Background
Hepatoblastoma is the most common primary liver tumor in children, accounting for just over 1% of pediatric cancers and 79% of liver cancers in children under the age of 15 [
1]. Most of these tumors are purely derived from epithelium composed exclusively of immature hepatocytic elements, known as fetal and embryonal types. The fetal type consists of smaller than normal hepatocytes that are arranged in irregular laminae, recapitulating those of the fetal liver. The embryonal type is comprised of smaller cells as compared to the fetal type. It has a more immature appearance and pattern of growth. Some of the tumors, referred to as mixed type, are characterized by epithelial patterns and spindled mesenchymal cells. A much rarer variant of such mixed type tumor harbors teratoid features, which contains foci of mature cartilage, intestinal-type or keratinized epithelium, melanin pigment, or skeletal muscle in addition to the elements mentioned above. To date, several genetic and epigenetic features have been observed in hepatoblastoma (reviewed in [
2]). The most recurrent cytogenetic abnormalities include the presence of extra copies of chromosomes 2, 8, 20, and the loss of chromosome 4. Mutations or upregulation of the genes involved in embryonic development have been reported. For example,
APC,
CTNNB1,
AXIN1, and
AXIN2 (key factors involved in the Wnt signaling pathway) are frequently mutated, suggesting that aberration of this pathway occurs as an early event during tumorigenesis. Mutation of
PIK3CA, amplification of
PIK3C2B, and upregulation of hedgehog ligands and their target genes have also been reported. Epigenetic silencing by promoter hypermethylation occurs at several tumor suppressor genes, such as
SFRP1,
APC,
HHIP,
SOCS1,
CASP8, and
RASSF1A. In addition, several imprinted genes, including
IGF2,
DLK1,
PEG3,
PEG10,
MEG3, and
NDN, have been reported to be overexpressed in hepatoblastoma [
2].
Imprinted genes are expressed in a parent-of-origin-specific manner. They are usually clustered in subchromosomal regions called imprinting domains. The human genome contains more than 30 imprinting domains (
http://www.geneimprint.com). Imprinting domains have at least one DMR that are characterized by DNA methylation on one of the two parental alleles. There are maternally methylated DMRs and paternally methylated DMRs. In addition, two classes of imprinted DMRs, gametic and somatic, have been described. Gametic DMRs acquire methylation during gametogenesis and the methylation is maintained from zygote to somatic cells during all the developmental stages. Most gametic DMRs are known as imprinting control regions (ICRs) that regulate the imprinted expression of the genes in the domain. By contrast, methylations of somatic DMRs are established during early embryogenesis after fertilization under the control of nearby ICRs [
3]. Somatic DMRs also regulate the expression of the imprinted genes.
Many imprinted genes regulate cell growth and differentiation, and, thus, disruption of imprinting, mainly due to aberrant DNA methylation at the responsible DMR, is implicated in pre- and/or post-natal growth disorders and in the pathogenesis of cancers [
4]. For example, hypermethylation of
H19-DMR, which is the ICR of the
IGF2/
H19 imprinting domain at the 11p15.5 locus, is a cause of Beckwith-Wiedemann syndrome (BWS), the most common overgrowth syndrome characterized by occasional development of embryonal tumors, including hepatoblastoma [
5]. The hypermethylation leading to biallelic expression of
IGF2 is seen in a range of tumors, also including hepatoblastoma [
6,
7]. The LOH of 11p15.5, especially the loss of the maternal allele, is found in approximately 20% of hepatoblastoma cases, and it is reported to be a risk factor for the relapse of this tumor [
7,
8]. Furthermore, several imprinted genes are overexpressed in hepatoblastoma as mentioned above. Thus, it is speculated that aberrant DNA methylation at imprinted DMRs is a key mechanism during malignant transformation of progenitor cells in a variety of tissues, including the liver [
2,
9]. However, the methylation status of imprinted DMRs scattered through the human genome has yet to be analyzed comprehensively in hepatoblastoma.
In this study, we performed comprehensive methylation analyses and polymorphism analyses of 33 imprinted DMRs in hepatoblastoma. We therefore describe some epigenetic and genetic characteristics of hepatoblastoma. These findings collectively aid in the understanding of the development of hepatoblastoma.
