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Genetics, Vol. 178, 35-45, January 2008, Copyright © 2008
doi:10.1534/genetics.107.081562

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Parental Effect of DNA (Cytosine-5) Methyltransferase 1 on Grandparental-Origin-Dependent Transmission Ratio Distortion in Mouse Crosses and Human Families

Lanjian Yang*, Moises Freitas Andrade{dagger},1, Stephane Labialle{dagger}, Sanny Moussette{dagger}, Geneviève Geneau{ddagger}, Donna Sinnett{ddagger}, Alexandre Belisle{ddagger}, Celia M. T. Greenwood§ and Anna K. Naumova*,{dagger},2

* Department of Human Genetics, Faculty of Medicine, McGill University, Montreal, Quebec H3A 1B1, Canada, {dagger} Department of Obstetrics and Gynecology, Faculty of Medicine, McGill University, and the Research Institute of the McGill University Health Centre, Montreal, Quebec H3A 1A1, Canada, {ddagger} McGill University and Genome Quebec Innovation Centre, Montreal, Quebec H3A 1A4, Canada and § Hospital for Sick Children, Research Institute, Program in Genetics and Genome Biology, Toronto, Ontario M5G 1L7 and the Department of Public Health Sciences, University of Toronto, Toronto, Ontario M5T 3M7, Canada

2 Corresponding author: Department of Obstetrics and Gynecology, Royal Victoria Hospital, Women's Pavilion, F3.32, 687 Pine Ave. West, Montreal, QC H3A 1A1, Canada.
E-mail: anna.naumova{at}muhc.mcgill.ca

Manuscript received September 5, 2007. Accepted for publication October 21, 2007.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Transmission ratio distortion (TRD) is a deviation from the expected Mendelian 1:1 ratio of alleles transmitted from parents to offspring and may arise by different mechanisms. Earlier we described a grandparental-origin-dependent sex-of-offspring-specific TRD of maternal chromosome 12 alleles closely linked to an imprinted region and hypothesized that it resulted from imprint resetting errors in the maternal germline. Here, we report that the genotype of the parents for loss-of-function mutations in the Dnmt1 gene influences the transmission of grandparental chromosome 12 alleles. More specifically, maternal Dnmt1 mutations restore Mendelian transmission ratios of chromosome 12 alleles. Transmission of maternal alleles depends upon the presence of the Dnmt1 mutation in the mother rather than upon the Dnmt1 genotype of the offspring. Paternal transmission mirrors the maternal one: live-born offspring of wild-type fathers display 1:1 transmission ratios, whereas offspring of heterozygous Dnmt1 mutant fathers tend to inherit grandpaternal alleles. Analysis of allelic transmission in the homologous region of human chromosome 14q32 detected preferential transmission of alleles from the paternal grandfather to grandsons. Thus, parental Dnmt1 is a modifier of transmission of alleles at an unlinked chromosomal region and perhaps has a role in the genesis of TRD.

TRANSMISSION ratio distortion (TRD) is a deviation from the Mendelian 1:1 ratio of allelic transmission. It may result from disruption/modification of different stages of several essential developmental processes, such as oogenesis (meiotic drive) (WU et al. 2005), spermatogenesis (reviewed in LYON 2003) and fertilization (different fertilizing potential of sperm that carry different genotypes) (ZHENG et al. 2001), or embryonic development. TRD has been observed in the human (MUNIER et al. 1994; NAUMOVA and SAPIENZA 1994; NAUMOVA et al. 1998, 2001; EAVES et al. 1999; PATERSON and PETRONIS 1999; ZOLLNER et al. 2004; HANCHARD et al. 2006) and in the mouse (ARANHA and MARTIN-DELEON 1991; PARDO-MANUEL DE VILLENA et al. 1997; CROTEAU et al. 2002), and the known causes of TRD include mutations (allele-specific TRD) (MUNIER et al. 1994) and chromosomal rearrangements (ARANHA and MARTIN-DELEON 1991; PARDO-MANUEL DE VILLENA and SAPIENZA 2001). More complex types of TRD are associated with groups of alleles, such as trinucleotide repeats of a certain length range (CHAKRABORTY et al. 1996; RUBINSZTEIN and LEGGO 1997; DEAN et al. 2006) or grandparental-origin-dependent TRD (NAUMOVA et al. 1995, 1998, 2001; CROTEAU et al. 2002). The underlying mechanisms of these types of TRD remain elusive. We hypothesized that grandparental-origin-dependent TRD resulted from epigenetic errors in the process of imprint resetting in the germline of the parents and/or in the maintenance of parental imprints in early embryos (Figure 1). For example, if imprint resetting failed in a proportion of maternal germ cells, embryos would inherit a normally imprinted allele from the father and a maternal allele that carries incorrect paternal imprints. Such embryos would have an androgenote-like epigenotype at this particular locus and loss of imprinting (LOI). If the LOI at this locus is incompatible with embryonic survival, the embryos that inherited the incorrectly imprinted allele would die and grandparental-origin-dependent TRD would occur (NAUMOVA et al. 2001). On the basis of this hypothesis we predicted that TRD should be found in genomic regions containing imprinted genes whose correct imprinting is essential for embryo survival. We tested this prediction in three-generation Centre d'Etude de Polymorphisme Humain (CEPH) families and in mouse crosses. In CEPH families, we found grandparental-origin-dependent and sex-of-offspring-specific TRD for alleles residing in chromosomal regions 11p15, 7p11, and 6q26-27 (NAUMOVA et al. 2001). We also observed familial heterogeneity with respect to TRD for 11p15 alleles. Independent studies confirmed TRD in chromosomal regions 11p15 and 6q26-27 (LEMIRE et al. 2004; IMBODEN et al. 2006). Thus, data from human families provide support for the association between TRD and genomic imprinting. In mice, preferential transmission of grandmaternal alleles occurs in the distal part of chromosome 12 (CROTEAU et al. 2002). This TRD results from dynamic adjustment of allelic ratios during embryonic development, including a loss of postimplantation embryos that carry the alleles of the maternal grandfather. We mapped the region of maximum TRD to the distal 10-cM portion of chromosome 12 (CROTEAU et al. 2002) that contains at least one imprinted region (CATTANACH and BEECHEY 1990). Aberrant expression of imprinted genes in the distal part of mouse chromosome 12 leads to developmental anomalies and fetal loss (GEORGIADES et al. 2001; LIN et al. 2003). In the human, uniparental disomy (UPD) for the chromosomal region 14q32, which is homologous to the imprinted region of mouse chromosome 12, is associated with intrauterine growth retardation, developmental delay, small stature, and early onset of puberty (maternal UPD) or with mental retardation and skeletal anomalies (paternal UPD) (reviewed in GEORGIADES et al. 1998; KOTZOT 2001, 2004; STEVENSON et al. 2004).


