Inheritance of a Cancer-Associated MLH1 Germ-Line Epimutation
Megan P. Hitchins, Ph.D., Justin J.L. Wong, B.Sc., Graeme Suthers, M.B., B.S., Catherine M. Suter, Ph.D., David I.K. Martin, M.D., Nicholas J. Hawkins, Ph.D., and Robyn L. Ward, Ph.D.
Persons who have hypermethylation of one allele of MLH1 in somaticcells throughout the body (a germ-line epimutation) have a predispositionfor the development of cancer in a pattern typical of hereditarynonpolyposis colorectal cancer. By studying the families oftwo such persons, we found evidence that the epimutation wastransmitted from a mother to her son but was erased in his spermatozoa.The affected maternal allele was inherited by three other siblingsfrom these two families, but in those offspring the allele hadreverted to the normal active state. These findings demonstratea novel pattern of inheritance of cancer susceptibility andare consistent with transgenerational epigenetic inheritance.
Hereditary nonpolyposis colorectal cancer results from germ-linesequence mutations in mismatch-repair genes, particularly MLH1and MSH2. Somatic inactivation of the remaining normal alleleby genetic or epigenetic events leads to the development ofmicrosatellite instability in tumors of the colorectum or endometriumat a young age.1 Methylation of cytosines that precede a guanosinein the DNA sequence (the CpG dinucleotide) is a key epigeneticmodification, and hypermethylation of gene promoters is associatedwith transcriptional silencing.2 In sporadic colorectal cancer,methylation of both alleles of the promoter of MLH1 occurs asa somatic event early in tumorigenesis and is seen in approximately15% of patients.3
Studies have shown that hypermethylation of MLH1 is not limitedto neoplastic cells. Rather, in some persons, hypermethylationof a single allele of MLH1 originates in the germ line and isthus widespread in normal somatic cells.4,5,6,7 This phenomenonof germ-line epimutation silences the affected allele in theabsence of intragenic sequence mutations. Persons with a germ-lineepimutation, like those with hereditary nonpolyposis colorectalcancer, have only one functional allele of the MLH1 gene fromconception, and cancers typical of the hereditary nonpolyposiscolorectal cancer syndrome have developed in all such casesdescribed to date.4,5,6,7 Colorectal and other tumors in personswith MLH1 germ-line epimutations do not express the MLH1 proteinand have microsatellite instability (the hallmark of failedmismatch-repair function), accompanied in some cases by somaticloss of the wild-type allele.4,5,6,7
Although germ-line sequence mutations are faithfully transmittedfrom one generation to the next in a mendelian pattern, epimutationsdo not involve changes in the DNA sequence and are relativelyunstable, perhaps as a result of epigenetic reprogramming, inprimordial germ cells and gametes,8 in the male genome in thezygote,9,10 and in the preimplantation embryo.11 This processremoves and resets epigenetic marks between generations. Nevertheless,nonmendelian patterns of transgenerational epigenetic inheritancehave been reported in mice.12,13,14 In the eight previouslyreported cases of germ-line MLH1 epimutation in humans, no intergenerationaltransmission was found, although in one patient the epimutationwas reported in a low proportion of spermatozoa.4
Since the presence of an MLH1 epimutation in the germ line impliesa potential for inheritance, we sought evidence for transmissionbetween generations. In this study, we identified additionalpersons with germ-line MLH1 epimutations and in one family showedmaternal transmission of the epimutation to her son but itserasure in his spermatozoa. Three other siblings from the twofamilies we studied also inherited the affected maternal alleles,but in these cases the epimutation had reverted to the normalstate, with concomitant allelic reactivation. These findingsare consistent with germ-line transmission of a silent epigeneticstate that confers disease susceptibility in humans.
Methods
Patients and Family Members
Our study was approved by the ethics committee at St. Vincent'sHospital in Sydney. Tissues were obtained with written informedconsent from probands and family members at St. Vincent's Hospitaland Women's and Children's Hospital in Adelaide, Australia.
