Human Molecular Genetics Advance Access originally published online on June 22, 2005
Human Molecular Genetics 2005 14(15):2135-2143; doi:10.1093/hmg/ddi218
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Interindividual variability and parent of origin DNA methylation differences at specific human Alu elements
1Fels Institute for Cancer Research and Molecular Biology, 2Department of Pathology and Laboratory Medicine, Temple University School of Medicine, 3307 North Broad Street, Philadelphia, PA 19140, USA, 3The Research Institute of McGill University Health Centre, Montreal, Quebec H3G 1A4, Canada, 4Eccles Institute of Human Genetics and Department of Human Genetics, University of Utah, 15 N 2030 E, Salt Lake City, UT 84112, USA, 5Bucknell University, 701 Moore Avenue, Lewisburg, PA 17837, USA and 6College of Science and Technology, Temple University, 1900 North 13th Street, Philadelphia, PA 19122, USA
* To whom correspondence should be addressed. Tel: +1 2157077373; Fax: +1 2157071454; Email: sapienza{at}temple.edu
Received April 7, 2005; Revised May 30, 2005; Accepted June 14, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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We investigated the CpG methylation of 19 specific members of Alu sub-families in human DNA isolated from whole blood, using an assay based on methylation-sensitive restriction endonuclease digestion of genomic DNA and ‘hot-stop’ polymerase chain reaction. We found significant interindividual variability in the level of methylation for specific Alu elements among the members of 48 three-generation families. Surprisingly, some of the elements also displayed quantitative parent of origin methylation differences; i.e. the mean level of methylation differed significantly when the insertions were transmitted through paternal versus maternal meiosis. Bisulfite sequence analysis of individual elements at such loci suggests, further, that maternal and paternal elements differ in the propensity of particular CpG sites to become unmethylated. Some individuals who exhibited high levels of methylation at specific Alu elements came from families in which more than one member also exhibited abnormal patterns of methylation at the differentially methylated regions of the IGF2/H19 or IGF2R loci, suggesting that there may be heritable differences between individuals in the fidelity with which allelic DNA methylation differences are established or maintained. Quantitative parental origin differences in methylation were identified only for Alu elements that lie in sub-telomeric or sub-centromeric bands of human chromosomes, whereas those assayed at intermediate positions did not exhibit any significant differences. The centromere/telomere restricted location of the methylation differences and the fact that none of these differences occur in regions of chromosomes known to contain transcriptionally imprinted genes suggest that maternal/paternal epigenetic modifications may play additional roles in processes other than transcriptional control.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Alu elements have been accumulating in the human genome throughout primate evolution, reaching a present copy number of over a million per genome (1
). Alu elements are dimeric 300 bp sequences; however, they are not identical in sequence and can be classified into several sub-families of different ages (reviewed in 2). Some of the recently integrated sub-families (termed Ya to Yi sub-families) are polymorphic (i.e. some chromosomes carry the Alu insertion, whereas others do not) within the genomes of human populations and more than 600 such polymorphic Alu repeats have been identified to date (3–6).
Alu repeats are rich in CpG dinucleotides, the principal target sites for DNA methylation in eukaryotes (7). Although Alu elements are almost completely methylated in most somatic tissues, some Alu repeats are differentially methylated in germ cells, with at least a subset of the recently integrated Alus being almost completely unmethylated in sperm DNA (8,9). The methylation state of Alu repeats has been investigated previously by genomic blot hybridization and more recently by bisulfite polymerase chain reaction (PCR) techniques that assay global methylation of Alu elements (10–12). However, recently described Alu insertion/deletion polymorphisms allow us to analyze DNA methylation of individual elements in a locus-specific manner. In this report, we describe the results of the DNA methylation analysis of 19 polymorphic human Alu repeats in a panel of 48 three-generation families.
