An analysis of the demographic history of the risk allele R4810K in RNF213 of moyamoya disease.

BACKGROUND: Ring finger protein 213 (RNF213) is a susceptibility gene of moyamoya disease (MMD). A previous case-control study and a family analysis demonstrated a strong association of the East Asian-specific variant, R4810K (rs112735431), with MMD. Our aim is to uncover evolutionary history of R4810K in East Asian populations. METHODS: The RNF213 locus of 24 MMD patients in Japan were sequenced using targeted-capture sequencing. Based on the sequence data, we conducted population genetic analysis and estimated the age of R4810K using coalescent simulation. RESULTS: The diversity of the RNF213 gene was higher in Africans than non-Africans, which can be explained by bottleneck effect of the out-of-Africa migration. Coalescent simulation showed that the risk variant was born in East Asia 14,500-5100 years ago and came to the Japanese archipelago afterward, probably in the period when the known migration based on archaeological evidences occurred. CONCLUSIONS: Although clinical data show that the symptoms varies, all sequences harboring the risk allele are almost identical with a small number of exceptions, suggesting the MMD phenotypes are unaffected by the variants of this gene and rather would be more affected by environmental factors.

Comprehensive methylation analysis of imprinting-associated differentially methylated regions in colorectal cancer.

BACKGROUND: Imprinted genes are regulated by DNA methylation at imprinting-associated differentially methylated regions (iDMRs). Abnormal expression of imprinted genes is implicated in imprinting disorders and tumors. In colorectal cancer (CRC), methylation and imprinting status of the IGF2/H19 domain have been studied. However, no comprehensive methylation analysis of iDMRs in CRC has been reported. Furthermore, the relationship between iDMR methylation status and other methylation-related issues, such as CpG island methylator phenotype (CIMP) and long interspersed element-1 (LINE-1) methylation, remains unclear. RESULTS: We analyzed the methylation status of 38 iDMRs in 106 CRC patients. We also investigated CIMP, LINE-1 methylation, KRAS and BRAF gene mutations, and loss of imprinting (LOI) of IGF2. We further examined the relationship between these factors and clinicopathological factors. The overall trend in iDMR methylation was towards hypermethylation, and iDMRs could be grouped into three categories: susceptible, resistant, and intermediate-to-aberrant methylation. The susceptible and resistant iDMRs consisted of all types of iDMR (gametic and somatic, maternally and paternally methylated). Hypermethylation of multiple iDMRs (HyMiD)-positive status was statistically associated with CIMP-positive status, but not associated with mutations in the BRAF and KRAS genes. HyMiD-positive status was inversely associated with LINE-1 methylation. Among four iDMRs within the IGF2/H19 domain, IGF2-DMR0 hypomethylation occurred most frequently, but was not associated with IGF2 LOI. Finally, we statistically calculated predictive prognostic scores based on aberrant methylation status of three iDMRs. CONCLUSION: In CRC tissues, some iDMRs were susceptible to hypermethylation independent of the type of iDMR and genomic sequence. Although HyMiD-positive status was associated with CIMP-positive status, this was independent of the BRAF and KRAS pathways, which are responsible for CIMP. Since IGF2-DMR0 hypomethylation and aberrant methylation of other iDMRs within the IGF2/H19 domain were not associated with IGF2 LOI, dysfunction of any of the molecular components related to imprinting regulation may be involved in IGF2 LOI. The prognostic score calculated based on aberrant methylation of three iDMRs has potential clinical applications as a prognostic predictor in patients. Further study is required to understand the biological significance of, and mechanisms behind, aberrant methylation of iDMRs and IGF2 LOI in CRCs.

Growing oocyte-specific transcription-dependent de novo DNA methylation at the imprinted Zrsr1-DMR.

BACKGROUND: Zrsr1 is a paternally expressed imprinted gene located in the first intron of Commd1, and the Zrsr1 promoter resides in a differentially methylated region (DMR) that is maternally methylated in the oocyte. However, a mechanism for the establishment of the methylation has remained obscure. Commd1 is transcribed in the opposite direction to Zrsr1 with predominant maternal expression, especially in the adult brain. RESULTS: We found Commed1 transcribed through the DMR in the growing oocyte. Zrsr1-DMR methylation was abolished by the prevention of Commd1 transcription. Furthermore, methylation did not occur at the artificially unmethylated maternal Zrsr1-DMR during embryonic development when transcription through the DMR was restored in the zygote. Loss of methylation at the maternal Zrsr1-DMR resulted in biallelic Zrsr1 expression and reduced the extent of the predominant maternal expression of Commd1. CONCLUSIONS: These results indicate that the establishment of methylation at Zrsr1-DMR occurs in a transcription-dependent and oocyte-specific manner and caused Zrsr1 imprinting by repressing maternal expression. The predominant maternal expression of Commd1 is likely caused by transcriptional interference by paternal Zrsr1 expression.

