Analysis of Gene Mutations in Children With Cholestasis of Undefined Etiology

Abstract

Background: 

The discovery of genetic mutations in children with inherited syndromes of intrahepatic cholestasis allows for diagnostic specificity despite similar clinical phenotypes. Here, we aimed to determine whether mutation screening of target genes could assign a molecular diagnosis in children with idiopathic cholestasis.

Patients and Methods: 

DNA samples were obtained from 51 subjects with cholestasis of undefined etiology and surveyed for mutations in the genes SERPINA1, JAG1, ATP8B1, ABCB11, and ABCB4 by a high-throughput gene chip. Then, the sequence readouts for all 5 genes were analyzed for mutations and correlated with clinical phenotypes. Healthy subjects served as controls.

Results: 

Sequence analysis of the genes identified 14 (or 27%) subjects with missense, nonsense, deletion, and splice site variants associated with disease phenotypes based on the type of mutation and/or biallelic involvement in the JAG1, ATP8B1, ABCB11, or ABCB4 genes. These patients had no syndromic features and could not be differentiated by biochemical markers or histopathology. Among the remaining subjects, 10 (or ∼20%) had sequence variants in ATP8B1 or ABCB11 that involved only 1 allele, 8 had variants not likely to be associated with disease phenotypes, and 19 had no variants that changed amino acid composition.

Conclusions: 

Gene sequence analysis assigned a molecular diagnosis in 27% of subjects with idiopathic cholestasis based on the presence of variants likely to cause disease phenotypes.

The evaluation of children with syndromes of intrahepatic cholestasis remains a clinical challenge despite advances in etiology and pathogenesis of diseases. Although the prevalence of individual syndromes is low, collectively they are frequent causes of chronic cholestasis. The best-characterized syndromes have been linked to mutations in genes that disrupt critical cellular processes. Among these genes, disease-causing mutations have been reported in SERPINA1 (responsible for deficiency in α-1-antitrypsin), JAG1 (for Alagille syndrome [AGS]), and 3 genes associated with progressive familial intrahepatic cholestasis (PFIC): ATP8B1 (type 1, encoding the familial intrahepatic cholestasis 1 [FIC1] protein), ABCB11 (type 2, encoding the bile salt export pump [BSEP]), and ABCB4 (type 3, encoding multidrug resistance protein 3 [MDR3]) ⁽¹⁾. Notably, the spectrum of phenotypes associated with mutations in ATP8B1, ABCB11, and ABCB4 is now broader and includes intrahepatic cholestasis of pregnancy ^(2–7), gallstone formation ^(8,9), and hepatobiliary tumors ^(10–12).

Despite the lack of predominant mutational hot spots, sequence analysis of the entire coding sequence can be performed by standard capillary sequencing methods or by a hybridization-based high-throughput gene chip ⁽¹³⁾. These techniques facilitate the potential screening of mutations in the clinical setting. In 1 report, a mutation analysis of ATP8B1 supported the phenotype of patients with benign recurrent intrahepatic cholestasis who were treated with a new approach to improve severe pruritus by nasobiliary drainage ⁽¹⁴⁾. Another mutation survey reported a previously unrecognized association between mutations in ABCB4 and fibrosing cholestatic liver disease in adults ⁽¹⁵⁾. Here, we performed a comprehensive multigene sequence analysis to determine whether a molecular diagnosis can be assigned to children with idiopathic cholestasis. Using a chip-based resequencing methodology, we identified sequence variants in JAG1, ATP8B1, ABCB11, or ABCB4 likely to cause disease phenotypes in 27% of the children with cholestasis of undefined etiology based on the type and/or biallelic involvement of the sequence variants.

PATIENTS AND METHODS

Patients

We performed high-throughput nucleotide sequence analyses using peripheral blood DNA from children with idiopathic cholestasis, which was defined by the presence of persistent cholestasis (high conjugated bilirubin and high serum bile acids), normal serum levels of α-1-antitrypsin, absence of syndromic features, and no family history of chronic liver disease. Subjects with high serum γ-glutamyl transpeptidase (γ-GTP) also had negative evaluation for main syndromic features of AGS (facial features, ophthalmologic examination, vertebral body anomalies, or structural cardiac defects). All of the subjects were either evaluated in the Pediatric Liver Care Center of Cincinnati Children's Hospital Medical Center or had blood samples, clinical data, and histopathology reports sent to our laboratory. For control subjects, DNA was also obtained from peripheral blood and used to determine the allele frequencies of new nonsynonymous nucleotide variants. The controls consisted of a cohort of 50 subjects without liver disease from southern Ohio (race: 83% white, 16% black or African American, 1% Asian; ethnicity: 5% Hispanic or Latino). The study protocol was approved by the institutional review board of Cincinnati Children's Hospital Medical Center, and informed consent (and assent when appropriate) was obtained from legal guardians.