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RESULTS |
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DNA methylation analysis of specific Alu insertion/deletion polymorphisms
We selected a panel of 26 specific Alu elements that were reported previously having an intermediate frequency of heterozygous individuals in populations of European ancestry (3
,4). These Alu polymorphisms have a diverse localization in the human genome with regard to both chromosome number and sub-chromosomal location. Nineteen of the Alu insertions were found to be polymorphic in the set of 48 three-generation CEPH families analyzed, with a level of heterozygosity varying from 
6 to 50% (Supplementary Material, Table S1). Each Alu element examined contains 22 or more CpG dinucleotides and three or, in some cases, four of these are found at HpaII recognition sites. We analyzed the methylation state of the HpaII sites in Alu inserts (there are no HpaII sites in the PCR fragments amplified from the allele lacking the Alu element) (Fig. 1) only from heterozygous individuals, for whom the parental origin of the two alleles was established by pedigree analysis (possible only for individuals from the second and third generations, when one of their parents was homozygous for one allele and the other heterozygous, or both parents were homozygous for the opposite alleles).
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To test the reproducibility of the assay, we performed a triplicate test for a subset of 100 samples (sets of 10 samples for 10 different loci). We found a high degree of reproducibility of the ratios (correlation coefficient for paired measurements r2=0.9954), indicating that intraindividual variation between assays is low (Fig. 2).
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DNA from all individuals informative for maternal/paternal origin of the Alu element was digested with an excess of HpaII methylation-sensitive restriction endonuclease (Fig. 3A and B) (see Materials and Methods). Control digestions using MspI, a methylation-insensitive isoschisomer of HpaII, induced complete digestion of the alleles containing the Alu insertions (Fig. 3C, lane 2), showing that resistance to digestion with HpaII is due to the presence of methyl groups at the CpG dinucleotide in the HpaII recognition sequence. After digestion, locus-specific PCR primers that do not flank any other HpaII sites outside the Alu repeat were added and a modified ‘hot-stop’ PCR procedure (13
) (see Materials and Methods) was performed. Hot-stop PCR avoids visualization of heteroduplexes as well as minor products characteristically observed in standard PCR assays and allows a more accurate quantification of the individual alleles.
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The ratio of the intensity of the allele containing the insertion (‘I’, insertion) to that without the insertion (‘D’, deletion) was computed for each individual at each locus. Even though we observed low between-assay variance for any individual (Fig. 2), all of the Alu insertions analyzed showed significant interindividual variability in methylation (Fig. 3A and B) (Supplementary Material, Fig. S1).
Parental origin effects on the methylation of Alu insertions
Because hemizygous transgene loci in the mouse (which are also insertion/deletion polymorphisms) often show parental origin differences in methylation (14–16) and because we were able to determine the parental origin of the alleles at each insertion/deletion locus examined, we tested whether parental origin could account for some of the interindividual variation in methylation of the Alu elements. The empirical distribution of the I/D ratio varied from symmetric to moderately skewed (Fig. 3A and B) (Supplementary Material, Fig. S1), therefore, significance of the difference between the means (according to parental origin) was assessed by resampling (see Materials and Methods).
Although none of the Alu elements exhibited complete or nearly complete parental origin-dependent methylation (on the scale of the IGF2/H19 DMR, for example), some of the Alu polymorphisms did show quantitative parent of origin methylation effects; i.e. the mean values of the I/D ratios of informative individuals are significantly higher when the insertions were transmitted through paternal (six cases) or maternal (one case) meiosis (Table 1; Fig. 3). A higher paternal I/D ratio indicates that a significantly higher fraction of cells contain an Alu element that is methylated at all three (or four) HpaII sites, when the element is paternally derived than when maternally derived, whereas the reverse is true in the single case of a higher maternal I/D ratio. Neither the I/D ratio nor their parental-specific methylation differences seem to depend on age or sex (Supplementary Material, Table S2).
