Imprinted segments in the human genome: different DNA methylation patterns in the Prader-Willi/Angelman syndrome region as determined by the genomic sequencing method
Imprinted segments in the human genome: different DNA methylation patterns in the Prader-Willi/Angelman syndrome region as determined by the genomic sequencing methodMichael Zeschnigk, Birgit Schmitz, Bärbel Dittrich1, Karin Buiting1, Bernhard Horsthemke1 and Walter Doerfler*
Institute for Genetics, University of Cologne, D-50931 Cologne, Germany and 1Institute for Human Genetics, University of Essen, D-45122 Essen, Germany
Received September 17, 1996;Revised and Accepted December 4, 1996
A deletion of 15q11-q13 and uniparental disomy 15 lead to Prader-Labhart-Willi syndrome (PWS) or Angelman syndrome (AS) because this region contains genes expressed exclusively from the paternal (PWS) or maternal (AS) chromosome, respectively. DNA methylation plays a role in the control of imprinted gene expression, but so far only a few 5'-CG-3' dinucleotides within the recognition sites of the methylation sensitive enzymes have been studied. As part of a study on DNA methylation patterns in the human genome, we have applied the bisulfite protocol of genomic sequencing to study all 5'-CG-3' dinucleotides around exon 1 of SNRPN and at the D15S63 locus, which contains a start site for alternative SNRPN transcripts possibly involved in imprint switching during gametogenesis. At least 17 PCR products derived from single chromosomes of normal individuals as well as PWS and AS patients have been sequenced. We have found that cytosine residues outside 5'-CG-3' dinucleotides are always unmethylated. However, >96% of all of the 23 5'-CG-3' dinucleotides around SNRPN exon 1 are methylated on the maternal chromosome and completely devoid of methylation on the paternal chromosome. This finding is in contrast to the D15S63 locus, where only the two CfoI/HhaI sites are methylated on the maternal chromosome at the same frequency as seen for the SNRPN segment. At the other five 5'-CG-3' dinucleotides, differential methylation is less pronounced, i.e. 45-70% on the maternal chromosome and 5-14% on the paternal chromosome. The differences between SNRPN and D15S63 methylation may reflect different biological functions of the alternative SNRPN transcripts. The systematic investigation of 5'-CG-3' methylation patterns as reported here will provide the basis for a PCR-based methylation test to diagnose PWS and AS.
The nucleotide sequence on human chromosome 15q11-13 is functionally non-equivalent on the paternal and maternal chromosomes and represents one of the well characterized genetically imprinted segments in the human genome. Deletions in this genome region lead to clinically completely different phenotypes depending on whether the deletion is located on the paternally or the maternally inherited chromosome. The Prader-Labhart-Willi syndrome (PWS) (1 ) is characterized by a deletion in 15q11-13 on the paternal chromosome. Deletions of the same region on the maternal chromosome cause the Angelman syndrome (AS) (2 -5 ). About 25% of the PWS and 2% of the AS cases are due to uniparental disomy with two maternal and paternal chromosomes, respectively (6 ). From the paternally inherited chromosome only, at least four transcripts from the regions PAR-1, PAR-5, IPW, and SNRPN have so far been identified (7 -9 ). Genes involved in AS have not yet been characterized.
In ~1% of PWS and 4% of AS patients, the syndromes are associated with abnormal patterns of methylation in the segments D15S63, ZNF127, and SNRPN (10 -12 ), possibly due to the impairment of an imprinting (methylation) control element termed imprinting center which is located 5' of the SNRPN gene and its 5'-CG-3' rich region (12 ).
