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Mechanisms for differences in monozygous twins
Early Human Development 64 (2001) 105 – 117 www.elsevier.com/locate/earlhumdev
Mechanisms for differences in monozygous twins Paul Gringrasa,*, Wai Chenb,c a
The Multiple Births Foundation, Hammersmith House, Level 4, Queen Charlotte’s and Chelsea Hospital, Du Cane Road, London W12 OHS, UK b Bloomfield Clinic, Department of Child and Adolescent Psychiatry, Guy’s, St. Thomas’s and King’s School of Medicine, Guy’s Hospital, St. Thomas Street, London SE1 9RT, UK c Centre of Social, Genetic and Developmental Psychiatry Research, Institute of Psychiatry, KCL London SE5, UK Received 1 July 2000; received in revised form 23 March 2001; accepted 14 April 2001
Abstract Monozygous (MZ) twins are often described as being physically and genetically identical. Clinical determination of zygosity relies on the assumption that any physical differences between a pair of twins imply they are dizygous. Most twin research relies on the assumption that dizygous twins share approximately 50% of the same genes, whereas monozygous twins share 100%. There is, however, increasing evidence to challenge both these assumptions. In this review, we describe a number of intrauterine effects and genetic mechanisms that may result in phenotypic, genotypic, and epigenetic differences between monozygous twins. Newer molecular techniques are resulting in such differences being increasingly commonly recognised. The potential for differences in monozygotic twin pairs is an important consideration for both clinicians and researchers involved in twin work. D 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Twin; Monozygous; Genetic mechanisms
1. Introduction Over 200 pairs of twins are assessed each year at the Multiple Births Foundation, London. Despite often appearing indistinguishable to strangers, no ‘identical’ twins assessed are so alike that their mothers fail to distinguish them accurately. Physical differences may be as subtle as one small mole, or a differently positioned hair crown; *
Corresponding author. Tel.: +44-208-383-3519. E-mail address: [email protected] (P. Gringras).
0378-3782/01/$ – see front matter D 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 3 7 8 2 ( 0 1 ) 0 0 1 7 1 - 2
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but still, they exist and are unmistakable once identified. Many parents can also differentiate their ‘identical’ twins by their personalities, some even claim from a very early age. Physical similarities between MZ twins are well recognised; and these similarities have long formed the basis of many instruments and clinical methods designed to classify zygosity, such as questionnaires and physical examinations. Even the most experienced practitioners can, however, ‘misclassify’ zygosity in about 6% of cases [1], and molecular genetic methods are now the preferred method for establishing zygosity [2]. The term ‘identical’—although frequently used—is not synonymous with ‘monozygous’ (MZ). Most MZ twins are phenotypically very similar, yet there are significant numbers of MZ pairs who are neither phenotypically nor genotypically identical. Even if one assumes a completely equal ‘apportioning’ of genetic endowment when twinning occurs, the twin pair will only remain identical if post-zygotic genetic, post-zygotic epi-genetic and post-zygotic environmental factors affect each twin equally. Given the extent of these influences and many potential opportunities for disruption during the long and complex intrauterine development, it is perhaps surprising that so many MZ twins do turn out to be so alike. Nevertheless, it is these anomalous cases of discordant twins that have taught us much about human genetics, development and twinning in the past. It is likely that they will continue to do so when new technologies are applied to future research in this area. This review summarises some past findings of well established studies, and also some from more recent exploratory studies using more experimental techniques and designs. We will first consider the ante-natal environmental factors and their effects, and then the genetic factors that contribute to discordance in MZ twins. Some examples of discordancy do not necessarily fit into the above neat categories. For convenience, they have been grouped together and discussed in the final section on ‘discordancies of unknown origin’.
2. Timing of monozygous twinning Monozygous (MZ) twinning occurs when one single fertilised egg gives rise to two separate embryos. The timing of this division can be an important contributory factor in determining the post-zygotic discordance in MZ twins. This timing can be characterised by the differences in amniotic sac, chorionic and placental anatomical formation [3]. In principle, the earlier twinning occurs, the less the twins will share common supportive structures; and the later, the more. The extreme example of late twinning are conjoint twins who even share some somatic organs. If twinning takes place prior to the first 4 days after conception, two separate placentas and sets of membranes are formed: that is, one set for each embryo. Such twins are called dichorionic (DC) MZ twins, and they account for about one third of all MZ twins. After the ‘fourth’ day, the progenitor cells of the placenta become separated from the inner cell mass of the embryo. As a result, for twinning occurring after this, only one single placenta will develop. This single monochorionic (MC) placenta serves both
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Table 1 Timing of twinning Days post conception
Amnionicity
Chorionicity
Frequency
0–4 4–8
Diamniotic Diamniotic
Dichorionic Monochorionic
8 – 12
Monoamniotic
Monochorionic twins
One-third of monozygous twins Approximately two-thirds monozygous twins Five percent of monozygous twins Conjoined twins
12 + a a
Timing for conjoint twins is theoretical and only suggested by animal models.
embryos, and in the majority of cases, contains anastomoses of blood vessels that connect the embryos. After about the eighth day, the MC MZ pair will share a common amniotic sac, in addition to the common MC placenta [4]. About 5% of MZ twins are monochorionic (MC) and monoamniotic (MA). Twinning after the second week results in the very rare phenomenon of conjoined twins (see Table 1). All MC twins are MZ by definition, and this is still the ‘gold standard’ when defining monozygosity. Although often seen in animals, vascular communications in dichorionic placentae in man are extremely rare [5]. The combination of monochorionicity and arterioarterial anastomoses is a better proof of monozygosity than any genetic test currently available. If placentation has not already been established by ultrasound in the first trimester, it relies on placental examination by pathologists; unfortunately, this still has not become routine clinical practice in most hospitals, despite numerous pleas in the literature [6,7].