We selected 24 patients in whom colorectal or endometrial cancerhad developed before the age of 50 years and who lacked deleteriousgerm-line sequence mutations in MSH2 or MLH1. In each case,the tumors had microsatellite instability, with complete lossof MLH1 protein expression and retention of MSH2. Additionaltissues were collected from patients shown to carry an MLH1germ-line epimutation and from their first-degree relatives.The "swim-up" procedure was used to isolate motile spermatozoa.15To remove contaminating somatic cells, spermatozoa that wereused for DNA analyses were additionally sorted by flow cytometry(FACSVantage DiVa, Becton Dickinson).16 DNA was extracted fromspermatozoa, hair follicles, and buccal mucosa with the useof QuickExtract solution (Epicentre Biotechnologies) and fromperipheral-blood leukocytes with the use of phenol–chloroform.
Methylation Analyses
Methylation of the promoter of MLH1 (A and C regions) and ofthe EPM2AIP promoter on the opposite strand was identified withthe use of combined bisulfite restriction analyses (Figure 1A)and confirmed by allelic bisulfite sequencing.7
Figure 1. Germ-Line Epimutation and Allelic Loss of Expression of MLH1 in Patients A and B.
Panel A shows a map of the CpG island encompassing the MLH1 and EPM2AIP promoters. The transcriptional start site is indicated by +1. Individual CpG doublets are depicted as circles. Horizontal bars show the regions amplified for combined bisulfite restriction analysis and bisulfite allelic sequencing. Primer positions are indicated by short arrows. Sites for restriction enzymes that exclusively cleave combined bisulfite restriction analysis amplicons from methylated templates are shown. Panel B shows bisulfite allelic sequencing of the C region of the MLH1 promoter in somatic tissues from Patient A and Patient B. Each horizontal line represents a single allele, and circles depict individual CpG dinucleotides as numbered, with black circles indicating methylated dinucleotides and white circles indicating unmethylated dinucleotides. Single-nucleotide polymorphism (SNP) rs1800734 is indicated with G in orange and A in green. Panel C shows representative sequence-based fluorescent electropherograms showing the SNP within exon 8 of MLH1 in the genomic DNA (gDNA) and messenger RNA (mRNA) from peripheral-blood leukocytes of Patient A; gDNA shows heterozygosity (arrow), whereas transcription is restricted to the G allele. Panel D is a schematic diagram of the effect of the germ-line epimutation on allelic activity of MLH1 in Patient A. Uniparental methylation of the promoter A allele (SNP rs1800734) causes allelic inactivation, so that only the allele with the G SNP at exon 8 (SNP rs1799977) is expressed (arrow).
Quantitative real-time methylation-specific polymerase-chain-reaction(PCR) assays were performed on bisulfite-treated DNA with theuse of primers specific to methylated templates for the C regionof MLH1 and the imprinted SNRPN gene.17 Primers that amplifythe control gene MyoD regardless of its methylation status wereused to normalize for DNA input. Real-time methylation-specificPCR assays with iQ SYBR Green Supermix reagent were analyzedwith the use of a real-time PCR system (MyiQ, BioRad). Absolutevalues for experimental samples were calculated from the PCRcycle number at which the fluorescence crossed the thresholdwith the use of a standard curve. The percentage of methylatedalleles in the C region of MLH1 and SNRPN was calculated againstMyoD with reference to 100% in vitro methylated human DNA (Chemicon).18All primer sequences are available on request.
Haplotyping
Single-nucleotide polymorphism (SNP) typing was performed byPCR amplification of constitutional DNA followed by restrictiondigestion or direct sequencing of the purified amplicons. Markersof sequence-tagged sites were typed by PCR amplification withthe use of fluorescent-labeled primers,19 separated by capillaryelectrophoresis on an automated DNA sequencer, and sized withthe use of LIZ markers (ABI 3700, Applied Biosystems).
Allelic Expression
For each patient or family member, heterozygous polymorphismswithin exons of MLH1 and EPM2AIP were used to identify the allelesbeing transcribed.20 RNA was extracted from peripheral-bloodleukocytes, lymphoblastoid cells, and spermatozoa with the useof Trizol reagent (Invitrogen), treated with DNaseI, and convertedto complementary DNA (cDNA). Allelic expression was determinedby sequencing cDNA at I219V SNP within exon 8 (rs1799977) andat polymorphisms within exon 16 and EPM2AIP.