Figure 1
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  		FIGURE 1.— Failure to erase imprints on maternal alleles. The solid lines correspond to maternally imprinted chromosomes, and the dashed lines correspond to paternally imprinted chromosomes. The offspring that inherited the grandmaternal (GM) correctly imprinted chromosome from the mother has normal imprinting. The offspring that inherited the incorrectly paternally imprinted grandpaternal (GP) chromosome from the mother has two paternally imprinted chromosomes and shows LOI.

 
Imprinting in the distal imprinted region of mouse chromosome 12 depends to a large extent upon two paternal differentially methylated regions (DMRs): the intragenic DMR (IG DMR) (Figure 2A), which is fully methylated in sperm and unmethylated in mature oocytes, and the Gtl2 DMR, which acquires its differential methylation only after fertilization (TAKADA et al. 2002; LIN et al. 2003; STESHINA et al. 2006).


Figure 2
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  		FIGURE 2.— Effect of DNMT1 on transmission ratios in mouse chromosome 12 and human chromosome 14q32. (A) Genetic map of the distal region of chromosome 12. Positions of markers used for genotyping are based on the Mouse Genome Informatics integrated map. The approximate position of the IG DMR is indicated by an open oval. (B) Genetic (top) and physical (bottom) maps of the human chromosomal region 14q32 that harbors the imprinted domain. Male-specific genetic map positions of markers used for genotyping are based on the Marshfield genetic map. Physical distance between the same markers is also based on data from the Ensembl database. The approximate position of the IG DMR is indicated by an open oval. (C) Diagrams of the mouse crosses used in the study. The presence of the Dnmt1 mutation in heterozygous state is indicated as "Dnmt1."

 
To further explore the mechanism of grandparental-origin-dependent TRD and its link to imprinting, we used the grandparental-origin-dependent TRD at chromosome 12 as a model for identifying modifiers of TRD. We used a candidate gene approach. We expected that a reduced supply of the major maintenance DNA methyltransferase, DNMT1 that is essential for genomic imprinting, would modify transmission ratios of maternal chromosome 12 alleles. However, if DNMT1 were not involved in the genesis of TRD, changes would not be observed.

We conducted mouse crosses in which the C57BL/6 strain was replaced with the Dnmt1n or Dnmt1c strains that carry targeted mutations of Dnmt1 (LI et al. 1992). Both mutations are associated with reduced production of DNMT1 and reduced methylation of the genome (LEI et al. 1996); however, the viability and fertility of the heterozygous mutant mice are not compromised (LI et al. 1992). We found that parental Dnmt1 mutations modify transmission ratios of alleles in the chromosome 12 distal region, suggesting a major role for Dnmt1 in grandparental-origin-dependent TRD.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Mice and crosses:
C57BL/6 and BALB/c mice were purchased from Charles River Laboratories. The mutant Dnmt1c mice were a kind gift of En Li and Jacquetta Trasler. The Dnmt1n mice were purchased from the Jackson Laboratories. The mutant mice were mated to BALB/c mice to generate breeders for the experimental crosses. The following crosses were generated (see Table 1 for number of mice involved in crosses):