3. Ante-natal environmental factors 3.1. Chorionicity, twin –twin transfusion syndrome and discordant birth weight Anastomotic connections between foetal circulations are present in around 90% of MC placentas. These anastomoses can result in the ‘twin to twin transfusion syndrome’ (TTTS) [8]. This can result either in a chronic ante-partum transfusion or acute intrapartum transfusion. In the former event, growth discordance occurs and there are risks for both the donor and recipient. These include the possibility of the donor becoming malnourished and growth retarded, while the recipient is at risk of cardiac hypertrophy, polycythaemia and hydramnios. In general, the mortality and morbidity rate for both twins in this situation is high without intervention [9]. The acute transfusion syndrome occurs intrapartum and causes increased mortality and morbidity, through both hypovolaemia and hypotension in one twin, and polycythaemia in the other. Even without TTTS, discordant birth weight in MZ twins remains common as a result of: (1) unequal in-utero blood supply, and hence growth; and perhaps (2) in theory, unequal division of inner cell mass at twinning. Although such differences may diminish
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with age, there is a growing body of evidence that significant discrepancy in birth weight may lead to long-lasting physiological changes in both twins. The concept of ‘foetal programming’ proposes that intrauterine growth affects long-term growth and metabolism in later life. Epidemiological studies linking low birth weight with hypertension and coronary artery disease in adult life suggest that undernutrition before birth ‘programmes’ later cardiovascular outcome [10]. Associations between ‘small for dates’ babies with later insulin resistance and cardiovascular disease are consistent with the hypothesis that late gestation may be a window of sensitivity to nutrition in terms of its influence on later cardiovascular disease. In twins discordant for the development of non-insulin dependant diabetes (NIDDM), birth weight has been found to be lower in the affected twin [11]. Investigators continue to use twins with discordant birth weight as a means to test the ‘foetal programming’ hypothesis, while assuming the twin pair would share common confounding variables such as social class, genetic endowment and post-natal environments. Two teams have recently reported the importance of birth weight in twins, independent of genetic differences, in influencing their blood pressure as adults [12]. Evidence for ‘foetal programming’ has even been found in early infancy: in a small cohort of MZ twins, where a twin – twin transfusion had occurred, differences in arterial distensibility were found in the donor twin when compared to the recipient [13]. Appealing though the findings from twin studies may be, the extent to which they are generalisable to singleton population is unknown. It is not fully understood if either the mechanism for growth discordancy or the responses to undernutrition differ between twins and singletons. 3.2. Chorionicity, cognition and personality Many twin studies have demonstrated the high heritability of cognitive abilities. It has been reported that MZ twins’ IQ test scores are almost as highly correlated as those obtained from testing and re-testing the same person. This finding is striking, given the very high test and re-test reliability of IQ tests [14]. Whether placentation in MZ twins affects cognitive function remains a controversial subject. MC MZ twins (where twinning occurs later when compared to DC MZ twins) have been suggested to be more similar cognitively and behaviourally than DC MC twins. Although there is some agreement that MC MZ twins are more alike in personality, the findings are not consistent for concordance in intellectual abilities [15]. Chorionicity (perhaps a surrogate marker for the timing of twinning) may be an important factor that underlies cognitive and personality differences between MZ twins, but plausible biological mechanisms are still yet to be elucidated. A prospective cohort study on a large MZ twin sample, where chorionicity are accurately and reliably determined, would be required to fully answer some of these questions. 3.3. Differential infections and teratogens Ascending infections (chorioamnionitis) are more likely to affect the lower gestation sac [3]. Differential congenital infections are thus probably more common than congenital infections, where both twins are equally infected. Infective agents include
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the usual list of toxoplasma, rubella, herpes and cytomegalovirus; all can adversely affect MZ twins to varying degrees. Where congenital HIV infections are concerned, birth order emerges as a major risk factor. Firstborn twins tend to contract HIV infection from their affected mothers more often than second born twins [16]. In cases where the infection is perinatally acquired during delivery, it has been suggested that the twin closer to the cervix may experience greater exposure to infected blood than twins located higher in the uterus. Maybe due to a higher rate of obstetric complications in births of twins or reasons not fully understood, the rates of HIV infection in twins are higher than that of singletons [17].
4. Genetic mechanisms It is a widely held belief that MZ twins are genetically identical; and that subsequent phenotypical discordances are attributable to environmental influences alone (shared or nonshared), altering and modifying the expression of the otherwise identical genetic endowment. Twin studies have long been used to estimate the genetic component in a range of conditions, from medical and psychiatric disorders to variations in somatic and psychological phenotypes. The central assumption of these studies is that MZ twins, formed from the division of a single fertilised egg, are genetically identical, whereas dizygous (DZ) twins, which result from two fertilised ova are genetically no more similar than siblings born from separate pregnancies. The degree of similarity between MZ pairs (concordance) over that of DZ pairs is taken as evidence of genetic contribution in the aetiology with respect to the phenotype studied. The validity of this assumption and the ability of twin studies to fully allow both ante-natal environmental effects and mutational genetic effects are still being debated [18]. There is now an ever-growing body of evidence that MZ twins are not always genetically identical. A number of phenotypic discordances in MZ twins have been demonstrated to be caused by genetic differences alone—that is, without having to invoke ante-natal environmental factors to account for their phenotypic differences. Furthermore, divergent epi-genetic modifications within a MZ twin pair, can lead to differential expression of inherited diseased genes. Genetically, MZ twins can be different at the level of (1) chromosomes or (2) DNA: 1. at the level of karyotype (cytogenetics)—i.e. the number or morphology of chromosomes may vary; and 2. at the level of molecular genetics—there may be (i) DNA mutations or (ii) epi-genetic modifications, such as differences in promoter region methylation, that suppress the expression of the DNA coding regions, and skewed X-chromosome inactivation by methylation ‘shut-down’ of the whole X chromosome. It is worth bearing in mind that the examples cited in this review are based on the findings detected by laboratory techniques currently available. This means the detected
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differences are limited by the resolution power of current technologies. As more advanced technologies are becoming available for more detailed genomic comparison and are applied to study MZ differences, it is possible that more MZ phenotypical discordance may be found to have a genetical or epi-genetical basis. 4.1. Heterokaryotypical divergence and chromosomal mosaicism MZ twins can have different chromosomal composition (heterokaryotypia). A well documented example for this is MZ discordance for gender and Ullrich – Turner syndrome (UTS)—that is, one twin is phenotypically male and the co-twin, a phenotypically female Turner [20]. This is thought to be the consequence of an early postzygotic mitotic error, resulting in heterokaryotic twinning that involves nondisjunction or anaphase lag of the Y chromosome. As a result, one twin is 47,XYY or 46,XY [20] or mosaic 45,X/46,XY [22] (resulting in a phenotypical male); and the co-twin is 45,X or mosaic 46,XY/45,X (resulting in a phenotypical Ullrich –Turner female). The timing of this mitotic nondisjunction determines the presence and degree of mosaicism in one or both twins. There have been more than nine reported cases of such discordant MZ twin. MZ discordance for Downs syndrome (trisomy 21) or Klinefelter syndrome (47,XXY) are uncommon, though Rogers et al. [23] reported a single case of MC MZ male twins who were discordant for trisomy 21. Interestingly, each was non-mosaic in fibroblast lines, but have mixture of cells in blood derived from 46,XY/47,XY,1 [21] cell lines. The theoretical explanation is that Downs and Klinefelter syndrome commonly result from a pre-zygotic nondisjunction, whereas UTS from post-zygotic nondisjunction pairs [21]. 4.2. Mosaicism, chimerism and pseudo-mosaicism For singletons, chimera is an organism with tissues of two or more different genotypes, often as a result of grafting or introduction of very early embryo stem cells from another genetically different individual(s). A chimera is often constructed experimentally in animal studies. In contrast to this, mosaicism denotes an organism comprising clones of cells with different genotypes (often caused by mutations or mitotic errors) deriving from the same zygote. For MZ twins, such a distinction is more problematic—as by definition, both embryos are derived from the same zygote. So strictly speaking, any genetic heterogeneity in MZ twins should be classified as ‘mosaicism’. However, some authors used the term ‘chimerism’ to describe transplantation of stem cells from one twin to its MZ co-twin, if genetic divergence has already occurred prior to twinning, i.e. twin A is subtly different from twin B genetically; and twin B has some stem cells from twin A in certain tissues. ‘Mosaicism’ is reserved to describe divergent cell lines as a result of mutations occurring within one twin after twinning. However, such distinction is not strictly applied within the twins literature. Often, it is difficult to make such distinction retrospectively. In monochorionic twins who share the same placenta, cross-placental transfusion enable blood and stem cells to be transferred from one twin to the other—due to