Studies on chromosomally integrated viral and on many mammalian promoter sequences have supported the concept that the sequence-specific methylation of promoters causes their long-term silencing (13 , contributions in ref. 14 ). The nucleotide positions decisive for methylation inactivation cannot be predicted but must be experimentally determined for each promoter. The proven association of promoter methylation with gene inactivation has played a major conceptual role in studies on the molecular mechanism underlying genetic imprinting. Additional factors may, of course, also be responsible for imprinting (15 ). The methylation status of some positions in the imprinted region 15q11-13, as defined by methylation sensitive restriction enzymes, correlates well with the parent-of-origin-specific expression of the SNRPN gene and has been developed as a diagnostic test for PWS and AS (16 -18 ). In all tissues analyzed the 5'-CG-3' rich region of the expressed paternally derived allele of the SNRPN gene is unmethylated, and the repressed maternally derived allele is highly methylated in these positions, except in intron 5 (18 ).
As methylation-sensitive restriction endonucleases provide only limited insights into the actually existing patterns of DNA methylation, we have analyzed the putative promoter and exon 1 regions of the SNRPN gene by genomic sequencing. In addition, we have investigated the D15S63 locus which maps 100 kb upstream of SNRPN. This locus contains a region (PWCFOA) from which an alternative SNRPN transcript is initiated (19 ). A protocol of genomic sequencing has been applied that treats the DNA with bisulfite and subsequently amplifies the relevant DNA segment by PCR using single strand-specific primers (20 -22 ). Individual PCR generated DNA molecules are then cloned and their nucleotide sequences are determined. Sodium bisulfite converts cytosine (C)-residues to uracil. Under the conditions employed, 5-methylcytosine (5-mC) does not react and remains unaltered (23 -25 ). In the ensuing PCR amplification, all unmethylated C-residues will be represented as thymine (T)-residues; only the original 5-mC positions will produce C-residues in the PCR products. Thus, the C-pattern in the nucleotide sequence of each PCR molecule reflects the 5-mC distribution in this segment on one of the human chromosomes.
Here, we report the exact patterns of DNA methylation on both strands for a number of 5'-CG-3' dinucleotides in imprinted regions on human chromosome 15. To the best of our knowledge, this analysis is the first in this imprinted region of the human genome based on the genomic sequencing method. The patterns have been determined for healthy control probands, for PWS and AS patients with deletions, uniparental disomy or imprinting center mutations. In PWS patients of any etiology, nearly all 5'-CG-3' dinucleotides in the SNRPN gene segment analyzed are methylated. The same dinucleotides are unmethylated in the AS patients studied. In contrast to the SNRPN region, the DNA in the PWCFOA segment is hypermethylated (60-75%) in PWS patients and hypomethylated (5-14%) in AS patients. In imprinting center mutations, the PWCFOA segment exhibits lower overall methylation frequencies in AS and PWS patients.
The plasmid p71.13.6 (Fig. 1 ) was in vitro premethylated by using the HhaI (5'-GCGC-3') or the SssI (5'-CG-3') DNA methyltransferase to test the reliability of the bisulfite modification and PCR methods as used in this laboratory. The methylated or the unmethylated p71.13.6 plasmid preparation was treated with bisulfite as described in Materials and Methods, and the DNA segment to be analyzed was amplified by PCR. The amplified nucleotide sequences and the primers used for PCR are indicated in Figure 2 b and Table 1 , respectively. PCR products were cloned, and the nucleotide sequence was determined in five clones from each of the in vitro premethylated plasmids. The results are schematically presented in Figure 3 . The horizontal bar in the center of the graph represents the investigated nucleotide sequence with the centromeric and telomeric termini. The letters A to G indicate the 5'-CG-3' positions in the nucleotide sequence; A and B correspond to the two HhaI sites in the p71.13.6 sequence. In a number of clones investigated, the nucleotide sequences in both strands were determined. Each horizontal row of filled (methylated 5'-CG-3') or open (unmethylated 5'-CG-3') squares refers to the results of sequencing in one isolated clone. The data indicate that the HhaI enzyme methylates only the 5'-GCGC-3' sites; the SssI DNA methyltransferase modifies almost all 5'-CG-3' dinucleotides. The few exceptions at random locations in this and all subsequently presented experiments could be explained by variance in DNA methyltransferase activity, in the bisulfite reaction or by mutations artificially introduced by the Taq polymerase.