Methods
Samples
Twelve hepatoblastomas and their paired adjacent normal liver tissues were analyzed. Eleven sporadic hepatoblastoma samples (HB01 - HB11) were obtained from the Department of Pediatric Surgery, Faculty of Medicine, Kyushu University, Japan. One hepatoblastoma developed in a BWS patient (BWS109) was obtained from Toho University, Omori Medical Centre, Japan. Histochemical analyses of the tumor tissues indicated that the average of the tumor cell contents was approximately 70%. Ten of the patients were treated based on the Japanese Study Group for Pediatric Liver Tumor-2 (JPLT-2) protocol (HB08 and HB09 were not). Clinical information of the hepatoblastoma cases is shown in Table
1. Three livers (CL7, CL16, CBD1) were used as normal controls. CL7 (a 7-year-old who died from spinal muscular atrophy type I-C with chronic respiratory insufficiency) and CL16 (a 16-year-old who died after head trauma) were provided by the non-profit organization, Human & Animal Bridging Research Organization (Chiba, Japan). CBD1 (a 7-month-old who had congenital biliary dilatation) was obtained from the Department of Pediatric Surgery, Faculty of Medicine, Kyushu University. Written informed consents were obtained from the parents or the guardians of the participants, because the participants were children or dead. This study was approved by the Ethical Committee for Human Genome and Gene Analyses of the Faculty of Medicine, Saga University.
Table 1
Clinical information of hepatoblastoma cases
HB01 | F/1y3m | Combined fetal and embryonal type | III | CITA4 | III | Alive | |
HB02 | F/3y2m | Fetal typec
| III | CITA4 | III | Aive | |
HB03 | F/7y11m | Hepatoblastoma (NOS)d
| III | CITA5 | III | Alive | Small for gestational age |
HB04 | M/1y4m | Mixed epithelial and mesenchymal with teratoid featurec
| IV | CITA4 + ITEC2 | IV | Alive | |
HB05 | M/1y2m | Mixed epithelial and mesenchymal with teratoid feature | III | CITA5 | II | Alive | |
HB06 | M/10m | Mixed epithelial and mesenchymal with teratoid feature | III | CITA4 | III | Alive | |
HB07 | M/8m | Combined fetal and embryonal type | II | CITA2 | II | Alive | |
HB08 | F/28d | Combined fetal and embryonal type | II | | | Alive | |
HB09 | M/1y6m | Combined fetal and embryonal type | II | | | Treatment related death | Small for gestational age |
HB10 | F/6y6m | Fetal type | II | CITA2 | II | Alive | |
HB11 | F/3m | Combined fetal and embryonal type | IV | CITA7 | III | Treatment related death | |
BWS109 | F/1y0m | Hepatoblastoma (NOS)d
| IV,M(+) | CITA7 + ITEC1 | IV | Alive | Beckwith-Wiedemann syndrome, liver transplantation at 1 year old |
DNA isolation and bisulphite conversion
Genomic DNA was extracted from each sample using the QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. One microgram of genomic DNA was subjected to bisulfite conversion using the EZ DNA Methylation KitTM (Zymo Research, CA), and then the converted DNA was eluted in 100 μl of water.
MALDI-TOF MS analysis
The methylation status of imprinted DMRs was screened by MALDI-TOF MS analysis with a MassARRAY system (Sequenom, CA) [
10], according to the manufacturer’s instructions. MALDI-TOF MS analysis produced signal pattern pairs indicative of non-methylated and methylated DNA. Epityper software analysis of the signals yielded the methylation index which ranged from 0 (no methylation) to 1 (full methylation) of each CpG unit, which contained one or more CpG sites measured as one unit in the MALDI-TOF MS analysis. Aberrant methylation of a CpG unit was defined as when the difference of methylation indexes between two samples exceeded 0.15, which was based on the fact that we have previously found that the differences of
H19-DMR hypermethylation or
KvDMR1 hypomethylation in BWS patients were at least more than 0.15 (data not shown). Since analyzed DMRs included several CpG units, aberrant methylation of a DMR was defined as when more than 60% of total number of analyzed CpG units showed aberrant methylation (with the difference exceeding 0.15). We used CL7 and CBD1 as normal controls in MALDI-TOF MS analysis.