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placental vascular anastomoses. This results in pseudomosaicism or chimerism (i.e. transfer of the co-twin’s stem cell into the bone marrow during foetal development). For genomic comparison studies of monochorionic MZ twins, it is therefore important to conduct tissue (i.e. skin fibroblasts) genotyping, rather than relying on blood lymphocytes alone [24]. An example is the case report the pair of MC twins discordant for trisomy 21 by Rogers et al., as cited above. They found only one karyotypes in fibroblast cell lines from each twin (one with normal karyotype; another with Down’s karotype), but in blood, both karyotypes are found in both twins, indicating the presence of ‘chimerism’ in blood (and in bone marrow), but not in somatic cell lines. ‘Mosaicism’ can arise from mitotic error in early development—resulting in a mixture of cells with divergent karyotypes (e.g. a mixture of 45,X and 46,XY cell lines in the UTS co-twin of gender discordant MZ twin). In some cases, the presence of ‘mosaicism’ may vary in different tissues from one single individual. In such cases, multiple tissue cultures are required to establish mosaicism. Edwards et al. [25] reported a pair of MZ heterokaryotic twins with a UTS female and a normal male phenotype. The male, despite normal sexual development to age 21, had two skin fibroblast cultures and two lymphocyte culture showing a nonmosaic 45,X karyotype [25]. The authors concluded that their patient would probably show mosaicism, and if gonadal tissue was sampled, the presence of Y chromosomal material would be found. Reindollar et al. [22] reported another pair of MZ twins with a UTS female and a normal male. They examined two separate cultures of penile skin of the male co-twin, showing one single 45,X cell; with 49 cells of 46,XY; while the cultures of gonadal tissue showing 100% 46,XY. This suggests that low level ‘mosaicism’ may exist in the phenotypically normal co-twin in these cases. 4.3. Epi-genetic modification, methylation and skewed X-inactivation Methylation is an epi-genetic mechanism by which the expression of an sequence of DNA can be modified by becoming ‘silenced’ or ‘switched off’. This is achieved by attaching a methyl group to the cytosine nucleotide in CpG islands [26]. CpG islands are clusters of cytosine and guanine repeats, commonly found near a gene coding DNA region. Methylation of CpG islands adjacent to a gene often leads to its inactivation [27]. In addition to the ‘silencing’ of a specific gene, methylation is also involved in the whole-scale inactivation of the surplus X chromosome in human females—a process called X-inactivation or lyonization. In the majority of cases, X-inactivation is a random process. That is, both X chromosomes in a female have an equal chance of being ‘switched off’ by methylating the whole X chromosome. However, for reasons not yet fully understood, X-inactivation can be ‘skewed’ or ‘non-random’ in a minority of individuals, that is, the X chromosome of one parental origin is preferentially inactivated [28]. Some authors report that skewed X-activation is more common in female MZ twins than predicated by chance, though this view is still subjected to debate. They propose that skewed X-inactivation may give rise to clonal differences within the post-zygotic inner
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cell mass and may trigger the very process of twinning [29,30], thus accounting for an excess of females among MZ twins, especially among monochorionic MZ twins. This view remains controversial. Derom et al. [31] did not find an excess of female twins in an epidemiological study of multiple births. Along with the development of more robust technology in detecting skewed Xinactivation, a number of cases with non-random X-inactivation and MZ phenotypic discordance have been identified. They consist of expressed X-linked diseases in females. Examples reported include fragile-X syndrome [32], colour blindness [24], Duchenne muscular dystrophy [33], haemophilia B, G6PD deficiency, Aicardi’s syndrome, Hunter and Fabry’s disease in discordant female MZ twins [34]. The clinically affected twin has non-random inactivation predominantly of the X-chromosome carrying the wild gene, while the unaffected twin either has predominant inactivation of the Xchromosome carrying the mutant gene or has random X-inactivation. 4.4. Post-zygotic genetic mutation Theoretically, post-zygotic DNA mutation in discordant MZ twins can occur at the level of (1) (2) (3) (4)
Dominant genes; Recessive genes; Imprintable genes; or Genes involved in quantitative trait loci.
Confirmed and proven molecular genetic evidence are still lacking, maybe in part due to the limitation of currently available genomic comparison techniques. A number of technical innovations are currently under development, but not yet sufficiently mature for routine application. The authors are unaware of any report demonstrating proven DNA mutations that account for discordant phenotypes in MZ twins. However, there exist reports on MZ discordance for conditions transmitted by well documented autosomal dominant inheritance. Some include conditions in which imprinting is implicated. For autosomal dominant conditions, Vaughn et al. [35] reported a pair of MZ twins with neurofibromatosis, showing markedly different density of cafe´-au-lait spots, assuming the aetiology to be a de novo mutation. Easton et al. [36], however, found striking concordance of 0.97 correlation in the exact counts of neurofibromas for five of the six MZ twin pairs. A number of MZ twins discordant for the severity of tuberous sclerosis have been reported. Among those documenting the phenotypical differences, Brilliant et al. [37] demonstrated a single discordant hybridizing fragment using high-resolution Southern blotting technique. However, the discordant fragment has not been pinpointed on the specific gene sequence responsible for tuberous sclerosis. The molecular evidence therefore remains inconclusive. A number of reports on MZ twins concordant for Williams syndrome, also an autosomal dominant condition, found differences in timing and severity in the develop-
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ment of abnormalities, especially inguinal herniae and facial anomalies and other signs attributable to connective tissue abnormalities [38]. Wiedemann –Beckwith syndrome (WBS) is a congenital anomaly syndrome, consisting of exomphalos, macroglossia and gigantism, with an increased risk of developing rare embryonal cell tumours. Though autosomal dominant inheritance (preferentially through the mother) is well recognised [39], sporadic cases with paternal uniparental disomy of 11p15 loci may implicate that imprinting is involved in WBS. A number of discordant MZ twins for WBS have been reported, but the precise molecular mechanisms for their discordance have not been clearly established [40]. There are two recent reports examining MZ twins discordant for schizophrenia. Tsujta et al. [41] analysed genomic DNA of a pair of MZ twins discordant for schizophrenia by using two-dimensional electrophoresis display, following PCR and restriction enzyme digest. They used both methylation sensitive and non-sensitive enzymes. They found discrepancies of at least two spots out of approximately 2000 spots on the autoradiograms of the pair. Both of the spots were estimated to represent 300 – 400 base pairs. The study can be criticised for lacking specificity, validity as well as statistical power; and contamination by errors or artefacts cannot be ruled out. On the other hand, it can be regarded as providing preliminary evidence that MZ twins may differ at the levels of DNA or methylation. Smith et al. [19] used targeted genomic differential display method to evaluate, quantitatively, the level of genome similarity in a group of twin presented to them as ‘MZ twins’ concordant and discordant for schizophrenia. At the DNA level, they found three distinct patterns. One type had DNA similarity levels equivalent to that of unselected sibling pairs, suggesting these to be dizygous twins misidentified as monozygous twins. At the other extreme was a group with a very high level of similarity, suggesting these are true monozygous twins. The surprising finding was an intermediate group, lying between true monozygous and dizygous twins. They found all concordant twin pairs were true monozygotic. The mixed discordant and concordant twin pairs belonged to the intermediate group. They concluded that their findings brought into question a large number of experiments that depended on accurate twin zygosity determinations for measuring the heritability and penetrance of the condition.
