Premature termination of reprogramming in vivo leads to cancer development through altered epigenetic regulation.

Cancer is believed to arise primarily through accumulation of genetic mutations. Although induced pluripotent stem cell (iPSC) generation does not require changes in genomic sequence, iPSCs acquire unlimited growth potential, a characteristic shared with cancer cells. Here, we describe a murine system in which reprogramming factor expression in vivo can be controlled temporally with doxycycline (Dox). Notably, transient expression of reprogramming factors in vivo results in tumor development in various tissues consisting of undifferentiated dysplastic cells exhibiting global changes in DNA methylation patterns. The Dox-withdrawn tumors arising in the kidney share a number of characteristics with Wilms tumor, a common pediatric kidney cancer. We also demonstrate that iPSCs derived from Dox-withdrawn kidney tumor cells give rise to nonneoplastic kidney cells in mice, proving that they have not undergone irreversible genetic transformation. These findings suggest that epigenetic regulation associated with iPSC derivation may drive development of particular types of cancer.

Whole-exome sequencing reveals causative genetic variants for several overgrowth syndromes in molecularly negative Beckwith-Wiedemann spectrum.

Background Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder caused by (epi)genetic alterations at 11p15. Because approximately 20% of patients test negative via molecular testing of peripheral blood leukocytes, the concept of Beckwith-Wiedemann spectrum (BWSp) was established to encompass a broader cohort with diverse and overlapping phenotypes. The prevalence of other overgrowth syndromes concealed within molecularly negative BWSp remains unexplored. Methods We conducted whole-exome sequencing (WES) on 69 singleton patients exhibiting molecularly negative BWSp. Variants were confirmed by Sanger sequencing or quantitative genomic PCR. We compared BWSp scores and clinical features between groups with classical BWS (cBWS), atypical BWS or isolated lateralised overgrowth (aBWS+ILO) and overgrowth syndromes identified via WES. Results Ten patients, one classified as aBWS and nine as cBWS, showed causative gene variants for Simpson-Golabi-Behmel syndrome (five patients), Sotos syndrome (two), Imagawa-Matsumoto syndrome (one), glycosylphosphatidylinositol biosynthesis defect 11 (one) or 8q duplication/9p deletion (one). BWSp scores did not distinguish between cBWS and other overgrowth syndromes. Birth weight and height in other overgrowth syndromes were significantly larger than in aBWS+ILO and cBWS, with varying intergroup frequencies of clinical features. Conclusion Molecularly negative BWSp encapsulates other syndromes, and considering both WES and clinical features may facilitate accurate diagnosis.

Possible regulation of ganglioside GD3 synthase gene expression with DNA methylation in human glioma cells.

Gangliosides are expressed in nervous systems and some neuroectoderm-derived tumors at high levels and play pivotal roles. However, mechanisms for the regulation of glycosyltransferase genes responsible for the ganglioside synthesis are not well understood. In this study, we analyzed DNA methylation patterns of promoter regions of GD3 synthase (ST8SIA1) as well as mRNA levels and ganglioside expression using human glioma cell lines. Among 5 cell lines examined, 4 lines showed changes in the expression levels of related genes after treatment with 5-aza-dC. LN319 showed up-regulation of St8sia1 and increased b-series gangliosides after 5-aza-dC treatment, and an astrocytoma cell line, AS showed high expression of ST8SIA1 and b-series gangliosides persistently before and after 5-Aza-2'-deoxycytidine treatment. Using these 2 cell lines, DNA methylation patterns of the promoter regions of the gene were analyzed by bisulfite-sequencing. Consequently, 2 regions that were methylated before 5-Aza-2'-deoxycytidine treatment were demethylated in LN319 after the treatment, while those regions were persistently demethylated in AS. These 2 regions corresponded with sites defined as promoter regions by Luciferase assay. Taken together, it was suggested that ST8SIA1 gene is regulated by DNA methylation at the promoter regions, leading to the regulation of tumor phenotypes.

A novel role of helix-loop-helix transcriptional factor Bhlhe40 in osteoclast activation.

