Human Molecular Genetics, 2000, Vol. 9, No. 18 2767-2775
© 2000 Oxford University Press
Gender of the embryo contributes to CAG instability in transgenic mice containing a Huntington’s disease gene
1Departments of Molecular Pharmacology and Experimental Therapeutics, 2Health Sciences Research, Section of Biostatistics, 3Biochemistry and Molecular Biology and 4Molecular Neuroscience Program, Mayo Clinic and Foundation, Rochester, MN 55905, USA
Received 11 August 2000; Revised and Accepted 14 September 2000.
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ABSTRACT |
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Gender is known to influence the transmission of trinucleotide repeats in human disease. However, the molecular basis for the parent-of-origin effect associated with trinucleotide repeat expansion is not known. We have followed, during transmission, the fate of the CAG trinucleotide repeat in a transgene containing the exon 1 portion of the human Huntington’s disease (HD) gene. Similar to humans, the mouse transmits expansions predominantly through the male germ line. Surprisingly, we find that the CAG repeat size of the mutant human HD gene is different in male and female progeny from identical fathers. Males predominantly expand the repeat whereas females predominantly contract the repeat. In contrast to the classic definition of imprinting, CAG expansion is influenced by the gender of the embryo. Our results raise the possibility that there are X- or Y-encoded factors that influence repair or replication of DNA in the embryo. Gender dependence in the embryo may explain why expansion in HD from premutation to disease primarily occurs through the paternal line.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Huntington’s disease (HD) is one of eight progressive neurodegenerative disorders in which the underlying mutation is a CAG expansion encoding a polyglutamine tract (1–3). Many aspects of the mechanism for expansion are poorly understood. One particularly puzzling feature of expansion is the imprinting that is associated with transmission. It has been well documented that alterations in the length of trinucleotide repeat after transmission are distinctly dependent on the gender of the transmitting parent. For example, CAG expansion from premutation to disease range in HD occurs primarily through the paternal line (4,5). Similarly, large changes in repeat number are associated with paternal transmission in HD (1–3,6–9), spinocerebellar ataxia type 1 (SCA1) (10,11), dentatorubral–pallidoluysian atrophy (12,13), Machado–Joseph disease (14,15) and spinal and bulbar muscular atrophy (SBMA) (16). For all of these, the CAG repeat lies in the coding sequence of the gene. In contrast, large expansions in the non-coding region are associated with maternal transmission. These include fragile X syndrome (FRAX) (17–19), myotonic dystrophy (20,21) and SCA8 (22).
The molecular basis for the parent-of-origin effect associated with trinucleotide repeat expansion is poorly understood. For example, it is not clear whether the gender dependence arises in the germ cell or the embryo. Analysis of the repeat tract sizes in DNA derived from identical twins in affected families has not provided a definitive answer. In one FRAX family, a mother who carried a premutation tract length of 68 repeats gave birth to identical twins where mutant alleles had 1000–1600 repeats in one brother and 1400–1800 repeats in the other (23). These studies indicate that substantial expansion can occur post-zygotically. Tissue mosaicism, frequently observed in trinucleotide expansion disease patients (8,24) and in transgenic mouse models for the disorders (25,26), supports the notion that expansion and the observed gender dependence might arise, in part, in somatic cells. Although the germ cell cannot be excluded, it is possible that gender dependence and large expansions occur during a single intergenerational transmission in the early embryo.
However, recent studies of the FRAX locus reveal that CGG expansion from a premutation to the full mutation occurs in the germ cells of both male and female progeny, but efficient contraction to the premutation allele size occurs during male germ cell development (19). Consequently, males harboring expansions in their somatic tissues would pass on the premutation allele rather than the expanded allele in the next generation. Although expansion may have occurred in the early embryo, these studies suggest that contraction events during male germ cell proliferation may underlie the gender dependence of transmission in FRAX.
Factors governing the paternal transmission bias in CAG repeat diseases are less clear. The basis for the gender effect may be preferential expansion in the germ cell of the father. In support of this notion, instability in repeat sizes at disease loci in sperm of affected patients has been reported for HD, SCA1 and SBMA (24,27–29). A great variety of sizes in mature sperm have been reported to result from small mitotic changes that have built up in the primary spermatogonia over the lifespan of the affected individual (30). Furthermore, CAG repeat length in somatic tissues of four monozygotic HD twins were identical (27). The lack of variability during somatic growth has suggested that expansion occurs during gametogenesis and that events in the germ cell may mediate gender-dependent aspects of expansion disease. However, experimental data cannot definitively distinguish germ cell events from those in the early embryo.