5. Differences of unknown origin 5.1. Mirror twinning Mirroring in MZ twins is a fascinating, but poorly understood and poorly defined cause of phenotypic and perhaps behavioural discordance. It has been estimated to occur in 25% of MZ twins [42]. The spectrum extends from those twins with just oppositesided occipital hair whorls, to those where one twin has complete reversal of body organs (situs inversus). The term is perhaps most commonly used for those MZ twins with discordant handedness (right and left handedness). In itself, this suggests some degree of asymmetry of cerebral hemisphere dominance. Researchers have recently confirmed using functional magnetic resonance brain imaging on MZ twins with discordant
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handedness that hemispheric lateralisation is indeed also present [43]. The same team suggests that this finding may complicate interpretation of twin research in illnesses where cerebral lateralisation is important to their pathology (such as schizophrenia, dyslexia, autism, depression) [44]. 5.2. Hair, eyes and fingerprints Zygosity examinations and questionnaires rely on finding differences between twins. Although very minor degrees of phenotypic discordance have been recognised in MZ twins, it is apparent the spectrum is widening. Different hair and eye colour would automatically qualify for a DZ label in all established questionnaires, but both have recently been described in male MZ twins [45,46]. Putative mechanisms for such discordances range from unequal division of the inner cell mass, to post-zygotic mutations. It is likely that complex gene –environment interactions play a part in such cases. Similar mechanisms where the ante-natal environment impacts on a genetically predetermined trait may account for the fingerprint differences seen in MZ twins. Police departments have long been grateful for this phenomenon, which ensures that MZ twins cannot commit the ‘perfect crime’ so often presented in works of fiction. Forensic studies in fact show that although MZ twins have different fingerprints the pattern, ridge count and other minutiae are nevertheless much more similar than those seen in DZ twins [47]. Variation in vascular supply to each twin can occur during the second prenatal trimester— the critical period when fingertip dermal cells migrate to form ridges. This is also the period when neural cells migrate to the cortex and several investigators have tried to exploit this temporal relationship in an attempt to use fingerprints as marker for prenatal anatomical insults that may have affected the twins differently. 5.3. Major malformation discordances Foetal pathologists have long recognised discordances for major malformations in MZ twins [48]. Many of these malformations do not have simple genetic origins, but are likely to be multifactorial. Examples commonly described include neural tube defects, cleft lip and palate, and symmelia [4]. While it is not clear how these discordant events arise, in many cases, the apparently unaffected co-twin also has a less severe manifestation of the same disorder. The implication in these cases is that the predisposition for the malformation is expressed in both twins.
6. Summary This review highlights some of the important findings since Francis Galton, the father of twin research, first thought of using twins to estimate the impact of genes in 1876 [49]. His paper on the use of twins ‘as a criterion of the relative powers of nature and nurture’ launched the whole field, which has since seen major advances in twin foetal development and twin genetics. This review has demonstrated the problems in assuming that being monozygous is synonymous with being identical.
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Apart for academic interest and implications for twin research, these facts also have clear implications for clinical practice. In the case of a twin pair found to be discordant for a major malformation, the assumption should not be that they are automatically DZ. They may be MZ and, more importantly, MC. In a case of heterochromia iridum (different colour irises) in MZ twins described by St. Clair et al. [45], immunosuppressive therapy following a renal transplant was needlessly continued for 15 years because dizygosity had been assumed. In their case discussion, the authors of this paper emphasised that the heterochromia discordance must fall ‘within the acceptable spectrum of MZ status’. This review demonstrates that as our understanding of MZ twinning increases, the spectrum will continues to widen, and the term ‘identical twins’ should be replaced by the more accurate term ‘monozygous twins’. The importance of establishing the zygosity of twins has been stressed in the literature. Keith and Machin [50] have summarised many of the issues: ‘For reasons of personal rights to identity, medico-legal responsibility, potential for transplantation, early education, and concordance/discordance for genetic disease, many feel that routine determination of twin zygosity at birth is needed and should be implemented in the near future. Apart from the benefits to the twins and their parents, the consequences for research in twins and genetics would be enormous, since there remain many puzzling aspects of twinning.’ To this list is now also added the importance of establishing chorionicity, preferably by ultrasound in very early pregnancy.
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[14] R. Plomin, Nature and Nurture, 1990, Brooks/Cole, Pacific Grove, CA. [15] Sokol DK, Moore CA, Rose RJ, Williams CJ, Reed T, Christian JC. Intrapair differences in personality and cognitive ability among young monozygotic twins distinguished by chorion type. Behav Genet 1995;25: 457 – 66. [16] Goedert JJDAM, Amos C, Felton S, Bigger RJ. High risk of HIV-1 infection for first-born twins. Lancet 1991;338:1471 – 5. [17] Thomas PA, Ralston SJ, Bernard M, Williams R, O’Dornell R. Pediatric acquired imunodeficiency syndrome: an unusually high incidence of twinning. Pediatrics 1990;86:774 – 7. [18] Philips DIW. Twin studies in medical research: can they tell us whether diseases are genetically determined? Lancet 1993;341:1008 – 9. [19] Smith CL, Nguyen G, Bouchard J, Foulon C, Keith L. Twin zygosity and discordance for schizophrenia. Mol Psychiatry 1999;4(Suppl. 1):S29. [20] Kurosawa K, Kuromaru R, Imaizymi K, Nakamura Y, Ishikawa F, Ueda K, et al. Monozygotic twins with discordant sex. Acta Genet Med Gemellol 1992;41:301 – 10. [21] Perlman EJ, Steeten G, Tuck-Muller CM, Farber RA, Neuman WL, Blakemore KJ, et al. Sexual discordance in monozygotic twins. Am J Med Genet 1990;37:551 – 7. [22] Reindollar RH, Byrd JR, Hahn DH, Haseltine FP, McDonough PG. A cytogenetic and endocrinologic study of a set of monozygotic isokaryotic 45X/46XY twins discordant for sex: mosaicism versus chimerism. Fertil Steril 1987;47:626 – 33. [23] Rogers JG, Voullaire L, Gold H. Monozygotic twins discordant for trisomy 21. J Med Genet 1982;11:143 – 6. [24] Jorgenwen AL, Philip J, Raskind WH, Matsushita M, Christensen B, Dreyer V, et al. Different patterns of X inactivation in MZ twins discordant for red-green color-vision deficiency. Am J Hum Genet 1992;51: 291 – 8. [25] Edwards JH, Dent K, Kahn J. Monozygotic twins of different sex. J Med Genet 1996;3:117 – 23. [26] Razin A, Shermer R. DNA methylation in early development. Hum Mol Genet 1995;4:1751 – 5. [27] Cedar H. DNA methylation and gene activity. Cell 1988;53:3 – 4. [28] Goto T, Monk M. Regulation of X-chromosome inactivation in development in mice and humans. Microbiol Mol Biol Rev 1998;62(2):362 – 78. [29] Lupski JR, Garcia CA, Zoghbi HY, Hoffman EP, Fenwick RG. Discordance of muscular dystrophy in monozygotic female twins: evidence supporting asymmetric splitting of the inner cell mass in a manifesting carrier of Duchenne dystrophy. Am J Med Genet 1991;40:354 – 64. [30] Goodship J, Carter J, Burn J. X-inactivation patterns in monozygotic and dizygotic female twins. Am J Med Genet 1996;61(3):205 – 8. [31] Derom R, Orlebeke J, Eriksson A. The epidemiology of multiple births. In: Kurjac A, editor. Textbook of perinatal medicine. London: Parthenon Publishing; 1998. p. 1463 – 80. [32] Kruyer H, Mila M, Glover G, Carbonell P, Ballestra B, Estvill X. Fragile X syndrome and the (CGG)n mutation: two families with discordant MZ twins. Am J Hum Genet 1994;54:437 – 42. [33] Zneimer SM, Schneider NR, Richards CS. In situ hybridization shows direct evidence of skewed X inactivation in one of monozygotic twin females manifesting Duchenne Muscular Dystrophy. Am J Med Genet 1993;45:601 – 5. [34] Tiberio G. MZ female twins discordant for X-linked diseases: a review. Acta Genet Med Gemellol 1994; 43:207 – 14. [35] Vaughn AJ, Bachman D, Sommer A. Neurofibromatosis in twins: a case report of spontaneous mutation. Am J Med Genet 1981;8:155 – 8. [36] Easton DF, Cox GM, Huson SM, Ponder BJ. An analysis of variation in expression of neurofibromatosis (NF) type I (NFI): evidence for modifying genes. Am J Hum Genet 1993;53:305 – 13. [37] Brilliant MH, Cleve H, Edwards JH, Waldinger D. Cell lines from monozygotic twins discordant for tuberous sclerosis display protein and DNA differences. Am J Hum Genet 1990;47:A210. [38] Castorina P, Selicorni A, Bedeschi F, Dalpra L, Larizza L. Genotype – phenotype correlation in two sets of monozygotic twins with Williams Syndrome. Am J Med Genet 1997;69(1):107 – 11. [39] Orstavik RE, Tommerup N, Eiklid K, Orstavik KH. Non-random X chromosome inactivation in an affected twin in a monozygotic twin pair discordant for Wiedemann – Beckwith Syndrome. Am J Med Genet 1995; 56:210 – 4.