DNMA: (C57BL/6 Dnmt1–/+ x BALB/c) F1 female x C57BL/6 male;
DNMB: (BALB/c x C57BL/6 Dnmt1–/+) F1 female x C57BL/6 male;
DNFA: (C57BL/6 x BALB/c) F1 female x C57BL/6 Dnmt1–/+ male;
DNFB: (BALB/c x C57BL/6) F1 female x C57BL/6 Dnmt1–/+ male;
C: C57BL/6 female x (C57BL/6 x BALB/c) F1 male;
D: C57BL/6 female x (BALB/c x C57BL/6) F1 male;
DNFC: C57BL/6 female x (C57BL/6 Dnmt1n/+ x BALB/c) F1 male;
DNFD: C57BL/6 female x (BALB/c x C57BL/6 Dnmt1n/+) F1 male.


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  		TABLE 1 Characteristics of the crosses

 
For analysis of IG DMR methylation, we generated male mice that were heterozygous for the chromosome 12 imprinted region and for the Dnmt1 mutation. These mice were derived by reciprocal matings between congenic B6.MOLF12 (CROTEAU et al. 2005) and Dnmt1n/+ mice. As a control, sperm from Dnmt1+/+ littermates were used. All experiments were conducted according to the Guide to the Care and Use of Experimental Animals from the Canadian Council on Animal Care.

Mouse genotyping:
Tail biopsies were collected from the offspring at weaning. DNA was extracted from tails using a standard proteinase K and phenol–chloroform procedure. The offspring of mouse crosses were genotyped for the microsatellite marker D12Nds2 that is polymorphic between BALB/c and C57BL/6 strains. Genotyping was done by PCR using primers synthesized by Integrated DNA Technologies, Taq polymerase (Fermentas), and dNTPs (Invitrogen, San Diego). PCR products were separated in agarose gels. The offspring of the DNM crosses that included the Dnmt1c mutation were genotyped using a PCR assay described in KELLY et al. (2003). The offspring of crosses involving the Dnmt1n mutation were genotyped using a PCR assay described in CORMIER and DOVE (2000).

Quantitative Dnmt1 expression analysis:
To evaluate the abundance of Dnmt1 RNA isoforms in oocytes from mutant and wild-type mice, MII oocytes were collected from 8- to 10-week-old Dnmt1n/+ (57 oocytes) and Dnmt1+/+ (52 oocytes) females after superovulation. RNA was extracted from oocytes as described in MOHAMED et al. (2001).

Tubules were obtained from five heterozygous mutant males and four wild-type littermates following the protocol described in NAGANO et al. (1999). Total RNA was extracted using Trizol (Invitrogen). Reverse transcription (RT) reactions were performed on 1 µg of total RNA treated by DNase I Amplification Grade (Invitrogen), using MMLV-Reverse Transcriptase and RNase Out (Invitrogen) following company instructions. RT reactions were primed using oligo(dT) primers (Invitrogen). Real-time PCR experiments were performed using the ABI Prism 7900HT (PE Applied Biosystems). The reactions were done using the SYBR Green PCR master mix kit (Applied Biosystems, Foster City, CA) following the manufacturer's instructions. Relative quantification was achieved following a standard curve method. We used 4 ng of cDNA generated from total RNA samples as templates for the PCR reactions. For the standard curves, we used 10-fold serial dilutions of cDNA. Reactions were performed over 40 cycles of 20 sec at 95°, 30 sec at 60°, and 45 sec at 72° and followed by a dissociation stage analysis. All the reactions, including the standards and nontemplate control (H2O) were run in triplicate. RNA levels of Hprt1 were used as the normalization control. Dnmt1o and Dnmt1s were detected using isoform-specific primers (KO et al. 2005). The Dnmt1p isoform was amplified using the primers Dnmt1p-F 5'-CCCCGCCCTATTATTTTAGC-3' and Dnmt1p-R 5'-CCAAGTCACACAACTGGCTTT-3'.

Collection of mouse sperm and sodium bisulfite sequencing methylation analysis of the IG DMR:
Collection of mouse sperm was performed using a modified protocol based on the method described by TASH and BRACHO (1998). An aliquot of 2 µl was mixed with 2 µl of 10% formalin (Sigma, St. Louis) and analyzed under the microscope to control quality of purification. DNA was purified using a standard proteinase K and phenol–chloroform protocol. DNA from two or three males with the same Dnmt1 genotype was pooled for analysis.