To better understand the parent-of-origin effects in expansion, we followed paternal transmission of a single integrated copy of a transgene containing a CAG repeat in exon 1 of the human HD gene (hHD) (26) in R6/1 mice. We report that male progeny expand the repeat and female progeny contract the repeat even though the progeny arise from the same father with the same germ cell population. In contrast to the classic definition of imprinting, our results suggest that expansion and contraction during transmission are influenced by gender of the embryo. The bias towards expansion in the male embryos may be a predisposing factor contributing to paternal transmission of a disease allele from a premutation allele in HD.
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RESULTS |
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R6/1 transgenic mice harbor an hHD CAG expansion and have been shown to display both intergenerational and somatic instability (25,31). In these experiments, individual founder males that were hemizygous for the hHD mutation were bred with normal female partners in order to produce a large number of progeny. A representative pedigree is shown in Figure 1A. In this family, the founding male had 54 offspring with 11 of 24 males and 13 of 30 females carrying the mutation. For all founders, we typically observe in the offspring that 65–84% of the mutant CAG tracts differ in repeat number relative to the transmitting father (Table 1). The degree of instability observed in mice is similar to that observed during transmission among HD patients with expanded alleles (8,9,30). Similar to humans and the R6/2 mouse line (25), transmission through the male germ line frequently results in intergenerational expansion (Fig. 1). The size of the intergenerational expansion in the R6/1 line is typically 1–5 repeats. This number is close to the repeat change observed in paternal transmissions in HD patients (6,9,32). The average intergenerational repeat change reported for male transmissions in HD patients varies in different studies: 2.92 repeats (9), 3.98 repeats (32) and 9.0 repeats (6). Therefore, the mouse represents a reasonable model for exploring the intergenerational instability and gender dependence during male transmission.
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Expansion and contraction of the CAG repeat in transgenic mice depend on the gender of the embryo
Gender of the transmitting parent is known to be a factor influencing trinucleotide expansion in humans. Therefore, we tested the hypothesis that germ cells of the parent determined the repeat size in the progeny and accounted for the gender-dependent effects during male transmission in mice. Breeding experiments were designed so that: (i) male and female progeny in each line had the same father and, therefore, arose from the identical germ cell population; (ii) transmission of the mutant hHD allele always occurred through the male germ cell; and (iii) the process of zygote formation was constant in each line. In other words, male and female progeny always arose from a zygote formation comprising a sperm harboring the mutation and a normal oocyte. These criteria precluded germ cell selection and zygote selection as a source of imprinting effects.
If the germ cell determines the repeat size in the progeny, two predictions can be made: (i) male and female progeny from identical fathers will have the same distribution of repeat sizes since they arise from the same germ cell population; and (ii) the distribution of repeat sizes in the progeny will reflect the frequency found in the sperm of the father. If parental germ cells encode the repeat size that is transmitted to the progeny, then we should not observe repeat sizes in the progeny that are not found in the sperm of the father.
To test the first prediction, DNA was isolated from the tail of each offspring and the CAG repeat size was evaluated (Fig. 2). Since somatic expansion can occur between 6 and 30 weeks of age (25), tail DNA of the progeny was evaluated at 3 weeks to reflect only repeat alterations occurring during transmission. In contrast to our first prediction, we found that CAG repeats tend to expand in male and contract in female offspring even though the disease allele is inherited from the same father (Fig. 2A and C; family 3 in Table 1). Despite a difference in direction, neither the magnitude nor the frequency of the repeat changes significantly differed between male and female progeny (Fig. 2B and D). The frequency of instability in male and female progeny is almost identical [85.6 and 81.8%, respectively (Table 1)] and comparable to that observed in HD patients (8,9,30).