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[40] Leonard NJ, Bernier FP, Rudd N, Machin GA, Bamforth F, Bamforth S, et al. Two paris of male monozygotic twins discordant for Wiedemann – Beckwith Syndrome. Am J Med Genet 1996;61:253 – 7. [41] Tsujita T, Niikawa N, Yamashita H, Imamura A, Hamada A, Nakane Y, et al. Genomic discordance between monozygotic twins discordant for schizophrenia. Am J Psychiatry 1998;155(3):422 – 4. [42] Springer S, Searleman A. Laterality in twins: the relationship between handedness and hemispheric asymmetry for speech. Behav Genet 1978;8:349 – 57. [43] Somer IEC, Ramsey NF, Bouma A, Kahn RS. Cerebral mirror-imaging in a monozygotic twin. Lancet 1999; 354:1445 – 6. [44] Bruder GE. Cerebral laterality and psychopathology: perceptual and event-related potential asymmetries in affective and schizophrenic disorders. In: Davidson D, Hughdahl K, editors. Brain asymmetry. Cambridge: MIT Press; 1995. [45] St. Clair DM, St. Clair JB, Swainson CP, Bamforth F, Machin G. Twin zygosity testing for medical purposes. Am J Med Genet 1998;77:412 – 4. [46] Gringras P. Identical differences—monozygotic twins with different hair colour. Lancet 1999;353(9152):562. [47] Lin CH, Liu JH, Osterburg JW, Nicol JD. Fingerprint comparison: similarity of fingerprints. J Forensic Sci 1982;27(2):290 – 304. [48] Cameron AH, Edwards JH, Derom R, Thiery M, Boelaert R. The value of twin surveys in the study of malformations. Eur J Obstet Gynecol Reprod Biol 1983;14:347 – 56. [49] Galton F. The history of twins as a criterion of the relative powers of nature and nurture. R Anthropol Inst G B Irel J 1876;6:391 – 406. [50] Keith L, Machin G. Zygosity testing: current status and evolving issues. J Reprod Med 1997;42:699 – 707.
Mechanisms for differences in monozygous twins Paul Gringrasa,*, Wai Chenb,c a
The Multiple Births Foundation, Hammersmith House, Level 4, Queen Charlotte’s and Chelsea Hospital, Du Cane Road, London W12 OHS, UK b Bloomfield Clinic, Department of Child and Adolescent Psychiatry, Guy’s, St. Thomas’s and King’s School of Medicine, Guy’s Hospital, St. Thomas Street, London SE1 9RT, UK c Centre of Social, Genetic and Developmental Psychiatry Research, Institute of Psychiatry, KCL London SE5, UK Received 1 July 2000; received in revised form 23 March 2001; accepted 14 April 2001
Abstract Monozygous (MZ) twins are often described as being physically and genetically identical. Clinical determination of zygosity relies on the assumption that any physical differences between a pair of twins imply they are dizygous. Most twin research relies on the assumption that dizygous twins share approximately 50% of the same genes, whereas monozygous twins share 100%. There is, however, increasing evidence to challenge both these assumptions. In this review, we describe a number of intrauterine effects and genetic mechanisms that may result in phenotypic, genotypic, and epigenetic differences between monozygous twins. Newer molecular techniques are resulting in such differences being increasingly commonly recognised. The potential for differences in monozygotic twin pairs is an important consideration for both clinicians and researchers involved in twin work. D 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Twin; Monozygous; Genetic mechanisms
1. Introduction Over 200 pairs of twins are assessed each year at the Multiple Births Foundation, London. Despite often appearing indistinguishable to strangers, no ‘identical’ twins assessed are so alike that their mothers fail to distinguish them accurately. Physical differences may be as subtle as one small mole, or a differently positioned hair crown; *
Corresponding author. Tel.: +44-208-383-3519. E-mail address: [email protected] (P. Gringras).
0378-3782/01/$ – see front matter D 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 3 7 8 2 ( 0 1 ) 0 0 1 7 1 - 2
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but still, they exist and are unmistakable once identified. Many parents can also differentiate their ‘identical’ twins by their personalities, some even claim from a very early age. Physical similarities between MZ twins are well recognised; and these similarities have long formed the basis of many instruments and clinical methods designed to classify zygosity, such as questionnaires and physical examinations. Even the most experienced practitioners can, however, ‘misclassify’ zygosity in about 6% of cases [1], and molecular genetic methods are now the preferred method for establishing zygosity [2]. The term ‘identical’—although frequently used—is not synonymous with ‘monozygous’ (MZ). Most MZ twins are phenotypically very similar, yet there are significant numbers of MZ pairs who are neither phenotypically nor genotypically identical. Even if one assumes a completely equal ‘apportioning’ of genetic endowment when twinning occurs, the twin pair will only remain identical if post-zygotic genetic, post-zygotic epi-genetic and post-zygotic environmental factors affect each twin equally. Given the extent of these influences and many potential opportunities for disruption during the long and complex intrauterine development, it is perhaps surprising that so many MZ twins do turn out to be so alike. Nevertheless, it is these anomalous cases of discordant twins that have taught us much about human genetics, development and twinning in the past. It is likely that they will continue to do so when new technologies are applied to future research in this area. This review summarises some past findings of well established studies, and also some from more recent exploratory studies using more experimental techniques and designs. We will first consider the ante-natal environmental factors and their effects, and then the genetic factors that contribute to discordance in MZ twins. Some examples of discordancy do not necessarily fit into the above neat categories. For convenience, they have been grouped together and discussed in the final section on ‘discordancies of unknown origin’.