The methylation pattern of the IG DMR in sperm was determined as described in TAKADA et al. (2002) with modifications. A minimum of two independent nested PCR reactions were done for each sodium-bisulfite-treated DNA sample to ensure representation of alleles. Products of each of these reactions were ligated and cloned independently using the TopoTA cloning kit (Invitrogen). A minimum of 10 clones were sequenced for each of the reactions. The sequencing was performed with BigDye Terminator (version 3.1) and analyzed on ABI 3730XL sequencers (Applied BioSystems). Maternal and paternal origin of the alleles was determined on the basis of genotypes of two SNPs between the MOLF and C57BL/6 DNAs (CROTEAU et al. 2005).

CEPH family genotypes for the chromosome 14q32 imprinted region:
The grandparental origin of the IG DMR in the 14q32 region was determined in 397 third-generation individuals from 51 three-generation CEPH families. Markers D14S65, rs1955897, D14S1426, D14S1006, D14S985, and D14S543, which are closely linked to the imprinted region, were chosen for this purpose (Figure 2B). The physical distance between markers D14S1426 and D14S985 is ~700 kb (Ensembl database http://www.ensembl.org); however, in the male-specific genetic map, they are assigned to the same genetic map position (100.71 cM; Marshfield maps, http://research.marshfieldclinic.org/genetics/GeneticResearch/compMaps.asp) (BROMAN et al. 1998) (Figure 2B). Genotypes of the members of the CEPH families for the 14q32 chromosomal region were determined by genotyping for microsatellite markers D14S1426, D14S611, and D14S118 or were acquired from public databases (HapMap database: http://hapmap.org; CEPH database: http://www.cephb.fr/cephdb; GenLink database: http://www.genlink.wustl.edu; and Marshfield database: http://research.marshfieldclinic.org/genetics/genotypingDataStatistics/CEPHFamilyGenoData.asp). For microsatellite genotyping, PCR reactions were performed using 10.0 ng of genomic DNA in a total volume of 8.0 µl containing 1.0 mM of MgCl2 (QIAGEN, Valencia, CA), 1x PCR buffer containing 1.5 mM of MgCl2 (QIAGEN), 50 µM of dNTPs (GE Healthcare), 0.04 unit/µl of HotstarTaq DNA polymerase (QIAGEN), and 150 nM of each primer. Touchdown PCR was initiated by denaturing the samples at 96° for 10 min followed by three cycles containing a denaturation at 94° for 30 sec, an annealing at 60° for 30 sec, and an extension at 72° for 60 sec. Second cycling corresponds to a denaturation at 94° for 30 sec, an annealing at 59° for 30 sec, and an extension at 72° for 60 sec, cycled two times. Principal cycling, repeated 35 times, corresponds to a denaturation at 94° for 30 sec, an annealing at 54° for 30 sec, and an extension at 72° for 60 sec. Final extension was done at 72° for 10 min. A reading mixture was prepared with 2 µl of PCR products, 0.15 µl of Genescan 500 Liz size standard (Applied Biosystems), and 8.5 µl of Hi-Di formamide (Applied Biosystems) and migrated on Applied Biosystems 3730xl DNA Analyzer. The genotypes were analyzed with Applied Biosystems Genemapper analysis program (Applied Biosystems, release 3.7, October 12, 2004). Grandparental origin of the IG DMR alleles in third-generation individuals was inferred on the basis of genotypes of the closest microsatellite markers, D14S1426, D14S1006, or D14S985. We assumed that the IG DMR genotype was the same as the genotype for D14S1426, D14S1006, or D14S985. The grandparental origin of the alleles in individuals who were not informative for D14S1426, D14S1006, and D14S985 was inferred on the basis of the grandparental origin of the flanking markers D14S611, D14S65, rs1955897, D14S1006, D14S543, D14S544, and D14S118 (Figure 2B). Several recombinant chromosomes were not genotyped for D14S985 or D14S1426. For several families we could not infer the grandparental origin of IG DMR alleles due to homozygosity of the parent for two of the three genotyped markers or similar genotypes of the grandparents. In total, grandparental origin genotypes were missing for the paternal chromosome of 43 CEPH offspring. To include these individuals in our analyses, we conducted the same analyses by assigning grandpaternal or grandmaternal origin to all unknown alleles.