Chip Hybridization and Analysis

DNA was isolated from peripheral blood using the Puregene Purification Kit (Gentra Systems, Minneapolis, MN) according to the manufacturer's protocol. Then, DNA samples served as template in long- and short-range high-fidelity polymerase chain reaction to amplify selected domains of target genes (amplicons), followed by hybridization with the JaundiceChip, detection of biotin-labeled signals by the GeneChip 3000 Scanner (Affymetrix, Santa Clara, CA), capture with the Affymetrix GeneChip Operating Software, and analysis with the Affymetrix GeneChip Sequence Analysis Software as described by us previously ⁽¹³⁾.

Sequence Analysis

The nucleotide sequence readout for all subjects was exported into an Excel (Microsoft, Redmond, WA) spreadsheet that also displayed the reference sequence for each gene (obtained from GenBank at www.ncbi.nlm.nih.gov/entrez) and a list of mutations associated with disease phenotypes as reported in the Human Genome Mutation Database (www.hgmd.cf.ac.uk) or in the published English-language literature (www.ncbi.nlm.nih.gov/sites/entrez), herein referred to as previously described nucleotide changes. All of the new nonsynonymous variants were analyzed by the computational methods Grantham Score ⁽¹⁶⁾, SIFT (Sorting Intolerant From Tolerant) ⁽¹⁷⁾, and PolyPhen ⁽¹⁸⁾ to assess the likelihood of significantly modifying (or “damaging”) the biological properties of encoded proteins ⁽¹⁹⁾. Nucleotide changes within 10 bp of the intron/exon boundaries (splice sites) were checked using NetGene (http://www.cbs.dtu.dk/biolinks/pserve2.php), a gene finder and intron splice site prediction algorithm hosted by the Center for Biological Sequence Analysis in Denmark.

Capillary Sequencing

To validate nucleotide variants identified by the JaundiceChip, we performed capillary sequencing for every nonsynonymous nucleotide substitutions, deletions, and splice site changes. Automated capillary sequencing was performed using ABI Prism 3730 DNA Analyzer (Applied Biosystems, Carlsbad, CA) at the Gene Expression and Sequence Core at Cincinnati Children's Hospital Medical Center. Results of nucleotide sequence readouts are presented according to the nomenclature suggested by the Human Genome Variation Society (www.hgvs.org/mutnomen).

RESULTS

Survey of Mutations in Subjects With Cholestasis of Undefined Etiology

The amplification of gene fragments, probe labeling, hybridization with the chip, and analysis of the signal intensity generated nucleotide sequences for all exons and intron-exon boundaries of SERPINA1, JAG1, ATP8B1, ABCB11, and ABCB4 in all of the subjects. To detect nucleotide sequence changes of potential relevance to clinical phenotypes, we analyzed the sequence output for missense variants that resulted in amino acid changes, nonsense variants, deletions/insertions, or splice site nucleotide substitutions. To be sure that sequence variants were reproducible, we reanalyzed individual variants in the patient's DNA using standard capillary sequencing. All of the sequence variants reported below were reproduced by capillary sequencing. From the 51 subjects with idiopathic cholestasis (or cholestasis of undefined etiology), we found 2 general groups of patients. One group consisted of 14 subjects who had gene sequence variants likely to cause disease phenotypes, and 10 additional subjects in whom the variations in nucleotide sequence affected only 1 allele of genes involved in autosomal recessive traits (thus presumably not likely to cause disease phenotypes) (Table 1). The remaining subjects did not have sequence variations that changed amino acid composition (N = 19; see Supplemental Digital Content table, https://links.lww.com/MPG/A19), had variants with high prevalence in controls, or produced amino acid changes that were predicted not to adversely affect the function of the encoded protein according to SIFT, Polyphen, and Grantham scores (N = 8, Table 2). Thus, from a cohort of 51 subjects with cholestasis of undefined etiology, mutation analysis enabled the assignment of a molecular diagnosis in 14 (or 27%) of the subjects.