2. Timing of monozygous twinning Monozygous (MZ) twinning occurs when one single fertilised egg gives rise to two separate embryos. The timing of this division can be an important contributory factor in determining the post-zygotic discordance in MZ twins. This timing can be characterised by the differences in amniotic sac, chorionic and placental anatomical formation [3]. In principle, the earlier twinning occurs, the less the twins will share common supportive structures; and the later, the more. The extreme example of late twinning are conjoint twins who even share some somatic organs. If twinning takes place prior to the first 4 days after conception, two separate placentas and sets of membranes are formed: that is, one set for each embryo. Such twins are called dichorionic (DC) MZ twins, and they account for about one third of all MZ twins. After the ‘fourth’ day, the progenitor cells of the placenta become separated from the inner cell mass of the embryo. As a result, for twinning occurring after this, only one single placenta will develop. This single monochorionic (MC) placenta serves both
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Table 1 Timing of twinning Days post conception
Amnionicity
Chorionicity
Frequency
0–4 4–8
Diamniotic Diamniotic
Dichorionic Monochorionic
8 – 12
Monoamniotic
Monochorionic twins
One-third of monozygous twins Approximately two-thirds monozygous twins Five percent of monozygous twins Conjoined twins
12 + a a
Timing for conjoint twins is theoretical and only suggested by animal models.
embryos, and in the majority of cases, contains anastomoses of blood vessels that connect the embryos. After about the eighth day, the MC MZ pair will share a common amniotic sac, in addition to the common MC placenta [4]. About 5% of MZ twins are monochorionic (MC) and monoamniotic (MA). Twinning after the second week results in the very rare phenomenon of conjoined twins (see Table 1). All MC twins are MZ by definition, and this is still the ‘gold standard’ when defining monozygosity. Although often seen in animals, vascular communications in dichorionic placentae in man are extremely rare [5]. The combination of monochorionicity and arterioarterial anastomoses is a better proof of monozygosity than any genetic test currently available. If placentation has not already been established by ultrasound in the first trimester, it relies on placental examination by pathologists; unfortunately, this still has not become routine clinical practice in most hospitals, despite numerous pleas in the literature [6,7].
3. Ante-natal environmental factors 3.1. Chorionicity, twin –twin transfusion syndrome and discordant birth weight Anastomotic connections between foetal circulations are present in around 90% of MC placentas. These anastomoses can result in the ‘twin to twin transfusion syndrome’ (TTTS) [8]. This can result either in a chronic ante-partum transfusion or acute intrapartum transfusion. In the former event, growth discordance occurs and there are risks for both the donor and recipient. These include the possibility of the donor becoming malnourished and growth retarded, while the recipient is at risk of cardiac hypertrophy, polycythaemia and hydramnios. In general, the mortality and morbidity rate for both twins in this situation is high without intervention [9]. The acute transfusion syndrome occurs intrapartum and causes increased mortality and morbidity, through both hypovolaemia and hypotension in one twin, and polycythaemia in the other. Even without TTTS, discordant birth weight in MZ twins remains common as a result of: (1) unequal in-utero blood supply, and hence growth; and perhaps (2) in theory, unequal division of inner cell mass at twinning. Although such differences may diminish
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with age, there is a growing body of evidence that significant discrepancy in birth weight may lead to long-lasting physiological changes in both twins. The concept of ‘foetal programming’ proposes that intrauterine growth affects long-term growth and metabolism in later life. Epidemiological studies linking low birth weight with hypertension and coronary artery disease in adult life suggest that undernutrition before birth ‘programmes’ later cardiovascular outcome [10]. Associations between ‘small for dates’ babies with later insulin resistance and cardiovascular disease are consistent with the hypothesis that late gestation may be a window of sensitivity to nutrition in terms of its influence on later cardiovascular disease. In twins discordant for the development of non-insulin dependant diabetes (NIDDM), birth weight has been found to be lower in the affected twin [11]. Investigators continue to use twins with discordant birth weight as a means to test the ‘foetal programming’ hypothesis, while assuming the twin pair would share common confounding variables such as social class, genetic endowment and post-natal environments. Two teams have recently reported the importance of birth weight in twins, independent of genetic differences, in influencing their blood pressure as adults [12]. Evidence for ‘foetal programming’ has even been found in early infancy: in a small cohort of MZ twins, where a twin – twin transfusion had occurred, differences in arterial distensibility were found in the donor twin when compared to the recipient [13]. Appealing though the findings from twin studies may be, the extent to which they are generalisable to singleton population is unknown. It is not fully understood if either the mechanism for growth discordancy or the responses to undernutrition differ between twins and singletons. 3.2. Chorionicity, cognition and personality Many twin studies have demonstrated the high heritability of cognitive abilities. It has been reported that MZ twins’ IQ test scores are almost as highly correlated as those obtained from testing and re-testing the same person. This finding is striking, given the very high test and re-test reliability of IQ tests [14]. Whether placentation in MZ twins affects cognitive function remains a controversial subject. MC MZ twins (where twinning occurs later when compared to DC MZ twins) have been suggested to be more similar cognitively and behaviourally than DC MC twins. Although there is some agreement that MC MZ twins are more alike in personality, the findings are not consistent for concordance in intellectual abilities [15]. Chorionicity (perhaps a surrogate marker for the timing of twinning) may be an important factor that underlies cognitive and personality differences between MZ twins, but plausible biological mechanisms are still yet to be elucidated. A prospective cohort study on a large MZ twin sample, where chorionicity are accurately and reliably determined, would be required to fully answer some of these questions. 3.3. Differential infections and teratogens Ascending infections (chorioamnionitis) are more likely to affect the lower gestation sac [3]. Differential congenital infections are thus probably more common than congenital infections, where both twins are equally infected. Infective agents include
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the usual list of toxoplasma, rubella, herpes and cytomegalovirus; all can adversely affect MZ twins to varying degrees. Where congenital HIV infections are concerned, birth order emerges as a major risk factor. Firstborn twins tend to contract HIV infection from their affected mothers more often than second born twins [16]. In cases where the infection is perinatally acquired during delivery, it has been suggested that the twin closer to the cervix may experience greater exposure to infected blood than twins located higher in the uterus. Maybe due to a higher rate of obstetric complications in births of twins or reasons not fully understood, the rates of HIV infection in twins are higher than that of singletons [17].
