Diagnosis of twin zygosity by mailed questionnaire

A deterministic questionnaire method for zygosity determination is developed for use in epidemiological studies of adult twins. It is based on the answers of both members of a twin pair to two questions on similarity and confusion in childhood. The algorithm of the method is used to determine the zygosity status of a twin pair at two different levels of certainty. The validity of the method is tested by making blood marker determinations of 11 polymorphic marker systems fro a random sample of 104 twin pairs. The agreement between questionnaire and blood marker diagnosis was 100%, but the stricter level of certainty left 8.7% in the nonclassified group. The genetical representativeness of the sample is tested by the allele distribution of the markers as compared to the Finnish population data as well as by the distribution of the number of intra-pair differences in blood markers.     

Bias of heritability estimates from twin data in presence of errors of zygosity determination

Simple heritability estimators of continuous as well as discrete traits from twin data are known to overestimate the degree of genetic determination of the measured traits for several reasons. Errors of zygosity determination will, however, underestimate the true heritability. The bias due to wrong assignment of dizygous twin pairs into monozygous type is evaluated here, and the results indicate that this negative bias has a compensatory effect on the estimate of the degree of genetic determination when other factors of similarity between twin pairs are taken into account. It is shown that when an estimate of zygosity error is available, the bias due to this factor can be evaluated quantitatively, and hence the adjustment for zygosity error can be incorporated in the estimation of the degree of genetic determination of a trait. Although this theory is explicitly developed here for twin studies, the general principle also applies for other types of errors of determining the degree of biological relationships for estimation of heritability, in which case this type of error may be more important than the simple zygosity error.     

Determination of twin zygosity: a comparison of DNA with various questionnaire indices.

This study examined cross-validation and test-retest reliability of questions and questionnaire indices commonly used for twin zygosity classification. Mothers of 58 monozygotic (MZ) and 52 dizygotic (DZ) same sex twin pairs were interviewed by telephone to answer questions regarding the similarity of their twins (mean age = 14.6 +/- 2.8 years). A logistic regression equation correctly classified 91% of both MZ and DZ twin pairs in our sample using 7 of the 12 zygosity questions. The internal consistency for the total questionnaire (Cronbach's alpha) was 0.88. The median two month temporal stability estimate for the individual questions was r = .56 and r = .79 for the test total. For the cross-validation, zygosity classification indices taken from 9 previous studies were applied to our sample and compared to classification according to DNA microsatellite analyses (agreement range = 44 to 100%). The accuracy of the classification indices was significantly lower than the original studies for 62% of the comparisons. If zygosity determination with DNA markers or blood group typing for all subjects is not feasible, rather than using classification indices based on other studies, an optimal classification scheme can be achieved by using a zygosity questionnaire of which the reliability and validity of the questions is established in a random subsample of the same twin cohort.     

The influence of zygosity and chorion type on fat distribution in young adult twins consequences for twin studies.

An adverse intra-uterine environment has been associated with abdominal fat distribution in singletons. Twins often have a low birth weight and a short gestation. Therefore, they may have an increased risk to develop abdominal obesity. Furthermore, monozygotic monochorionic twins (MZ MC) have a larger intra-pair birth weight difference compared to monozygotic dichorionic twins (MZ DC). If adult anthropometry is programmed in utero, this may affect the intra-pair correlations in adulthood and, consequently, also the results from the classic twin method to estimate genetic and environmental influences. In the present study, we compared the absolute values, the intra-pair differences, and the intra-pair correlations of body mass, height, BMI, and abdominal fat distribution of 424 MZ MC, MZ DC and dizygotic (DZ) twin pairs (aged 18-34 yrs). DZ, MZ DC and MZ MC twins did not differ for most anthropometric characteristics. Only MZ women tended (p = 0.03) to accumulate more abdominal fat compared to DZ twins. Overall, the contribution of zygosity and chorion type to adult anthropometry was rather low (< or = 1.7%). Although the intra-pair birth weight difference of MZ MC pairs (10.5% in men, 12.3% in women) was significantly larger compared to that of MZ DC pairs (6.9% and 9.2% resp.), the intra-pair differences in adult anthropometry were similar for both MZ twin types. Also the intra-pair correlations of MZ MC and MZ DC pairs were strikingly alike, suggesting no significant influence of the prenatal environment on adult concordance. In conclusion, the substantial difference in the prenatal environment of MZ MC and MZ DC twins did not result in a difference in intra-pair concordance of adult anthropometry and fat distribution. Therefore, we suggest that the chorion type of MZ twins does not bias the twin design and the estimation of the genetic contribution to adult anthropometry.     