Results
By studying the peripheral blood of 24 patients, we identifiedtwo unrelated women (Patient A and Patient B) who had the typicalmolecular and clinical characteristics of persons with germ-lineMLH1 epimutations — namely, multiple MLH1-negative cancersof the colorectum and endometrium and hemiallelic methylationof MLH1 in all somatic cells. In both women, there was densemethylation of one allele of the MLH1 and EPM2AIP promotersin somatic cells from the three embryonic germ layers (Figure 1B,and Fig. 1A of the Supplementary Appendix, available with thefull text of this article at www.nejm.org). Both women had metachronouscarcinomas that had microsatellite instability and lacked MLH1expression. (Patient A received a diagnosis of cancer of theendometrium at the age of 45 years, of the colon at 59 years,and of the rectum at 60 years; Patient B received a diagnosisof cancer of the colon at 41 years and of the rectum at 45 years.)Patient A was heterozygous for a SNP (rs1800734) within theMLH1 promoter, with methylation confined to the A allele. Inboth Patient A and Patient B, the methylated allele was transcriptionallysilent, as evidenced by monoallelic expression of MLH1 and EPM2AIPtranscripts in their messenger RNA (mRNA) (Figure 1C and 1D,and Fig. 1B of the Supplementary Appendix).
To identify MLH1 epimutations within families of the probands,combined bisulfite restriction analysis was performed on constitutionalDNA from nine first-degree relatives, none of whom had a historyof cancer (Figure 2B and 2C). Partial methylation of MLH1 wasfound in one of Patient A's four sons (family member II6-A)(Figure 2C). Methylation of the A allele (SNP rs1800734) onapproximately 50% of chromosomes was confirmed by bisulfitesequencing (Figure 3A). We identified an expressible CT SNPwithin MLH1 exon 16 in family member II6-A, which was used todemonstrate that he was transcribing RNA only from the MLH1allele inherited from his father (Figure 3A). These data areconsistent with transmission of the MLH1 epimutation from PatientA to her son.
Figure 2. Pedigrees of Patients A and B Showing Intergenerational Transmission of the MLH1 Epimutation and Haplotypes.
Panel A shows a map of SNPs within the MLH1 and EPM2AIP genes, used to determine inheritance patterns of the epimutant alleles and to analyze allelic expression. Panels B and C show pedigrees for Patient A and Patient B, with large black circles denoting Patient A and Patient B and with the current age of each family member given. Generations are listed I to III, and patients are identified by number. In Panels B and C, combined bisulfite restriction analysis of the C region of MLH1 (digested with MluI) is shown, with the lanes corresponding to data for family members shown in the pedigrees directly above. Combined bisulfite restriction analysis was performed on DNA extracted from peripheral-blood leukocytes from all patients except family member II7-A and family member II8-A, from whom only hair-follicle DNA was available. Lane M indicates the pUC19/MspI DNA ladder, lane C– indicates DNA extracted from peripheral-blood leukocytes from a control with an unmethylated MLH1 promoter, and lane C+ indicates DNA extracted from the biallelically methylated RKO colorectal-carcinoma cell line (ATCC). Band sizes (in base pairs) are shown at the right. At the bottom of Panels B and C, haplotypes generated from informative SNPs are listed according to the key for each pedigree. Alleles associated with the epimutation are highlighted in yellow, and the presence of methylation (Me) is indicated. Maternally inherited alleles are shown in red, and paternally derived alleles are shown in blue; black letters indicate unknown parental origin. Although the haplotypes associated with the epimutation were inherited by several children, only family member II6-A retained the epimutation.
Figure 3. Maternal Inheritance of the MLH1 Germ-Line Epimutation by Family Member II6-A and the Erasure of the Epimutation in His Spermatozoa.
Panel A shows methylation and monoallelic expression of MLH1 in the somatic tissues of a son of Patient A, family member II6-A. Bisulfite allelic sequencing of the C region of the MLH1 promoter in somatic tissues from family member II6-A appears at the top of the panel. Each horizontal line represents a single allele, and circles depict individual CpG dinucleotides, with black circles indicating methylated dinucleotides and white circles indicating unmethylated dinucleotides. family member II6-A is homozygous for the rs1800734 SNP (indicated by green boxes). Sequence electropherograms show the exon 16 CT SNP (arrow). The SNP is at nucleotide position 1890 of the MLH1 messenger RNA sequence (GenBank accession number NM000249) and does not confer any amino acid change. Family member II6-A and his father are heterozygous for this SNP. Family member II6-A expresses MLH1 from only the paternally derived (T) allele. Panel B shows erasure of the germ-line MLH1 epimutation in spermatozoa obtained from family member II6-A. At the top of the panel, the map shows the region of the MLH1 promoter amplified by real-time methylation-specific PCR. Below the map, a graph shows the percentage of alleles methylated at either MLH1 or the differentially methylated region of the imprinted SNRPN gene, as determined by real-time methylation-specific PCR. SNRPN is an imprinted gene methylated specifically on the maternal allele; it is unmethylated in mature spermatozoa,21 providing a control for somatic contamination of the spermatozoa samples. As expected, SNRPN shows approximately 50% methylation in somatic DNA and negligible methylation in sperm DNA. In family member II6-A, MLH1 shows 42% methylation in peripheral-blood leukocytes (PBLs) but no methylation in spermatozoa. In the right portion of the panel, sequence electropherograms of the exon 16 CT SNP from DNA and RNA in spermatozoa from family member II6-A show the presence of both MLH1 alleles in the spermatozoa DNA and biallelic expression in the RNA. Spermatozoa for the RNA analysis were subjected to the "swim-up" technique only and may contain a proportion of somatic cells.
