Epigenetic variation illustrated by DNA methylation patterns of the fragile-X gene FMR1
Epigenetic variation illustrated by DNA methylation patterns of the fragile-X gene FMR1 Reinhard Stöger1,*, Tina M. Kajimura1, W. Ted Brown2 and Charles D. Laird1,3,*
1Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, C3-168, Seattle, WA 98109, USA, 2Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA and 3Department of Zoology, University of Washington, Seattle, WA, USA
Received May 5, 1997;Revised and Accepted July 2, 1997
Genomic methylation patterns of mammals can vary among individuals and are subject to dynamic changes during development. In order to gain a better understanding of this variation, we have analyzed patterns of cytosine methylation within a 200 bp region at the CpG island of the human FMR1 gene from leukocyte DNA. FMR1 is normally methylated during inactivation of the X chromosome in females and it is also methylated and inactivated upon expansion of CGG repeats in fragile-X syndrome. Patterns of methylation (epigenotypes) were determined by the sequencing of bisulfite-treated alleles from normal males and females and alleles from a family of five brothers who are methylation mosaics and are affected to various degrees by the fragile-X syndrome. Our data indicate that: (i) methylation of individual CpG cytosines is strikingly variable in hypermethylated epigenotypes obtained from a single individual, suggesting that maintenance of cytosine methylation is a dynamic process; (ii) methylation of non-CpG cytosines in the region studied may occur but is rare; (iii) mosaicism of methylation in the analyzed fragile-X males is remarkably similar to that found for the active X and inactive X alleles in normal females, suggesting that the methylation mosaicism of some fragile-X males reflects similar on and off states of FMR1 expression that exist in normal females;(iv) hypermethylation is slightly more pronounced on fragile-X alleles than on normal inactive X alleles of females;(v) the general dichotomy of hypo- and hypermethylated alleles persisted over the 5 year period that separated samplings of the fragile-X males; (vi) methylation variability was most pronounced at a consensus binding sequence for the [alpha]-PAL transcription factor, a sequence that may play a role in regulating expression of FMR1.
The DNA sequence of an individual human is generally found to be invariant throughout development and life, with the exception of somatic mutations and rearrangements at the immunoglobulin loci in a minority of cells. With most current sequencing methods, however, only the four major bases in DNA are detected. A fifth base, 5-methylcytosine (5-MeC), is present in the DNA sequence of many organisms, including humans. It is usually found in the symmetrical sequence 5-MeCpG. Like the primary nucleotide sequence, the overall methylation blueprint can differ among individuals and is inherited in Mendelian fashion (1 ). Stability of methylation is apparent in the propagation of clonal patterns that maintain the inactive X chromosome in its silenced state in somatic cells of female mammals (reviewed in 2 -4 ).
Other aspects of DNA methylation are more dynamic. For example, fluctuations in cytosine methylation occur during early development of the mouse, where genome-wide demethylation is followed by an extensive wave of de novo methylation prior to gastrulation (5 ,6 ). Reduced levels of methyltransferase activity in mice result in an embryonic lethal phenotype and this result indicates that an intact DNA modification system plays a vital role in mammals (7 ). Aberrant methylation patterns and their clonal propagation in cell lineages are correlated with certain conditions in humans, such as the Prader-Willi, Angelman and Fragile-X syndromes (8 -14 ).
The 200 bp sequence whose methylation profile is described here contains 22 CpG dinucleotides and 45 non-CpG cytosines (Fig. 1 ). Sodium bisulfite catalyzes the conversion of cytosine to uracil residues in single-stranded DNA, whereas methylated cytosines remain unreactive under these conditions. The specificity of this reaction is remarkably high, as will be apparent in the presentation of data from females, where both hyper- and hypomethylated alleles are normally present in the same cell. Upon PCR amplification of the genomic region of interest the converted uracil residues are replicated as thymines instead of cytosines (35 ,36 ). A remaining cytosine in the sequence of the PCR product therefore indicates that this site was methylated on the template DNA. We cloned and sequenced individual PCR products from the coding strand; because methylation is usually symmetrical at CpG dinucleotides (37 ,38 ), we interpret our data as representing methylation patterns of individual alleles. Each methylation pattern thus represents the epigenetic profile of an allele in one cell, or an `epigenotype'.
DNA from six brothers of a fragile-X family was used to analyze methylation patterns of normal and fragile-X alleles in males (Fig. 2 ). We determined 180 epigenotypes from these six brothers, representing for each brother 15 epigenotypes from each of two samples collected 5 years apart. Male II-4 inherited a normal X chromosome and was shown to have a completely unmethylated EagI site at FMR1, as expected for a CpG island of an X chromosomal locus in males (31 ). DNA from individual II-4 can thus be used to assess the methylation status for normal, transcriptionally active FMR1 alleles in males (39 ). Our results confirm that this region of the FMR1 CpG island is markedly hypomethylated in normal male DNA. Twenty seven of 30 alleles were unmethylated at all sites, two alleles were methylated at one site each (sites 19 and 22) and one allele was methylated at three sites (sites 19, 21 and 22; Fig. 3 ). Similar hypomethylation was observed for epigenotypes of a normal male, unrelated to the fragile-X family (data not shown).