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Bisulfite sequence analysis of individual Alu elements
The I/D ratio measures the fraction of elements at which all three (or four) HpaII–CpG sites are methylated. We note that each individual I/D ratio shown in Figure 3 is based on the analysis of
15 000 to 45 000 chromosomes (assuming a diploid cell contains 6x109 bp of DNA and each assay contains 100 ng of DNA; i.e. 15 000 chromosomes per assay, repeated up to three times) (see Materials and Methods), so the precision with which quantitative differences between individuals can be measured is relatively high. In this regard, one does not expect to be able to distinguish quantitative differences in methylation of individual CpG sites of the scale observed (Fig. 3; Table 1) by bisulfite sequence analysis, because the power to detect such differences requires the analysis of a large number of individual elements (17). However, the unexpected observation of maternal/paternal differences in the methylation of HpaII sites at some loci prompted us to examine the methylation of all CpG sites in a sample of maternally and paternally derived elements. We performed bisulfite sequence analysis on individual chromosomes from subjects in which element Ya5NBC345 was either maternally (46 chromosomes from six individuals, Fig. 4A) or paternally (34 chromosomes from six individuals, Fig. 4B) derived. Overall, the great majority of CpG sites are methylated, regardless of whether the element is maternal or paternal and there is no significant difference in the overall level of CpG site methylation detected by this method. There is also no significant difference in the frequency at which all three HpaII sites are observed to be methylated (the basis of the assay is shown in Fig. 3) by this method. However, an analysis of the frequency with which individual CpG sites are not methylated reveals some differences between maternal and paternal elements (Fig. 4C). There are two sites in the middle of the element (sites 11 and 12) that are methylated on all 46 maternal chromosomes analyzed, whereas one or both sites were methylated on only 26 of the 36 paternal chromosomes analyzed (Fig. 4C). An additional site (site 10) also appears to exhibit differences in the frequency at which the site is not methylated but in the opposite direction; this site is methylated in 35 of the 36 paternal clones analyzed, but it is the most frequently unmethylated site in maternal clones (Fig. 4C). We note that our estimate of the frequency that particular CpG sites were unmethylated is biased against the possibility of an overestimate for any particular site by including only a single clone with a particular pattern of methylated/unmethylated CpGs from any single individual. Thus, the only way for an individual CpG site to be scored as unmethylated multiple times was for that site to be unmethylated in multiple clones with different bisulfite sequence patterns or to be scored as unmethylated in multiple individuals.
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Familial clustering of extreme I/D ratios
In an earlier report, we described the interindividual variation in allelic DNA methylation at the IGF2/H19 and IGF2R loci (17
). Because a substantial fraction of the variability in methylation at those loci appears to be due to unlinked genetic factors, we wished to determine whether such factors might play a role in the interindividual variability in methylation of individual Alu elements. To identify familial clustering of extreme I/D ratios, we calculated the mean I/D ratio of the population of informative individuals at each locus, as well as the standard deviation (SD), and designated individuals with I/D ratios more than 2 SD from the mean as extreme (‘low’ or ‘high’). This stratification identified 19 instances of ‘low’ I/D ratios (0.79%) and 88 instances of ‘high’ I/D ratios (3.64%), out of the 2415 ratios summarized in Table 1. We applied two selective criteria to identify families in which abnormal I/D ratios might reflect the action of unlinked genetic factors: (1) three or more family members exhibited abnormal I/D ratios at some locus and (2) abnormal I/D ratios occurred at three or more loci in members of the same family. Although these selective criteria are somewhat arbitrary, they are sufficiently stringent to exclude all but a few familial associations. Families in which both criteria were fulfilled are summarized in Table 2.