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We repeated the analysis in two additional pedigrees generated from two different male founders. Genescan images for one of these are shown (Fig. 3) and results from all three founder transmissions are summarized (Table 1). We found that males consistently exhibit a high degree of expansion although contractions are also present (Table 1). The majority of female animals from the same fathers exhibit contractions. Since all offspring arise from the same pool of sperm, the gender dependence of the repeat change cannot originate from events in the germ cell. Rather, the data support the hypothesis that gender-dependent changes in repeat length are influenced by events taking place post-zygotically in the embryo.
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Repeat distribution in progeny does not represent repeats in sperm of the transmitting father
We cannot determine precisely which sperm combined with the oocyte to generate a zygote. However, if the germ cell of the father provides the information for the repeat length in the progeny, then the second prediction should hold. The repeat size in the progeny should reflect the frequency and distribution of repeat sizes found in the sperm of the father. On the other hand, if repeat expansion and contraction are derived from events in the embryo, the distribution in the progeny might be skewed from that of the paternal sperm. To distinguish between these two possibilities, the male founders for two pedigrees were sacrificed after breeding at 15 weeks of age and their sperm was typed for repeat size distribution. The relationship of CAG repeat size between the mature epididymal sperm of transmitting father and his progeny is summarized (Table 2). Consistent with a prominent role for the embryo, we found that distribution of repeat lengths in the progeny did not faithfully reflect the distribution of repeats present in the sperm of the founding fathers. For example, in one transgenic family with a founding father with 117 repeats (Fig. 3; family 2 in Table 2), 11.9% of the founder sperm contained 116 repeats whereas 29% of the total progeny had 116 repeats. Thus, the contractions in the progeny were 2.5 times the expected frequency. Roughly 25% of the founder sperm was between 110 and 114 repeats yet there were no occurrences of progeny at these shorter repeat lengths. As expected from Table 1, the pattern in the progeny was more skewed relative to the paternal sperm when female and male offspring were analyzed separately (Table 2, male and female). Although the germ cell must transmit the repeat, expansion and contraction in the progeny is influenced by the gender of the embryo.
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An in-depth statistical analysis confirms that the entire distribution of repeat sizes in any progeny was significantly different from the distribution of repeats in its sire (Fig. 4). The analysis was performed by computing the Wilcoxon statistic Pr (parent > child) for each father–child pair (33). The statistic measures the probability that the father’s repeat count is greater than that of the child if a random cell were drawn from the parent and another from the child. Assuming that the two distributions are Gaussian, then Pr (parent > child) is directly related to the difference in the mean repeat counts. For non-Gaussian or variable width distributions, the Wilcoxon statistic provides a more general definition of one group being larger than the other (34). Adding half the probability of a tied value, Pr (parent = child), the resulting set of numbers should be randomly distributed near 0.5 if there is no expansion or contraction in effect. A representative data set for female progeny of one father is shown in Figure 4A. The distribution of repeats for any female progeny tends to be skewed to the left of the parent distribution and tends to have Wilcoxon probabilities smaller than 0.5. These data indicate that, for most of the female progeny, the probability of any repeat in the entire distribution within each female is smaller than its father. The analysis is valid since our data are not ordered or selected by their ‘significance’. Rather the data are tested as an ensemble with the results presented also as an unselected ensemble (35).
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The distribution of values for the four sire–progeny subsets is shown (Fig. 4B), along with the result of a one-sample t-test applied to each (36). The results reveal that females tend to have probabilities of <0.5, whereas males tend to have values of >0.5, consistent with contraction and expansion, respectively.