Genotyping of parents for DNMT1:
Fathers from 51 CEPH families were genotyped for two SNP markers that reside within the DNMT1 locus. DNA samples were obtained from Coriell Cell Repositories. Genotyping was performed using the fluorescent polarization (FP)–single-base extension (SBE) assay (CHEN et al. 1999). PCR primers were designed using the FastPCR software (http://www.biocenter.helsinki.fi/bi/programs/fastpcr.htm). The PCR primers used were rs6511685_AT-F 5'-GCCGAGATCGTGCCACTG-3'; rs6511685_AT-R 5'-GGCGTCACGTGGCAATTCT-3'; rs7253062_AG-F 5'-GCAAGGGGTACCCACA CAG-3'; and rs7253062_AG-R 5'-GCACTGCACCGGCTATTGT-3' with SBE detection primers rs6511685_AT-P 5'-GATCTCATTGTCTAGAAATATACTAAACTAT-3'; rs6511685_AT-Pa 5'-CCCCACCTGGATTACTTTGTCCATA-3'; rs7253062_AG-P 5'-CCAGGAGTTTGCCTGGGAAATC-3'; and rs7253062_CT-Pa 5'-GCTACTAATTGTCAG TAATCAAATCAGACCA-3'. PCR reactions were carried out as follows: 3.0 ng of genomic DNA was added to an 8.0-µl reaction mixture containing 1.0 mM of MgCl2 (QIAGEN), 35 µM of dNTPs (GE Healthcare), 0.04 unit of HotstartTaq DNA polymerase (QIAGEN), and 200 nM of each primer. PCR was initiated by denaturing the samples at 95° for 10 min followed by 45 cycles of denaturation at 95° for 30 sec, annealing at 57° for 30 sec, and extension at 72° for 30 sec. Final extension was done at 72° for 7 min. PCR products were treated with exonuclease I and shrimp alkaline phosphatase as recommended by the manufacturer (AcycloPrime-FP SNP detection kit, Perkin-Elmer, Norwalk, CT). SBE detection primers were designed in both orientations for each SNP using FastPCR. FP–SBE reactions were performed in both orientations as suggested by the manufacturer (AcycloPrime-FP SNP detection kit, Perkin-Elmer). After addition of reading buffer, we read the plates using an Analyst HT reader (Molecular Devices) as described previously (HSU et al. 2001).

Statistical analysis:
Confidence intervals for transmission ratio distortion were estimated assuming the binomial distribution for the proportion of grandmaternal alleles transmitted (Tgm). For analyses of mouse crosses, logistic regressions predicting the grandparental source of inherited alleles were fit, including variables for sex, cross, and their interactions. The best model was chosen by using Akaike's information criterion (AIC), which assists in the comparison of non-nested models. For analyses of CEPH families, similarly, logistic regressions predicting grandparental source were fit, including variables for sex, the combined genotype at rs6511685 and rs7253062, and their interaction. The genotype at these two markers was coded as either homozygous at both loci or heterozygous at both loci; individuals heterozygous at only one locus were excluded from analysis. Reported P-values have not been corrected for multiple testing, but when multiple models were fit, we report the number of models examined.

Web resources:
The following web resources were used in this study: CEPH genotype database, http://www.cephb.fr/cephdb/; GenLink database, http://www.genlink.wustl.edu/; International HapMap Project, http://www.hapmap.org/; Mouse Genome Informatics database, http://www.informatics.jax.org/; Mouse imprinting database, http://www.mgu.har.mrc.ac.uk/research/imprinting; SNP database, http://www.ncbi.nlm.nih.gov/SNP/; and The Center for Human Genetics, Marshfield Clinic Research Foundation, http://research.marshfieldclinic.org/genetics/genotypingData_Statistics/CEPHFamilyGenoData.asp.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Loss of transmission ratio distortion of chromosome 12 alleles among the offspring of DNMT1 mutant mothers:
To determine if Dnmt1 mutations modify the transmission of grandparental chromosome 12 alleles in mice, we conducted backcrosses with heterozygous Dnmt1 mutant mothers. We determined the genotypes of the offspring for the distal region of chromosome 12 (microsatellite marker D12Nds2) and at the Dnmt1 locus. In this work, we used Tgm as a characteristic of transmission ratios.

Crosses involving maternal Dnmt1c and Dnmt1n mutations produced similar results (supplemental Table 1S at http://www.genetics.org/supplemental/). Therefore, the results from all crosses with maternal Dnmt1 mutations were combined (Table 2). To determine if the effect of Dnmt1 mutations on TRD depended upon the Dnmt1 genotype of the offspring or the genotype of the mother (maternal effect), we conducted two additional backcrosses between F1 wild-type mothers and C57BL/6 fathers that carried the Dnmt1n mutation. We observed that a slight excess of grandpaternal alleles was transmitted to female offspring in the DNM crosses; however, the deviation from 1:1 ratios was not statistically significant (95% C.I. 0.41–0.52). The DNFA + DNFB crosses displayed preferential transmission of grandmaternal alleles from mothers to daughters, as did the original crosses A and B (Table 2). However, unlike in the A and B crosses, the TRD was apparent among the male offspring, too.


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  		TABLE 2 Transmission of alleles of maternal grandparents to 3-week-old offspring in mouse crosses

 
We examined the data in Table 2 with three hypotheses in mind:
  1. The Dnmt1 mutations do not have an effect on TRD. The expectation here is that all three groups of crosses will show similar transmission ratios.
  2. The Dnmt1 mutation has a maternal effect on TRD. The expectation is that the DNM crosses will have different transmission ratios compared to A + B and DNFA + DNFB crosses.
  3. The effect of the Dnmt1 mutation is modulated by the Dnmt1 genotype of the offspring; i.e., it is offspring specific. The expectation is that DNM and DNF crosses will show similar transmission ratios, but will be different from A + B.