An ancestral haplotype of the human PERIOD2 gene associates with reduced sensitivity to light-induced melatonin suppression.

Humans show various responses to the environmental stimulus in individual levels as "physiological variations." However, it has been unclear if these are caused by genetic variations. In this study, we examined the association between the physiological variation of response to light-stimulus and genetic polymorphisms. We collected physiological data from 43 subjects, including light-induced melatonin suppression, and performed haplotype analyses on the clock genes, PER2 and PER3, exhibiting geographical differentiation of allele frequencies. Among the haplotypes of PER3, no significant difference in light sensitivity was found. However, three common haplotypes of PER2 accounted for more than 96% of the chromosomes in subjects, and 1 of those 3 had a significantly low-sensitive response to light-stimulus (P < 0.05). The homozygote of the low-sensitive PER2 haplotype showed significantly lower percentages of melatonin suppression (P < 0.05), and the heterozygotes of the haplotypes varied their ratios, indicating that the physiological variation for light-sensitivity is evidently related to the PER2 polymorphism. Compared with global haplotype frequencies, the haplotype with a low-sensitive response was more frequent in Africans than in non-Africans, and came to the root in the phylogenetic tree, suggesting that the low light-sensitive haplotype is the ancestral type, whereas the other haplotypes with high sensitivity to light are the derived types. Hence, we speculate that the high light-sensitive haplotypes have spread throughout the world after the Out-of-Africa migration of modern humans.

The allele frequency of ALDH2*Glu504Lys and ADH1B*Arg47His for the Ryukyu islanders and their history of expansion among East Asians.

OBJECTIVES: A cline of frequencies of the derived allele of the ALDH2 gene, which causes a deficiency of an enzyme and "facial flushing" in humans who drink alcohol, has been known among the people of the Japanese archipelago. This cline is conventionally explained by admixture with immigrants from the Asian continent occurring during the Yayoi period. Previous studies lack sufficient data from the peripheral regions of the indigenous Jomon people, and those data the ADH1B gene that is involved in the Class I ADH gene cluster and contains another variant leading to a functional change. METHODS: We focused on the southwestern-most people from the Ryukyu Islands (n = 218) and those from northern Kyushu (n = 21) where the Yayoi immigrants likely arrived. We investigated both the Class I ADH and ALDH2 loci, as well as neutral genetic markers. RESULTS: In the Ryukyu Islands, the frequencies of the ancestral alleles in both loci were always higher than those in mainland Japan, while the frequencies of ADH1B were less than those of the derived allele. A haplotype block was not observed in ALDH2 but was in Class I ADH. DISCUSSION: Our data suggest that the derived allele of ALDH2 came with the Yayoi immigrants from the Asian continent to the Japanese archipelago. However, the derived allele of ADH1B is unlikely to be related to the Yayoi migration. Therefore, we postulate that the expansion of the derived allele of ADHIB in East Asia could be traced back to the last glacial period.

The HUS1B promoter is hypomethylated in the placentas of low-birth-weight infants.

Aberrant DNA methylation is associated with a range of human disorders. To identify differences in DNA methylation of gene promoters between placentas of low-birth-weight (LBW) and normal-birth-weight (NBW) infants, we screened 8091 genes for aberrant methylation in placentas using microarray-based integrated analysis of methylation by isoschizomers (MIAMI). Seven candidate genes for hypomethylation in the placentas of LBW infants were selected. Among these candidates, COBRA analyses suggested that the HUS1B gene was hypomethylated in some of the placentas. Quantitative methylation analyses by bisulfite-pyrosequencing indicated that the promoter region of the gene was hypomethylated in three of the 86 placentas analyzed. The HUS1B promoter was highly methylated in two cell lines derived from trophoblastic cells. Gene expression increased when the promoter was demethylated by 5Aza-dC treatment. This suggests that hypomethylation of HUS1B alters gene expression in the placenta and that this dysregulated gene expression may contribute to the pathogenesis of LBW by affecting placental functions involved in fetal growth.

Identification of consensus motifs associated with mitotic recombination and clinical characteristics in patients with paternal uniparental isodisomy of chromosome 11.

