct epithelial edema, vacuolation, and small bile duct hyperplasia; (iii) second hepatic histopathology: G2S2, interlobular bile duct stenosis, and small bile duct hyperplasia. C Patient 19: (i-iv) first hepatic histopathology: (i) lobular structure disorder (rete); (ii) bile duct absence, CK7+, chronic bile salt stasis; (iii) mild interstitial fibrosis in the junction area; (iv) spot-like necrosis, decreased CD10 expression in some areas (vanishing cholangiography syndrome, biliary liver fibrosis, G2S1); (v) second hepatic histopathology: atrophy and loss of interlobular bile duct epithelium, typical interlobular bile duct is rare in some convergence areas, small bile duct hyperplasia is obvious, mild interlobular inflammation (G2S2)

Patient 18 showed G2S4 in the first hepatic histopathology (28 years old). After continued UDCA treatment, the second hepatic histopathology presented as G1S2 (32 years of age), indicating that the disease was alleviated. Patient 19 underwent the hepatic histopathology at 23 years and 30 years, indicating increased hepatic fibrosis (Fig. 1C).

The pathological features patient 1 (10 months old) was shown in Fig. 2, indicating the disease is serious.

Histological findings of liver biopsy in representative patients with early onset of disease (patient 1). A G2S4, the hepatic lobule structure is disturbed, and false lobules have formed; (B) enlarged manifold area, fibrous tissue hyperplasia, interlobular bile duct hyperplasia; (C) numerous inflammatory cells infiltrated, mild fine bile duct reaction (CK7+)

In this study, 30 ABCB4 gene mutations were detected in 23 patients, including 20 missense mutations, five small deletions (< 21 bp), four splicing mutations, and one nonsense mutation (Table S2). The frameshift, nonsense, canonical ±1 or 2 splicing-site variants and exon deletions were defined as null variants, while missense and non-canonical splicing sites, as non-null variants, according to the ACMG standards [11].

Eighteen mutation sites were novel, whereas the remaining sites were reported as disease-causing mutations in the Human Gene Mutation Database. Except for c.2394 + 82C > T, the other VUS were predicted to be pathogenic using the in silico algorithm (Table 2). For novel missense mutations, PyMOL was used to visualize the three-dimensional structure of the ABCB4 protein, showing the impact of the variants on the protein structure (Fig. 3, Fig. S1). The multispecies conservative prediction of novel missense mutations indicated that the mutation sites were highly conserved (Fig. 4). In this case, the mutation may have affected the protein function.

Novel missense mutations that predict changes in crystal structure. The mutation sites and surrounding residues are shown in yellow and green sticks, respectively. A p.C353Y: predicted to produce polar interaction with SER992. B p.G319V: predicted to produce repulsion. C p.G384R: predicted to produce new polar interactions with ARG461, ARG384, and ARG383. D p.L55S: predicted to produce a new polar interaction with TRP51. E p.R831S: the polar interaction with SER992 disappears, and polar interactions with ALA827 and GLY826 are generated

Conservation analysis of the nine missense variants among different species. Multispecies alignments of ABCB4 protein are shown. The mutation sites are labeled by blue rectangle

In this study, 11 children and two adults with biallelic ABCB4 mutations presented the phenotype of PFIC3. One adult with compound heterozygous mutations presented with ICP cirrhosis caused by a benign mutation. Four children and five adults with non-biallelic ABCB4 mutations were phenotypically diverse, ranging from DILI, ICP, and LPAC, to liver transplantation due to severe liver fibrosis (one child). In conclusion, patients with biallelic mutations in ABCB4 have a young age of onset and rapid disease progression. Their clinical phenotypes were mostly consistent with that of PFIC3, which is a common phenotype in children. The diseases in non-biallelic mutation carriers are relatively mild and late onset and are significant in adults. Although the clinical phenotype of most ABCB4 mutation carriers conforms to the genotype-phenotype relationship mentioned above, there are exceptions. The age of onset in Patient 9 (child), who carried a heterozygous variant (c.2525 T > C, p.L842P), was 6 years. His clinical phenotypes included cirrhosis and cholestasis, which progressed rapidly. He underwent liver transplantation for end-stage liver disease at the age of 12 years.

In the present study, biallelic mutations played a dominant role in the children and non-null variants (missense) were more. Non-biallelic mutations were predominant in adults, and most of them were null various. The types of gene mutations were diverse, and the clinical phenotypes of deletion mutations were relatively severe.

All patients with ABCB4-related cholestatic liver diseases were treated with UDCA, glycyrrhein, glutathione, and butyldisulfonate methionine during hospitalization. Except for Patient 3, 9, 17, and 20, all patients responded partially or fully to UDCA treatment. The follow-up time was 6.7 (0.5–13.5) years. One adult patient (Patient 20) died of liver failure while four children underwent liver transplantation. Cirrhosis continued to progress in six patients (four children and two adults). The conditions of the remaining 12 patients remained stable.