To determine methylation patterns of normal FMR1 alleles from active and inactive X chromosomes we examined DNA from two females (L2 and L7) who are not related to the fragile-X family (Fig. 4 ). In addition, DNA was analyzed from the mother (I-2) of the six brothers (Fig. 4 ). I-2 has one normal X chromosome and one X with an expanded CGG repeat in the pre-mutation range (~65 repeats) and is referred to as a `carrier'; she is not affected by the fragile-X syndrome and does not exhibit aberrant methylation of the EagI site in the FMR1 promoter (31 ).
Most hypermethylated alleles were not methylated at all 22 CpG sites (Fig. 6 ). This was significantly more pronounced (P < 0.001, t-test) on normal, inactive X chromosomes from females, where hypermethylated alleles have an average of 2.9 +- 0.9 (+-2 SE) unmodified CpG sites. In contrast, hypermethylated alleles from fragile-X chromosomes in males are found to have a mean of only 1.1 +- 0.4 unmethylated CpG sites (Figs 5 and 6 ). Complete methylation of all 22 CpGs occurred in 32% of hypermethylated alleles from the five fragile-X brothers, but only in 17% of the normal, inactive X alleles. Fewer than 1% (1/150) of the fragile-X alleles from the five males had intermediate levels of methylation (defined as >= 7 and <= 15 sites modified), whereas 5% (4/82) of alleles derived from females had intermediate methylation levels. Thus both the completeness of methylation of hypermethylated alleles and the frequency of alleles with intermediate methylation status differed between fragile-X and normal alleles, with the fragile-X alleles exhibiting higher levels of methylation.
The hypomethylated alleles from normal female DNA and from DNA of the six males of the fragile-X family did not differ noticeably in their methylation states. Most hypomethylated alleles were completely devoid of 5-MeC; the frequency of single-site methylation was ~10-fold lower than for hypomethylated alleles with no methylation (Fig. 5 ). A small subset of hypomethylated alleles had two to six unconverted C residues at particular CpG sites that may represent rare cytosine methylation in active alleles (for example sites 19 and 22; Fig. 6 ).
Sequencing provided complete information for all 22 CpG sites on 246 of the 262 epigenotypes, representing 5412 CpG cytosines. Complete information for all 45 non-CpG cytosines was obtained for 188 epigenotypes, representing 8460 non-CpG cytosines. This data set permits accurate estimation of the specificity of the bisulfite reaction in terms of the efficiency of conversion of non-methylated cytosines to uracil and the degree of protection from conversion by methylation.
Non-CpG cytosines are expected to be rarely methylated and thus provide a good estimate of the efficiency of conversion: 99.4% of the non-CpG cytosines were converted in female DNA samples, irrespective of whether the alleles were hyper- or hypomethylated at CpG sites; conversion of non-CpG cytosines in DNA samples from the males of the fragile-X family was 99.6% efficient. Does the remaining 0.6 and 0.4% protection represent bona fide methylation of non-CpG cytosines? Inheritance of cytosine methylation at non-CpG sites, especially CpNpG trinucleotides in mammalian cells, has been reported previously (41 ). We therefore examined the location of the 43 events of unconverted non-CpG cytosines. These were found at highest frequency (16/43) to be at trinucleotides CpNpT, in particular at the CpApT (9/43) positioned immediately 3' of CpG site 19 in a palindromic sequence. Resistance to conversion of this cytosine was most pronounced on hypomethylated alleles of males (5/9) in which one or more of the flanking CpG sites (sites 18-22) was also methylated. In addition, two hypermethylated male alleles and two intermediately methylated alleles (one female and one male) had this cytosine protected. Non-CpG cytosine protection was also detected on 14 CpNpG trinucleotides. Eleven of these cytosines were positioned at CCG triplets, with four being at the CCG identified by CpG site 10 [here referred to as C(CpG)-10], three at C(CpG)-7, two at C(CpG)-11, one at C(CpG)-5 and one at C(CpG)-6. The rest of the unconverted non-CpG cytosines occurred at CpNpC (12/43) and CpNpA (1/43) trinucleotides sites.
The apparent specificity of the bisulfite reaction under appropriate conditions, as described above, together with the large number of alleles analyzed, permits analysis of the frequencies of methylation of individual sites. The incomplete methylation of most hypermethylated alleles allows us to determine which particular CpG sites have unusual frequencies of methylation in comparison with the general methylation status. This frequency varies from site to site for both fragile-X and inactive X alleles (Fig. 6 ). Among the hypermethylated fragile-X and inactive X alleles, representing 65 epigenotypes, the highest degree of modification was observed at sites 8 and 13, which were always methylated (Fig. 6 ). In contrast, site 18 had the lowest frequency of methylation on both inactive X and fragile-X hypermethylated alleles (0.45 and 0.8 respectively, compared with averages of 0.87 and 0.95 for these two allele classes). This cytosine marks the 5'-end of a 14 bp palindromic DNA sequence (Fig. 1 ) containing CpG sites that, as mentioned above, are occasionally methylated on hypomethylated alleles from males (Fig. 6 ).
Sites 2-4 in female hypermethylated alleles are also unusually low in their frequency of methylation, averaging 0.72 (Fig. 6 ). A palindrome spans the first two of these sites, each of which was found to be methylated in three hypomethylated male and two hypomethylated female alleles.