Pyrosequencing
Pyrosequencing was conducted using QIAGEN PyroMark Q24 according to the manufacturer’s instruction (Qiagen, Germany). Some of the primers for DMR analysis were described by Woodfine et al. [
11]. We designed other primers by using PyroMark Assay Design 2.0 (Qiagen, Germany). The primers for LINE-1 (GenBank accession no. X58075) analyses were described by Bollati et al. [
12]. The criterion for MALDI-TOF MS analysis was also employed to define the aberrant methylation of each CpG site and an analyzed region. We used three livers, i.e. CL7, CL16, and CBD1, as normal controls in pyrosequencing. The control livers were analyzed in triplicate for LINE-1 and once for DMRs.
DNA Polymorphism analysis
LOH, UPD, and copy number abnormalities were investigated with DNA polymorphisms. For quantitative analyses, tetranucleotide repeat markers near the imprinted DMRs were amplified and separated by electrophoresis on an Applied Biosystems 3130 genetic analyzer. Data were then quantitatively analyzed with GeneMapper software (Applied Biosystems, CA). The peak height ratios of two parental alleles were calculated. A single nucleotide polymorphism (SNP) of KCNQ1DN (rs229897) was also analyzed.
All primers used in this study are shown in Additional file
1: Table S1.
Statistical analysis
The methylation statuses of the samples were compared in three pairs: adjacent normal liver tissue (A) and control livers (C), denoted as AxC; tumors (T) and control livers, denoted as TxC; tumors and adjacent normal liver tissue, denoted as TxA. The binomial distribution test was performed to compare aberrant hypomethylation and aberrant hypermethylation within each comparison pair (AxC, TxC, and TxA). The Chi squared test or Fisher’s exact test was used for comparison between maternally methylated and paternally methylated DMRs and between gametic and somatic DMRs in aberrant hypomethylation and aberrant hypermethylation cases for each comparison pair. For LINE-1 methylation, a paired t-test was used to compare tumor and adjacent normal liver tissue, and an independent t-test (Welch’s t-test) was used for comparing tumor or adjacent normal liver tissue with control liver. A p-value less than 0.05 was considered to be statistically significant. Bonferroni correction was performed when needed.
Discussion
In this study, we found many imprinted DMRs methylated aberrantly in hepatoblastomas and paired adjacent normal liver tissue. An important finding was that the aberrant hypomethylation occurred not only in tumor tissue but also in adjacent normal liver tissue. One possible explanation is that the occurrence of the aberrant hypomethylation at certain DMRs may be a very early and specific event prior to tumor development, although there is another possibility that the tumor may induce methylation changes in the adjacent tissues. Okamoto et al. have previously reported a similar phenomenon with respect to aberrant hypermethylation of
H19-DMR that was frequently found in normal tissues adjacent to Wilms’ tumors, which carried the same aberrant methylation [
23]. Based on the results, it was hypothesized that the preceding aberrant methylation may be a constitutional aberration in the onset of embryonal tumors. In contrast to the hypomethylation, the aberrant hypermethylation of the DMR occurred only in tumors. These results indicated that the hypermethylation of the DMRs, especially for
INPP5Fv2-DMR,
RB1-DMR, and
GNASXL-DMR, was a specific event for tumor development; this suggested that the pre-cancerous cells did not carry hypermethylation at the DMRs, but acquired the aberrant methylation during tumor development.
We also analyzed the genome-wide methylation level, represented by LINE-1 methylation, and we did not find large difference among three sample groups as a whole. LINE-1 is usually hypomethylated in many adult tumors, and its methylation level correlates with clinicopathological features of the tumors [
19]. The different situation concerning LINE-1 methylation between hepatoblastoma and adult tumors may reflect a different mechanism of tumorigenesis in embryonal tumors as compared to adult tumors.
Hypermethylation in tumors was frequently observed at three DMRs,
INPPF5v2-DMR,
RB1-DMR, and
GNASXL-DMR.
INPPF5v2-DMR controls the expression of
INPP5F transcript variant 2, which encodes a protein of an unknown function [
24,
25].