Our analysis of the data suggests that the second hypothesis may be true. We compared models that constructed particular comparisons among the six groups of Table 2 on the basis of the above hypotheses. The best model (based on AIC) contained an interaction between sex of offspring and an indicator for whether the Dnmt1 mutation was carried in the mother. This model suggests that Tgm in female offspring from DNM mothers is different from (i.e., lower than) Tgm in other crosses (DNM vs. other crosses interacting with sex of the offspring: P = 0.0196; DNM vs. other crosses: P = 0.0147). Therefore, the maternal Dnmt1 genotype is a modifier of grandparental-origin-dependent TRD of maternal alleles in the distal region of mouse chromosome 12. The model also suggests that the inheritance pattern in male offspring is different in the DNF cross compared to the DNM and A + B crosses.

The P-values presented have not been altered to account for multiple testing. We coded the comparisons in two ways corresponding to the hypotheses stated above, and then within each coding we evaluated whether interaction terms with sex were important. In total, we fit six different models and chose the best one via the AIC criterion.

Paternal DNMT1 mutation causes TRD in the mouse chromosome 12 distal region:
To further explore the modifying effect of Dnmt1 mutations on transmission ratios, we investigated the transmission of D12Nds2 alleles from fathers to offspring. We generated four crosses, including wild type (crosses C and D) and Dnmt1n/+ (crosses DNFC and DNFD) mice and genotyped the offspring from these crosses at the age of 3 weeks (Figure 2, Tables 1 and 3). The offspring were genotyped using microsatellite marker D12Nds2 that showed non-Mendelian transmission of maternal alleles. In our crosses, the wild-type mice show preferential transmission of D12Nds2 alleles from the paternal grandmother, which just fails to reach 95% significance level (Tgm = 0.54; P = 0.068). In contrast, the offspring of the mutant fathers more often inherit the alleles of the paternal grandfather (Tgm = 0.43; P = 0.016; Table 3), and the difference between these two Tgm estimates is strongly significant (P = 0.0027). In the DNFC and DNFD crosses, the paternal transmission ratios do not depend upon the Dnmt1 genotype or sex of the offspring (data not shown), suggesting a paternal effect of the mutation.


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  		TABLE 3 Transmission of alleles of paternal grandparents to offspring

 
mRNA levels of Dnmt1 isoforms in oocytes and testicular tubules of heterozygous mutant mice:
It has been previously demonstrated by others that the Dnmt1 mutations affect transcription and abolish the production of the DNMT1 protein in embryonic somatic cells (LEI et al. 1996). To determine if the Dnmt1n mutation affected the abundance of the gamete-specific RNA isoforms in heterozygous mice, we compared the RNA levels of the oocyte-specific (Dnmt1o) and the pachytene spermatocyte-specific (Dnmt1p) isoforms in mature oocytes and testicular tubules, respectively. Dnmt1 RNA levels were compared in samples from heterozygous mutant mice and their wild-type littermates using quantitative real-time PCR. The levels of the somatic-specific RNA isoform (Dnmt1s) were also assayed. In oocytes, the levels of Dnmt1o were lower in heterozygous mutant mice than in their wild-type littermates (Figure 3). The Dnmt1s mRNA isoform was not detected in oocytes. The average mRNA levels of the pachytene-specific Dnmt1p and the somatic Dnmt1s isoforms in the tubules of the five heterozygous mutant males were about half of those found in their four wild-type littermates (Figure 3). However, a high inter-individual variability in the Dnmt1 RNA levels was observed, rendering the difference between the two groups nonsignificant (supplemental Figure 1S at http://www.genetics.org/supplemental/). Thus, our data are consistent with an overall reduced transcription of Dnmt1 in the germ cells of heterozygous mice.


Figure 3
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  		FIGURE 3.— Reduced mRNA abundance of Dnmt1 isoforms in oocytes and testicular tubules of heterozygous mutant mice. Each graph represents the results of quantitative RT–PCR assays in oocytes or testicular tubules. The isoform assayed in each experiment is indicated in the top left corner of each graph. Solid bars correspond to RNA levels in heterozygous mutant mice; shaded bars correspond to RNA levels in wild type (wt) mice. The mean RNA levels in wild-type mice were assigned a 100% value and RNA levels in heterozygous mutant mice were calculated as a percentage of the wild-type levels.

 
Lack of major methylation defects in the IG DMR from sperm of Dnmt1n/+ males:
The imprinting control region that carries gametic marks in the distal part of mouse chromosome 12 is the IG DMR. Therefore, we tested the possibility that the maternal alleles of the IG DMR were hypomethylated in the sperm of Dnmt1 mutant males. The maternal alleles of these males had slightly lower methylation levels than the paternal alleles; however, this pattern was independent of the presence of the Dnmt1 mutation as it was similar to that found in sperm of the wild-type mice (Figure 4).