The basic helix-loop-helix transcriptional factor, Bhlhe40 has been shown as a crucial regulator of immune response, tumorigenesis, and circadian rhythms. We identified Bhlhe40 as a possible regulator of osteoclast differentiation and function by shRNA library screening and found that Bhlhe40 was required for osteoclast activation. Bhlhe40 expression was induced in bone marrow macrophages (BMMs) by RANKL, whereas the expression of its homolog Bhlhe41 was decreased in osteoclastogenesis. µCT analysis of tibias revealed that Bhlhe40 knockout (KO) mice exhibited increased bone volume phenotype. Bone morphometric analysis showed that osteoclast number and bone resorption were decreased in Bhlhe40 KO mice, whereas significant differences in the osteoblast parameters were not seen between wild-type (WT) and Bhlhe40 KO mice. In vitro culture of BMMs showed that Bhlhe40 deficiency did not cause difference in osteoclast formation. In contrast, bone resorption activity of Bhlhe40 KO osteoclasts was markedly reduced in comparison with that of WT osteoclasts. Analysis of potential target genes of Bhlhe40 using data-mining platform ChIP-Atlas (http://chip-atlas.org) revealed that predicted target genes of Bhlhe40 were related to proton transport and intracellular vesicle acidification. We then analyzed the expression of proton pump, the vacuolar (V)-ATPases which are responsible for bone resorption. The expression of V-ATPases V1c1 and V0a3 was suppressed in Bhlhe40 KO osteoclasts. In addition, Lysosensor yellow/blue DND 160 staining demonstrated that vesicular acidification was attenuated in vesicles of Bhlhe40 KO osteoclasts. Furthermore, analysis with pH-sensitive fluorescent probe showed that proton secretion was markedly suppressed in Bhlhe40 KO osteoclasts compared to that in WT osteoclasts. Our findings suggest that Bhlhe40 plays a novel important role in the regulation of acid production in osteoclastic bone resorption.

Short-term running exercise alters DNA methylation patterns in neuronal nitric oxide synthase and brain-derived neurotrophic factor genes in the mouse hippocampus and reduces anxiety-like behaviors.

Running exercise has beneficial effects on brain health. However, the effects of relatively short-term running exercise (STEx) on behavior, and its underlying signaling pathways, are poorly understood. In this study, we evaluated the possibility that the regulation by STEx of brain-derived neurotrophic factor (BDNF) and neuronal nitric oxide synthase (nNOS, encoded by NOS1), which are important molecules for anxiety regulation, might involve mechanisms of epigenetic modification, such as DNA methylation. C57BL/6J male mice were divided into sedentary (SED, n = 12) and STEx (EX, n = 15) groups; STEx was conducted with the mice for a duration of 11 days. STEx reduced anxiety-like behaviors, and STEx reduced Nos1α and increased Bdnf exon I and IV mRNA levels in the hippocampus. Interestingly, behavioral parameters were associated with Bdnf exon I and IV and Nos1α mRNA levels in the ventral, but not dorsal, hippocampal region. However, STEx had no effect on peroxisome proliferator-activated receptor-γ coactivator 1α (Pgc-1α) or fibronectin type III domain-containing 5 (Fndc5) mRNA levels, which are relatively long-term exercise-induced upstream regulators of BDNF. In parallel with gene expression changes, we found, for the first time, that STEx downregulated Bdnf promoter IV and upregulated Nos1 DNA methylation levels in the hippocampus, and these patterns were partially different between the dorsal and ventral regions. These findings suggest that the beneficial effects of running exercise on mood regulation may be controlled by alterations in epigenetic mechanisms, especially in the ventral hippocampus. These effects occur even after a relatively short-term period of exercise.

Hypomethylation of a centromeric block of ICR1 is sufficient to cause Silver-Russell syndrome.

Silver-Russell syndrome (SRS) is a representative imprinting disorder. A major cause is the loss of methylation (LOM) of imprinting control region 1 (ICR1) within the IGF2/H19 domain. ICR1 is a gametic differentially methylated region (DMR) consisting of two repeat blocks, with each block including three CTCF target sites (CTSs). ICR1-LOM on the paternal allele allows CTCF to bind to CTSs, resulting in IGF2 repression on the paternal allele and biallelic expression of H19 We analysed 10 differentially methylated sites (DMSs) (ie, seven CTSs and three somatic DMRs within the IGF2/H19 domain, including two IGF2-DMRs and the H19-promoter) in five SRS patients with ICR1-LOM. Four patients showed consistent hypomethylation at all DMSs; however, one exhibited a peculiar LOM pattern, showing LOM at the centromeric region of the IGF2/H19 domain but normal methylation at the telomeric region. This raised important points: there may be a separate regulation of DNA methylation for the two repeat blocks within ICR1; there is independent control of somatic DMRs under each repeat block; sufficient IGF2 repression to cause SRS phenotypes occurs by LOM only in the centromeric block; and the need for simultaneous methylation analysis of several DMSs in both blocks for a correct molecular diagnosis.