Next, we have investigated the PWCFOA region, from which an alternative SNRPN transcript is initiated (19 ). The sequence (Fig. 2 b) presents two HhaI restriction sites (positions A and B) which have been successfully used for methylation-detecting molecular diagnostics of PWS and AS in clinical genetics (29 ). The frequency of 5'-CG-3' dinucleotides in this sequence segment is considerably lower when compared to the sequence of the putative promoter and exon 1 regions in the SNRPN gene. In these experiments, we have genomically sequenced the same DNA from the same patients as used for the analyses of the SNRPN putative promoter and exon 1 regions. The sequences of at least eight PCR products from each of the patients and each pair of primers are compiled in Figure 5 .
The highest density of 5-mC residues in this DNA segment derived from PWS patients is observed in the two HhaI restriction sites (positions A and B in Fig. 5 a, panels I, II, and III). These C-residues are almost completely methylated, while local frequencies of C-methylation in the other 5'-CG-3' dinucleotides (positions C to G in Fig. 5 a) vary between 33% (panel III, position E) and 85% (panel I, positions C and F). The overall frequency of DNA methylation in positions C to G is 68% for the PWS patient with a chromosome 15q deletion (Fig. 5 a, panel I), 60% for the PWS patient with uniparental disomy (panel II), and 44% for the PWS patient with an imprinting center mutation (panel III). The results of our control experiment (Fig. 3 ) suggest that the genomic sequencing procedure as applied in these experiments might underestimate the 5-mC levels by ~3.3%. Since the frequency of methylation in several 5'-CG-3' dinucleotide positions (position D in panel I, and position E in panel III) of one DNA strand is not identical to the methylation pattern in the same positions on the complementary strand, it is likely that these positions are hemimethylated (Fig. 5 a).
The same DNA segment close to the marker PW71 has been analyzed in DNA preparations obtained from blood cells from Angelman patients (Fig. 5 b). Although the frequency of 5'-CG-3' methylation is markedly lower than in DNA from PWS patients, the region is not completely unmethylated as seen in the promoter region of the SNRPN gene in the same AS patients (cf. Fig. 4 b). The local frequencies in methylation of individual 5'-CG-3' dinucleotides can reach ~40% (panel II, position C). The overall frequency of methylation in this DNA segment is 13% for the AS patients with a deletion, and for the patient with uniparental disomy, whereas only 5% of all 5'-CG-3' dinucleotides have been found to be methylated in the AS patient with an imprinting center mutation. Considering the patterns observed in all patients, 85% of all 5-mC residues are located in positions A to D (Fig. 5 b).
In AS patients, except for two cases (clones 2 and 5 in panel I of Fig. 5 b), at least one out of the two HhaI restriction sites (positions A and B) has remained unmethylated on the same DNA strand resulting in a cleavable CfoI or HhaI restriction site. This finding confirms the reliability of the DNA methylation test developed for the molecular diagnostics of PWS and AS patients (29 ). The results of genomic sequencing with the DNA from a male healthy donor are reminiscent of those in Figure 4 c (data not shown).
Patterns of DNA methylation in the human genome are characterized by a high degree of specificity and, at least in several segments, by interindividual concordance (30 -32 ). There is much evidence that sequence-specific methylation is part of the mechanism of genomic imprinting in mammalian genomes and also in the human genome (33 -35 ). One of the most intensely investigated imprinted regions in the human genome is the region on chromosome 15q11-13 with a medically relevant, highly interesting differentiation in clinical phenotypes depending on the parental origin of the deletion, of uniparental disomy or imprinting center mutation (3 ,6 ,12 ,29 ). Differences in the levels of DNA methylation in restriction sites in the PWS/AS segment have been documented (16 ,18 ) in that the maternal chromosome seems to be hypermethylated in this region. A very reliable test based on Southern blotting has been developed which exploits these findings to assess unequivocally the clinical diagnoses (11 ,16 ,18 ).