To ascertain the possibility of transmission of the MLH1 epimutationfrom family member II6-A to his offspring, we studied the levelof allelic methylation in his pure motile spermatozoa with theuse of a sensitive quantitative real-time methylation-specificPCR assay within the C region of MLH1 (Figure 1A). In DNA fromperipheral-blood leukocytes obtained from family member II6-A,approximately half of the MLH1 alleles (mean ±SD, 42.0±4.6%)were methylated. In contrast, his sperm had no trace of MLH1methylation, despite containing equal proportions of allelesderived from his mother and father (Figure 3B). Furthermore,analysis of the RNA in his sperm at the MLH1 exon 16 CT SNPshowed reactivation of the maternally derived MLH1 allele (Figure 3B).These results indicate reversion of the MLH1 epimutation tonormality during spermatogenesis, suggesting a negligible riskof transmission from family member II6-A (Figure 4).
Figure 4. Schematic Representation of Inheritance of MLH1 Alleles in Three Sons of Patient A.
The diagram depicts MLH1 alleles within the nucleus of somatic cells and sperm, with pink representing maternal origin and blue representing paternal origin. Active transcription is indicated by an arrow, and the RNA transcripts from the alleles are shown within cytoplasm. In the mother, one allele was methylated in the promoter (red band) and therefore not transcribed; only RNA from her yellow allele was expressed. In her second son (family member II6-A), the maternally inherited allele (pink, red band) was silent, and the paternal allele (pale blue) was expressed. Haploid DNA within spermatozoa from the son contained alleles from both the father and mother, and both were transcribed. There was no evidence of methylation of either allele. The first and third sons (family members II5-A and II7-A) also inherited the affected maternal allele, but in these offspring methylation had been erased and biallelic expression of MLH1 was found, indicating that the epimutation was reversed to the normal state. This model shows transgenerational inheritance of an epigenetically mutated tumor-suppressor gene and subsequent reversal of the epimutation within spermatozoa.
To investigate allelic inheritance patterns and the possibilitythat the epimutations were caused by alterations in the DNAstrand carrying the allele that could influence methylationof the allele (in cis defects), we used SNPs within MLH1,EPM2AIP,and flanking sequence-tagged site markers to construct a haplotypemap of a region of approximately 8 Mb around MLH1 for all membersof both families (Figure 2B and 2C, and Fig. 2 of the Supplementary Appendix).Patient A, her sister (family member I1-A), and three of herfour sons (family members II5-A, II6-A, and II7-A) all shareda haplotype in this region, even though MLH1 was methylatedonly in Patient A and her son (family member II6-A). In thecase of Patient B, haplotype and expression analysis (Figure 2B,and Fig. 1 of the Supplementary Appendix) confirmed that theMLH1 epimutation resided on her maternally derived allele. Thisis likely to have arisen spontaneously, since we found no evidenceof methylation in her mother (Figure 2B). Notably, the haplotypein Patient B differed from the one on which the epimutationoccurred in Patient A's family. Patient B transmitted the haplotypeto one of her two sons (family member III2-B), but there wasno evidence of MLH1 methylation in his case (Figure 2B). Forboth family member II5-A and family member III2-B, absence ofmethylation from their respective maternal alleles correlatedwith biallelic expression from the MLH1 locus (Fig. 3 of theSupplementary Appendix), indicating reversion of the epimutationin these family members or in their mothers' germ cells. Norecombinations were observed in the vicinity of MLH1, indicatingthat the affected alleles were identical in family members bearingthe epimutation and in those in whom it had reverted (Fig. 2of the Supplementary Appendix).