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.

Mbf1 ensures Polycomb silencing by protecting E(z) mRNA from degradation by Pacman.

Under stress conditions, the coactivator Multiprotein bridging factor 1 (Mbf1) translocates from the cytoplasm into the nucleus to induce stress-response genes. However, its role in the cytoplasm, where it is mainly located, has remained elusive. Here, we show that Drosophila Mbf1 associates with E(z) mRNA and protects it from degradation by the exoribonuclease Pacman (Pcm), thereby ensuring Polycomb silencing. In genetic studies, loss of mbf1 function enhanced a Polycomb phenotype in Polycomb group mutants, and was accompanied by a significant reduction in E(z) mRNA expression. Furthermore, a pcm mutation suppressed the Polycomb phenotype and restored the expression level of E(z) mRNA, while pcm overexpression exhibited the Polycomb phenotype in the mbf1 mutant but not in the wild-type background. In vitro, Mbf1 protected E(z) RNA from Pcm activity. Our results suggest that Mbf1 buffers fluctuations in Pcm activity to maintain an E(z) mRNA expression level sufficient for Polycomb silencing.

DNA methylation analysis of multiple imprinted DMRs in Sotos syndrome reveals IGF2-DMR0 as a DNA methylation-dependent, P0 promoter-specific enhancer.

Haploinsufficiency of NSD1, which dimethylates histone H3 lysine 36 (H3K36), causes Sotos syndrome (SoS), an overgrowth syndrome. DNMT3A and DNMT3B recognizes H3K36 trimethylation (H3K36me3) through PWWP domain to exert de novo DNA methyltransferase activity and establish imprinted differentially methylated regions (DMRs). Since decrease of H3K36me3 and genome-wide DNA hypomethylation in SoS were observed, hypomethylation of imprinted DMRs in SoS was suggested. We explored DNA methylation status of 28 imprinted DMRs in 31 SoS patients with NSD1 defect and found that hypomethylation of IGF2-DMR0 and IG-DMR in a substantial proportion of SoS patients. Luciferase assay revealed that IGF2-DMR0 enhanced transcription from the IGF2 P0 promoter but not the P3 and P4 promoters. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) revealed active enhancer histone modifications at IGF2-DMR0, with high enrichment of H3K4me1 and H3 lysine 27 acetylation (H3K27ac). CRISPR-Cas9 epigenome editing revealed that specifically induced hypomethylation at IGF2-DMR0 increased transcription from the P0 promoter but not the P3 and P4 promoters. NSD1 knockdown suggested that NSD1 targeted IGF2-DMR0; however, IGF2-DMR0 DNA methylation and IGF2 expression were unaltered. This study could elucidate the function of IGF2-DMR0 as a DNA methylation dependent, P0 promoter-specific enhancer. NSD1 may play a role in the establishment or maintenance of IGF2-DMR0 methylation during the postimplantation period.

Phenotypically concordant but epigenetically discordant monozygotic dichorionic diamniotic twins with Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder caused by (epi)genetic alterations. The incidence of monozygotic (MZ) twins in BWS is higher than in the general population. Most MZ twins with BWS are female and have phenotypical discordance: one twin is clinically diagnosed with BWS, while the other shows a mild or normal phenotype. The most frequent (epi)genetic alteration in MZ twins is loss of methylation of imprinting control region 2 (ICR2-LOM) at 11p15.5. Intriguingly, ICR2-LOM is usually found in the peripheral blood leukocytes (PBL) of both twins, even if they are clinically discordant. Here, we present a rare pair of MZ dichorionic diamniotic female twins with BWS and concordant phenotypes (a Beckwith-Wiedemann spectrum score of 5 in each twin). Molecular analysis of genomic DNA from PBL revealed ICR2-LOM in one twin but not the other. Our analyses suggest that ICR2-LOM occurred between days 1 and 3 after fertilization, followed by twinning. We speculate that during embryogenesis, ICR2-LOM cells were distributed to the hematopoietic stem cells in different ratios in the two fetuses, and also to commonly affected tissues, such as the tongue, in similar ratios, although we were unable to analyze any tissues other than PBL.