As detailed in this report, one subsegment of the PWS/AS region, i.e., the 5'-part of the SNRPN region, exhibits almost complete methylation on the maternally derived chromosome, as evidenced by studies on the DNA from patients with PWS caused by any of the three etiologic factors discussed (Fig. 4 a). In AS patients with an intact paternal chromosome, the same DNA segment has been shown to be almost completely unmethylated (Fig. 4 b). In healthy control donors ~50% of the cloned PCR products are methylated, 50% unmethylated (Fig. 4 c). This finding is consistent with the data derived from PWS and AS patients. A less uniform methylation pattern has been found on the maternal and paternal alleles in a second segment, close to the marker PW71, in the PWS/AS part of the genome (Fig. 5 a-c). This finding may reflect different biological functions of the different SNRPN transcripts. The SNRPN gene is ubiquitously expressed at a high level. In contrast, the alternative SNRPN transcripts initiated at D15S63 and another site 30 kb centromeric to D15S63 are much less abundant, present in fewer tissues and possibly involved in imprint switching during gametogenesis (19 ). The diagnostically exploited HhaI sites in the PWCFOA segment, however, conform to strict uniformity in complete methylation on the maternal, and absence of methylation on the paternal chromosome. It is possible that the HhaI sites are the most important part of a genetic element controlling the parent-of-origin specific expression of the alternative SNRPN transcripts.
Slight deviations in methylation from a generally distinct pattern might be due to some low degree of polymorphism, which appears likely in biology, or to technical imperfections which cannot be completely ruled out. None of the chemical or enzymatic reaction steps, the bisulfite reaction, the Taq polymerase-mediated PCR amplification or the cloning and sequencing steps in the protocol can be guaranteed to be devoid of minor deviations.
This study presents the first analysis of DNA methylation patterns of this imprinted region in the human genome by applying the genomic sequencing technique. Before one can interpret the biological significance of DNA methylation patterns in complex genomes, like the human, and assess their role in the mechanism of genetic imprinting, these patterns have to be determined in detail. It will no longer suffice to base, sometimes far-reaching, conclusions on the results of restriction enzyme analyses which may not encompass more than 20-30% of the methylatable, 5'-CG-3' containing sequences.
By using the bisulfite genomic sequencing protocol in the mouse Igf2 upstream region, which contains 12 5'-CG-3' dinucleotides, Feil et al. (36 ) have found that the expressed paternal allele is more methylated than the repressed maternal allele, but individual chromosomes revealed diverse patterns of DNA methylation. The results of the single-chromosome analyses have been taken as evidence against simple clonality of methylation patterns in this region.
It is interesting to note that a well established imprinted region in the human genome is uniformly and completely methylated on one and unmethylated on the other allele. Again, distinct patterns of methylation characterize this DNA segment in the human genome. In terms of the long-term silencing of the imprinted region on one of the chromosomes, it will be useful to recall that it is not necessarily the complete methylation of a eukaryotic promoter region that is responsible for promoter inactivation, but a highly promoter-specific methylation pattern in a 5'-CG-3'-rich imprinted region. As a case in point, we recall findings on a late frog virus 3 (FV3) promoter that is methylated in all of its 5'-CG-3' dinucleotides and transcriptionally active. The same promoter can, however, be experimentally inactivated by the selective methylation of its eight 5'-CCGG-3' (HpaII) sites, at least when a construct of this promoter has been used in transfection experiments (37 ).
All patients with typical PWS or AS phenotypes were molecularly classified by DNA methylation and polymorphism studies (16 ,39 ). The imprinting mutation patients (PWS-S and AS-D) were described in references (12 ) and (40 ). The description of PWS deletion patient (M0049) was included in reference (38 ). The AS patient with uniparental disomy (G1244) was the patient presented in (41 ). The PWS patient with uniparental disomy (G1478) was introduced by Knoblauch (42 ). The AS deletion patient (AS23) was not reported before. A DNA sample from this patient was kindly provided by K. Schmidt, Berlin.