Epimutations are meiotically reversible and often show somaticmosaicism. We therefore considered whether some family membershad low levels of allelic MLH1 methylation, suggesting incompletesomatic erasure of an epimutation, or a susceptibility of theallele to subsequent somatic methylation. With the use of real-timemethylation-specific PCR, we found no evidence of allelic mosaicismfor MLH1 methylation in any patient in our study, except thosewith epimutations (38±9% for Patient B, 42±7%for Patient A, and 42±4.6% for family member II6-A).
Discussion
We have found evidence of germ-line epimutation of MLH1 in awoman with cancer and in her son (Figure 4), which supportsthe concept of transgenerational epigenetic inheritance. TheMLH1 epimutation that predisposed the mother to multiple tumorswith microsatellite instability has increased the risk of cancerin her son (family member II6-A).
The findings from the two families in this study, as well asprevious studies, offer insights into the pattern of inheritanceof germ-line epimutations. With reference to the parent of origin,the mother of family member II6-A, like the mothers of two patientswith MLH1 epimutations reported previously, had cancer in apattern typical of hereditary nonpolyposis colorectal cancer.6Furthermore, in two other cases in which germ-line epimutationswere shown to arise spontaneously (Patient B and a patient whosecase was reported previously7), the methylated allele was maternallyderived. Taken together, these data raise the possibility thatepigenetic errors may arise more frequently during oogenesisor are more likely to be maintained during this process. Ourfinding of erasure of the epimutation during spermatogenesisin family member II6-A is consistent with this hypothesis. However,paternal inheritance cannot be excluded, given our previousfinding of low-level MLH1 methylation in sperm of an affectedperson.6
Another characteristic of the inheritance pattern in the familiesin this study is that four sons inherited their mother's MLH1haplotype, yet in three of the sons, the maternally derivedallele had undergone demethylation and transcriptional reactivation.It appears that the normal process of gametogenesis allowedcorrection of the MLH1 epimutation, perhaps contemporaneouslywith erasure of parent-specific methylation of imprinted genesin primordial germ cells.11 If so, then transmission of an epimutationto family member II6-A must reflect resistance to reprogramming,either through incomplete erasure or by retention of an epigeneticmemory. Although at present we have limited information, theoverall cancer risk for families with germ-line MLH1 epimutationsappears to be lower than for those with germ-line sequence mutations.
An alternative explanation for our findings is that epimutationsare not inherited per se. Rather, they are erased in gametogenesisbut reestablished in successive generations because of cis-actingor even trans-acting genetic factors that increase susceptibilityto MLH1 epimutations.21,22 Examples of epigenetic silencingthat are driven by genetic events in cis include deletion ofimprint-control centers in imprinted disorders23 and expansionof triplet repeats within the FMR1 promoter in the fragile Xsyndrome.24 Such a mechanism may also explain the recently reportedstrongly heritable pattern of epimutation in MSH2,25 since themethylation state segregated faithfully with the genetic haplotype.In contrast, in the two families described in our study, wefound no evidence of a fully penetrant in cis defect. Rather,they showed epimutations that were meiotically reversible andtransmitted in a nonmendelian fashion. A simple explanationfor this pattern is that epimutations can occur on any haplotype,and although they usually are cleared in the germ line, theymay be retained at low but uncertain frequency.
Regardless of uncertainties about patterns, frequencies, andmechanisms of inheritance, offspring of patients with MLH1 epimutationsmust be regarded as being at risk for cancer until proven otherwise.The broader implication of this study is that disease statesin humans may be the consequence of nonmendelian inheritanceof epigenetic changes in one or more genes.
Supported by the National Health and Medical Research Council,the Cancer Council New South Wales, and the Justin O'ConnorFoundation. Mr. Wong is the recipient of a University of NewSouth Wales International Postgraduate Research Scholarship.
No potential conflict of interest relevant to this article wasreported.
We thank Kay Fong Cheong, Deborah Packham, Vita Ap Lin, andChau-To Kwok for their technical assistance and Rachel Williamsand Kerry Phillips for their coordination of sample collection.