Uniparental disomy (UPD) is defined as the inheritance of both homologs of a given genomic region from only one parent. The majority of UPD includes an entire chromosome. However, the extent of UPD is sometimes limited to a subchromosomal region (segmental UPD). Mosaic paternal UPD (pUPD) of chromosome 11 is found in approximately 20% of patients with Beckwith-Wiedemann syndrome (BWS) and almost all pUPDs are segmental isodisomic pUPDs resulting from mitotic recombination at an early embryonic stage. A mechanism initiating a DNA double strand break (DSB) within 11p has been predicted to lead to segmental pUPD. However, no consensus motif has yet been found. Here, we analyzed 32 BWS patients with pUPD by SNP array and searched for consensus motifs. We identified four consensus motifs frequently appearing within breakpoint regions of segmental pUPD. These motifs were found in another nine BWS patients with pUPD. In addition, the seven motifs found in meiotic recombination hot spots could not be found within pUPD breakpoint regions. Histone H3 lysine 4 trimethylation, a marker of DSB initiation, could not be found either. These findings suggest that the mechanism(s) of mitotic recombination leading to segmental pUPD are different from that of meiotic recombination. Furthermore, we found seven patients with paternal uniparental diploidy (PUD) mosaicism. Comparison of clinical features between segmental pUPDs and PUDs showed that developmental disability and cardiac abnormalities were additional characteristic features of PUD mosaicism, along with high risk of tumor development. We also found that macroglossia was characteristic of segmental pUPD mosaicism.

Comprehensive and quantitative multilocus methylation analysis reveals the susceptibility of specific imprinted differentially methylated regions to aberrant methylation in Beckwith-Wiedemann syndrome with epimutations.

PURPOSE: Expression of imprinted genes is regulated by DNA methylation of differentially methylated regions (DMRs). Beckwith-Wiedemann syndrome is an imprinting disorder caused by epimutations of DMRs at 11p15.5. To date, multiple methylation defects have been reported in Beckwith-Wiedemann syndrome patients with epimutations; however, limited numbers of DMRs have been analyzed. The susceptibility of DMRs to aberrant methylation, alteration of gene expression due to aberrant methylation, and causative factors for multiple methylation defects remain undetermined. METHODS: Comprehensive methylation analysis with two quantitative methods, matrix-assisted laser desorption/ionization mass spectrometry and bisulfite pyrosequencing, was conducted across 29 DMRs in 54 Beckwith-Wiedemann syndrome patients with epimutations. Allelic expressions of three genes with aberrant methylation were analyzed. All DMRs with aberrant methylation were sequenced. RESULTS: Thirty-four percent of KvDMR1-loss of methylation patients and 30% of H19DMR-gain of methylation patients showed multiple methylation defects. Maternally methylated DMRs were susceptible to aberrant hypomethylation in KvDMR1-loss of methylation patients. Biallelic expression of the genes was associated with aberrant methylation. Cis-acting pathological variations were not found in any aberrantly methylated DMR. CONCLUSION: Maternally methylated DMRs may be vulnerable to DNA demethylation during the preimplantation stage, when hypomethylation of KvDMR1 occurs, and aberrant methylation of DMRs affects imprinted gene expression. Cis-acting variations of the DMRs are not involved in the multiple methylation defects.

Comprehensive analyses of imprinted differentially methylated regions reveal epigenetic and genetic characteristics in hepatoblastoma.

BACKGROUND: Aberrant methylation at imprinted differentially methylated regions (DMRs) in human 11p15.5 has been reported in many tumors including hepatoblastoma. However, the methylation status of imprinted DMRs in imprinted loci scattered through the human genome has not been analyzed yet in any tumors. METHODS: The methylation statuses of 33 imprinted DMRs were analyzed in 12 hepatoblastomas and adjacent normal liver tissue by MALDI-TOF MS and pyrosequencing. Uniparental disomy (UPD) and copy number abnormalities were investigated with DNA polymorphisms. RESULTS: Among 33 DMRs analyzed, 18 showed aberrant methylation in at least 1 tumor. There was large deviation in the incidence of aberrant methylation among the DMRs. KvDMR1 and IGF2-DMR0 were the most frequently hypomethylated DMRs. INPP5Fv2-DMR and RB1-DMR were hypermethylated with high frequencies. Hypomethylation was observed at certain DMRs not only in tumors but also in a small number of adjacent histologically normal liver tissue, whereas hypermethylation was observed only in tumor samples. The methylation levels of long interspersed nuclear element-1 (LINE-1) did not show large differences between tumor tissue and normal liver controls. Chromosomal abnormalities were also found in some tumors. 11p15.5 and 20q13.3 loci showed the frequent occurrence of both genetic and epigenetic alterations. CONCLUSIONS: Our analyses revealed tumor-specific aberrant hypermethylation at some imprinted DMRs in 12 hepatoblastomas with additional suggestion for the possibility of hypomethylation prior to tumor development. Some loci showed both genetic and epigenetic alterations with high frequencies. These findings will aid in understanding the development of hepatoblastoma.

