Progressive familial intrahepatic cholestasis (PFIC) is a heterogeneous group of severe autosomal recessive liver disorders of childhood and can result in cholestasis and progress to end-stage liver disease (1). PFIC can be divided clinically into 2 phenotypes: those with elevated γ-glutamyltransferase (GGT) and those with normal or low GGT in the serum. Recent molecular and genetic studies have allowed the identification of 2 genes involved in 2 subtypes of PFIC with normal or low serum GGT activity (1). The first type, called PFIC1, is caused by mutations in ATP8B1 gene, which encodes a P4-subfamily P-type adenosine triphosphatase, that is, FIC1 (2). ATP8B1 gene is expressed in various organs, including liver, pancreas, and small intestine (3,4). How mutations of this gene cause cholestasis is not clear (4). The second type, called PFIC2, is caused by mutations in the adenosine triphosphatase–dependent canalicular bile salt export pump gene (BSEP, ABCB11). Mutations in ABCB11 gene resulting in changes in BSEP protein are responsible for the decreased biliary bile salt secretion described in affected patients (5). Mutations in ATP8B1 and ABCB11 genes are also responsible for benign recurrent intrahepatic cholestasis type 1 (BRIC1) and BRIC2, respectively (6). Genotype-phenotype studies show that it remains difficult to distinguish patients with PFIC1 or PFIC2 before genetic studies because of a significant clinical overlap (1). ATP8B1 and ABCB11 gene mutations have been reported in certain populations including American, European Arabic, and Taiwan Chinese (2,5,7,8), but never in mainland Chinese. The aim of the present study was to elucidate the role and characteristics of ATP8B1 mutations in mainland Chinese patients with progressive intrahepatic cholestasis and low GGT.
PATIENTS AND METHODS
Between January 2004 and July 2007, 24 infants referred to the Children's Hospital of Fudan University (a tertiary referral hospital and key specialist pediatric hospital in eastern China) for the management of conjugated hyperbilirubinemia or pruritus were enrolled in the study. The inclusion criteria included the onset of conjugated jaundice or pruritus before 3 months of age; serum total bilirubin <5 mg/dL and conjugated bilirubin >1 mg/dL, or total bilirubin >5 mg/dL and conjugated bilirubin >20%; the period of jaundice lasting beyond the age of 1, or death due to liver failure before the age of 1; serum GGT levels consistently <50 U/L for at least 6 months during the follow-up period; no obvious causes of liver disease identified after an extensive workup as described earlier (9), which involved detailed investigations to exclude infective, drug-related, metabolic, and surgical causes of conjugated jaundice in this age group; and written informed consent obtained from the parents. Infants were excluded if they had an international normalized ratio >1.2 and could not be fully corrected after vitamin K intravenous injection at presentation, and if they presented with concomitant multiple organ dysfunction or congenital malformations. The study was approved by the ethics committee of the Children's Hospital of Fudan University, and written informed consents were obtained from the parents of all of the patients.
Peripheral blood samples were taken from the 24 patients. Genomic DNA of peripheral blood leukocytes was extracted. Mutations in the ABCB11 gene were then analyzed to detect homozygotes or compound heterozygotes by polymerase chain reaction (PCR) and direct sequencing, according to the process described by Lang et al (10). Subsequently, all encoding exons and flanking areas of the ATP8B1 gene were sequenced in the patients, in whom only 1 or no mutation of ABCB11 gene was found. Owing to insufficient DNA in the samples, genomic DNA from the blood of cases 7, 11, 12, 15, 16, 17, 19, 21, 23, and 24 was amplified first by using GenomiPhi V2 DNA Amplification Kit (GE Healthcare, Chalfont St Giles, UK) before analysis of the ATP8B1 gene. Twenty-five microliters of genomic DNA extracted directly from the blood of each of the 24 patients was reserved for further study. Primers to amplify coding exons 2 to 28 and at least 100 bp of adjacent intronic sequences of ATP8B1 gene were designed (Table 1). ATP8B1 fragments were generated by using AmpliTaq Gold DNA Polymerase (Applied Biosystems [ABI], Foster City, CA). Thirty-five cycles of PCR reaction were carried out in the following sequence: denaturing for 30 seconds at 94°C, annealing for 30 seconds at 55°C, and extending for 30 seconds at 72°C. The purified PCR fragments were sequenced directly by laser-induced fluorescence on an ABI Prism 3730 Genetic Analyzer (ABI) and then analyzed using BioEdit software (North Carolina State University, Raleigh). All of the sequences were blasted to gene bank to find the variations, and single nucleotide polymorphisms were excluded. NG_007148.1 was used as the ATP8B1 reference sequence. Singletons were confirmed by generating a second, independent PCR fragment and direct sequencing from both ends by using the genomic DNA extracted from blood as template.
Liver Histology and Clinical Features
Liver biopsy was performed in 17 patients after informed consent was obtained from the parents. The histopathological features to be noted by the pathologists in the present study included presence of cholestasis in the hepatocyte and cholangioles, degeneration of the hepatocytes, number of ductules and presence of proliferation or paucity, inflammation and fibrosis at the portal area, and integrity of lobular architecture. The pathological features were correlated with the clinical features.