Source Information
From the Department of Medical Oncology, St. Vincent's Hospital (M.P.H., J.J.L.W., C.M.S., R.L.W.); the Schools of Medicine (M.P.H., C.M.S., R.L.W.) and Medical Sciences (J.J.L.W., N.J.H.), University of New South Wales; and the Victor Chang Cardiac Research Institute (C.M.S., D.I.K.M.) — all in Sydney; the Department of Genetic Medicine, Women's and Children's Hospital, North Adelaide, Australia (G.S.); and the Children's Hospital Oakland Research Institute, Oakland, CA (D.I.K.M.).
Address reprint requests to Dr. Ward at the Department of Medical Oncology, St. Vincent's Hospital, Victoria Street, Darlinghurst, NSW 2010, Australia, or at robyn{at}unsw.edu.au.
References
Lynch HT, Lynch JF. What the physician needs to know about Lynch syndrome: an update. Oncology 2005;19:455-463. [ISI][Medline]
Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 2003;349:2042-2054. [Free Full Text]
Herman JG, Umar A, Polyak K, et al. Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci U S A 1998;95:6870-6875. [Free Full Text]
Gazzoli I, Loda M, Garber J, Syngal S, Kolodner RD. A hereditary nonpolyposis colorectal carcinoma case associated with hypermethylation of the MLH1 gene in normal tissue and loss of heterozygosity of the unmethylated allele in the resulting microsatellite instability-high tumor. Cancer Res 2002;62:3925-3928. [Free Full Text]
Miyakura Y, Sugano K, Akasu T, et al. Extensive but hemiallelic methylation of the hMLH1 promoter region in early-onset sporadic colon cancers with microsatellite instability. Clin Gastroenterol Hepatol 2004;2:147-156. [CrossRef][Medline]
Suter CM, Martin DI, Ward RL. Germline epimutation of MLH1 in individuals with multiple cancers. Nat Genet 2004;36:497-501. [CrossRef][ISI][Medline]
Hitchins M, Williams R, Cheong K, et al. MLH1 germline epimutations as a factor in hereditary nonpolyposis colorectal cancer. Gastroenterology 2005;129:1392-1399. [CrossRef][ISI][Medline]
Hajkova P, Erhardt S, Lane N, et al. Epigenetic reprogramming in mouse primordial germ cells. Mech Dev 2002;117:15-23. [CrossRef][ISI][Medline]
Mayer W, Niveleau A, Walter J, Fundele R, Haaf T. Demethylation of the zygotic paternal genome. Nature 2000;403:501-502. [Medline]
Oswald J, Engemann S, Lane N, et al. Active demethylation of the paternal genome in the mouse zygote. Curr Biol 2000;10:475-478. [CrossRef][ISI][Medline]
Morgan HD, Santos F, Green K, Dean W, Reik W. Epigenetic reprogramming in mammals. Hum Mol Genet 2005;14:R47-R58. [Free Full Text]
Morgan HD, Sutherland HG, Martin DI, Whitelaw E. Epigenetic inheritance at the agouti locus in the mouse. Nat Genet 1999;23:314-318. [CrossRef][ISI][Medline]
Rakyan VK, Chong S, Champ ME, et al. Transgenerational inheritance of epigenetic states at the murine Axin(Fu) allele occurs after maternal and paternal transmission. Proc Natl Acad Sci U S A 2003;100:2538-2543. [Free Full Text]
Roemer I, Reik W, Dean W, Klose J. Epigenetic inheritance in the mouse. Curr Biol 1997;7:277-280. [CrossRef][ISI][Medline]
Morales P, Vantman D, Barros C, Vigil P. Human spermatozoa selected by Percoll gradient or swim-up are equally capable of binding to the human zona pellucida and undergoing the acrosome reaction. Hum Reprod 1991;6:401-404. [Free Full Text]
Schoell WM, Klintschar M, Mirhashemi R, Pertl B. Separation of sperm and vaginal cells with flow cytometry for DNA typing after sexual assault. Obstet Gynecol 1999;94:623-627. [Free Full Text]
Kubota T, Das S, Christian SL, Baylin SB, Herman JG, Ledbetter DH. Methylation-specific PCR simplifies imprinting analysis. Nat Genet 1997;16:16-17. [Medline]