Twins, chorionicity and zygosity.

Accurate determination of zygosity and chorionicity is essential in all multiple maternities. The parents and the multiples themselves ask it. It is of medical importance and now considered as a prerequisite in several domains of twin research, especially when perinatal data are analysed. It helps the multiples and their parents and teachers to ascertain identity. The methods are briefly described and a plea is made to obstetricians and paediatricians to use them systematically at the time of birth.     

Fingerprints and the determination of zygosity in twins

Zygosity was determined in 183 pairs of twins living in Aberdeen City, Scotland, by blood group studies, general appearance and fingerprints. The usefulness of simple methods of fingerprint analysis to determine zygosity was examined and it was found that comparisons using ridge counts were more efficient than those of pattern types.     

A twin study of mitochondrial DNA polymorphisms shows that heteroplasmy at multiple sites is associated with mtDNA variant 16093 but not with zygosity

The mitochondrial theory of ageing proposes that damage to mitochondria and diminished mitochondrial DNA (mtDNA) repair are major contributors to cellular dysfunction and age-related diseases. We investigate the prevalence of heteroplasmy in the mtDNA control region in buccal swab and blood derived samples for 178 women from the TwinsUK cohort (41 DZ pair 39 MZ pairs, 18 singletons, mean age 57.5 range 28–82) and its relationship to age, BMI and fasting insulin and glucose serum levels. The overall estimated prevalence of heteroplasmy for both tissues in the control region measured for 37 sites was 17%. The prevalence of heteroplasmy was higher among the older half of the study subjects than in the younger half (23% vs 10% p<0.03), primarily reflecting the increase in the prevalence of a heteroplasmic dinucleotide CA repeat in variable region II (VRII) with age. The VRII 523–524 heteroplasmic site (heteroplasmic in 25 subjects) was also associated with a decrease in BMI. In addition, concordance rates for common heteroplasmy were observed to be near complete for both dizygotic (DZ = 94%) and monozygotic twin pairs (MZ = 100%), consistent with previous reports that suggest variation in heteroplasmy rates between generations are determined by bottlenecks in maternal transmission of mitochondria. Differences in the prevalence of heteroplasmy were observed overall between samples derived from buccal swabs (19%) and blood (15%, p<0.04). These were particularly marked at position 16093 of hypervariable region I (HVI, 7% vs 0%, respectively, p<4×10⁻¹¹). The presence of the C allele at position 16093 in blood was associated with the presence of heteroplasmy in buccal swabs at this position (p = 3.5×10⁻¹⁴) and also at VRII (p = 2×10⁻⁴) suggesting a possible predisposing role for this site in the accumulation of heteroplasmy. Our data indicate that BMI is potentially associated with control region heteroplasmy.     

Influence of maternal-fetal histocompatibility and MHC zygosity on maternal microchimerism

