Robert W. Rapkins

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.

Intravitreal Triamcinolone Acetonide Inhibits Breakdown of the Blood-Retinal Barrier through Differential Regulation of VEGF-A and its Receptors in Early Diabetic Rat Retinas

OBJECTIVE To elucidate the mechanism of the unique beneficial effect of intravitreal steroid therapy on diabetic macular edema, we investigated the effect of locally administered triamcinolone acetonide (TA) on the expression of vascular endothelial growth factor (VEGF)-A and its receptors in retinas of rats with streptozotocin (STZ)-induced diabetes. We then correlated the expression of these proteins with breakdown of the blood-retinal barrier (BRB). RESEARCH DESIGN AND METHODS Thirty-two eyes of 16 diabetic and nondiabetic rats were divided into four groups. TA was injected into the vitreous of the right eye, and saline was injected into the left eye (control) 3.5 weeks after induction of diabetes. Retinas were harvested 48 h following treatment. mRNA and protein expression of VEGF-A, VEGF-A receptor 1 (fms-like tyrosine kinase [FLT]-1), and VEGF-A receptor 2 (fetal liver kinase [FLK]-1) were determined by real-time RT-PCR and immunohistochemistry. BRB permeability was quantitated by measuring extravasated endogenous albumin and retinal thickness. RESULTS Diabetes-induced retinal thickness and albumin extravasation were significantly reduced in TA-treated diabetic retinas to a level similar to that in sham-treated nondiabetic eyes. A close correlation between albumin leakage and increased expression of both Vegf-a and Flk-1 was noted in the diabetic retinas. TA downregulated the expression of Vegf-a and Flk-1 but upregulated the expression of Flt-1. TA did not alter the expression of these genes in nondiabetic retinas. CONCLUSIONS Intravitreal injection of TA stabilizes the BRB in association with regulation of Vegf-a, Flk-1, and Flt-1 expression in retinas in the early stages of diabetes.

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.

Characterizing the chromosomes of the Australian model marsupial Macropus eugenii (tammar wallaby)

Marsupials occupy a phylogenetic middle ground that is very valuable in genome comparisons of mammal and other vertebrate species. For this reason, whole genome sequencing is being undertaken for two distantly related marsupial species, including the model kangaroo species Macropus eugenii (the tammar wallaby). As a first step towards the molecular characterization of the tammar genome, we present a detailed description of the tammar karyotype, report the development of a set of molecular anchor markers and summarize the comparative mapping data for this species.

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

The T genotype of the MGMT C>T (rs16906252) enhancer single-nucleotide polymorphism (SNP) is associated with promoter methylation and longer survival in glioblastoma patients

Clinical studies in patients with newly diagnosed glioblastoma treated with temozolomide have shown that the methylation status of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is both predictive and prognostic of outcome. Methylation of the promoter region of MGMT is the most clinically relevant measure of MGMT expression and its assessment has become integral in current and planned clinical trials in glioblastoma. Our study confirmed that MGMT methylation, assessed by pyrosequencing, is associated with a significant survival benefit in glioblastoma patients treated with temozolomide (either concurrently with radiotherapy or sequential treatment). More interestingly, our study demonstrated that a promoter variant, the c.-56C>T (rs16906252) single nucleotide polymorphism (SNP) located within a cis-acting enhancer element at the proximal end of MGMT, is associated with the presence of MGMT promoter methylation in de novo glioblastoma. Furthermore, we show that the overall survival of patients carrying both the SNP and MGMT methylation showed a strong survival benefit when compared to either molecular event on their own. Promoter reporter experiments in MGMT methylated glioblastoma cell lines showed the T allele conferred a ∼30% reduction in normalised MGMT promoter activity compared to the wild-type haplotype. This might account for the propensity of the T allele to undergo promoter methylation, and in turn, the improved survival observed in carriers of the T allele. An independent validation on larger cohorts is required to confirm the prognostic and predictive value of individuals carrying the T allele.

The MLH1 c.-27C>A and c.85G>T variants are linked to dominantly inherited MLH1 epimutation and are borne on a European ancestral haplotype.