Trinh BN, Long TI, Laird PW. DNA methylation analysis by MethyLight technology. Methods 2001;25:456-462. [CrossRef][ISI][Medline]
Hegde M, Blazo M, Chong B, Prior T, Richards C. Assay validation for identification of hereditary nonpolyposis colon cancer-causing mutations in mismatch repair genes MLH1, MSH2, and MSH6. J Mol Diagn 2005;7:525-534. [Free Full Text]
El-Maarri O, Buiting K, Peery EG, et al. Maternal methylation imprints on human chromosome 15 are established during or after fertilization. Nat Genet 2001;27:341-344. [CrossRef][ISI][Medline]
Blewitt M, Vickaryous N, Paldi A, Koseki H, Whitelaw E. Dynamic reprogramming of DNA methylation at an epigenetically sensitive allele in mice. PLoS Genet 2006;2:e49-e49. [CrossRef][Medline]
Dittrich B, Buiting K, Korn B, et al. Imprint switching on human chromosome 15 may involve alternative transcripts of the SNRPN gene. Nat Genet 1996;14:163-170. [CrossRef][ISI][Medline]
Stoger R, Kajimura TM, Brown WT, Laird CD. Epigenetic variation illustrated by DNA methylation patterns of the fragile-X gene FMR1. Hum Mol Genet 1997;6:1791-1801. [Free Full Text]
Chan TL, Yuen ST, Kong CK, et al. Heritable germline epimutation of MSH2 in a family with hereditary nonpolyposis colorectal cancer. Nat Genet 2006;38:1178-1183. [CrossRef][ISI][Medline]
Zhang, L.
(2008). Immunohistochemistry versus Microsatellite Instability Testing for Screening Colorectal Cancer Patients at Risk for Hereditary Nonpolyposis Colorectal Cancer Syndrome: Part II. The Utility of Microsatellite Instability Testing. J. Mol. Diagn.
10: 301-307
[Abstract][Full Text]
Joensuu, E. I., Abdel-Rahman, W. M., Ollikainen, M., Ruosaari, S., Knuutila, S., Peltomaki, P.
(2008). Epigenetic Signatures of Familial Cancer Are Characteristic of Tumor Type and Family Category. Cancer Res.
68: 4597-4605
[Abstract][Full Text]
Nilsson, E. E, Anway, M. D, Stanfield, J., Skinner, M. K
(2008). Transgenerational epigenetic effects of the endocrine disruptor vinclozolin on pregnancies and female adult onset disease. Reproduction
135: 713-721
[Abstract][Full Text]
Matouk, C. C., Marsden, P. A.
(2008). Epigenetic Regulation of Vascular Endothelial Gene Expression. Circ. Res.
102: 873-887
[Abstract][Full Text]
Imai, K., Yamamoto, H.
(2008). Carcinogenesis and microsatellite instability: the interrelationship between genetics and epigenetics. Carcinogenesis
29: 673-680
[Abstract][Full Text]
Raptis, S., Mrkonjic, M., Green, R. C., Daftary, D., Pethe, V., Dicks, E., Younghusband, B. H., Parfrey, P. S., Gallinger, S. S., McLaughlin, J. R., Knight, J. A., Bapat, B.
(2007). Response: Re: MLH1 93G>A Promoter Polymorphism and the Risk of Microsatellite-Unstable Colorectal Cancer. JNCI J Natl Cancer Inst
99: 1490-1491
[Full Text]
Ogino, S., Hazra, A., Tranah, G. J., Kirkner, G. J., Kawasaki, T., Nosho, K., Ohnishi, M., Suemoto, Y., Meyerhardt, J. A., Hunter, D. J., Fuchs, C. S.
(2007). MGMT germline polymorphism is associated with somatic MGMT promoter methylation and gene silencing in colorectal cancer. Carcinogenesis
28: 1985-1990
[Abstract][Full Text]
Lynch, H. T., Boland, C. R., Rodriguez-Bigas, M. A., Amos, C., Lynch, J. F., Lynch, P. M.
(2007). Who Should Be Sent for Genetic Testing in Hereditary Colorectal Cancer Syndromes?. JCO
25: 3534-3542
[Abstract][Full Text]
Gosden, R. G., Feinberg, A. P.
(2007). Genetics and Epigenetics -- Nature's Pen-and-Pencil Set. NEJM
356: 731-733
[Full Text]
T SNP within MLH1 exon 16 in family member II6-A, which was used to demonstrate that he was transcribing RNA only from the MLH1 allele inherited from his father (Figure 3A). These data are consistent with transmission of the MLH1 epimutation from Patient A to her son. 
Previous