Clinical manifestations of placental mesenchymal dysplasia in Japan: A multicenter case series.

AIM: This study aimed to evaluate the clinical features and pregnancy outcomes of placental mesenchymal dysplasia (PMD) in Japan. METHODS: We requested detailed clinical information and placental tissue of PMD cases in 2000-2018 from Japanese facilities with departments of obstetrics and gynecology and analyzed the pregnancy course and neonatal outcomes. RESULTS: We collected 49 cases of PMD. Of 18 patients with measured maternal serum alpha-fetoprotein (MSAFP) levels, 15 (83.3%) had elevated levels. Maternal serum human chorionic gonadotropin (MShCG) levels were transiently elevated in five (17.8%) of 28 patients. Forty-seven patients continued their pregnancies. All pregnancies were singleton and 40 (85.1%) were associated with adverse events including fetal growth restriction (FGR), threatened premature delivery, fetal demise, and hypertensive disorder of pregnancy in 34 (72.3%), 14 (29.8%), eight (17.0%), and six (12.8%) patients, respectively. Of 47 infants, there were eight stillbirths. There were 40 (85.1%) female infants, and eight (17.0%) had Beckwith-Wiedemann syndrome. Of 39 live births, 23 (59.0%) were associated with premature induction of labor or cesarean section for obstetric indications related to FGR. Eighteen (46.2%) neonates had complications. PMD-affected placentas were pathologically heterogeneous in both grossly PMD-affected and non-affected areas. CONCLUSIONS: Our study included the largest number of PMD cases with detailed clinical information. PMD is a high-risk condition for both the mother and the child. Elevated MSAFP levels with normal MShCG levels indicate PMD. Conventional perinatal management of FGR in Japan might be effective in reducing the fetal mortality rate.

One week, but not 12 hours, of cast immobilization alters promotor DNA methylation patterns in the nNOS gene in mouse skeletal muscle.

KEY POINTS: DNA methylation may play an important role in regulating gene expression in skeletal muscle to adapt to physical activity and inactivity. Neuronal nitric oxide synthase (nNOS) in skeletal muscle is a key regulator of skeletal muscle mass; however, it is unclear whether nNOS expression is regulated by DNA methylation. We found that 1 week of cast immobilization increased nNOS DNA methylation levels and downregulated nNOS gene expression in atrophic slow-twitch soleus muscle from the mouse leg. These changes were not detected in non-atrophic fast-twitch extensor digitorum longus muscle. Twelve hours of cast immobilization decreased nNOS gene expression, whereas nNOS DNA methylation levels were unchanged, suggesting that downregulation of nNOS gene expression by short-term muscle inactivity is independent of the DNA methylation pattern. These findings contribute to a better understanding of the maintenance of skeletal muscle mass and prevention of muscle atrophy by epigenetic mechanisms via the nNOS/NO pathway. ABSTRACT: DNA methylation is a mechanism that controls gene expression in skeletal muscle under various environmental stimuli, such as physical activity and inactivity. Neuronal nitric oxide synthase (nNOS) regulates muscle atrophy in skeletal muscle. However, the mechanisms regulating nNOS expression in atrophic muscle remain unclear. We hypothesized that nNOS expression in atrophic muscle is regulated by DNA methylation of the nNOS promotor in soleus (Sol; slow-twitch fibre dominant) and extensor digitorum longus (EDL; fast-twitch fibre dominant) muscles. One week of cast immobilization induced significant muscle atrophy in Sol but not in EDL. We showed that 1 week of cast immobilization increased nNOS DNA methylation levels in Sol, although only a minor change was detected in EDL. Consistent with the increased DNA methylation levels in atrophic Sol, the gene expression levels of total nNOS and nNOSµ (i.e. the major splicing variant of nNOS in skeletal muscle) decreased. The abundance of the nNOS protein and cell membrane (especially type IIa fibre) immunoreactivity also decreased in atrophic Sol. These changes were not observed in EDL after 1 week of cast immobilization. Furthermore, despite the lack of significant atrophy, 12 h of cast immobilization decreased gene expression levels of total nNOS and nNOSµ in Sol. However, no association was detected between nNOS DNA methylation and gene expression. The expression of the nNOSß gene, another splicing variant of nNOS, in EDL was unchanged by cast immobilization, whereas its expression was not detected in Sol. We concluded that chronic adaptation of nNOS gene expression in cast immobilized muscle may involve nNOS DNA methylation.

