Recent Assembly of an Imprinted Domain from Non-Imprinted Components

Genomic imprinting, representing parent-specific expression of alleles at a locus, raises many questions about how—and especially why—epigenetic silencing of mammalian genes evolved. We present the first in-depth study of how a human imprinted domain evolved, analyzing a domain containing several imprinted genes that are involved in human disease. Using comparisons of orthologous genes in humans, marsupials, and the platypus, we discovered that the Prader-Willi/Angelman syndrome region on human Chromosome 15q was assembled only recently (105–180 million years ago). This imprinted domain arose after a region bearing UBE3A (Angelman syndrome) fused with an unlinked region bearing SNRPN (Prader-Willi syndrome), which had duplicated from the non-imprinted SNRPB/B′. This region independently acquired several retroposed gene copies and arrays of small nucleolar RNAs from different parts of the genome. In their original configurations, SNRPN and UBE3A are expressed from both alleles, implying that acquisition of imprinting occurred after their rearrangement and required the evolution of a control locus. Thus, the evolution of imprinting in viviparous mammals is ongoing

Veliparib in combination with radiotherapy for the treatment of MGMT unmethylated glioblastoma

BackgroundThe O 6 -methylguanine methyltransferase (MGMT) gene is frequently unmethylated in patients with glioblastoma (GBM), rendering them non-responsive to the standard treatment regime of surgery followed by concurrent radiotherapy (RT) and temozolomide. Here, we investigate the efficacy of adding a PARP inhibitor, veliparib, to radiotherapy to treat MGMT unmethylated GBM.MethodsThe inhibition of PARP with veliparib (ABT-888), a potent and orally bioavailable inhibitor in combination with RT was tested on a panel of patient derived cell lines (PDCLs) and patient-derived xenografts (PDX) models generated from GBM patients with MGMT unmethylated tumors.ResultsThe combination of veliparib and RT inhibited colony formation in the majority of PDCLs tested. The PDCL, RN1 showed significantly reduced levels of the homologous repair protein, Mre11 and a heightened response to PARP inhibition measured by increased apoptosis and decreased colony formation. The oral administration of veliparib (12.5 mg/kg, twice daily for 5 days in a 28-day treatment cycle) in combination with whole brain RT (4 Gy) induced apoptosis (Tunel staining) and decreased cell proliferation (Ki67 staining) in a PDX of MGMT unmethylated GBM. Significantly longer survival times of the PDX treated with the combination treatment were recorded compared to RT only or veliparib only.ConclusionsOur results demonstrate preclinical efficacy of targeting PARP at multiple levels and provide a new approach for the treatment of MGMT unmethylated GBM.<br /

Identification of constitutional MLH1 epimutations and promoter variants in colorectal cancer patients from the colon cancer family registry

Purpose:Constitutional MLH1 epimutations manifest as promoter methylation and silencing of the affected allele in normal tissues, predisposing to Lynch syndrome-Associated cancers. This study investigated their frequency and inheritance.Methods:A total of 416 individuals with a colorectal cancer showing loss of MLH1 expression and without deleterious germline mutations in MLH1 were ascertained from the Colon Cancer Family Registry (C-CFR). Constitutive DNA samples were screened for MLH1 methylation in all 416 subjects and for promoter sequence changes in 357 individuals.Results:Constitutional MLH1 epimutations were identified in 16 subjects. Of these, seven (1.7%) had mono-or hemi-Allelic methylation and eight had low-level methylation (2%). In one subject the epimutation was linked to the c.-27C>A promoter variant. Testing of 37 relatives from nine probands revealed paternal transmission of low-level methylation segregating with a c.+27G>A variant in one case. Five additional probands had a promoter variant without an MLH1 epimutation, with three showing diminished promoter activity in functional assays.Conclusion:Although rare, sequence changes in the regulatory region of MLH1 and aberrant methylation may alone or together predispose to the development of cancer. Screening for these changes is warranted in individuals who have a negative germline sequence screen of MLH1 and loss of MLH1 expression in their tumor.Genet Med 2013:15(1):25-35

Dominantly inherited constitutional epigenetic silencing of MLH1 in a cancer-affected family is linked to a single nucleotide variant within the 5'UTR