TABLE 1: Type and frequency of gene sequence variants in a cohort of 51 subjects with CUE

TABLE 2: Description of subjects with CUE^* found to have heterozygous variants with high incidence in normal controls or encoding amino acid changes not predicted to alter the function of the encoded protein^†

High γ-GTP Cholestasis–Sequence Variants in JAG1 or ABCB4

The assignment of a molecular diagnosis in 27% of the subjects was based on the presence of sequence variants in 1 of the genes JAG1, ATP8B1, ABCB11, or ABCB4 (Fig. 1). In the entire cohort, 16 subjects had high γ-GTP (≥100 IU/mL) and 34 had low γ-GTP (<100 IU/mL); γ-GTP was not available in 1 subject. Among those with high γ-GTP, 2 subjects displayed JAG1 variants. They had no evidence of typical facial features or ocular, cardiovascular, or vertebral body abnormalities. Liver biopsy was done in 1 of them at 3.5 months of age and showed canalicular cholestasis, giant cell transformation, and small bile ducts (Table 3). In these patients, the JAG1 variants introduced a premature stop codon (p.C251X) or resulted in an amino acid substitution that is predicted to be damaging to the function of the encoded protein (p.V1086E; Fig. 2). Both variants involved 1 allele, which were consistent with the autosomal dominant mode of inheritance for subjects with AGS. One other patient without syndromic features and with high γ-GTP underwent liver biopsy at 1.5 years of age, which showed pseudoacinar transformation of hepatocytes, portal inflammation, and moderate fibrosis. This patient had 1 sequence variant that introduced a premature stop codon (p.Q945X) in ABCB4 and a second missense variant predicted to be damaging to the encoded protein (p.Y1171C; Table 3 and Fig. 2), which together are likely to result in MDR3 deficiency.

FIGURE 1: Percentage of subjects displaying sequence variants in a cohort of 51 subjects with cholestasis of undefined etiology. All variants in the red portion were homozygous or compound heterozygous, except for those involving JAG1 (see percentage in vertical bar), whereas those in the blue portion were heterozygous for ATP8B1 or ABCB11. Percent in the white portion corresponds to subjects without sequence variants or with variants of high prevalence in controls.

TABLE 3: Gene sequence variants likely to cause disease phenotypes in subjects with CUE^*

FIGURE 2: Categorization of new nucleotide variants identified in subjects with cholestasis of undefined etiology according to the Grantham score, SIFT, and PolyPhen (damaging predicted by scores of >100, <0.05, and >1.5, respectively). The green color predicts the variant to be “benign” and the red color as “damaging” to the function of the encoded protein when the nucleotide substitutions affect both alleles for ATP8B1, ABCB11, and ABCB4 or 1 allele for JAG1.

Low γ-GTP Cholestasis–Sequence Variants in ATP8B1 or ABCB11

The remaining 11 subjects had γ-GTP <100 IU/μL. In this group, 3 had ATP8B1 biallelic variants that included homozygous deletions and compound heterozygous missense, nonsense, and splice site changes consistent with deficiency of the encoded FIC1 protein (Table 3). One patient with compound heterozygous variants (c.1819+1g>a/p.R930X) subsequently had electron microscopy of a liver biopsy with features consistent with Byler bile in the canaliculi, whereas the other 2 subjects had canalicular and cytoplasmic cholestasis, portal inflammation, and fibrosis (Table 3). The remaining 8 subjects, which comprised the majority of the subjects in this group (Fig. 1), had ABCB11 sequence variants that included homozygous or compound heterozygous deletions, and missense and nonsense changes; these variants are in keeping with deficiency of the encoded BSEP protein. In these subjects, liver histopathology reports did not allow differentiation from the patients with ATP8B1 variants (Table 3). Thus, mutation survey in the low γ-GTP group enabled the potential assignment of subjects into specific diagnosis despite similar clinical, biochemical, and histological features.

Heterozygous Variants and Intrahepatic Cholestasis

Mutation survey also identified heterozygous sequence variants in ATP8B1 and ABCB11 in 10 of 51 (or ∼20%) subjects with intrahepatic cholestasis (Fig. 1). The involvement of only 1 allele does not support a direct link with disease phenotype due to the autosomal recessive pattern of inheritance of mutations in both genes. Two of the sequence variants in ATP8B1 and 3 in ABCB11 were reported previously in subjects with intrahepatic cholestasis of pregnancy or benign recurrent intrahepatic cholestasis (for ATP8B1^[20,21]) or PFIC2 (for ABCB11^[10,22,23]), whereas the new variants had low allele incidence in the control population (Table 4). Of note, the presence of high levels of γ-GTP in 1 subject with an ATP8B1 variant and in 2 with ABCB11 variants is consistent with a lack of causality with liver disease secondary to deficiencies of these canalicular transporters.