The inactive state of X chromosomes is stable and inherited in a clonal fashion to daughter cells after DNA replication (42 ); methylation patterns are thought to be similarly inherited because of the properties of the maintenance methyltransferase (37 ,38 ). We thus looked for evidence of stable methylation patterns for CpG sites within the hypermethylated alleles derived from an individual. DNAs from the six brothers were isolated in both 1986 and 1991, thus providing the opportunity to determine if cytosine methylation patterns change in lymphocytes of affected individuals over a 5 year period. The marked bimodal methylation status of alleles was maintained over these years in all of the DNA samples. The same distributions of hypo- and hypermethylated alleles were observed for both sets of samples from individuals II-2, II-3 and II-5 (Fig. 3 ). The 1991 DNA samples from II-1 and II-6 have higher proportions of hypomethylated alleles compared with the 1986 samples (Fig. 3 ). The decrease in the fraction of hypermethylated clones is marginally significant for II-6 and for II-6 and II-1 taken together, using a one-tailed [chi]2 test (P < 0.05). More epigenotypes would need to be characterized to assess the significance of this finding.
As mentioned above, the broad methylation patterns of hypo- or hypermethylation were maintained over a 5 year period. Most hypermethylated alleles, however, were characterized by unique modification patterns that did not indicate strict clonal propagation of detailed methylation patterns. In only one case did we observe an identical, detailed pattern in which the same three sites lacked methylation (1986-6 and 1991-9 from individual II-5; Fig. 3 ). Several examples of completely methylated alleles from an individual were observed (for example 1986-2, 1986-6 and 1991-10 from individual II-1; Fig. 3 ), but complete methylation patterns provide less sensitivity to address questions of clonality than do partially methylated alleles. Thus only a small subset of the hypermethylated epigenotypes characterized are compatible with a model of strict clonal propagation of methylation patterns.
We analyzed in vivo methylation patterns at the resolution of individual DNA molecules, using the CpG island of FMR1 as a model to study epigenetic heterogeneity in humans. Our findings underline the complexity of epigenetic variations within a mammalian species (1 ,43 ). Some of these variations may affect the phenotype of an individual, as exemplified by the mosaicism of methylation in the fragile-X family described here. The description of cytosine methylation and its variations is thus of interest for a better understanding of epigenetic control of human phenotype and the rules that govern the propagation of methylation patterns.
Genomic DNAs from the fragile-X family were isolated from peripheral blood samples as described by McConkie-Rosell et al. (31 ). The 1986 DNA samples were used to determine the percentage of cytosine methylation at the EagI site in the McConkie-Rosell et al. study after double digestion with EcoRI and EagI and subsequent hybridization with probe StB12.3 (31 ). The following data on FMR1 CGG repeats from males of the fragile-X family are taken from table 2 of the McConkie-Rosell et al. study (31 ): II-1 is mosaic with unmethylated alleles of ~66 and 300 repeats and methylated alleles of ~300 repeats; II-2 is mosaic with unmethylated alleles ranging between ~130 and 200 repeats and methylated alleles of ~170 repeats; II-3 is mosaic with unmethylated alleles ranging between ~100 and 270 repeats and methylated alleles of ~170 repeats; II-4 has a normal allele with ~29 repeats; II-5 is mosaic with methylated alleles ranging between ~130 and 670 repeats; II-6 is mosaic with unmethylated alleles ranging between 170 and 200 repeats and methylated alleles of ~200 and 530 repeats; I-2 has a normal allele with ~29 CGG repeats and an allele with an expanded repeat in the pre-mutation range (~65 repeats). Leukocyte DNAs from females L2 and L7 were isolated following a standard phenol/chloroform extraction method. L2 has two alleles with normal CGG repeats (~29 repeats), as determined by double digestion of DNA with EcoRI and EagI and subsequent hybridization with probe StB12.3 (data not shown). L7 has two alleles with normal CGG repeats (~29 repeats), as determined by amplification of the FMR1 CGG repeat by PCR (data not shown).
With a few minor modifications, the bisulfite conversion of genomic DNA was carried out following the protocol developed and described by Clark et al. (36 ). Approximately 10 ng unsheared, genomic DNA and 2 [mu]g yeast tRNA (carrier nucleic acid) were denatured by adding freshly prepared NaOH to a final concentration of 0.3 M in a 10 [mu]l reaction volume and incubated at 42oC for 30 min. Increasing the stringency of the denaturing conditions (42oC/30 min instead of 37oC/15 min) improved the conversion rate of non-CpG cytosines. The more stringent denaturing conditions are perhaps necessary because the FMR1 promoter sequence has a high GC content (66% GC-rich) and therefore an overall higher Tm. Fresh solutions of 3.8 M sodium bisulfite (S-8890; Sigma), adjusted to pH 5 with NaOH, and 20 mM hydroquinone (H-9003; Sigma) were prepared by gentle mixing at 37oC. Final concentrations of 3.4 M sodium bisulfite and 1 mM hydroquinone were added to the denatured DNA to a final volume of 100 [mu]l. The DNA was gently mixed in this sodium bisulfite/hydroquinone solution, overlaid with mineral oil and incubated in the dark at 55oC for 6 h. After recovering the aqueous phase from under the oil, unbound bisulfite was removed from the DNA by use of MicroSpin S-200 HR columns (Pharmacia Biotech). The purified DNA sample was subsequently mixed and incubated with freshly prepared NaOH (0.3 M final concentration) at 37oC for 20 min. NaOH was removed by use of MicroSpin S-200 HR columns and the flow-through (~100 [mu]l), containing the converted DNA, was frozen and stored until aliquots were used as PCR templates.