Constitutional epimutations of tumor suppressor genes manifest as promoter methylation and transcriptional silencing of a single allele in normal somatic tissues, thereby predisposing to cancer. Constitutional MLH1 epimutations occur in individuals with young-onset cancer and demonstrate non-Mendelian inheritance through their reversal in the germline. We report a cancer-affected family showing dominant transmission of soma-wide highly mosaic MLH1 methylation and transcriptional repression linked to a particular genetic haplotype. The epimutation was erased in spermatozoa but reinstated in the somatic cells of the next generation. The affected haplotype harbored two single nucleotide substitutions in tandem; c.-27C > A located near the transcription initiation site and c.85G > T. The c.-27C > A variant significantly reduced transcriptional activity in reporter assays and is the probable cause of this epimutation

Assembly of the PWS-AS Imprinted Region in Placental Mammals during Vertebrate Evolution

<p>Relationships between fish, birds/reptiles, and the three mammal groups are presented as a phylogeny (top). In the ancestral arrangement, shared by marsupials, monotremes, birds, and fish, the block of imprinted human 15q genes (pink) is flanked by human X (green) and human 2 (purple) blocks. These three blocks were separated by at least two fissions and were rearranged next to an unlinked block of genes (on chick Chromosome 10, pale blue) to make up the present regions of human Chromosome 15q (and with two more inversions, of mouse Chromosome 7). <i>SNRPB′</i> (brown) is present on a different chromosome in fish, birds, and marsupials, but its duplicate <i>SNRPN</i> (orange) is transposed next to <i>UBE3A</i> in placentals. Other human 15q genes absent from non-placental vertebrates (orange) were independently added to the imprinted region in the placental lineage.</p

Biallelic Expression Demonstrated by Sequencing Brain cDNA from Heterozygous Animals for Alleles of <i>SNRPN</i> and <i>UBE3A</i> in Tammar Wallaby and <i>UBE3A</i> in Platypus

<p>Alleles differ at an A/T polymorphism at base pair 67 of the 3′ UTR of tammar <i>SNRPN,</i> a C/T polymorphism at base pair 247 of exon 5 in tammar <i>UBE3A,</i> and an insertion polymorphism of a C at base pair 179 of the 3′ UTR in platypus <i>UBE3A</i> (marked by boxes).</p

FISH Localization of BACs Containing Tammar Homologs of PWS-AS Genes

<p><i>UBE3A</i>/<i>CNGA3, GABRB3,</i> and <i>HERC2</i> localize to tammar wallaby Chromosome 5p and <i>SNRPN</i>/<i>SNRPB</i> to Chromosome 1q.</p

SNP rs16906252C>T is an expression and methylation quantitative trait locus associated with an increased risk of developing MGMT-methylated colorectal cancer

Methylation of the MGMT promoter is the major cause of O6-methylguanine methyltransferase deficiency in cancer and has been associated with the T variant of the promoter enhancer SNP rs16906252C>T. We sought evidence for an association between the rs16906252C>T genotype and increased risk of developing a subtype of colorectal cancer featuring MGMT methylation, mediated by genotype-dependent epigenetic silencing within normal tissues.By applying a molecular pathologic epidemiology case-control study design, associations between rs16906252C>T and risk for colorectal cancer overall, and colorectal cancer stratified by MGMT methylation status, were estimated using multinomial logistic regression in two independent retrospective series of colorectal cancer cases and controls. The test sample comprised 1,054 colorectal cancer cases and 451 controls from Sydney, Australia. The validation sample comprised 612 colorectal cancer cases and 245 controls from the Australasian Colon Cancer Family Registry (ACCFR). To determine whether rs16906252C>T was linked to a constitutively altered epigenetic state, quantitative allelic expression and methylation analyses were performed in normal tissues.An association between rs16906252C>T and increased risk of developing MGMT-methylated colorectal cancer in the Sydney sample was observed [OR, 3.3; 95% confidence interval (CI), 2.0-5.3; P T represents an expression and methylation quantitative trait locus.We provide evidence that rs16906252C>T is associated with elevated risk for MGMT-methylated colorectal cancer, likely mediated by constitutive epigenetic repression of the T allele. Clin Cancer Res; 22(24); 6266-77. ©2016 AACR