Five of the 24 patients were homozygous or compound heterozygous for mutations of the ABCB11 gene, so the remaining 19 children were investigated for the ATP8B1 gene, including 2 children with heterozygous mutation for the ABCB11 gene. 9 of 19 patients had ATP8B1 gene mutations: 4 homozygotes, 3 compound heterozygotes, and 2 heterozygotes. Nine mutations were detected in the 9 patients, including 2 nonsense mutations, 1 nucleotide insertion, 1 nucleotide deletion, 1 splicing mutation, 2 missense mutations, 1 synonymous mutation, and 1 linkage mutation (Table 2). Figure 1 illustrates the locations of the 8 allele mutations within the coding region of the ATP8B1 gene. The linked mutations 625C>A(P209T) and IVS6+5G>T were detected in 4 patients, with 2 homozygotes and 2 heterozyotes. Sequence analysis of genomic DNA in 171 children with nonprogressive intrahepatic cholestasis and elevated GGT levels as controls revealed no mutation in the linkage site. Each of the remaining 8 mutations was detected in only 1 child. Ten patients had no mutations in analyzed ATP8B1 sequences, including the 2 with 1 ABCB11 gene mutation and 8 without mutations in the ABCB11 or ATP8B1 gene sequences.
Among the 24 patients with progressive intrahepatic cholestasis and low GGT, liver biopsy had been performed in 17 patients, including 6 with ATP8B1 gene mutations, 5 with ABCB11 gene mutations, and 6 without either. Minimal to mild cholestasis and lymphocytic infiltration in portal areas were the primary liver histological features of the patients with ATP8B1 gene mutations. Cases 4 (10 months old) and 17 (7 months old) also had moderate portal fibrosis. In cases 23 and 24, hepatocytic impairments were noted: hydropic degeneration in case 23 and ballooning degeneration in case 24. Mild giant cell transformation of hepatocytes was found in case 6. In comparison, 4 of 5 children with ABCB11 gene mutations and 3 of 6 patients without mutations showed marked giant cell transformation. Histologically, with the exception of giant cell transformation, there was no significant difference between patients with ATP8B1 gene mutations and those without. Patients without the genetic mutations seemed to have more severe hepatic pathology, with 2 presenting as early cirrhosis and 4 with varying degrees of fibrosis in the portal areas.
Besides the manifestation of jaundice, 10 of the children had pruritus, including 4 with ATP8B1 mutations, 4 with ABCB11 mutations, and 2 without detectable mutations. Serum total bile acid (TBA) had been determined in 18 of 24 children and was found elevated in all of them. The other 6 patients, including 2 with ATP8B1 mutations (cases 1 and 2), 2 with ABCB11 mutations (1 with compound heterozygous mutations, 1 with a heterozygous nonsense mutation), and 2 without mutations in the coding sequences of ABCB11 and ATP8B1, had no serum TBA data because serum TBA determination was not available in our hospital at that time. Among the 6 children, 2 had pruritus, including 1 with compound heterozygous mutations of ABCB11 and 1 without mutations in the 2 genes.
Most of the 24 patients did not present with extrahepatic features. However, 1 child with ABCB11 gene mutations had gallstones, 1 with ATP8B1 gene mutations had loose diarrhea, and 2 (1 with ABCB11 gene mutations and 1 with ATP8B1 gene mutations) had mild growth retardation. Five of the 8 patients without mutations in either ABCB11 or ATP8B1 died before the age of 1 year. In comparison, 1 child with ABCB11 mutations died at 11 months of age and another child with ATP8B1 mutations (case 1) died at the age of 4½ years. Ten patients were lost in follow-up. At present, only 7 children are still being monitored, including 3 with ATP8B1 mutations, 3 with ABCB11 mutations, and 1 without mutations in either gene. Six of the 7 children had pruritus, except 1 with compound heterozygous mutations of ABCB11, although his TBA levels remain elevated. All 7 children have fluctuating bilirubin and aminotransferase levels. Most of them are taking traditional Chinese medicine, and none received liver biopsy recently.
ATP8B1 gene mutations have been found in some areas of the world (2,5,7,8), but no common mutation has been reported in patients with PFIC1 until now. Furthermore, there was no previous knowledge of whether ATP8B1 mutations existed in mainland Chinese people, the largest population group in the world as well. The present study is the first, to our knowledge, to report the characteristic of ATP8B1 mutation and a hot-linked mutation in mainland Chinese patients with progressive intrahepatic cholestasis and low GGT. The linked mutation is also the first reported hot spot mutation found in patients with PFIC1 worldwide.