TABLE 4: Gene sequence variants involving 1 allele in subjects with CUE^*

DISCUSSION

We found sequence variants in 1 of the JAG1, ATP8B1, ABCB11, or ABCB4 genes in 14 of 51 (or 27%) subjects with chronic cholestasis of undefined etiology. The variants were homozygous or compound heterozygous for ATP8B1, ABCB11, and ABCB4 or heterozygous for JAG1; all were reported previously in subjects with well-defined clinical phenotypes, or represent new variants predicted to be damaging to the encoded mutant protein. Although another 20% of the cohort carried heterozygous variants in ATP8B1 or ABCB11 likely to impair the function of the encoded protein, the involvement of only 1 allele does not support a causative association based on the autosomal recessive mode of inheritance for mutations in these genes. Most interesting was that, for the entire cohort, 50 of 51 subjects with idiopathic chronic cholestasis had no clinical, biochemical, or histological features that enabled the clinical diagnosis of either AGS or 1 of the syndromes caused by deficiency of FIC1, BSEP, or MDR3. Thus, these data suggest that mutation surveys of candidate genes may enable a molecular diagnosis based on the presence of nucleotide sequence variants likely to be associated with disease phenotypes, even when a predominant clinical, biochemical, or histological pattern is not obvious.

A careful analysis of clinical features, biochemical markers (eg, the levels of serum γ-GTP in children with other markers of cholestasis), and histopathology often narrows the diagnosis to a small number of syndromes of intrahepatic cholestasis ^(1,24). If typical facial features associated with ocular, cardiovascular, and vertebral abnormalities are present, the diagnosis of AGS is in order. However, the diagnosis may not be obvious in young infants with few or incomplete syndromic features or with a biopsy without paucity of bile ducts. In our cohort, 2 infants with these features (ages 1 and 3.5 months) were found to carry JAG1 variants. The finding of these 2 patients probably represents an underestimation because the chip-based gene sequencing may not detect heterozygous deletions or insertions ⁽¹³⁾, which may account for a substantial percent of mutations reported in subjects with AGS ⁽²⁵⁾. In contrast, the finding of ABCB4 variants in only 1 of 16 subjects with high γ-GTP levels is consistent with a low incidence of MDR3 deficiency in the cohort because nucleotide changes are detected by the chip and are the most frequent types of mutations in these patients.

Among the subjects with low γ-GTP levels, the most common biallelic variants affected ABCB11, which encodes for BSEP. These patients were indistinguishable from those with ATP8B1 variants. The spectrum of sequence variants included missense and nonsense mutations, splice site changes, and homozygous deletions, all reported previously in patients with the diagnosis of PFIC1 or 2, respectively, or were new and predicted to affect the function of the mutant protein. The finding that 19 of 51 subjects displayed no candidate mutation in JAG1, ATP8B1, ABCB11, or ABCB4 suggests that a molecular diagnosis cannot be ascertained in a substantial portion of children with undefined cholestasis. These patients may constitute a population that is most suitable for gene sequencing studies to identify new cholestasis-related genes. Before such an endeavor, it would be important to complement chip-based sequencing with complementary sequencing technologies (eg, genome and cDNA capillary sequencing, fluorescence in situ hybridization, real-time polymerase chain reaction) in the same subjects to precisely show the absence of other nucleotide sequence changes in these genes.

The cholestasis phenotype in the subjects with heterozygous mutations of ATP8B1 and ABCB11 cannot be explained solely by the nucleotide changes reported here. It is possible that a second mutation may reside in promoter regions or intron domains not sequenced by the chip, or that insertions or deletions in the other allele were not detected by the chip. Another possibility is the coexistence of a heterozygous mutation in 1 of the other 4 related gene sequenced by the chip. Our experimental strategy formally rejected this scenario. However, it remains possible that mutations in other genes not included in the chip may contribute to the clinical phenotype.

Although mutation survey may represent a powerful ancillary test to improve specificity of diagnostic algorithms, it is important to recognize that no 1 single technology reported to date is 100% accurate in identifying all possible mutations. In 1 study ⁽²⁵⁾, the combination of 4 different techniques was necessary to increase accuracy to 94% for mutations in JAG1 in subjects with carefully defined features of AGS. Without a highly prevalent mutation in most patients with inherited syndromes of intrahepatic cholestasis involving JAG1, ATP8B1, ABCB11, and ABCB4, the findings of new mutations spread across the entire genes would benefit from functional analysis of the mutant protein or complementary immunohistochemical analysis to more precisely assess the effect of candidate mutations on the function of the protein. Despite these limitations, our data suggest that an analysis of the nucleotide composition of candidate genes identifies gene sequence variants associated with disease phenotypes. Whether this approach is used in clinical practice or as an investigational tool, it has the potential to broaden our knowledge of the genetic basis of cholestatic syndromes and the design of patient-based studies that take into account the genetic makeup of the individual patient.

Acknowledgments

The authors acknowledge the support of Susan Krug and the staff of the Pediatric Liver Care Center at Cincinnati Children's Hospital Medical Center with patient recruitment.