The primers were designed to amplify bisulfite-converted DNA (top strand) of the FMR1 promoter and have the following sequences: 5'-GGAATTTTAGAGAGGTC/TGAATTGGG-3' (1F), positions 2246-2270; 5'-GTTATTGAGTGTATTTTTGTAGAAATGGG-3' (2F), positions 2296-2325; 5'-CCCTCTCTCTTCAAATAACCTAAAAAC-3' (3R), positions 2492-2466. (The numbering of the base residues corresponds to the unconverted, genomic human FMR1 DNA sequence, accession no. X61378.) The fragment of interest was amplified by semi-nested PCR in 25 [mu]l reaction mixtures. The first PCR contained 10 [mu]l bisulfite-treated genomic DNA, 200 [mu]M dNTPs, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 0.5 mM primers (1F and 3R) and 1.25 UTaq polymerase, overlaid with 25 [mu]l mineral oil. The amplification was performed in a Hybaid Omn-E thermal cycler under the following conditions: 94oC for 2 min for one cycle; 94oC for 15 s, 57oC for 15 s, 72oC for 1 min for 29 cycles; 72oC for 2 min for one cycle. The second PCR contained 1 [mu]l reaction mixture from the first PCR, 200 [mu]M dNTPs, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 3 mM MgCl2, 0.01% gelatin, 0.5 mM primers (2F and 3R) and 1.25 U Taq polymerase and was overlaid with 25 [mu]l mineral oil. The amplification was performed in a Hybaid Omn-E thermal cycler under the following conditions: 94oC for 2 min for one cycle; 94oC for 15 s, 65oC for 15 s, 73oC for 30 s for 29 cycles; 72oC for 2 min for one cycle.
This semi-nested PCR is extremely sensitive; minute amounts of template DNA can be amplified. Special care was taken during the sodium bisulfite conversion and the subsequent PCR procedures in order to avoid contamination. At least two DNA-negative PCR controls were performed for every DNA sample that was amplified.
The PCR products generated with primer pair 2F/3R were run on a 2% agarose gel and isolated using the MERmaid kit (Bio 101). Isolated fragments were ligated into plasmid pCR2.1 and the ligation product transformed into competent Escherichia coli (INVaF') according to the protocols of the manufacturer (TA cloning kit; Invitrogen). DNA of randomly picked clones was isolated (QIAprep; Qiagen) and sequenced using either the Sequenase version 2.0 DNA sequencing kit (US Biochemials) or the ABI PRISM dye terminator cycle sequencing ready reaction kit (Perkin Elmer). Sequence analysis was performed with computer programs of the Wisconsin Package, V.8.1-Unix (Genetics Computer Group, Madison, WI) and SIGNAL SCAN (64 ). DNA samples from the fragile-X family were coded and the epigenotypes matched with the individuals only after the bisulfite conversion and cloning had been performed.
We thank Scott Hansen, Stan Gartler, Douglas Kim and members of the Laird laboratory for critical comments on the manuscript, Susan Clark for helpful advice on the bisulfite sequencing method and Tracy Stapp for high quality sequencing. R.S. was supported by an Erwin-Schrödinger fellowship from the Austrian Science Foundation and a fellowship from the FRAXA Research Foundation. This work was supported by National Institutes of Health grant GM53805.
1 Silva,A.J. and White,R. (1988) Inheritance of allelic blueprints for methylation patterns. Cell, 54, 145-152.MEDLINE Abstract
2 Gartler,S.M., Dyer,K.A., Graves,J.A.M. and Rocchi,M. (1985) A two step model for mammalian X-chromosome inactivation. In Cantoni,G.L. and Razin,A. (eds), Biochemistry and Biology of DNA Methylation. Alan R.Liss, New York, NY, pp. 223-235.
3 Lyon,M.F. (1988) X-chromosome inactivation and the location and expression of X-linked genes. Am. J. Hum. Genet., 42, 8-16.MEDLINE Abstract
4 Riggs,A.D. and Pfeifer,G.P. (1992) X-chromosome inactivation and cell memory. Trends Genet., 8, 169-174.MEDLINE Abstract
5 Monk,M., Boubelik,M. and Lehnert,S. (1987) Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development, 99, 371-382.MEDLINE Abstract
6 Kafri,T., Ariel,M., Brandeis,M., Shemer,R., Urven,L., McCarrey,J., Cedar,H. and Razin,A. (1992) Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line. Genes Dev., 6, 705-714.MEDLINE Abstract
7 Li,E., Bestor,T.H. and Jaenisch,R. (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell, 69, 915-926.MEDLINE Abstract
8 Dittrich,B., Robinson,W.P., Knoblauch,H., Buiting,K., Schmidt,K., Gillessen-Kaesbach,G. and Horsthemke,B. (1992) Molecular diagnosis of the Prader-Willi and Angelman syndromes by detection of parent-of-origin specific DNA methylation in 15q11-13. Hum. Genet., 90, 313-315.MEDLINE Abstract