Homozygous deletion of DIS3L2 exon 9 due to non-allelic homologous recombination between LINE-1s in a Japanese patient with Perlman syndrome.

Perlman syndrome is a rare, autosomal recessive overgrowth disorder. Recently, the deletion of exon 9 and other mutations of the DIS3L2 gene have been reported in patients; however, the mechanism behind this deletion is still unknown. We report the homozygous deletion of exon 9 of DIS3L2 in a Japanese patient with Perlman syndrome. We identified the deletion junction, and implicate a non-allelic homologous recombination (NAHR) between two LINE-1 (L1) elements as the causative mechanism. Furthermore, the deletion junctions were different between the paternal and maternal mutant alleles, suggesting the occurrence of two independent NAHR events in the ancestors of each parent. The data suggest that the region around exon 9 might be a hot spot of L1-mediated NAHR.

Antisense transcription occurs at the promoter of a mouse imprinted gene, commd1, on the repressed paternal allele.

The Commd1 gene is imprinted in the adult mouse brain and is predominantly expressed from the maternal allele. A paternally expressing imprinted gene, U2af1-rs1, resides in the first intron of Commd1 in an antisense orientation. We found that RNA polymerase II phosphorylated at serine 2 of the carboxyl-terminal domain repeats, a marker of transcription elongation, is enriched on the paternal allele than on the maternal allele in the Commd1 promoter. The Commd1 promoter harbours no allelic differences in DNA methylation and histone modifications. These results strongly suggested that imprinting of Commd1 is generated by interference with paternal Commd1 transcription by the oppositely directed U2af1-rs1 transcription.

MeCP2 knockdown reveals DNA methylation-independent gene repression of target genes in living cells and a bias in the cellular location of target gene products.

MeCP2, a methyl-CpG binding domain (MBD) protein, is known to bind to methylated CpG sites via a conserved MBD, leading to transcriptional repression. However, studies in cell-free system for gene repression and MeCP2 binding have suggested that DNA methylation-independent repression also occurs in living cells. It has been difficult to characterize the target genes of MeCP2 because a limited number have been identified to date. In this context, we screened for MeCP2 target genes using knockdown (KD) experiments combined with microarray gene expression analyses. Of the 49 genes that showed a more than three-fold increase in expression in two independent KD experiments conducted with different siRNA sets, unexpectedly, half (24 genes) did not contain promoter CpG islands (CGIs). Of seven selected genes that did contain CGIs, only two were methylated at the CGI, bound MeCP2 before KD, and reduced MeCP2 after KD. For three, MeCP2 was observed to bind to the unmethylated CGI before KD, and for one MeCP2 was reduced after KD. Another two genes neither had DNA methylation nor bound MeCP2 before KD. Gene ontology analysis suggested that MeCP2 represses a certain group of genes. These results suggest that in addition to the canonical gene repression function, MeCP2 can repress gene expression by binding to unmethylated DNA in particular genes in living cells.

Japanese and North American/European patients with Beckwith-Wiedemann syndrome have different frequencies of some epigenetic and genetic alterations.

Beckwith-Wiedemann syndrome (BWS) is an imprinting-related human disease. The frequencies of causative alterations such as loss of methylation (LOM) of KvDMR1, hypermethylation of H19-DMR, paternal uniparental disomy, CDKN1C gene mutation, and chromosome abnormality have been described for North American and European patients, but the corresponding frequencies in Japanese patients have not been measured to date. Analysis of 47 Japanese cases of BWS revealed a significantly lower frequency of H19-DMR hypermethylation and a higher frequency of chromosome abnormality than in North American and European patients. These results suggest that susceptibility to epigenetic and genetic alterations differs between the two groups.

Retinoic acid receptor beta2 is epigenetically silenced either by DNA methylation or repressive histone modifications at the promoter in cervical cancer cells.

To elucidate the silencing mechanism of retinoic acid receptor beta2 (RAR beta2) in cervical carcinogenesis, we investigated RAR beta2 expression and the status of both DNA methylation and histone modifications at the promoter in cervical cancer cell lines. RAR beta2 was frequently repressed in cancer cell lines and in primary cancers of the cervix. Although the majority of RAR beta2-negative cancers had methylated promoter, RAR beta2 was repressed with hypomethylated promoter in a substantial fraction of the cancers. The RAR beta2-negative cells with hypomethylated promoters showed a repressive histone modification pattern at the promoter. RAR beta2 was reactivated by a histone deacetylase inhibitor, accompanied by formation of active histone modifications. The repressive modification was also observed in cells repressed with hypermethylated promoter, but RAR beta2 was reactivated only by DNA demethylating agent and not by histone deacetylase inhibitor. Our results suggest that RAR beta2 is silenced by either of the two key epigenetic pathways, DNA methylation or repressive histone modifications, depending on the individual cancer cells.