4. Genetic mechanisms It is a widely held belief that MZ twins are genetically identical; and that subsequent phenotypical discordances are attributable to environmental influences alone (shared or nonshared), altering and modifying the expression of the otherwise identical genetic endowment. Twin studies have long been used to estimate the genetic component in a range of conditions, from medical and psychiatric disorders to variations in somatic and psychological phenotypes. The central assumption of these studies is that MZ twins, formed from the division of a single fertilised egg, are genetically identical, whereas dizygous (DZ) twins, which result from two fertilised ova are genetically no more similar than siblings born from separate pregnancies. The degree of similarity between MZ pairs (concordance) over that of DZ pairs is taken as evidence of genetic contribution in the aetiology with respect to the phenotype studied. The validity of this assumption and the ability of twin studies to fully allow both ante-natal environmental effects and mutational genetic effects are still being debated [18]. There is now an ever-growing body of evidence that MZ twins are not always genetically identical. A number of phenotypic discordances in MZ twins have been demonstrated to be caused by genetic differences alone—that is, without having to invoke ante-natal environmental factors to account for their phenotypic differences. Furthermore, divergent epi-genetic modifications within a MZ twin pair, can lead to differential expression of inherited diseased genes. Genetically, MZ twins can be different at the level of (1) chromosomes or (2) DNA: 1. at the level of karyotype (cytogenetics)—i.e. the number or morphology of chromosomes may vary; and 2. at the level of molecular genetics—there may be (i) DNA mutations or (ii) epi-genetic modifications, such as differences in promoter region methylation, that suppress the expression of the DNA coding regions, and skewed X-chromosome inactivation by methylation ‘shut-down’ of the whole X chromosome. It is worth bearing in mind that the examples cited in this review are based on the findings detected by laboratory techniques currently available. This means the detected
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differences are limited by the resolution power of current technologies. As more advanced technologies are becoming available for more detailed genomic comparison and are applied to study MZ differences, it is possible that more MZ phenotypical discordance may be found to have a genetical or epi-genetical basis. 4.1. Heterokaryotypical divergence and chromosomal mosaicism MZ twins can have different chromosomal composition (heterokaryotypia). A well documented example for this is MZ discordance for gender and Ullrich – Turner syndrome (UTS)—that is, one twin is phenotypically male and the co-twin, a phenotypically female Turner [20]. This is thought to be the consequence of an early postzygotic mitotic error, resulting in heterokaryotic twinning that involves nondisjunction or anaphase lag of the Y chromosome. As a result, one twin is 47,XYY or 46,XY [20] or mosaic 45,X/46,XY [22] (resulting in a phenotypical male); and the co-twin is 45,X or mosaic 46,XY/45,X (resulting in a phenotypical Ullrich –Turner female). The timing of this mitotic nondisjunction determines the presence and degree of mosaicism in one or both twins. There have been more than nine reported cases of such discordant MZ twin. MZ discordance for Downs syndrome (trisomy 21) or Klinefelter syndrome (47,XXY) are uncommon, though Rogers et al. [23] reported a single case of MC MZ male twins who were discordant for trisomy 21. Interestingly, each was non-mosaic in fibroblast lines, but have mixture of cells in blood derived from 46,XY/47,XY,1 [21] cell lines. The theoretical explanation is that Downs and Klinefelter syndrome commonly result from a pre-zygotic nondisjunction, whereas UTS from post-zygotic nondisjunction pairs [21]. 4.2. Mosaicism, chimerism and pseudo-mosaicism For singletons, chimera is an organism with tissues of two or more different genotypes, often as a result of grafting or introduction of very early embryo stem cells from another genetically different individual(s). A chimera is often constructed experimentally in animal studies. In contrast to this, mosaicism denotes an organism comprising clones of cells with different genotypes (often caused by mutations or mitotic errors) deriving from the same zygote. For MZ twins, such a distinction is more problematic—as by definition, both embryos are derived from the same zygote. So strictly speaking, any genetic heterogeneity in MZ twins should be classified as ‘mosaicism’. However, some authors used the term ‘chimerism’ to describe transplantation of stem cells from one twin to its MZ co-twin, if genetic divergence has already occurred prior to twinning, i.e. twin A is subtly different from twin B genetically; and twin B has some stem cells from twin A in certain tissues. ‘Mosaicism’ is reserved to describe divergent cell lines as a result of mutations occurring within one twin after twinning. However, such distinction is not strictly applied within the twins literature. Often, it is difficult to make such distinction retrospectively. In monochorionic twins who share the same placenta, cross-placental transfusion enable blood and stem cells to be transferred from one twin to the other—due to
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placental vascular anastomoses. This results in pseudomosaicism or chimerism (i.e. transfer of the co-twin’s stem cell into the bone marrow during foetal development). For genomic comparison studies of monochorionic MZ twins, it is therefore important to conduct tissue (i.e. skin fibroblasts) genotyping, rather than relying on blood lymphocytes alone [24]. An example is the case report the pair of MC twins discordant for trisomy 21 by Rogers et al., as cited above. They found only one karyotypes in fibroblast cell lines from each twin (one with normal karyotype; another with Down’s karotype), but in blood, both karyotypes are found in both twins, indicating the presence of ‘chimerism’ in blood (and in bone marrow), but not in somatic cell lines. ‘Mosaicism’ can arise from mitotic error in early development—resulting in a mixture of cells with divergent karyotypes (e.g. a mixture of 45,X and 46,XY cell lines in the UTS co-twin of gender discordant MZ twin). In some cases, the presence of ‘mosaicism’ may vary in different tissues from one single individual. In such cases, multiple tissue cultures are required to establish mosaicism. Edwards et al. [25] reported a pair of MZ heterokaryotic twins with a UTS female and a normal male phenotype. The male, despite normal sexual development to age 21, had two skin fibroblast cultures and two lymphocyte culture showing a nonmosaic 45,X karyotype [25]. The authors concluded that their patient would probably show mosaicism, and if gonadal tissue was sampled, the presence of Y chromosomal material would be found. Reindollar et al. [22] reported another pair of MZ twins with a UTS female and a normal male. They examined two separate cultures of penile skin of the male co-twin, showing one single 45,X cell; with 49 cells of 46,XY; while the cultures of gonadal tissue showing 100% 46,XY. This suggests that low level ‘mosaicism’ may exist in the phenotypically normal co-twin in these cases. 4.3. Epi-genetic modification, methylation and skewed X-inactivation Methylation is an epi-genetic mechanism by which the expression of an sequence of DNA can be modified by becoming ‘silenced’ or ‘switched off’. This is achieved by attaching a methyl group to the cytosine nucleotide in CpG islands [26]. CpG islands are clusters of cytosine and guanine repeats, commonly found near a gene coding DNA region. Methylation of CpG islands adjacent to a gene often leads to its inactivation [27]. In addition to the ‘silencing’ of a specific gene, methylation is also involved in the whole-scale inactivation of the surplus X chromosome in human females—a process called X-inactivation or lyonization. In the majority of cases, X-inactivation is a random process. That is, both X chromosomes in a female have an equal chance of being ‘switched off’ by methylating the whole X chromosome. However, for reasons not yet fully understood, X-inactivation can be ‘skewed’ or ‘non-random’ in a minority of individuals, that is, the X chromosome of one parental origin is preferentially inactivated [28]. Some authors report that skewed X-activation is more common in female MZ twins than predicated by chance, though this view is still subjected to debate. They propose that skewed X-inactivation may give rise to clonal differences within the post-zygotic inner
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cell mass and may trigger the very process of twinning [29,30], thus accounting for an excess of females among MZ twins, especially among monochorionic MZ twins. This view remains controversial. Derom et al. [31] did not find an excess of female twins in an epidemiological study of multiple births. Along with the development of more robust technology in detecting skewed Xinactivation, a number of cases with non-random X-inactivation and MZ phenotypic discordance have been identified. They consist of expressed X-linked diseases in females. Examples reported include fragile-X syndrome [32], colour blindness [24], Duchenne muscular dystrophy [33], haemophilia B, G6PD deficiency, Aicardi’s syndrome, Hunter and Fabry’s disease in discordant female MZ twins [34]. The clinically affected twin has non-random inactivation predominantly of the X-chromosome carrying the wild gene, while the unaffected twin either has predominant inactivation of the Xchromosome carrying the mutant gene or has random X-inactivation. 4.4. Post-zygotic genetic mutation Theoretically, post-zygotic DNA mutation in discordant MZ twins can occur at the level of (1) (2) (3) (4)
Dominant genes; Recessive genes; Imprintable genes; or Genes involved in quantitative trait loci.
