1 Department of Epidemiology, Biostatistics and Occupational Health, Faculty of Medicine, McGill University, Montréal, Québec, Canada
2 Biostatistics Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC
Correspondence to Dr. Claire Infante-Rivard, Department of Epidemiology, Biostatistics, and Occupational Health, Faculty of Medicine, McGill University, 1130 Pine Avenue West, Montréal, Québec, Canada H3A 1A3 (e-mail: claire.infante-rivard{at}mcgill.ca).
Received for publication March 14, 2005. Accepted for publication June 1, 2005.
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ABSTRACT |
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alleles; factor XIII; fetal development; fetal growth retardation; genomic imprinting; polymorphism, genetic; thrombophilia
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INTRODUCTION |
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We carried out a study of the relation between thrombophilia and intrauterine growth restriction. Thrombophilia is a condition characterized by a tendency to develop thrombosis, mostly as a consequence of inherited polymorphisms (4). The most often studied thrombophilic polymorphisms include the C677T and A1298C variants in the methylenetetrahydrofolate reductase (MTHFR) gene; the G1691A variant of the factor V gene, now known as factor V Leiden; and the G20210A variant in the prothrombin (or factor II) gene. Other common polymorphic genes involved in the thrombotic process, such the plasminogen activator inhibitor-1 (PAI-1) gene and the factor XIII gene (5
), have been studied much less often. In the PAI-1 gene, a single nucleotide insertion/deletion polymorphism has been identified 675 base pairs upstream from the transcription site, leading to a sequence of four or five guanine nucleotides; it is referred to as the "4G/5G polymorphism." The 4G allele and the 4G/4G genotype have been associated with increased PAI-1 activity in plasma (6
). On the factor XIII gene, a G-to-T transition in exon 2 results in the substitution of leucine for valine at amino acid position 34; the polymorphism is identified as Val34Leu. It is located near the thrombin activation site and has been associated with an altered rate of factor XIII activation and an abnormal fibrin clot structure (7
). Both polymorphisms have been associated with outcomes such as coronary heart disease; the carriers of the 4G/4G genotype are at increased risk, while those with the Leu/Leu genotype seem to be protected (5
).
We had postulated that thrombophilic polymorphisms would lead to placental insufficiency, which is known to be an underlying cause of intrauterine growth restriction (8). This hypothesis was not supported by our data (9
, 10
). However, in studying the transmission of the variant alleles from parents to offspring, we had not considered the possibility of parent-of-origin effects; without explicit consideration of this possibility, paternal (maternal) effects could be diluted by noncontributory maternal (paternal) meioses. The functional activity of some genes or chromosomal regions is unequal and dependent on whether they have been inherited maternally or paternally. This epigenetic phenomenon is termed "genomic imprinting." The activity or silence of an imprinted gene or chromosomal region is set during gametogenesis but can be reset during stages of fetal life. Although only some imprinted genes have been identified to date, the known ones play an important role in the regulation of fetal growth and development in mammals (11
). This phenomenon is believed to have arisen in placental mammals because of competition between the somewhat asymmetric reproductive interests of the mother and the father (12
, 13
), where the father's interests may be advanced by promoting growth and those of the mother advanced by restricting growth. Some 600 genes are known to be imprinted, and the placenta is formed primarily under regulation of paternally expressed genes governing fetal access to nutrients, hence growth. Imprinting may also have an impact on other areas that may influence nutrient supply, such as fetal and maternal blood flow and transport of the nutrients themselves (12
). A role for particular allelic variants of a paternally expressed gene in the etiology of intrauterine growth restriction would be evidenced by an apparent distortion in transmission of the allele from heterozygous fathers to affected offspring. No distortion in transmission from mothers would be seen.
A nonrandom segregation of chromosomes during meiosis is termed "segregation distortion" or "meiotic drive" (14). Deviation from the expected Mendelian proportions in surviving offspring in the population at large can also be due to postmeiotic mechanisms (such as pregnancy loss), and the overall phenomenon is referred to as "transmission-ratio distortion" (14
). Little is known about transmission distortion in humans; most examples have occurred among rare variants (15
). Embryonic loss can be partly due to genetic factors. This form of selection would result in preferential transmission (via survival) of fitter genotypes to liveborn offspring (15
). To our knowledge, a formal evaluation of transmission-ratio distortion with the more frequently studied thrombophilic polymorphisms has not been published. However, because some of these polymorphisms are rare (such as factor V Leiden and factor II) and are known to affect function (5
), it is relevant to evaluate this possibility.