The extent of DNA methylation anticipation due to a genetic defect in ICR1 in Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is a representative imprinting disorder. Gain of methylation at imprinting control region 1 (ICR1-GOM), leading to the biallelic expression of IGF2 and silencing of H19, is one of the causative alterations in BWS. Twenty percent of BWS patients with ICR1-GOM have genetic defects in ICR1. Evidence of methylation anticipation in familial BWS patients with ICR1 genetic defects has been reported. However, the precise methylation pattern and extent of anticipation in these patients remain elusive. In addition, although age-related IGF2-DMR0 hypomethylation has been reported in the normal population, the period of its occurrence is unknown. In this study, we analyzed 10 sites (IGF2-DMR0, IGF2-DMR2, CTCF binding sites 1-7, and the H19 promoter) within the IGF2/H19 domain in familial BWS patients harboring a pathogenic variant in ICR1. We found that sites near the variant had relatively higher methylation in the first affected generation and observed methylation anticipation through maternal transmission in the next generation. The extent of anticipation was greater at sites far from the variant than nearby sites. The extended and severe GOM might be due to the insufficient erasure/demethylation of pre-acquired ICR1-GOM in primordial germ cells or during the preimplantation stage. In the normal population, age-related IGF2-DMR0 hypomethylation occurred; it became established by young adulthood and continued to old age. Further studies are needed to clarify (1) the precise mechanism of anticipation in patients with familial BWS and (2) the mechanism and biological significance of constitutive hypomethylation of IGF2-DMR0 and/or other imprinted differentially methylated regions.

Habitual Light-intensity Physical Activity and ASC Methylation in a Middle-aged Population.

Apoptosis-associated, speck-like protein containing a caspase recruitment domain (ASC) plays an important role in inflammatory cytokine synthesis in peripheral blood mononuclear cells (PBMCs), and the expression of ASC is suppressed by increased methylation of its CpG sites. The current study investigated the longitudinal association of replacing sedentary time with light-intensity physical activity (LPA) or moderate to vigorous-intensity physical activity (MVPA) on the ASC methylation in middle-aged people. We investigated 1 238 individuals who participated in baseline and 5-year follow-up surveys of a population-based cohort study. Sedentary, LPA and MVPA time were objectively measured using accelerometers. ASC methylation in PBMCs was measured by pyrosequencing. Using a multiple linear regression and employing an isotemporal substitution model, the longitudinal associations of changes in the sedentary time, LPA and MVPA on the changes in the ASC methylation were analyzed after adjusting for potential confounders. Substituting 60 min per day of LPA for sedentary time was associated with 1.17 times (95% confidence interval 1.07, 1.27) higher ASC methylation levels (mean of 7 CpG sites, P<0.001). However, such effects were not seen for MVPA. These results suggest that substituting LPA for sedentary time may be linked with increased (favorable) ASC methylation as a potential biomarker of systemic inflammation.

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.

CTCF deletion syndrome: clinical features and epigenetic delineation.

BACKGROUND: Heterozygous mutations in CTCF have been reported in patients with distinct clinical features including intellectual disability. However, the precise pathomechanism underlying the phenotype remains to be uncovered, partly because of the diverse function of CTCF. Here we describe extensive clinical and genetic investigation for two patients with a microdeletion encompassing CTCF. METHODS: We performed genetic examination including comprehensive investigation of X chromosome inactivation and DNA methylation profiling at imprinted loci and genome-wide. RESULTS: Two patients showed comparable clinical features to those in a previous report, indicating that haploinsufficiency of CTCF was the major determinant of the microdeletion syndrome. Despite the haploinsufficiency of CTCF, X chromosome inactivation was normal. DNA methylation at imprinted loci was normal, but hypermethylation at CTCF binding sites was demonstrated, of which PRKCZ and FGFR2 were identified as candidate genes. CONCLUSIONS: This study confirms that haploinsufficiency of CTCF causes distinct clinical features, and that a microdeletion encompassing CTCF could cause a recognisable CTCF deletion syndrome. Perturbed DNA methylation at CTCF binding sites, not at imprinted loci, may underlie the pathomechanism of the syndrome.