Figure 4
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  		FIGURE 4.— Methylation patterns of the IG DMR in sperm of heterozygous mutant males and their wild-type littermates. Each row corresponds to an allele with a specific methylation profile. Solid circles correspond to methylated CGs, and open circles correspond to unmethylated CGs. The number on the right shows the number of clones with the respective methylation pattern.

 
Lack of major differences between the mutant and wild-type littermates excludes abnormal methylation of the IG DMR as the molecular mechanism underlying TRD.

Transmission of 14q32 alleles in human families:
We examined the transmission of microsatellite marker alleles linked to the 14q32 imprinted region (homologous to the imprinted region of mouse chromosome 12) in three-generation CEPH families. Transmission of maternal alleles did not deviate from Mendelian 1:1 transmission ratios. However, a slight excess (Tgm = 0.469) of transmission of alleles from the paternal grandfather (Table 4) was detected. The excess of grandpaternal alleles was due to preferential transmission of alleles of the paternal grandfather to grandsons, but not to granddaughters (P = 0.0062). Interestingly, allelic inheritance patterns in human chromosome 14q32 bear resemblance to the patterns observed in mouse crosses involving Dnmt1 mutations (Figure 5).


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  		TABLE 4 Transmission of alleles linked to chromosome 14q32 imprinted region depends upon the genotype of the father

 

Figure 5
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  		FIGURE 5.— Comparison of grandparental-origin-dependent inheritance patterns in the homologous regions of human chromosome 14q32 and mouse chromosome 12. Solid areas indicate alleles of the grandmother, whereas open areas indicate the alleles of the grandfather. The percentage of transmitted alleles to offspring of both sexes is given.

 
To establish whether the DNMT1 genotype of the father influenced the transmission of his 14q32 alleles to his sons, we determined the genotypes of the fathers from 51 three-generation CEPH families for two intronic SNPs located within the DNMT1 locus. The two SNPs, rs6511685 and rs7253062, are part of a linkage disequilibrium block that spans the whole DNMT1 gene (INTERNATIONAL HAPMAP CONSORTIUM 2005; http://www.hapmap.org). Three DNMT1 haplotypes are found in the four world populations: AG (frequency in the CEPH families: 0.508), TA (corresponds to two extended haplotypes with combined frequency in the CEPH families: 0.441), and TG (frequency in the CEPH families: 0.05). None of the CEPH fathers was homozygous for the rare TG haplotype. The CEPH families were ascertained on the basis of the DNMT1 haplotype of the father, and transmission ratios of grandparental 14q32 alleles were determined for male offspring (Table 5). Among five genotypic groups, three showed preferential transmission of grandpaternal alleles. Next, we compared the transmissions of grandparental alleles among offspring of homozygous (AA/GG, TT/AA) and doubly heterozygous (AT/GA) fathers, as these were the groups with the largest number of informative families. A significant interaction was detected (P = 0.0034) between sex of offspring and heterozygosity/homozygosity of the father for the two DNMT1 SNPs in their influence on transmission of grandparental alleles. This result remains significant after using a Bonferroni correction for multiple testing; six different models were fit examining the interplay between DNMT1 haplotypes and gender.


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  		TABLE 5 Transmission of IG DMR alleles to male offspring and paternal DNMT1 genotype

 
In total, grandparental origin genotypes were missing for the paternal chromosome of 43 CEPH offspring. To evaluate the sensitivity of the test of interaction, we conducted the same analyses by assigning grandpaternal or grandmaternal origin to all unknown alleles. The unknown individuals did not change the transmission ratios, as the results remained statistically significant (P = 0.0044, when unknown genotypes were reassigned to be grandpaternal, and P = 0.0027, when unknown genotypes were reassigned to be grandmaternal).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Familial (in humans) or strain-specific (in mice) factors may influence allelic transmission ratios, suggesting that transmission depends upon the genetic background (GUMMERE et al. 1986; ALLEN et al. 1990; LYON 2003; RAKYAN et al. 2003); however, the identity of such genetic modifiers remains unknown. Here, we report that loss-of-function mutations in the gene encoding the maintenance methyltransferase, DNA (cytosine-5) methyltransferase 1 (Dnmt1), residing in the proximal region of mouse chromosome 9, modify transmission of alleles in the distal part of chromosome 12, an unlinked region. Therefore, our data implicate Dnmt1 as a trans-acting factor in the genesis of grandparental-origin-dependent TRD. We tested the effects of two mutations, Dnmt1c and Dnmt1n, on transmission of maternal alleles. The Dnmt1c is a complete loss-of-function mutation resulting from a deletion of two conserved motifs of the catalytic domain replaced by a Pgk-Neo cassette, whereas the Dnmt1n mutation is a partial loss of function mutation (LI et al. 1992; LEI et al. 1996). It was generated by a deletion of a part of the N-terminal regulatory domain of DNMT1 (LI et al. 1992). Although different parts of the gene were targeted in these knockout mice, both mutations completely (Dnmt1c), or almost completely (Dnmt1n), prevent transcription and expression of the DNMT1 protein in homozygous mice (LI et al. 1992; LEI et al. 1996; TRASLER et al. 1996) and therefore are likely to lead to a functional hemizygosity of heterozygous mice for Dnmt1. Interestingly, the Dnmt1n mutation generates multiple abnormal transcripts, most of which are not translated (LEI et al. 1996). The levels of whole-genome methylation are higher in Dnmt1n homozygous mutant mice compared to Dnmt1c mice (LEI et al. 1996); however, in our study, both mutations produced the same effect on maternal TRD, i.e., restored Mendelian 1:1 transmission ratios. DNMT1 is not considered to be a major player in the establishment of maternal genomic imprints because its somatic form is absent in the developing ovaries (RATNAM et al. 2002). The targeted mutation of the oocyte-specific isoform (Dnmt1°) has no effect on methylation of imprinted genes in oocytes, but it is essential for the maintenance of genomic imprints in eight-cell-stage embryos (HOWELL et al. 2001). Therefore, it is possible that maternal Dnmt1 mutations correct abnormal hypermethylation that occurs on the grandpaternal alleles of some of the embryos. The reduced Dnmt1o mRNA abundance in the oocytes of heterozygous mutant mice is consistent with this hypothesis and suggests that embryos from heterozygous mutant mothers are exposed to a reduced supply of Dnmt1 during preimplantation development. This reduced supply, however, does not affect the overall embryonic viability, as litter sizes are similar to those from wild-type mothers, but most likely modifies survival chances of embryos that carry certain alleles.