Comparative analyses of genomic imprinting and CpG island-methylation in mouse Murr1 and human MURR1 loci revealed a putative imprinting control region in mice.

Human MURR1 is an orthologue of mouse Murr1 gene, which was previously reported to be imprinted only in adult brain with a maternal allele-predominant expression and to contain another imprinted gene, U2af1-rs1, in the first intron. Human MURR1 was found not to harbor the U2af1-rs1 orthologue and to be expressed biallelically in tissues, including adult brain. Three genes identified around Murr1 and their orthologues around MURR1 were expressed biallelically. These findings suggest that the mouse imprinting locus is limited to a small region and the introduction of U2af1-rs1 in mouse causes the imprinting of this locus. The CpG island (CGI) at U2af1-rs1 with maternal methylation was the only differentially methylated region among CGIs found in these loci. Detailed methylation analyses of the U2af1-rs1 CGI in germ cells led to identification of a region with oocyte-specific methylation. These results suggest that this region is the imprinting control region of the Murr1/U2af1-rs1 locus in mouse.

The essential role of histone H3 Lys9 di-methylation and MeCP2 binding in MGMT silencing with poor DNA methylation of the promoter CpG island.

Silencing of the O (6)-methylguanine-DNA methyltransferase (MGMT) gene, a key to DNA repair, is involved in carcinogenesis. Recent studies have focused on DNA hypermethylation of the promoter CpG island. However, cases showing silencing with DNA hypomethylation certainly exist, and the mechanism involved is not elucidated. To clarify this mechanism, we examined the dynamics of DNA methylation, histone acetylation, histone methylation, and binding of methyl-CpG binding proteins at the MGMT promoter region using four MGMT negative cell lines with various extents of DNA methylation. Histone H3K9 di-methylation (H3me2K9), not tri-methylation, and MeCP2 binding were commonly seen in all MGMT negative cell lines regardless of DNA methylation status. 5Aza-dC, but not TSA, restored gene expression, accompanied by a decrease in H3me2K9 and MeCP2 binding. In SaOS2 cells with the most hypomethylated CpG island, 5Aza-dC decreased H3me2K9 and MeCP2 binding with no effect on DNA methylation or histone acetylation. H3me2K9 and DNA methylation were restricted to in and around the island, indicating that epigenetic modification at the promoter CpG island is critical. We conclude that H3me2K9 and MeCP2 binding are common and more essential for MGMT silencing than DNA hypermethylation or histone deacetylation. The epigenetic mechanism leading to silent heterochromatin at the promoter CpG island may be the same in different types of cancer irrespective of the extent of DNA methylation.

Silencing of imprinted CDKN1C gene expression is associated with loss of CpG and histone H3 lysine 9 methylation at DMR-LIT1 in esophageal cancer.

The putative tumor suppressor CDKN1C is an imprinted gene at 11p15.5, a well-known imprinted region often deleted in tumors. The absence of somatic mutations and the frequent diminished expression in tumors would suggest that CDKN1C expression is regulated epigenetically. It has been, however, controversial whether the diminution is caused by imprinting disruption of the CDKN1C/LIT1 domain or by promoter hypermethylation of CDKN1C itself. To clarify this, we investigated the CpG methylation index of the CDKN1C promoter and the differentially methylated region of the LIT1 CpG island (differentially methylated region (DMR)-LIT1), an imprinting control region of the domain, and CDKN1C expression in esophageal cancer cell lines. CDKN1C expression was diminished in 10 of 17 lines and statistically correlated with the loss of methylation at DMR-LIT1 in all but three. However, there was no statistical correlation between CDKN1C promoter MI and CDKN1C expression. Furthermore, loss of CpG methylation was associated with loss of histone H3 lysine 9 (H3K9) methylation at DMR-LIT1. Histone modifications at CDKN1C promoter were not correlated with CDKN1C expression. The data suggested that the diminished CDKN1C expression is associated with the loss of methylation of CpG and H3K9 at DMR-LIT1, not by its own promoter CpG methylation, and is involved in esophageal cancer, implying that DMR-LIT1 epigenetically regulates CDKN1C expression not through histone modifications at CDKN1C promoter, but through that of DMR-LIT1.