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No familial clustering of the small number of ‘low’ I/D ratios was observed (see Discussion). However, 32 of the ‘high’ ratios occurred in 26 individuals distributed among only six families (Table 2). Interestingly, four of these families were identified previously as families in which abnormal methylation of maternal IGF2/H19 or paternal IGF2R DMRs occurred (Table 2) (17
), suggesting the possibility of trans-acting genetic modifiers of methylation level, consistent with the conclusions of our earlier work (17). We exclude the possibility that these results are due to technical problems with particular DNA samples, such as non-specific resistance to restriction endonuclease digestion, because preamplification digestion using MspI induced complete cutting of alleles containing Alu insertions. In addition, the same individuals who exhibited abnormal ratios at some loci exhibited normal ratios at other loci (Note: each of the six families indicated in Table 2 was informative for 9–14 of the 19 Alu polymorphic loci; no family showed increased methylation at more than four of the loci). Moreover, we have shown previously that samples collected from the same person two decades apart have similar abnormal methylation ratios at IGF2/H19 or IGF2R, and such results were also obtained for a subset of 10 individuals with ‘high’ I/D ratios (correlation coefficient for paired measurements r2=0.8466) (data not shown), again consistent with the possibility of one or more factors that influence DNA methylation in trans.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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We report three findings from a family-based, locus-specific analysis of DNA methylation at 19 Alu elements that give rise to insertion/deletion (I/D) polymorphisms in the human genome. First, we observed significant interindividual variability (several-fold) in the I/D ratios at each particular locus. Secondly, we identified a parental origin effect on maintenance of DNA methylation at multiple HpaII sites for seven of the 19 Alu insertions. These quantitative methylation differences extended to several CpG sites that were not part of HpaII sites for one locus examined by bisulfite sequencing. Thirdly, we found familial clustering of the most extreme (‘high’) I/D methylation ratios among 26 members of six families. Four of these families had been identified previously in a screen for abnormal allelic methylation ratios at the IGF2/H19 or IGF2R differentially methylated regions, suggesting that trans-acting genetic factors influence the establishment or maintenance of allelic methylation differences and that these factors act globally rather than in a locus-specific manner. In this vein, a recent N-ethyl-N-nitrosourea-induced mutation screen in the mouse (18
) has identified at least five loci that appear to affect aspects of chromatin structure, including DNA methylation, on a global level.
We note that although we did not identify familial clustering of ‘low’ I/D ratios, we do not wish to imply that such clustering cannot or does not exist. It seems possible, and even likely, that genetic variants of trans-acting modifiers of methylation level will be analogous to other genetic modifiers of chromatin structure, such as enhancers and suppressors of position-effect variegation in Drosophila (reviewed in 19). The most likely explanation for our inability to detect familial clustering of ‘low’ I/D ratios is that our selection criteria to designate this variability as ‘familial’ were very stringent and there were only a small number of individuals with ‘low’ I/D ratios.
The fact that quantitative parental-origin influences on the methylation of Alu elements were found at only some insertion/deletion loci prompts the question of whether there is any common factor that distinguishes elements that have such differences from those that do not. There does not appear to be a relationship between element sub-family and parental origin differences (Table 1). In addition, none of the elements that show parental origin differences in methylation is found in regions of the genome known to contain imprinted genes (www.geneimprint.com). Interestingly, however, the distribution of Alu elements that exhibit these differences does not appear to be random (Fig. 5). All seven of the elements that show parental origin-dependent quantitative differences in CpG methylation map to the centromeric or telomeric regions of chromosomes (the cytogenetic band or sub-band closest to the centromere or telomere of the relevant chromosome). In fact, seven of the 10 elements examined that map to these regions show quantitative parental origin differences in methylation, whereas none of the nine elements examined that map to intermediate cytogenetic bands shows such differences (P=0.0031, Fisher's exact test) (Fig. 5).