Repeat length in the paternal sperm does not change during the mating period
It has been reported that advancing age of the transmitting parent can influence repeat stability during transmission in some mouse models (26,37). Although our mice were young at breeding, the conclusion that gender-dependent alterations in repeat sizes occur post-zygotically rests on the assumption that no alterations in the sperm of the father occur during the mating period. In our studies, progeny in each large pedigree were produced from mating male R6/1 founders over a short period of 8 weeks (between 7 and 15 weeks of age). We therefore tested whether any alterations in repeat length occurred in the paternal sperm during this period. The sperm of individual male animals were evaluated after partial castration at two ages that spanned the mating period (Fig. 5A). In these experiments, one testis and epididymis was removed between 6 and 8 weeks, the animal was allowed to recover and the other testis and epididymis was removed between 9 and 15 weeks. The CAG repeat size present in the sperm from one epididymis at 6–9 weeks of age was not significantly different from that in the sperm evaluated in the other epididymis of the same male at a later time (9–15 weeks) for any animal tested (Fig. 5A). Furthermore, we observed no difference in the frequency of expansion and contraction in male and female progeny with age of the father between 7 and 15 weeks (Fig. 5B), similar to observations in humans (9). Gender dependence of the repeat size could not, therefore, arise from age-related repeat alterations in the sperm of the father during the mating period.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Imprinting is generally defined as a phenotype that depends on the gender of the transmitting parent and gender is known to influence the transmission of trinucleotide repeats in human disease. In HD, new disease mutations arise from premutation alleles (29–34 CAG repeats) by small expansions (n = 1–4 repeats) that are paternally transmitted (4,5). Once the disease range has been reached, expansion can be passed on in either male or female. However, larger expansions (n > 7 repeats) arise almost exclusively through paternal transmission (1–3,6–9). Because sperm from human HD males show greater repeat variability and larger repeat sizes than somatic tissues (24,27–29), it has been assumed that gender dependence of transmission in HD was contributed by germ cells of the transmitting parent. Although there is a clear role for the germ cells in transmitting the expansion mutation to the next generation, it is not at all clear that gender dependence associated with transmission is actually an imprinted event.
We report here that the repeat size can be influenced by the gender of the embryo. We have created conditions under which many male and female progeny arise from the same germ cell population in which the mutation is carried in the male. Distinct gender-dependent changes are observed in repeat number of the progeny on transmission under conditions where there are no corresponding changes in the germ cell of the father. These gender-dependent changes are observed at an early age (3 weeks) before somatic cell expansion takes place (25). Therefore, the mutations appear to arise during early embryogenesis. As discussed below, the gender dependence during transmission that we observe in mice is not the result of sperm selection and is independent of the germ cell of the transmitting parent.
Gender dependence of repeat length in the mice cannot arise from sperm selection since both expansions and contractions are observed in the progeny from the identical fathers. Thus, no negative selection occurs during gametogenesis since sperm containing both expanded and contracted alleles exist in the sperm population. Furthermore, if there were selection for repeat length in the sperm then it is expected that the shorter repeats would survive better and be over-represented in the progeny. However, we find that the opposite is true in males. Similarly, selection for repeat size does not occur at fertilization since zygote formation is identical in each mating. Gender-dependent differences are observed despite the fact that all of the mutant alleles are transmitted through the paternal sperm and all zygotes are formed by a union of a sperm carrying a mutant hHD allele and an oocyte lacking the transgene. Similarly, gender dependence cannot arise from differences in repeats present in sperm and oocytes since only male germ cells contain an expanded allele. Finally, our results cannot be explained by differences in sperm repeat size due to the age of the father at mating (Fig. 5) or differences in the X- and Y-bearing sperm. The latter would be evident as increases in expansions and contractions in N1 cells after replication and meiotic reduction compared with N2 progenitor cells. We find that expansions but not contractions occur during germ cell development (I.V. Kovtun, unpublished data). Therefore, we cannot explain the gender dependence of expansion and contraction in our experiment by events that occur in the germ cell. Rather, our results suggest that this gender dependence arises post-zygotically in the embryo.
In humans, the small number of offspring from any individual affected parent has precluded the type of analysis that we have been able to perform in mice. However, if human transmission is similar to that in the mouse, then gender dependence of expansion may also have contributions from the embryo. These results may be important with respect to new mutations in HD. The initial expansion from premutation or carriers to full disease mutation arises almost exclusively through paternal line. Male children of a father with an intermediate allele have a higher risk of developing HD relative to their female siblings (4). At the premutation length, even a small increase in repeat length can significantly increase the probability of expansion in the next generation. Therefore, it is possible that a gender bias towards expansion in the male embryo is a contributing factor underlying paternal transmission in HD. If gender dependence occurs in the early embryo, then our results raise the possibility that there may be X- or Y-encoded factors that influence expansion and contraction. Since expansion is associated with male progeny and contraction with female progeny, it is possible that an Y-encoded factor increases the likelihood of expansion or that an X-encoded factor suppresses expansion. Among the possibilities for X- or Y-linked factors are the gender-linked expression of replication/repair enzymes that play a role at early stages of embryo division. Alternatively, it is known that the direction of replication can influence the frequency of expansion and contraction (38). Therefore, there may be gender-linked factors that differentially influence the firing of the origin of replication in the male and female progeny. It is interesting to note that, if males and females utilize different origins of replication, expansion in the male and contraction in the female might suggest that the transgene is positioned such that direction of replication is opposite in the two genders. Future experiments must determine whether gender dependence and expansion arise from replication or repair of CAG repeats in the embryo.