9 Driscoll,D.J., Waters,M.F., Williams,C.A., Zori,R.T., Glenn,C.C., Avidano,K.M. and Nicholls,R.D. (1992) A DNA methylation imprint, determined by the sex of the parent, distinguishes the Angelman and Prader-Willi syndromes. Genomics, 13, 917-924.MEDLINE Abstract
10 Reis,A., Dittrich,B., Greger,V., Buiting,K., Lalande,M., Gillessen-Kaesbach,G., Anvret,M. and Horsthemke,B. (1994) Imprinting mutations suggested by abnormal DNA methylation patterns in familial Angelman and Prader-Willi syndromes. Am. J. Hum. Genet., 54, 741-747.MEDLINE Abstract
11 Bell,M.V., Hirst,M.C., Nakahori,Y., MacKinnon,R.N., Roche,A., Flint,T.J., Jacobs,P.A., Tommerup,N., Tranebjaerg,L., Froster-Iskenius,U., Kerr,B., Turner,G., Lindenbaum,R.H., Winter,R., Pembrey,M., Thibodeau,S. and Davies,K.E. (1991) Physical mapping across the fragile X: hypermethylation and clinical expression of the fragile X syndrome. Cell, 64, 861-866.MEDLINE Abstract
12 Vincent,A., Heitz,D., Petit,C., Kretz,C., Oberlé,I. and Mandel,J. (1991) Abnormal pattern detected in fragile X patients by pulsed field gel electrophoresis. Nature, 349, 624-626.MEDLINE Abstract
13 Oberlé,I., Rousseau,F., Heitz,D., Kretz,C., Devys,D., Hanauer,A., Boué,J., Bertheas,M.F. and Mandel,J.L. (1991) Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome. Science, 252, 1097-1102.MEDLINE Abstract
14 Zeschnigk,M., Schmitz,B., Dittrich,B., Buiting,K., Horsthemke,B. and Doerfler,W. (1997) Imprinted segments in the human genome-different DNA methylation patterns in the Prader-Willi/Angelman syndrome region as determined by the genomic sequencing method. Hum. Mol. Genet., 6, 387-395.MEDLINE Abstract
15 Holliday,R. and Pugh,J.E. (1975) DNA modification mechanisms and gene activity during development. Science, 187, 226-232.MEDLINE Abstract
16 Riggs,A.D. (1975) X inactivation, differentiation, and DNA methylation. Cytogenet. Cell Genet., 14, 9-25.MEDLINE Abstract
17 Sager,R. and Kitchin,R. (1975) Selective silencing of eukaryotic DNA. Science, 189, 426-433.MEDLINE Abstract
18 Meehan,R., Lewis,J., Cross,S., Nan,X., Jeppesen,P. and Bird,A. (1992) Transcriptional repression by methylation of CpG. J. Cell. Sci., 16 (suppl.), 9-14.
19 Bird,A. (1986) CpG-rich islands and the function of DNA methylation. Nature, 321, 209-213.MEDLINE Abstract
20 Antequera,F. and Bird,A. (1993) Number of CpG islands and genes in human and mouse. Proc. Natl. Acad. Sci. USA, 90, 11995-11999.MEDLINE Abstract
21 Razin,A. and Cedar,H. (1994) DNA methylation and genomic imprinting. Cell, 77, 473-476.MEDLINE Abstract
22 Eden,S. and Cedar,H. (1994) Role of DNA methylation in the regulation of transcription. Curr. Opin. Genet. Dev., 4, 255-259.MEDLINE Abstract
23 Wöhrle,D. and Steinbach,P. (1991) Fragile X expression and X inactivation. II. The fragile site at Xq27.3 has a basic function in the pathogenesis of fragile X-linked mental retardation. Hum. Genet., 87, 421-424.MEDLINE Abstract
24 Hansen,R.S., Gartler,S.M., Scott,C.R., Chen,S.-H. and Laird,C.D. (1992) Methylation analysis of CGG sites in the CpG island of the human FMR1 gene. Hum. Mol. Genet., 1, 571-578.MEDLINE Abstract
25 Hornstra,I.K., Nelson,D.L., Warren,S.T. and Yang,T.P. (1993) High resolution methylation analysis of the FMR-1 gene trinucleotide repeat region in Fragile X syndrome. Hum. Mol. Genet., 2, 1659-1665.MEDLINE Abstract
26 Dietrich,A., Kioschis,P., Monaco,A.P., Gross,B., Korn,B., Williams,S.V., Sheer,D., Heitz,D., Oberlé,I., Toniolo,D., Warren,S.T., Lehrach,H. and Poustka,A. (1991) Molecular cloning and analysis of the fragile X region in man. Nucleic Acids Res., 19, 2567-2572.MEDLINE Abstract
27 Pieretti,M., Zhang,F.P., Fu,Y.H., Warren,S.T., Oostra,B.A., Caskey,C.T. and Nelson,D.L. (1991) Absence of expression of the FMR-1 gene in fragile X syndrome. Cell, 66, 817-22.MEDLINE Abstract
28 Hagerman,R., Kemper,M. and Hudson,M. (1985) Learning disabilities and attentional problems in boys with the fragile X syndrome. Am. J. Dis. Child., 139, 674-678.MEDLINE Abstract
29 Veenema,H., Veenema,T. and Geraedts,J.P.M. (1987) The fragile X syndrome in a large family. II Psychological investigations. J. Med. Genet., 24, 32-38.MEDLINE Abstract
30 Loesch,D.Z., Huggins,R., Hay,D.A., Gedeon,A.K., Mulley,J.C. and Sutherland,G.R. (1993) Genotype-phenotype relationships in fragile X syndrome: a family study. Am. J. Hum. Genet., 53, 1064-1073.MEDLINE Abstract