RB1-DMR, located in intron 2 of the
RB1 gene, leads to maternal expression of transcript variants from exon 2B with very low expression in normal tissues [
26]. The function of the variants in cell proliferation is not known. Thus, the effect of these hypermethylated DMRs on tumorigenesis would be little or unknown.
GNASXL-DMR is associated with the paternal expression of
GNASXL, which encodes a protein involved in signal transduction [
27‐
29]. The DMR was shown to be mostly unmethylated in control livers (Additional file
2: Figure S1). Thus, hypermethylation of GNASXL-DMR would reduce expression of
GNASXL. Unfortunately, the expression of genes linked to aberrantly methylated DMRs could not be analyzed due to poor RNA quality, which was probably due to effects of chemotherapy and a limited amount of samples. Therefore, we could not assess the involvement of hypermethylation in tumorigenesis of hepatoblastoma.
Another important finding was the frequent occurrence of both genetic and epigenetic alterations at the two chromosomal loci, 11p15.5 and 20q13.3. The 11p15.5 locus is a well-known imprinted locus responsible for BWS, a tumor-predisposing imprinting disorder. The locus was found to be altered genetically and/or epigenetically in 10 of 12 tumors. Hypermethylation at
H19-DMR and hypomethylation at
IGF2-DMR0 associated with biallelic expression of
IGF2 were reported in adult and embryological tumors, including hepatoblastoma [
6,
7]. Hypermethylation at
H19-DMR and the
H19 promoter also reduced the expression of
H19 in Wilms’ tumor [
30,
31]. In addition to epigenetic alterations, genetic alterations, such as the amplification of paternal alleles leading to overexpression of
IGF2 and LOH of the maternal allele leading to reduced expression of
H19, were observed in sporadic Wilms’ tumors [
32,
33]. In this study, in addition to the hypermethylations at
H19-DMR and the
H19 promoter in two tumors, hypomethylation at
IGF2-DMR0 occurred in another two adjacent normal liver tissues. Further, abnormal allelic copy number, paternal UPD, and maternal LOH of 11p15.5 were observed. The overexpression of
IGF2 and the reduced expression of
H19 would play an important role in tumorigenesis of hepatoblastoma.
The 20q13.3 locus was also altered genetically and/or epigenetically in 7 of 12 tumors. This locus is responsible for pseudohypoparathyroidism, a condition in which pathogenesis is attributed to the tissue specific imprinting of
Gsα, for example, which occurs in the proximal renal tubule. On the other hand, an extra copy of chromosome 20 has been known to be the most recurrent cytogenetic alteration in hepatoblastoma [
2,
34]. We found copy number differences of the alleles in this region, suggesting the existence of non-imprinted oncogenic gene(s) in this region.
Many epigenetic and genetic alterations were found at the loci linked to the 33 imprinted DMRs in 12 hepatoblastomas. However, since sample numbers in this study were small, more hepatoblastoma samples should be analyzed to confirm the present data and to evaluate the precise role of these alterations in tumorigenesis in addition to assessing their usefulness as markers for clinical characteristics, such as stage classification, response to chemotherapy, and prognosis. Also needed are the expression analyses of the genes linked to aberrantly methylated DMRs to assess their role in tumor development, although it is very difficult to obtain hepatoblastoma samples without any chemotherapeutic history.
Acknowledgements
We thank Prof. Yutaka Kondo, Division of Epigenomics, Aichi Cancer Center Research Institute, Japan for technical advices in LINE-1 methylation analysis. This study was supported, in part, by a Grant for Research on Intractable Diseases from the Ministry of Health, Labor, and Welfare; a Grant for Child Health and Development from the National Center for Child Health and Development; and, a Grant-in-Aid for Challenging Exploratory Research and a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JMR made significant contributions to the acquisition and analysis of data and also helped in manuscript preparation. TM, KH1, HY, and KN1 made contributions to technical supports and data analyses. RS, KM, RH, KK, YO, TS, TT3, and TT8 prepared the tissue samples. KN5 and KH5 performed technical support and statistical analyses. SA performed HE analyses of tumor samples. HS conceived the study, participated in its design and supervision and prepared the manuscript. KJ also participated in the design and supervision of the study and the preparation of the manuscript. All authors read and approved the final manuscript.