Our objective in this study was to investigate parent-of-origin effects in the transmission of common thrombophilic alleles to intrauterine growth-restricted newborns and transmission-ratio distortion for the same alleles in unaffected newborns.
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MATERIALS AND METHODS |
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Maternal and umbilical cord blood were collected from all case dyads. Approximately midway through the study, we started collecting buccal swabs from fathers. We obtained genetic material on 258 case fathers and 248 control fathers, representing a response rate of approximately 86 percent (of those invited to participate).
The project was approved by the hospital's ethics committee. An informed consent form was signed by the mother for collection of umbilical cord and maternal blood and by the father for collection of his own genetic material.
Genotyping
The presence of the MTHFR C677T and A1298C, factor V Leiden G1691A, and prothrombin G20210A gene polymorphisms was determined using polymerase chain reaction allele-specific oligonucleotide hybridization assays (9). Genotyping of the Val34Leu polymorphism on the factor XIII A-subunit was carried out according to the method reported by Elbaz et al. (17
). For genotyping of the PAI-1 gene, the region containing the 4G/5G polymorphism was amplified by polymerase chain reaction as previously described (18
); the products of amplification were visualized by allele-specific oligonucleotide hybridization assays (19
). The membranes were read using PhosphoImager (Molecular Dynamics, Sunnyvale, California) and an automatic scanning program; they were also read visually by two independent observers and again by three observers together. Disagreements remaining after this step were resolved by reamplification, digestion by appropriate restriction enzymes, and gel electrophoresis.
Statistical analysis
To evaluate parent-of-origin effects in the transmission of alleles, we used the likelihood-based approaches proposed by Weinberg (20). Weinberg et al. (21
) proposed an analytic approach based on extending a log-linear model to allow the relative risk conferred by a single inherited copy of an allele to depend on the parent of origin of that allele (note, however, that the model given in the original paper was overparameterized and requires reduction). Weinberg later cautioned that if the gene under study is in linkage disequilibrium with a different susceptibility allele (is a marker), the former log-linear method may not be strictly correct (20
), because the risk can then depend on the parental genotypes, even after conditioning on the inherited genotype. For this reason, she proposed a somewhat more robust alternative. The alternative method considers the three mating types in which the mother and the father carry unequally many copies of the variant alleles, with further stratification on the number of copies of the allele inherited by the child (20
). Like Weinberg, we will call this latter method the "parent-of-origin likelihood ratio test" (PO-LRT), whereas we will refer to the first one as the "log-linear likelihood ratio test" (LL-LRT).
Both models account for maternally mediated in-utero effects (maternal genotype effects on the pregnancy phenotype) which could otherwise confound the assessment of parent-of-origin effects (20). The LL-LRT approach has the advantage that although it is slightly less robust, it provides separate estimates of the relative risk conferred by a maternally derived versus a paternally derived single inherited copy of the variant allele, whereas the PO-LRT provides only the ratio of two relative risks. PO-LRT and LL-LRT are implemented in LEM software (22
), which also allows for missing parental genotype data, using an expectation-maximization algorithm.
Both methods presume that the trios in the population at large exhibit Mendelian proportions, so that, for example, parents who are both heterozygous will produce a child who is a homozygous carrier of the variant allele with probability 0.25, and so on. All transmission-based methods rely on this key assumption, against which one can estimate relative risks and test for apparent excess transmission of a putative susceptibility allele to affected offspring. If the allele under study affects gestational survival, this key assumption can break down; consequently, for a reproductive endpoint like intrauterine growth restriction, evaluation of apparent rates of transmission to unaffected children can enable the investigator to verify that the data support the implicit assumption that the inherited genotype is not related to prenatal attrition.