31 McConkie-Rosell,A., Lachiewicz,A.M., Spiridigliozzi,G.A., Tarleton,J., Schoenwald,S., Phelan,M.C., Goonewardena,P., Ding,X. and Brown,W.T. (1993) Evidence that methylation of FMR-1 locus is responsible for variable phenotypic expression of the fragile x syndrome. Am. J. Hum. Genet., 53, 800-809.MEDLINE Abstract
32 de Vries,B.B., Jansen,C.C., Duits,A.A., Verheij,C., Willemsen,R., van Hemel,J.O., van den Ouweland,A.M., Niermeijer,M.F., Oostra,B.A. and Halley,D.J. (1996) Variable FMR1 gene methylation of large expansions leads to variable phenotype in three males from one fragile X family. J. Med. Genet., 33, 1007-1010.MEDLINE Abstract
33 Heitz,D., Rousseau,R., Devys,D., Saccone,S., Abderrahim,H., LePaslier,D., Cohen,D., Vincent,A., Toniolo,D., Della Valle,G., Johnson,S., Schlessinger,D., Oberlé,I. and Mandel,J.L. (1991) Isolation of sequences that span the Fragile X and identification of a Fragile X-related CpG island. Science, 251, 1236-1239.MEDLINE Abstract
34 Brown,W.T., Gross,A., Goonewardena,P., Ferrando,C., Dobkin,C. and Jenkins,E.C. (1991) Detection of fragile X non-penetrant males by DNA marker analysis. Am. J. Med. Genet., 38, 292-297.MEDLINE Abstract
35 Frommer,M., McDonald,L.E., Millar,D.S., Collis,C.M., Watt,F., Grigg,G.W., Molloy,P.L. and Paul,C.L. (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. USA, 89, 1827-1831.MEDLINE Abstract
36 Clark,S.J., Harrison,J., Paul,C.L. and Frommer,M. (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res., 22, 2990-2997.MEDLINE Abstract
37 Gruenbaum,Y., Cedar,H. and Razin,A. (1982) Substrate and sequence specificity of a eukaryotic DNA methylase. Nature, 295, 620-622.MEDLINE Abstract
38 Bestor,T.H. and Ingram,V.M. (1983) Two DNA methyltransferases from murine erythroleukemia cells: purification, sequence specificity, and mode of interaction with DNA. Proc. Natl. Acad. Sci. USA, 80, 5559-5563.MEDLINE Abstract
39 Feng,Y., Lakkis,L., Devys,D. and Warren,S.T. (1995) Quantitative comparison of FMR1 gene expression in normal and premutation alleles. Am. J. Hum. Genet., 56, 106-113.MEDLINE Abstract
40 Rousseau,F. et al. (1991) Direct diagnosis by DNA analysis of the fragile X syndrome of mental retardation. New Engl. J. Med., 325, 1673-1681.MEDLINE Abstract
41 Clark,S.J., Harrison,J. and Frommer,M. (1995) CpNpG methylation in mammalian cells. Nature Genet., 10, 20-27.MEDLINE Abstract
42 Lyon,M. (1961) Gene action in the X chromosome of the mouse (Mus musculus L.). Nature, 190, 372-373.
43 McGowan,R., Campbell,R., Peterson,A. and Sapienza,C. (1989) Cellular mosaicism in the methylation and expression of hemizygous loci in the mouse. Genes Dev., 3, 1669-1676.MEDLINE Abstract
44 Rousseau,F., Heitz,D., Oberlé,I. and Mandel,J.-L. (1991) Selection in blood cells from female carriers of the fragile X syndrome: inverse correlation between age and proportion of active X chromosomes carrying the full mutation. J. Med. Genet., 28, 830-836.MEDLINE Abstract
45 Malter,H.E., Iber,J.C., Willemsen,R., de Graaff,E., Tarleton,J.C., Leisti,J., Warren,S.T. and Oostra,B.A. (1997) Characterization of the full fragile X syndrome mutation in fetal gametes. Nature Genet., 15, 165-169.MEDLINE Abstract
46 Reis,R.J. and Goldstein,S. (1982) Interclonal variation in methylation patterns for expressed and non-expressed genes. Nucleic Acids Res., 10, 4293-4304.MEDLINE Abstract
47 Pfeifer,G.P., Steigerwald,S.D., Hansen,R.S., Gartler,S.M. and Riggs,A.D. (1990) Polymerase chain reaction-aided genomic sequencing of an X chromosome-linked CpG island: methylation patterns suggest clonal inheritance, CpG site autonomy, and an explanation of activity state stability. Proc. Natl. Acad. Sci. USA, 87, 8252-8256.MEDLINE Abstract
48 Macleod,D., Charlton,J., Mullins,J. and Bird,A.P. (1994) Sp1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island. Genes Dev., 8, 2282-2292.MEDLINE Abstract
49 Silva,A.J., Ward,K. and White,R. (1993) Mosaic methylation in clonal tissue. Dev. Biol., 156, 391-398.MEDLINE Abstract
50 Selker,E.U., Fritz,D.Y. and Singer,M.J. (1993) Dense nonsymmetrical DNA methylation resulting from repeat-induced point mutation in Neurospora. Science, 262, 1724-1728.MEDLINE Abstract
51 Goyon,C., Nogueira,T.I. and Faugeron,G. (1994) Perpetuation of cytosine methylation in Ascobolus immersus implies a novel type of maintenance methylase. J. Mol. Biol., 240, 42-51.MEDLINE Abstract