Germline mutations of the DNA mismatch repair genes MLH1, MSH2, MSH6 or PMS2, and deletions affecting the EPCAM gene adjacent to MSH2, underlie Lynch syndrome by predisposing to early-onset colorectal, endometrial and other cancers. An alternative but rare cause of Lynch syndrome is constitutional epimutation of MLH1, whereby promoter methylation and transcriptional silencing of one allele occurs throughout normal tissues. A dominantly transmitted constitutional MLH1 epimutation has been linked to an MLH1 haplotype bearing two single-nucleotide variants, NM_000249.2: c.−27C>A and c.85G>T, in a Caucasian family with Lynch syndrome from Western Australia. Subsequently, a second seemingly unrelated Caucasian Australian case with the same MLH1 haplotype and concomitant epimutation was reported. We now describe three additional, ostensibly unrelated, cancer-affected families of European heritage with this MLH1 haplotype in association with constitutional epimutation, bringing the number of index cases reported to five. Array-based genotyping in four of these families revealed shared haplotypes between individual families that extended across ≤2.6–≤6.4 megabase regions of chromosome 3p, indicating common ancestry. A minimal ≤2.6 megabase founder haplotype common to all four families was identified, which encompassed MLH1 and additional flanking genes and segregated with the MLH1 epimutation in each family. Our findings indicate that the MLH1 c.−27C>A and c.85G>T variants are borne on a European ancestral haplotype and provide conclusive evidence for its pathogenicity via a mechanism of epigenetic silencing of MLH1 within normal tissues. Additional descendants bearing this founder haplotype may exist who are also at high risk of developing Lynch syndrome-related cancers.

The MGMT promoter SNP rs16906252 is a risk factor for MGMT methylation in glioblastoma and is predictive of response to temozolomide

BACKGROUND Promoter methylation of O(6)-methylguanine-DNA methyltransferase (MGMT) is an important predictive biomarker in glioblastoma. The T variant of the MGMT promoter-enhancer single nucleotide polymorphism (SNP; rs16906252) has been associated with the presence of MGMT promoter methylation in other cancers. We examined the association of the T allele of rs16906252 with glioblastoma development, tumor MGMT methylation, MGMT protein expression, and survival outcomes. METHODS Two independent temozolomide-treated glioblastoma cohorts-one Australian (Australian Genomics and Clinical Outcomes of Glioma, n = 163) and the other American (University of California Los Angeles/Kaiser Permanente Los Angeles, n = 159)-were studied. Allelic bisulphite sequencing was used to determine if methylation was specific to the T allele. Additionally, we compared the incidence of the T allele between glioblastoma cases and matched controls to assess whether it was a risk factor for developing MGMT methylated glioblastoma. RESULTS Carriage of the T allele of the rs16906252 SNP was associated with both MGMT methylation and low MGMT protein expression and predicted significantly longer survival in temozolomide-treated patients with both MGMT methylated and nonmethylated glioblastoma. Methylation was linked to the T allele, inferring that the T variant plays a key role in the acquisition of MGMT methylation. Carriage of the T allele was associated with a significantly elevated risk of developing glioblastoma (adjusted odds ratio, 1.96; P = .013), increasing further when glioblastoma was classified by the presence of MGMT methylation (adjusted odds ratio, 2.86; P = .001). CONCLUSIONS The T allele of the rs16906252 SNP is a key determinant in the acquisition of MGMT methylation in glioblastoma. Temozolomide-treated patients with the rs16906252 T genotype have better survival, irrespective of tumor methylation status.

SNP rs16906252C > T Is an Expression and Methylation Quantitative Trait Locus Associated with an Increased Risk of Developing MGMT-Methylated Colorectal Cancer

Purpose: 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. Experimental Design: 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. Results: 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 < 0.0001], which was replicated in the ACCFR sample (OR, 4.0; 95% CI, 2.4–6.8; P < 0.0001). The T allele demonstrated about 2.5-fold reduced transcription in normal colorectal mucosa from cases and controls and was selectively methylated in a minority of normal cells, indicating that rs16906252C>T represents an expression and methylation quantitative trait locus. Conclusions: 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.

Functional analysis of a germline promoter variant to determine its role in epigenetic silencing of MLH1 in a Lynch syndrome family

Resumen de la comunicacion presentada en 4th Biennial Meeting : International Society for Gastrointestinal Hereditary Tumours San Antonio, Texas, March 31-April 2, 2011.4th Biennial Meeting: International Society for Gastrointestinal Hereditary Tumours San Antonio, Texas, March 31–April 2, 2011 Springer Science+Business Media B.V. 2011 1 Basic Science Session I, Thursday, March 31, 2011,