The log-linear model also provides estimates of the relative risks for maternally mediated genetic effects. These correspond to effects on the fetus that are secondary to the effect of the maternal genotype on the maternal phenotype during pregnancy. We estimate S2 and S1, which correspond to the relative risk for a mother with two copies or one copy of the variant allele, respectively, relative to a mother with no copies. These are estimated among cases by assuming mating symmetry in the population at large, making use of the phenomenon that if mothers with two copies have increased risk of intrauterine growth restriction, then couples in which the mother carries two copies will be overrepresented among parents of cases, according to a pattern that is simple to characterize mathematically. There are corresponding relative risks R1 and R2 for the inherited fetal genotypes, where these are estimated on the basis of mathematical comparison against the Mendelian null pattern of inheritance. (See the article by Weinberg et al. (21) for further details.) The issue of parent-of-origin effects has to do with whether we need two different versions of R1, depending on whether a single inherited copy came from the father or the mother.
Although Weinberg et al. (21) did not include parameters corresponding to interaction between the mother's and the newborn's genotypes, these were considered by Sinsheimer et al. (23
). If one is ready to consider maternally mediated effects, it seems reasonable to think that there might also be interaction between the mother's and the newborn's genotypes. We tested a model including R1, R2, S1, S2, a maternal imprinting parameter (IM), and four interaction variables corresponding to the following mother-child genotype combinations (1 = 1 copy of the variant and 2 = 2 copies): 1-1, 1-2, 2-1, and 2-2. To test for interaction, we compared results from this model with those from a model without the interaction terms using a 4-df likelihood ratio test.
We checked for transmission-ratio distortion or departure from Mendelian transmission proportions in the unaffected control families. We again used a log-linear model implemented in the LEM software (22). Because the variant alleles have a low prevalence and homozygous variants are uncommon, the model applied was a gene-dosage model (where the ß2 coefficient for two copies of the variant in the child is assumed to be equal to 2 x ß1, the coefficient for one copy; that is, the relative survival rate for offspring with two copies is the square of the relative survival rate for those with one copy). For complete data, this yields a test asymptotically equivalent to the transmission disequilibrium test.
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RESULTS |
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Parent-of origin effects
Based on families with an affected newborn, both methods used (LL-LRT and PO-LRT) suggested an excess of paternal transmission and a deficit of maternal transmission of the Leu allele of the factor XIII gene to intrauterine growth-restricted newborns. A maximum of 488 case trios were included in the analysis of the factor XIII gene (482 mothers, 222 fathers, and 458 newborns were successfully genotyped). Table 1 shows the results for the factor XIII gene; relative risks associated with newborn alleles, maternal alleles, and maternal imprinting are given, as well as the results of likelihood ratio tests for imprinting. The parameter IM indicates the multiplicative parent-of-origin effect for a maternally derived copy, and 1/IM would be the corresponding imprinting parameter for a paternally derived copy. As parameterized, when IM is less than 1, the paternally derived copy of the allele is associated with greater risk (or less protection) than a maternally derived copy. A simple way to think of this is that IM is the ratio of the relative risk for a single maternally derived copy to the relative risk for a single paternally derived copy. With respect to factor XIII, we found no interaction between maternal and newborn genotypes (likelihood ratio test (4 df): 2 = 0.87; p = 0.92 (data not shown)).
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Transmission-ratio distortion in unaffected newborns
For the study of transmission-ratio distortion in unaffected families, a maximum of 471 control trios were included in the analysis of the factor V Leiden variant (471 mothers, 243 fathers, and 461 newborns). For the factor II variant, that maximum number was also 471 (471 mothers, 248 fathers, and 460 newborns). The maximum number for MTHFR A1298C was 467 (463 mothers, 233 fathers, and 457 newborns). Finally, the maximum number for the PAI-1 variant was 465 (466 mothers, 158 fathers, and 459 newborns). Because of the relative infrequency of informative trios in which the offspring was homozygous-variant, we used a parsimonious model where . This permits a Wald-type test for the single parameter or a 1-df likelihood ratio test based on the log-linear model. In studying transmissions to unaffected offspring, the parameter R1 is interpretable as the relative likelihood of surviving to birth without the condition under study for a fetus with one copy, relative to a fetus with no copies.