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We have proposed previously that the centromeric and telomeric regions of all chromosomes may be differentially marked in order to distinguish between homologs during the processes of homologous recombination in meiotic cells and DNA repair in mitotic cells (20; further discussed in 21). The data in Figures 3 and 5 are consistent with this hypothesis because they suggest chromosome position-dependent and parental origin-dependent differences between homologous chromosomes, even though the chromosomes examined are not known to harbor transcriptionally imprinted genes. We note that such quantitative differences in methylation are not peculiar to insertion/deletion polymorphisms, because we have also examined the methylation of a pseudogene member of the olfactory receptor gene family located at the telomere of chromosome 17p (22
) and have also found significant quantitative maternal/paternal differences in methylation at this locus (Supplementary Material, Fig. S2).
We note that not all sequences at the telomeres or centromeres of chromosomes exhibit differential methylation [e.g. the telomeric and sub-telomeric ‘similarly methylated regions’ described by Strichman-Almanshu et al. (23)], but further support for differential methylation of telomeric and centromeric sequences can be gleaned from the data of Yamada et al. (24). These authors reported a comprehensive analysis of 149 CpG islands on human chromosome 21q. They found that 31 of the CpG islands are fully methylated in normal peripheral blood cells. Three of the fully methylated CpG islands are located near the centromere (at 21q11.1) and 25 were found at 21q22.3 near the telomere. However, they also found seven CpG islands with a composite pattern of methylation (characteristic of CpGs islands that are methylated on one allele and unmethylated on the other) and five of these seven were located at 21q22.3 near the telomere. One of these telomeric CpG islands exhibited maternal-specific DNA methylation and another showed allele-specific, but parental origin-independent DNA methylation.
Although the pairing of homologous chromosomes is an important step in the successful completion of meiosis (25), the mechanisms by which maternal and paternal homologs find each other are not well-understood. Telomere-led bouquet formation during meiotic prophase is thought to facilitate homologous chromosome pairing and restrict ectopic interaction. This process is conserved in a wide range of species, from yeast to Drosophila to mouse and man (reviewed in 26–28). Comparison of telomere and centromere distribution patterns of mouse and human meiocytes reveals movements of centromeres and then telomeres to the nuclear envelope and subsequent bouquet formation as conserved motifs of the pairing process (29).
The mechanism by which initial pairing of the chromosomes is achieved embodies a fundamental biological question of distinguishing ‘self’ from ‘non-self’ (30). Our finding of parental origin-dependent methylation differences at regions that are involved in chromosome pairing supports the hypothesis that such epigenetic chromosomal marking might be selected for the common purpose of distinguishing homologous chromosomes from non-homologous chromosomes (20).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Subjects
DNA samples obtained from unfractionated nucleated peripheral blood cells (i.e. not lymphoblastoid cell lines) from the Salt Lake City collection of CEPH/Utah pedigrees (individuals from 48 three-generation families) were studied. All subjects gave informed consent under University of Utah I.R.B. approved protocol number 6090-96.
Methylation analysis of specific Alu insertions
We screened DNA samples for individuals who were heterozygous for 26 specific Alu insertion polymorphisms. Genotypes were determined by amplification of 25 ng of genomic DNA in a standard 35-cycle, three-step PCR, with the addition of dimethylsulfoxide (DMSO) to a final concentration of 1 M for resolution of preferential amplification of the allele without the insertion.
During the screening of the DNA samples for informative (heterozygous) individuals, we encountered preferential amplification of the non-insertion allele in heterozygotes. We improved significantly the amplification of the allele containing the Alu insertion by adding DMSO, known to facilitate the amplification of GC-rich templates, to a final concentration of 1 M (31). The degree to which the preferential amplification of the non-insertion allele was resolved was found to be very consistent for each particular locus, but there was significant variability between loci (data not shown) in the ability of DMSO to prevent preferential amplification of the non-insertion allele. Consequently, we have limited our analysis of individual variability in methylation to within-locus comparisons.
Appropriate primer annealing temperatures were optimized, empirically, for each locus. Primers used were previously reported (3,4) with the exception of cases in which primers had to be redesigned to exclude potential HpaII restriction sites from the amplified portion of the allele without the Alu element (Supplementary Material, Table S1). PCR samples were loaded onto multiple-combed 2% agarose gels and were separated by electrophoresis at 80 V for 2 h. Ethidium bromide-stained gels were visualized by ultraviolet and photographed. Parental origin of alleles of heterozygous individuals was determined by pedigree analysis, when possible.