Mutations in genes encoding polycomb repressive complex 2 subunits cause Weaver syndrome.

Weaver syndrome (WS) is a rare congenital overgrowth disorder caused by heterozygous mutations in EZH2 (enhancer of zeste homolog 2) or EED (embryonic ectoderm development). EZH2 and EED are core components of the polycomb repressive complex 2 (PRC2), which possesses histone methyltransferase activity and catalyzes trimethylation of histone H3 at lysine 27. Here, we analyzed eight probands with clinically suspected WS by whole-exome sequencing and identified three mutations: a 25.4-kb deletion partially involving EZH2 and CUL1 (individual 1), a missense mutation (c.707G>C, p.Arg236Thr) in EED (individual 2), and a missense mutation (c.1829A>T, p.Glu610Val) in SUZ12 (suppressor of zeste 12 homolog) (individual 3) inherited from her father (individual 4) with a mosaic mutation. SUZ12 is another component of PRC2 and germline mutations in SUZ12 have not been previously reported in humans. In vitro functional analyses demonstrated that the identified EED and SUZ12 missense mutations cause decreased trimethylation of lysine 27 of histone H3. These data indicate that loss-of-function mutations of PRC2 components are an important cause of WS.

Genome-wide parent-of-origin DNA methylation analysis reveals the intricacies of human imprinting and suggests a germline methylation-independent mechanism of establishment.

Differential methylation between the two alleles of a gene has been observed in imprinted regions, where the methylation of one allele occurs on a parent-of-origin basis, the inactive X-chromosome in females, and at those loci whose methylation is driven by genetic variants. We have extensively characterized imprinted methylation in a substantial range of normal human tissues, reciprocal genome-wide uniparental disomies, and hydatidiform moles, using a combination of whole-genome bisulfite sequencing and high-density methylation microarrays. This approach allowed us to define methylation profiles at known imprinted domains at base-pair resolution, as well as to identify 21 novel loci harboring parent-of-origin methylation, 15 of which are restricted to the placenta. We observe that the extent of imprinted differentially methylated regions (DMRs) is extremely similar between tissues, with the exception of the placenta. This extra-embryonic tissue often adopts a different methylation profile compared to somatic tissues. Further, we profiled all imprinted DMRs in sperm and embryonic stem cells derived from parthenogenetically activated oocytes, individual blastomeres, and blastocysts, in order to identify primary DMRs and reveal the extent of reprogramming during preimplantation development. Intriguingly, we find that in contrast to ubiquitous imprints, the majority of placenta-specific imprinted DMRs are unmethylated in sperm and all human embryonic stem cells. Therefore, placental-specific imprinting provides evidence for an inheritable epigenetic state that is independent of DNA methylation and the existence of a novel imprinting mechanism at these loci.

Long term survival of a patient with Perlman syndrome due to novel compound heterozygous missense mutations in RNB domain of DIS3L2.

Perlman syndrome is a rare overgrowth syndrome characterized by polyhydramnios, macrosomia, distinctive facial appearance, renal dysplasia, and a predisposition to Wilms' tumor. The syndrome is often associated with a high neonatal mortality rate and there are few reports of long-term survivors. We studied a 6-year-old Japanese female patient, who was diagnosed with Perlman syndrome, with novel compound heterozygous mutations in DIS3L2 (c.[367-2A > G];[1328T > A]), who has survived long term. Most reported DIS3L2 mutations have been the homozygous deletion of exon 6 or exon 9, and these mutations would certainly have caused the loss of both RNA binding and degradation activity. We have identified new compound heterozygous mutations in the DIS3L2 of this long-term survivor of Perlman syndrome. The reason our patient has survived long-term would be a missense mutation (c.1328 T > A, p.Met443Lys) having retained RNA binding in both the cold-shock domains and the S1 domain, and through partial RNA degradation. If partial exonuclease functions remain in at least one allele, long-term survival may be possible. Further studies of Perlman syndrome patients with proven DIS3L2 mutations are needed to clarify genotype-phenotype correlation.

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.