As table 2 shows, we detected no distortion in the transmission of the Leu allele of the factor XIII gene, the 4G allele of the PAI-1 gene, or the T allele of MTHFR C677T. However, there was evidence for transmission distortion with the variants of factor V Leiden and factor II and, to a lesser extent, with MTHFR A1298C. For all three genes, the variant allele was transmitted less often than expected among unaffected newborns.
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DISCUSSION |
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There is ample evidence suggesting that factor XIII (24) is involved in the thrombotic process and some evidence showing that the studied polymorphism changes the function of factor XIII (25
); however, the link between the studied variant and clinical thrombotic diseases is still uncertain. Moreover, the contribution of this polymorphism to intrauterine growth restriction is unexplored. Our own association studies with this polymorphism showed negative and similar results with both a case-control design and a family-based design (10
). However, we had not considered parent-of-origin effects.
A parental conflict hypothesis has been put forward by Haig and Westoby (26) to explain why imprinting occurs in mammals. It predicts that the paternal genome promotes growth, whereas the maternal genome limits it, with asymmetric expression achieved through epigenetic silencing of one of the two copies of the inherited allele. One example is the common class I variable nucleotide tandem repeat polymorphism in the insulin gene, where paternal transmission evidently confers susceptibility to obesity in the offspring (27
). In addition to an effect on the placental surface area for nutrient exchange, imprinting may have an impact on other functions, such as transport of nutrients and fetal and maternal blood flow (12
). In our data, excess paternal transmission of the Val34Leu allele of factor XIII, as compared with maternal transmission, was evident in the small babies. The relative risk of intrauterine growth restriction associated with paternal inheritance of that variant is estimated to be 1.69. Given the role of factor XIII, this finding may support the hypothesis that imprinting has an impact on blood flow.
Transmission distortion in unaffected subjects (resulting from an excess or a deficit in transmission of particular alleles) seems more widespread than believed in humans (15). Using the full potential of our data with the expectation-maximization algorithm, we observed a significant deficit in transmission among unaffected newborns for polymorphisms such as factor V Leiden, factor II, or even MTHFR A1298C, whose functions are well known (5
). These observations are particularly interesting in light of the inconsistency of results for these thrombophilic genes on adverse pregnancy outcomes (28
). In the face of true transmission distortion in trios with unaffected offspring, in settings where genetic population stratification can be neglected, the case-control comparisons give a more valid test than methods based on cases and their parents, because the trio methods rely on an assumption that Mendelian proportions would be seen in the population at large. Case-control methods do not rely on Mendelian transmission from parents to surviving children, so they should be much preferred when studying a gene with an allele related to fetal survival.
Alternatively, if a study includes both case-parent trios and control-parent trios, one can use the log-linear model to compare transmission rates and estimate relative risks without the need to assume Mendelian transmission in the population. When we used this technique to examine effects of the factor V Leiden variant, the estimated relative risk was 3.59 (95 percent confidence interval: 1.37, 9.41), suggesting that inheritance of this variant is associated both with fetal death and with intrauterine growth restriction among those fetuses that survive.
However, this is a complex picture; other investigators have reported that certain genotype combinations for MTHFR C677T and A1298C (combinations with three or four mutant alleles) are not found in live newborns, whereas they are found among spontaneously aborted fetuses (29). The authors proposed this as evidence that certain genotype combinations carry a selection disadvantage and lead to compromised viability. Removal of some fetuses with poor genotype combinations could lead to surviving intrauterine growth-restricted cases with more neutral combinations, and could explain why we do not find strong evidence for transmission distortion among affected surviving offspring.
In conclusion, we report novel results suggesting that a variant allele in a thrombophilic gene (factor XIII) may have a parent-of-origin effect and that there is departure from Mendelian transmission proportions among unaffected offspring for variants in other thrombophilic genes (factor V Leiden, factor II, and MTHFR A1298C). The implications of this study are complex, and the results will need replication.
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ACKNOWLEDGMENTS |
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The authors thank Drs. Emily Kistner and Dmitri Zaykin for their helpful comments.
Conflict of interest: none declared.
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References |
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