Four of 9 patients with ATP8B1 mutations had the linkage mutation of 625C>A(P209T) and IVS6+5G>T. 625C>A was located at the first nucleotide of the last codon in exon 6, resulting in the replacement of proline by threonine at residue 209 of the protein (P209T). 209P is a conservative amino acid residue of FIC1 among different species (Fig. 2); the amino acid substitute may influence the structure and/or function of the protein. IVS6+5G>T located in the splice site may disturb the messenger RNA splicing. All of this and the fact that the linked mutation was not found in 171 controls suggest that the linked mutation is probably a disease-causing mutation. Thus far, no hot mutation was found in ATP8B1 gene, except for the I661T mutation that is common in patients with BRIC1 (2,14). By analyzing the mutations in the present study and the 54 distinct mutations reported by Klomp et al (14) and others (2,7,8,15,16), it is shown that mutations are scattered in different regions of the ATP8B1 gene rather than clustered in a specific domain. Therefore, the hot-linked mutation is helpful in the diagnosis of PFIC1, which can only be identified by the gene studied at present.
Nonsense mutations R46X and R952X are thought to interrupt protein production in exons 2 and 23, respectively. R952X had been reported in patients with PFIC1 and patients with BRIC1, respectively (14). This nonsense mutation, in different compound heterozygous forms, can cause different phenotypic severities. An adenine insertion at nucleotide 607 produces a stop codon 9 bp away in the downstream sequence, resulting in a truncated protein product. The deletion of a deoxythymidine at 2532 nucleotide results in a frameshift and a stop codon in the 35th codon downstream. The above 4 mutations are disease-causing mutations.
Splicing mutation of IVS12+1G>A can result in exon skipping or intron insertion, leading to the production of a defective protein with totally or partially reduced activity. Future RNA-based study could further support the identity of splice sites as disease causing rather than rare normal variants. Nevertheless, the location of the splice site mutation in highly conserved or invariant splicing consensus residues further suggests that this change is a disease-causing mutation.
Both the missense mutations C265R and I694N occur in conserved sequences of FIC1 (Fig. 2). These changes may alter the structure and/or function of the protein. I694N (2081T>A) is located in the putative cytoplasmic domains of P-type adenosine triphosphatase. This has been reported in Taiwanese patients with PFIC1 (8), and the same allele mutation has been found in French patients with BRIC1 (14). However, in the patients with BRIC1, thymine is replaced by cytosine, which induces isoleucine being changed to threonine, that is, I694T. Therefore, the 2081 allele may be a common mutation point worldwide and can be substituted by different nucleotides. Whether the synonymous mutation V1159V is in association with the disease is unknown. Development of an in vitro assay for FIC1 function would permit further investigations on how the different genotypes may influence the FIC1.
According to the theory of molecular genetics, 6 patients with homozygous or compound heterozygous mutations in ATP8B1 gene can be diagnosed as having PFIC1, except case 24, who had homozygous synonymous mutation. Whether case 1 with the missense mutation C265R or case 21 with the nonsense mutation R46X had PFIC1 needs to be further investigated, but if the change of only 1 allele influences the protein significantly, then it may also cause PFIC1, for example, only 1 mutation in ABCB11 gene can cause PFIC2 (17).
Mutations for the ABCB11 or ATP8B1 gene were not detected in 8 of the 24 patients. However, they may have other mutations in regions that have not been analyzed, such as untranslated regions, upstream regulatory sequences, and introns, or they may have other disorders, the presentations of which overlap with those of BSEP or FIC1 defects. Compared with patients with ATP8B1 or ABCB11 gene mutations, the clinical features of the 8 patients are more severe. Therefore, the screened patients lacking mutations in both ATP8B1 and ABCB11 are good candidates for studies to identify mutations in additional loci in low GGT PFIC. Screening of TJP2 and BAAT may also be indicated because these genes are mutated in some cases of familial hypercholanemia, the presentation of which may overlap clinically with that of PFIC (18).
By comparing the liver histologic features of patients with ATP8B1 gene mutations and the patients with ABCB11 gene mutations, it was found that histologic examination is helpful in differentiating FIC1- from BSEP-related disease. In 1997, Bull et al (15) reported that patients with low GGT PFIC could be categorized as 2 groups by clinical and histologic features. Patients with nonspecific hepatitis were mapped to chromosome 18q21, and patients with giant cell transformation were later mapped to chromosome 2q24 (5). In 2001, Chen et al (8) confirmed the above speculations, but with only 6 genetically confirmed cases. Our results are in agreement with the above reports, but the histologic difference seems nonspecific, with giant cell hepatitis also found in 1 patient with ATP8B1 mutations.
Being a retrospective study, there are certain unavoidable limitations. We did not routinely evaluate developmental milestones in our patients; therefore, we can only report growth retardation when they are obviously noticeable. Hearing test was not routinely performed, and the lack of such clinical parameters reduces the quality of our study when compared with published studies from other centers (15,16,19).
In conclusion, the present study is the first to demonstrate that ATP8B1 gene mutations play an important role in the pathogenesis of low GGT PFIC in mainland Chinese patients, and that the mutation spectrum is different from that for other population groups. The present study is also the first to report a hot mutation in the ATP8B1 gene in patients with PFIC. Our results enrich the knowledge of ATP8B1 gene mutations, and the finding of a hot-linked mutation will, it is hoped, facilitate the genetic diagnosis of FIC1 deficiency.
We thank Prof Xi-qi Hu for assistance and expertise in liver pathology.
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