52 Meijer,H., de Graaff,E., Merckx,D.M., Jongbloed,R.J., de Die-Smulders,C.E., Engelen,J.J., Fryns,J.P., Curfs,P.M. and Oostra,B.A. (1994) A deletion of 1.6 kb proximal to the CGG repeat of the FMR1 gene causes the clinical phenotype of the fragile X syndrome. Hum. Mol. Genet., 3, 615-620.MEDLINE Abstract
53 Pfeifer,G.P., Tanguay,R.L., Steigerwald,S.D. and Riggs,A.D. (1990) In vivo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1. Genes Dev., 4, 1277-1287.MEDLINE Abstract
54 Hornstra,I.K. and Yang,T.P. (1994) High-resolution methylation analysis of the human hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes: correlation with binding sites for transcription factors. Mol. Cell. Biol., 14, 1419-1430.MEDLINE Abstract
55 Brandeis,M., Frank,D., Keshet,I., Siegfried,Z., Mendelsohn,M., Nemes,A., Temper,V., Razin,A. and Cedar,H. (1994) Sp1 elements protect a CpG island from de novo methylation. Nature, 371, 435-438.MEDLINE Abstract
56 Weiss,A., Keshet,I., Razin,A. and Cedar,H. (1996) DNA demethylation in vitro: involvement of RNA. Cell, 86, 709-718.MEDLINE Abstract
57 Jacob,W.F., Silverman,T.A., Cohen,R.B. and Safer,B. (1989) Identification and characterization of a novel transcription factor participating in the expression of eukaryotic initiation factor 2 alpha. J. Biol. Chem., 264, 20372-20384.MEDLINE Abstract
58 DeSimone,S.M. and White,K. (1993) The Drosophilaerect wing gene, which is important for both neuronal and muscle development, encodes a protein which is similar to the sea urchin P3A2 DNA binding protein. Mol. Cell .Biol., 13, 3641-3649.MEDLINE Abstract
59 Efiok,B.J., Chiorini,J.A. and Safer,B. (1994) A key transcription factor for eukaryotic initiation factor-2 alpha is strongly homologous to developmental transcription factors and may link metabolic genes to cellular growth and development. J. Biol. Chem., 269, 18921-18930.MEDLINE Abstract
60 Laayoun,A. and Smith,S.S. (1995) Methylation of slipped duplexes, snapbacks and cruciforms by human DNA(cytosine-5)methyltransferase. Nucleic Acids Res., 23, 1584-1589.MEDLINE Abstract
61 Antequera,F., Boyes,J. and Bird,A. (1990) High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell, 62, 503-514.MEDLINE Abstract
62 Frank,D., Keshet,I., Shani,M., Levine,A., Razin,A. and Cedar,H. (1991) Demethylation of CpG islands in embryonic cells. Nature, 351, 239-241.MEDLINE Abstract
63 Bestor,T.H. (1992) Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain. EMBO J., 11, 2611-2617.MEDLINE Abstract
64 Prestridge,D.S. (1991) SIGNAL SCAN: a computer program that scans DNA sequences for eukaryotic transcriptional elements. CABIOS, 7, 203-206.
D. P. Genereux, W. C. Johnson, A. F. Burden, R. Stoger, and C. D. Laird Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies
Nucleic Acids Res.,
December 1, 2008;
36(22):
e150 - e150.
[Abstract][Full Text][PDF]
P. D. Ladd, L. E. Smith, N. A. Rabaia, J. M. Moore, S. A. Georges, R. S. Hansen, R. J. Hagerman, F. Tassone, S. J. Tapscott, and G. N. Filippova An antisense transcript spanning the CGG repeat region of FMR1 is upregulated in premutation carriers but silenced in full mutation individuals
Hum. Mol. Genet.,
December 15, 2007;
16(24):
3174 - 3187.
[Abstract][Full Text][PDF]
S. J. Clark Action at a distance: epigenetic silencing of large chromosomal regions in carcinogenesis
Hum. Mol. Genet.,
April 15, 2007;
16(R1):
R88 - R95.
[Abstract][Full Text][PDF]
C. Dahl, K. Gronskov, L. A. Larsen, P. Guldberg, and K. Brondum-Nielsen A Homogeneous Assay for Analysis of FMR1 Promoter Methylation in Patients with Fragile X Syndrome
Clin. Chem.,
April 1, 2007;
53(4):
790 - 793.
[Abstract][Full Text][PDF]
M. P. Hitchins, J. J.L. Wong, G. Suthers, C. M. Suter, D. I.K. Martin, N. J. Hawkins, and R. L. Ward Inheritance of a Cancer-Associated MLH1 Germ-Line Epimutation
N. Engl. J. Med.,
February 15, 2007;
356(7):
697 - 705.
[Abstract][Full Text][PDF]
D. P. Genereux, B. E. Miner, C. T. Bergstrom, and C. D. Laird A population-epigenetic model to infer site-specific methylation rates from double-stranded DNA methylation patterns
PNAS,
April 19, 2005;
102(16):
5802 - 5807.
[Abstract][Full Text][PDF]
B. E. Miner, R. J. Stoger, A. F. Burden, C. D. Laird, and R. S. Hansen Molecular barcodes detect redundancy and contamination in hairpin-bisulfite PCR
Nucleic Acids Res.,
September 30, 2004;
32(17):
e135 - e135.
[Abstract][Full Text][PDF]
A. D. Riggs and Z. Xiong Methylation and epigenetic fidelity
PNAS,
January 6, 2004;
101(1):
4 - 5.