Bisulfite treatment (21 ). Genomic DNA (5 [mu]g) or plasmid DNA (10 pg mixed with 5 [mu]g of salmon sperm DNA) was cleaved with the restriction enzyme BamHI in a reaction volume of 100 [mu]l and was denatured for 15 min at 37oC by adding 11 [mu]l of 3 M NaOH. For complete denaturation, samples were incubated at 95oC for 3 min and immediately cooled on ice. The bisulfite solution was prepared by dissolving 8.1 g of sodium bisulfite (Sigma) in 15 ml of degassed water, 1 ml of 40 mM hydrochinone was added, and the pH was adjusted to 5 by adding 600 [mu]l of 10 M NaOH. The denatured DNA solution (110 [mu]l) was mixed with 1 ml of the bisulfite solution, overlaid with mineral oil and incubated at 55oC for 16 h in a water bath in the dark. The DNA was recovered from the bisulfite solution by using glassmilk (GeneClean II Kit, Bio 101 Inc.) and eluted in 100 [mu]l of H2O. Subsequently, 11 [mu]l of 3 M NaOH was added, and the sample was incubated for 15 min at 37oC. The solution was then neutralized by adding 110 [mu]l of 6 M ammonium acetate, pH 7, and the DNA was ethanol-precipitated, washed in 70% ethanol, dried and redissolved in 20 [mu]l H2O.PCR (polymerase chain reaction). The methylation patterns of all sequences were determined for both strands in separate reactions. For PCR, 5 [mu]l of bisulfite-treated DNA (~500 ng) was used in a 100 [mu]l reaction mixture: 50 mM KCl, 1.7 mM MgCl2, 10 mM Tris-HCl (pH 9.0 at 25oC), 0.1% Triton X-100, 0.2 mM of each of the four dNTPs, 1 [mu]M of each primer, and 2.5 U Taq DNA polymerase (Promega). PCR was performed in a Perkin Elmer Cetus DNA Thermal Cycler 480 under the following cycle conditions: 94oC for 1 min, for 1 cycle; subsequently 94oC for 1 min, 51oC for 1 min, and 72oC for 1 min, for 35 cycles. A 5 [mu]l fraction of the PCR products was reamplified by using nested primers under the same conditions, except that the reaction proceeded through 25 cycles. The nucleotide sequences of all primer oligodeoxyribonucleotides used in this study were indicated in Table 1 .Cloning and sequencing. PCR products were ethanol-precipitated and dissolved in 10 [mu]l of H2O. A portion of 1 [mu]l was used for ligation into the vector pGEM-T (Promega) or the vector pT7Blue (Novagene) and transformed into competent E.coliXL1BlueMRF'cells (44 ). Recombinant plasmid DNA was prepared from white colonies by standard methods. Nucleotide sequences were determined in an automated Applied Biosystems DNA Sequencer 373A by using the chain termination method (45 ) and fluorescence-labeled dNTP for color detection in the automated sequencer. Sequencing primers: SP6 (pGEM-T) and universal primer (pT7Blue) were used as recommended by Promega and Novagene, respectively.
To test the reliability of the bisulfite method, 1 [mu]g of the p71.13.6 plasmid DNA was in vitro methylated by using the SssI or HhaI DNA methyltransferase under the following conditions: 1 mM Tris-HCl, pH 7.9, 5 mM MgCl2, 5 mM NaCl, 1 mM dithiothreitol, 160 [mu]M S-adenosylmethionine, 10 U SssI- or HhaI-DNA methyltransferase (NEB). The mixture was incubated at 37oC for 16 h.
M.Z. thanks Peter Meyer for technical help. This research was supported by the Deutsche Forschungsgemeinschaft through SFB274-A1 to W.D., by a stipend to M.Z., Ze 379/1-1 and by grant Ho 949/12-1 to B.H.
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