Confirmed and proven molecular genetic evidence are still lacking, maybe in part due to the limitation of currently available genomic comparison techniques. A number of technical innovations are currently under development, but not yet sufficiently mature for routine application. The authors are unaware of any report demonstrating proven DNA mutations that account for discordant phenotypes in MZ twins. However, there exist reports on MZ discordance for conditions transmitted by well documented autosomal dominant inheritance. Some include conditions in which imprinting is implicated. For autosomal dominant conditions, Vaughn et al. [35] reported a pair of MZ twins with neurofibromatosis, showing markedly different density of cafe´-au-lait spots, assuming the aetiology to be a de novo mutation. Easton et al. [36], however, found striking concordance of 0.97 correlation in the exact counts of neurofibromas for five of the six MZ twin pairs. A number of MZ twins discordant for the severity of tuberous sclerosis have been reported. Among those documenting the phenotypical differences, Brilliant et al. [37] demonstrated a single discordant hybridizing fragment using high-resolution Southern blotting technique. However, the discordant fragment has not been pinpointed on the specific gene sequence responsible for tuberous sclerosis. The molecular evidence therefore remains inconclusive. A number of reports on MZ twins concordant for Williams syndrome, also an autosomal dominant condition, found differences in timing and severity in the develop-
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ment of abnormalities, especially inguinal herniae and facial anomalies and other signs attributable to connective tissue abnormalities [38]. Wiedemann –Beckwith syndrome (WBS) is a congenital anomaly syndrome, consisting of exomphalos, macroglossia and gigantism, with an increased risk of developing rare embryonal cell tumours. Though autosomal dominant inheritance (preferentially through the mother) is well recognised [39], sporadic cases with paternal uniparental disomy of 11p15 loci may implicate that imprinting is involved in WBS. A number of discordant MZ twins for WBS have been reported, but the precise molecular mechanisms for their discordance have not been clearly established [40]. There are two recent reports examining MZ twins discordant for schizophrenia. Tsujta et al. [41] analysed genomic DNA of a pair of MZ twins discordant for schizophrenia by using two-dimensional electrophoresis display, following PCR and restriction enzyme digest. They used both methylation sensitive and non-sensitive enzymes. They found discrepancies of at least two spots out of approximately 2000 spots on the autoradiograms of the pair. Both of the spots were estimated to represent 300 – 400 base pairs. The study can be criticised for lacking specificity, validity as well as statistical power; and contamination by errors or artefacts cannot be ruled out. On the other hand, it can be regarded as providing preliminary evidence that MZ twins may differ at the levels of DNA or methylation. Smith et al. [19] used targeted genomic differential display method to evaluate, quantitatively, the level of genome similarity in a group of twin presented to them as ‘MZ twins’ concordant and discordant for schizophrenia. At the DNA level, they found three distinct patterns. One type had DNA similarity levels equivalent to that of unselected sibling pairs, suggesting these to be dizygous twins misidentified as monozygous twins. At the other extreme was a group with a very high level of similarity, suggesting these are true monozygous twins. The surprising finding was an intermediate group, lying between true monozygous and dizygous twins. They found all concordant twin pairs were true monozygotic. The mixed discordant and concordant twin pairs belonged to the intermediate group. They concluded that their findings brought into question a large number of experiments that depended on accurate twin zygosity determinations for measuring the heritability and penetrance of the condition.
5. Differences of unknown origin 5.1. Mirror twinning Mirroring in MZ twins is a fascinating, but poorly understood and poorly defined cause of phenotypic and perhaps behavioural discordance. It has been estimated to occur in 25% of MZ twins [42]. The spectrum extends from those twins with just oppositesided occipital hair whorls, to those where one twin has complete reversal of body organs (situs inversus). The term is perhaps most commonly used for those MZ twins with discordant handedness (right and left handedness). In itself, this suggests some degree of asymmetry of cerebral hemisphere dominance. Researchers have recently confirmed using functional magnetic resonance brain imaging on MZ twins with discordant
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handedness that hemispheric lateralisation is indeed also present [43]. The same team suggests that this finding may complicate interpretation of twin research in illnesses where cerebral lateralisation is important to their pathology (such as schizophrenia, dyslexia, autism, depression) [44]. 5.2. Hair, eyes and fingerprints Zygosity examinations and questionnaires rely on finding differences between twins. Although very minor degrees of phenotypic discordance have been recognised in MZ twins, it is apparent the spectrum is widening. Different hair and eye colour would automatically qualify for a DZ label in all established questionnaires, but both have recently been described in male MZ twins [45,46]. Putative mechanisms for such discordances range from unequal division of the inner cell mass, to post-zygotic mutations. It is likely that complex gene –environment interactions play a part in such cases. Similar mechanisms where the ante-natal environment impacts on a genetically predetermined trait may account for the fingerprint differences seen in MZ twins. Police departments have long been grateful for this phenomenon, which ensures that MZ twins cannot commit the ‘perfect crime’ so often presented in works of fiction. Forensic studies in fact show that although MZ twins have different fingerprints the pattern, ridge count and other minutiae are nevertheless much more similar than those seen in DZ twins [47]. Variation in vascular supply to each twin can occur during the second prenatal trimester— the critical period when fingertip dermal cells migrate to form ridges. This is also the period when neural cells migrate to the cortex and several investigators have tried to exploit this temporal relationship in an attempt to use fingerprints as marker for prenatal anatomical insults that may have affected the twins differently. 5.3. Major malformation discordances Foetal pathologists have long recognised discordances for major malformations in MZ twins [48]. Many of these malformations do not have simple genetic origins, but are likely to be multifactorial. Examples commonly described include neural tube defects, cleft lip and palate, and symmelia [4]. While it is not clear how these discordant events arise, in many cases, the apparently unaffected co-twin also has a less severe manifestation of the same disorder. The implication in these cases is that the predisposition for the malformation is expressed in both twins.
6. Summary This review highlights some of the important findings since Francis Galton, the father of twin research, first thought of using twins to estimate the impact of genes in 1876 [49]. His paper on the use of twins ‘as a criterion of the relative powers of nature and nurture’ launched the whole field, which has since seen major advances in twin foetal development and twin genetics. This review has demonstrated the problems in assuming that being monozygous is synonymous with being identical.
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Apart for academic interest and implications for twin research, these facts also have clear implications for clinical practice. In the case of a twin pair found to be discordant for a major malformation, the assumption should not be that they are automatically DZ. They may be MZ and, more importantly, MC. In a case of heterochromia iridum (different colour irises) in MZ twins described by St. Clair et al. [45], immunosuppressive therapy following a renal transplant was needlessly continued for 15 years because dizygosity had been assumed. In their case discussion, the authors of this paper emphasised that the heterochromia discordance must fall ‘within the acceptable spectrum of MZ status’. This review demonstrates that as our understanding of MZ twinning increases, the spectrum will continues to widen, and the term ‘identical twins’ should be replaced by the more accurate term ‘monozygous twins’. The importance of establishing the zygosity of twins has been stressed in the literature. Keith and Machin [50] have summarised many of the issues: ‘For reasons of personal rights to identity, medico-legal responsibility, potential for transplantation, early education, and concordance/discordance for genetic disease, many feel that routine determination of twin zygosity at birth is needed and should be implemented in the near future. Apart from the benefits to the twins and their parents, the consequences for research in twins and genetics would be enormous, since there remain many puzzling aspects of twinning.’ To this list is now also added the importance of establishing chorionicity, preferably by ultrasound in very early pregnancy.
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