[Full Text][PDF]
C. D. Laird, N. D. Pleasant, A. D. Clark, J. L. Sneeden, K. M. A. Hassan, N. C. Manley, J. C. Vary Jr., T. Morgan, R. S. Hansen, and R. Stoger From The Cover: Hairpin-bisulfite PCR: Assessing epigenetic methylation patterns on complementary strands of individual DNA molecules
PNAS,
January 6, 2004;
101(1):
204 - 209.
[Abstract][Full Text][PDF]
E Pascale, E Battiloro, G C. Reale, R Pietrobono, M G Pomponi, P Chiurazzi, R Nicolai, M Calvani, G Neri, and E D'Ambrosio Modulation of methylation in the FMR1 promoter region after long term treatment with L-carnitine and acetyl-L-carnitine
J. Med. Genet.,
June 1, 2003;
40(6):
e76 - 76.
[Full Text][PDF]
R. Pietrobono, M. G. Pomponi, E. Tabolacci, B. Oostra, P. Chiurazzi, and G. Neri Quantitative analysis of DNA demethylation and transcriptional reactivation of the FMR1 gene in fragile X cells treated with 5-azadeoxycytidine
Nucleic Acids Res.,
July 15, 2002;
30(14):
3278 - 3285.
[Abstract][Full Text][PDF]
A. Bird DNA methylation patterns and epigenetic memory
Genes & Dev.,
January 1, 2002;
16(1):
6 - 21.
[Full Text][PDF]
C. Grunau, S. J. Clark, and A. Rosenthal Bisulfite genomic sequencing: systematic investigation of critical experimental parameters
Nucleic Acids Res.,
July 1, 2001;
29(13):
e65 - e65.
[Abstract][Full Text][PDF]
F Tassone, R J Hagerman, A K Taylor, and P J Hagerman A majority of fragile X males with methylated, full mutation alleles have significant levels of FMR1 messenger RNA
J. Med. Genet.,
July 1, 2001;
38(7):
453 - 456.
[Abstract][Full Text][PDF]
R. S. Hansen, R. Stoger, C. Wijmenga, A. M. Stanek, T. K. Canfield, P. Luo, M. R. Matarazzo, M. D'Esposito, R. Feil, G. Gimelli, et al. Escape from gene silencing in ICF syndrome: evidence for advanced replication time as a major determinant
Hum. Mol. Genet.,
November 1, 2000;
9(18):
2575 - 2587.
[Abstract][Full Text][PDF]
U. Salat, B. Bardoni, D. Wöhrle, and P. Steinbach Increase of FMRP expression, raised levels of FMR1 mRNA, and clonal selection in proliferating cells with unmethylated fragile X repeat expansions: a clue to the sex bias in the transmission of full mutations?
J. Med. Genet.,
November 1, 2000;
37(11):
842 - 850.
[Abstract][Full Text]
M. Kersting, C. Friedl, A. Kraus, M. Behn, W. Pankow, and M. Schuermann Differential Frequencies of p16INK4a Promoter Hypermethylation, p53 Mutation, and K-ras Mutation in Exfoliative Material Mark the Development of Lung Cancer in Symptomatic Chronic Smokers
J. Clin. Oncol.,
September 18, 2000;
18(18):
3221 - 3229.
[Abstract][Full Text][PDF]
H. Kinoshita, Y. Shi, C. Sandefur, L. F. Meisner, C. Chang, A. Choon, C. R. Reznikoff, G. S. Bova, A. Friedl, and D. F. Jarrard Methylation of the Androgen Receptor Minimal Promoter Silences Transcription in Human Prostate Cancer
Cancer Res.,
July 1, 2000;
60(13):
3623 - 3630.
[Abstract][Full Text]
B. Genc, H. Muller-Hartmann, M. Zeschnigk, H. Deissler, B. Schmitz, F. Majewski, A. v. Gontard, and W. Doerfler Methylation mosaicism of 5'-(CGG)n-3' repeats in fragile X, premutation and normal individuals
Nucleic Acids Res.,
May 15, 2000;
28(10):
2141 - 2152.
[Abstract][Full Text][PDF]
A. Hmadcha, F. J. Bedoya, F. Sobrino, and E. Pintado Methylation-Dependent Gene Silencing Induced by Interleukin 1{beta} via Nitric Oxide Production
J. Exp. Med.,
December 6, 1999;
190(11):
1595 - 1604.
[Abstract][Full Text][PDF]
D. J. Wong, S. A. Foster, D. A. Galloway, and B. J. Reid Progressive Region-Specific De Novo Methylation of the p16 CpG Island in Primary Human Mammary Epithelial Cell Strains during Escape from M0 Growth Arrest
Mol. Cell. Biol.,
August 1, 1999;
19(8):
5642 - 5651.
[Abstract][Full Text][PDF]
A. Aggerholm, P. Guldberg, M. Hokland, and P. Hokland Extensive Intra- and Interindividual Heterogeneity of p15INK4B Methylation in Acute Myeloid Leukemia
Cancer Res.,
January 1, 1999;
59(2):
436 - 441.
[Abstract][Full Text][PDF]
J. M. LaSalle, R. J. Ritchie, H. Glatt, and M. Lalande Clonal heterogeneity at allelic methylation sites diagnostic for Prader-Willi and Angelman syndromes
PNAS,
February 17, 1998;
95(4):
1675 - 1680.
[Abstract][Full Text][PDF]
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