To investigate the relationship between maternal-fetal histocompatibility and maternal microchimerism, we developed a sensitive quantitative PCR assay for the neomycin resistance gene (neoR), and, in a mouse model system, used neoR as a noninherited maternal allele marker of maternal cells to detect and quantitate maternal microchimerism in tissues of neoR(-/-) N2 backcross progeny of (neoR(+/-))F(1) females mated with neoR(-/-) males. Using this approach, we obtained evidence for the presence of chimeric maternal cells in the brain, spleen, and thymus of all weanling and adult mice so tested. The numbers of chimeric maternal cells present in the spleen did not differ significantly from those in the thymus regardless of age or maternal-fetal histocompatibility. At all ages, brain tissue had higher level of maternal microchimerism than lymphoid tissue in mice MHC identical with their mothers, but the levels were similar in mice MHC disparate with their mothers. The levels of chimeric maternal cells in both brain and lymphoid tissue of mice with homozygous syngenicity and maternal allogenicity were similar, and tended to be higher than tissue-specific levels in mice with either combined maternal-fetal allogenicity or heterozygous syngenicity. Thus, MHC homozygous progeny had higher levels of maternal microchimerism than MHC heterozygous progeny. We conclude that normal mice possess small numbers of maternal cells in spleen, thymus, brain, and probably most other tissues, and that maternal-fetal histocompatibility influences the levels of these cells by mechanisms related to MHC zygosity of the progeny.     

MHC zygosity in the frog,Rana temporaria

The major histocompatibility complex (MHC) zygosity of the field-collected frogs, Rana temporaria, was detected by progeny testing. Groups of sibling tadpoles were grafted with intrafamilial tail-tip allografts and the ratio of rapidly rejected allografts to slowly rejected ones was estimated. Twenty-five percent of parental frogs appeared to be MHC homozygotes. Thus, MHC homozygosity in natural frog populations seems to be considerably higher than in wild mouse populations.     

Differences in protocols of craniofacial development related to twinship and zygosity

Using 56 adult dental diameters as a subsystem model for craniofacial development, we show that monozygotic (MZ), dizygotic (DZ), and singleton groups differ significantly in developmental relationships assessed by multivariate statistical methods under commonly accepted assumptions. Given the differences observed, we suggest that any assumption of developmental equivalence between MZ and DZ twins, or between twins of either group and singletons, for variables of craniofacial or behavioral development, may be subject to serious doubt. Implications for twin study theory and methodology, and for study of early human development, are discussed.     

Why zygosity of multiple births is not always obvious: an examination of zygosity testing requests from twins or their parents.

This paper examines why parents of twins or adult twins themselves request zygosity testing. Of 405 multiples including 8 sets of triplets, the majority (93%) were monozygotic. Age of testing ranged from 0 days to 73 years. About 50% of requests came from parents or twins who were curious about, or expressed a need to be certain of, their zygosity. Other reasons included health concerns (current or future), other twins in the family, and misinformation about zygosity, frequently because of the erroneous assumption that all dichorionic twins are dizygotic. Parents of monozygotic twins may expect their twins to be 'identical' and believe their twins to be dizygotic because of minor phenotypic differences between them. Dizygotic twins like other siblings may share a phenotypic resemblance. Health professionals should be aware that zygosity of multiples may not always be obvious to parents and that accurate knowledge of zygosity may be justified.     

Rapid and accurate determination of zygosity in transgenic animals by real-time quantitative PCR

Successful identification of homozygous and heterozygous transgenic animals with currently available techniques demands tedious and time-consuming procedures with a high proportion of ambiguous results. Real-time PCR is a quantitative and extremely precise method with high throughput that could be applied to the analysis of large numbers of animals differing only by a factor of two in the amount of target sequences. We defined the technical conditions of real-time PCR to co-amplify a transgene and a reference gene using two fluorogenic probes and the comparative cycle threshold method. We applied these conditions to the analysis of zygosity in a line of transgenic rats. Real-time PCR allowed clear-cut identification of all transgenic animals analysed (n=45) as homozygous or heterozygous. Southern blot analysis of these animals using an internal quantitative control and PhosphorImager quantification showed ambiguous results in six of them and was concordant with real-time PCR in the rest. Mating of homozygous and heterozygous animals, as defined by real-time PCR, showed transgene transmission to the offspring following expected Mendelian laws. Real-time PCR allows rapid, precise, non-ambiguous and high throughput identification of zygosity in transgenic animals. This technique could be helpful in the establishment of breeding programs for transgenic colonies and in experiments in which gene dosage effects could have a functional impact.