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MATERIALS AND METHODS |
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Animals
Transgenic male B6CBA-TgN R6/1 mice (31) were purchased from the Jackson Laboratory (Bar Harbor, ME) and bred to negative B6CBA female partners. Litters were routinely screened for the presence of the hHD transgene by PCR (39).
Sperm isolation
The entire epididymis was excised from surrounding tissues and chopped in phosphate-buffered saline. Mature sperm cells were obtained by filtering the initial suspension through three layers of cheesecloth. The purity of the resulting sperm was evaluated by light microscopy.
CAG repeat sizing on gels
DNA was prepared from tail biopsies of animals at 3 weeks of age. Paternal sperm was collected from epididymis at 15 weeks of age. Tail or sperm DNA was prepared using a QIAamp Tissue kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The CAG repeat sizes were determined by PCR (39). In our procedure, 25 ng of DNA was amplified with specific primers, 5'-AAAAGCTGATGAAGGCCTTCGAG-3' and 5'-CGGCGGTGGCGGCTGTTG-3' (10 pM), using [32P]dCTP in 20 mM Tris–HCl, 10 mM (NH4)2SO4, 0.25 mM Mg(HSO4)2, 10 mM KCl, 0.2 mM dNTP, 0.1% Triton. The PCR products were resolved by electrophoresis on 6% polyacrylamide gel. Size of the products was evaluated by comparison with a sequencing ladder which was included on each gel. Gels were dried on a BioRad (Hercules, CA) 583 gel dryer and visualized by autoradiography using Kodak Biomax MR scientific imaging film.
Genescan analysis
To quantify CAG repeats, PCR was performed using identical conditions except that primer 1 was labeled with a fluorescence tag, [(3',6'-dipivaloyfluoresceinyl)-6-carboxamidohexyl]-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (6-FAM) (Glen Research, Sterling, VA). An internal size standard (2500-TAMPA; Perkin Elmer, Foster City, CA) was added to a 1.0 µl aliquot of amplified fluorescent product and resolved on a polyacrylamide gel. The traces of the PCR products were obtained and analyzed using GeneScan Analysis V3 (Perkin Elmer). Results from PCR analysis were accepted only if Genescan traces were reproduced in two to three independent reactions. The peak with the largest area was taken as the midpoint of the peak distributions in Genescan traces after normalization with respect to internal standards. Peak areas were calculated by program software.
Partial castration and intra-animal typing of sperm as a function of age
The CAG repeat sizes were evaluated as a function of age within individual animals by comparing sperm at two different ages by partial castration. In each male animal, one testis and epididymis was removed at an early age (either 6 or 9 weeks). The second was removed weeks later (between 9 and 15 weeks) and the intra-animal repeat pattern at DNA from both ages was evaluated by PCR and Genescan analysis (see above). This period spanned the mating interval of each male founder (between 7 and 15 weeks). All surgical procedures were performed by an approved animal protocol.
Statistical analysis
Statistical analysis of the Wilcoxon probabilities was performed as described in Results using S-PLUS (40). The comparison of the proportion with expansion/contraction between males and females was tested using Fisher’s exact test (36). Comparison of the collection of offspring with their sire was based on a one-sample t-test (36).
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ACKNOWLEDGEMENTS |
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We thank Drs Spiro, Trushina, Dyer, Gacy, Isaya, Maher and Toft for critical discussion. This work was supported by the Mayo Foundation, the Hereditary Disease Foundation, DK 43694-01A2 and MH-56207 from the National Institutes of Health, and IBN 9728120 from the National Science Foundation (to C.T.M.).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 507 284 1597; Fax: +1 507 284 9111; Email: mcmurray.cynthia@mayo.edu
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REFERENCES |
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