A 100 ng sample of genomic DNA from informative individuals (heterozygous with parental origin of the insertion established with certainty by pedigree analysis) was digested for 6 h at 37°C with an excess (5 UI) of HpaII methyl-sensitive restriction endonuclease (Roche), according to the manufacturer's instructions. As control for digestion efficiency, heterozygous samples were digested in similar conditions using the MspI restriction endonuclease, an isoschisomer of HpaII whose activity is not inhibited by DNA methylation. Digested DNA was then ethanol-precipitated and amplified in a modified ‘hot-stop’ PCR assay (13) using the same primers as the ones used for the screening of informative individuals. Briefly, this assay involves the addition, after 27 cycles, of 3 µCi of [
-32P]dCTP and allowing synthesis to take place for a final cycle. PCR products were separated on 5–8% polyacrylamide gels and the intensity of the bands (alleles) was quantified using a Fuji BAS 2000 phosphorimager (32).
Bisulfite-sequencing analysis at Ya5NBC345
Five micrograms of genomic DNA was digested with XbaI overnight and then denatured at 42°C for 30 min using freshly prepared 3 M sodium hydroxide (final concentration of 0.3 M). Denatured DNA was subsequently incubated at 55°C with 40.5% sodium metabisulfite and 10 mM hydroquinone in the dark, overnight (33). After purification with the QIAquick PCR purification kit (Qiagen), the converted DNA was used as a template for a nested PCR. The first set of primers was F1: 5'-GAGTATATTAAAATGGTGAGAGA-3' and R1: 5'-ATCTCCACCATCTCTACTA-3'. As a second set of primers, we used F2: 5'-AGTGGTTAGTATGTTTAGGGTTAATGTTTT-3' and R2: 5'-AAAATAAAAATATCCTAATTAACAAAACATTCTC-3'. The PCR products were separated in 1.5% agarose gels, purified using the QIAEX II gel extraction kit (Qiagen) and subsequently sub-cloned into a TA Cloning vector (Invitrogen), according to the manufacturer's instructions. The DNA from 10 to 15 individual clones was sequenced for each individual.
Statistical analysis
The mean values of I/D ratios were compared using t-test under the assumption of homoscedasticity with the P-values obtained by Monte Carlo simulation (10 000 bootstrapped replicates). The statistical program R (http://www.R-project.org) was used for this task. All the other statistics were generated by using Prism4.0 software (GraphPad).
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SUPPLEMENTARY MATERIAL |
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Supplementary Material is available at HMG Online.
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ACKNOWLEDGEMENTS |
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We are grateful to Laurie Mecham and Tena Varvil for administrative and technical assistance. We would like to extend our sincere thanks to all family members who participated in the Utah Genetic Reference Project. We also thank Andreas P. Peiffer, MD, PhD, UGRP Medical Director and Melissa M. Dixon, UGRP Study Coordinator. This research was supported by a grant from the National Institutes of Health (NIH R21ES/CA11607) (to C.S.), by a Public Health Services research grant to the Huntsman General Clinical Research Center at the University of Utah and by grant nos M01-RR00064 and C06-RR11234 from the National Center for Research Resources. It was also supported by generous gifts from the W.M. Keck Foundation and from the George S. and Delores Doré Eccles Foundation.
Conflict of Interest statement. None declared.
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FOOTNOTES |
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Present address: Laboratory of Developmental Genetics and Imprinting, Developmental Genetics Programme, The Babraham Institute, Babraham Research Campus, Cambridge CB2 4AT, UK. 
Present address: Department of Molecular, Cellular and Developmental Biology, KBT 1044, Yale University, 219 Prospect Street, New Haven, CT 06511, USA.
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