The Dnmt1n mutation present in the father induces grandparental-origin-dependent TRD of paternal alleles among his offspring. Again, as with the transmission of maternal alleles from wild-type females (CROTEAU et al. 2002), the TRD favors the transmission of alleles inherited from the grandparent of the same sex as the transmitting parent. We show that the average mRNA levels of both the Dnmt1p and Dnmt1s isoforms in the tubules of heterozygous mutant mice are lower than in wild-type mice. The mechanism by which a reduced supply of paternal DNMT1 may affect transmission ratios is not clear, because DNMT1 is not implicated in the establishment of paternal methylation patterns. This point of view is based on the absence of the protein in meiotic spermatocytes, where Dnmt1 expression occurs from an alternative promoter and the product is believed to be a nontranslated RNA (MERTINEIT et al. 1998). It would be interesting to know if paternally derived transcripts or protein contribute to the establishment of embryonic methylation patterns. An alternative possibility is that the effect of Dnmt1n mutation on TRD is mediated through the multiple abnormal transcripts that are generated from the mutant allele. In principle, such transcripts might be transmitted at the time of fertilization and target the normal Dnmt1 alleles of the embryo. Such a mechanism would be similar to the one described for the RNA-mediated epigenetic inheritance at the Kit locus in mice (RASSOULZADEGAN et al. 2006). Further analysis of the Dnmt1n mutation should clarify the mechanism involved.

Lack of methylation anomalies at the imprinting control region (IG DMR) of the chromosome 12 imprinted cluster suggests that either another DMR from the same domain or another imprinted domain is involved. The latter possibility is not an unlikely one because the distal part of chromosome12 harbors several imprinted regions, only one of which has been characterized in detail (TEVENDALE et al. 2006). Moreover, we cannot rule out the possibility that an epigenetic mechanism distinct from genomic imprinting is responsible for the grandparental origin effects on allelic transmission in the distal region of chromosome 12.

The transmission patterns of IG DMR alleles among the third-generation individuals in human CEPH families display a grandparental-origin-dependent and sex-of-offspring-specific TRD that favors inheritance of alleles of the paternal grandfather by his grandsons. Familial heterogeneity with respect to TRD depends upon the genotype of the father: heterozygous fathers do not show preferential transmission of their paternal alleles, whereas homozygous fathers preferentially transmit their paternal alleles to boys. This phenomenon should be taken into account when linkage analysis is used for genetic mapping in human families.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
The authors are grateful to Jacquetta Trasler and En Li for kindly providing the Dnmt1c mutant mice, to Makoto Nagano and Hugh Clarke for help with some of the methodologies, and to Andrei A. Verner for the artwork. This study was supported by a Canadian Institutes of Health Research (CIHR) operating grant (to A.K.N.) and by funding from Genome Canada/Genome Quebec and the Fraser endowment (to A.K.N.), the Ontario Genomics Institute (C.M.T.G.), and Genome Canada (C.M.T.G.). A.K.N. was supported by a CIHR New Investigator Award and a John R. and Clara M. Fraser Memorial Award of the Faculty of Medicine of McGill University.


   FOOTNOTES
 
1 Present address: Neurovascular Unit, National Research Council Canada, Institute for Biological Science, Ottawa, ON K1A 0R6, Canada. Back


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Communicating editor: T. R. MAGNUSON





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