American Journal of Medical Genetics Part C (Seminars in Medical Genetics) 151C:148– 151 (2009) A R T I C L E Clinical Significance of Chimerism DIANNE ABUELO* Twins have been previously classified as either monozygotic or dizygotic. In recent years, fascinating, nontraditional mechanisms of twinning have been uncovered. We define chimerism versus mosaicism, touch on chimerism in the animal world, and explain timing of chimerism in humans. In addition, we discuss when to suspect chimerism in patients, and how to proceed with diagnostic evaluation and confirmation. ß 2009 Wiley-Liss, Inc. KEY WORDS: chimerism; intermediate twinning How to cite this article: Abuelo D. 2009. Clinical significance of chimerism. Am J Med Genet Part C Semin Med Genet 151C:148–151. ‘‘She was the mother of Chimaera who breathed raging fire, a creature fearful, great, swift-footed and strong, who had three heads, one of a grim-eyed lion; in her hinderpart, a dragon; and in her middle, a goat, breathing forth a fearful blast of blazing fire. Her did Pegasus and noble Bellerophon slay.’’ Hesiod, Theogony INTRODUCTION Most of us have been taught that twins are either identical or non-identical [Hall, 2003], that is, either monozygotic (MZ), having arisen from one fertilized egg, or dizygotic (DZ), from two eggs fertilized by two sperm. However, reports of the occurrence of monozyDr. Dianne Abuelo is Associate Professor of Pediatrics at the Warren Alpert School of Medicine of Brown University and the Director of the Genetic Counseling Center at Rhode Island Hospital and Hasbro Children’s Hospital. She has authored over 60 publications, travels to Latin American to teach genetic courses, and she is actively involved in resident and fellowship education. *Correspondence to: Dianne Abuelo, M.D., Genetic Counseling Center, Rhode Island Hospital and Hasbro Children’s Hospital, Warren Alper School of Medicine of Brown University, 593 Eddy Street, Providence, Rhode Island 02903. E-mail: email@example.com DOI 10.1002/ajmg.c.30213 Published online 17 April 2009 in Wiley InterScience (www.interscience.wiley.com) ß 2009 Wiley-Liss, Inc. gous twins of unlike sex [Edwards et al., 1966] or unlike karyotypes [O’Donnell et al., 2004] have uncovered some fascinating, non-traditional mechanisms for twinning, which do not conform to either of the two traditional categories. These unusual cases are more common than have been appreciated up to now and have raised doubts about the validity of the accepted tests for zygosity testing, which divide results into simple MZ or DZ categories. Many of the cases of unusual types of twinning involve the formation of individuals who are chimeras. DEFINITION OF CHIMERISM Chimerism has been imagined since ancient times, for example, in Greek mythology, the chimera was a monstrous creature composed of the body of a lioness, the head of a goat and the tail of a snake. Nowadays, we know that chimerism, although not as dramatic in presentation, exists in animals and also in humans. They are fascinating individuals who are composed of two genetically different types of cells. Only recently with current technology, have we been able to understand the causes, clinical manifestations and complications. Here we present a simplified classification, with the aim of raising physicians’ awareness about when to suspect chimerism in their patients, and then once suspected, how to proceed with diagnostic evaluation and confirmation. First, chimerism must be distinguished from mosaicism. In chimerism cells derive from more than one genetically distinct zygote. In mosaicism there are also different cell types, but they come from a single zygote. First, chimerism must be distinguished from mosaicism. In chimerism cells derive from more than one genetically distinct zygote. In mosaicism there are also different cell types, but they come from a single zygote. For example, an individual who is mosaic for trisomy 21 has one cell line that is normal and another cell line with 47 chromosomes including an extra chromosome number 21. In the mosaic individual, both cell lines have arisen from a single fertilized egg. It is possible for a chimeric individual to have a similar karyotype, that is, a mixture of normal ARTICLE and trisomy 21 cells, but these cell lines can be shown to have originated from separate zygotes, as exemplified by the set of twins described below by O’Donnell et al. . CHIMERISM IN THE ANIMAL WORLD The phenomenon of freemartinism in cows and other animals has been recognized for many years [Lillie, 1917] and explained the observation of intersex in these animals. It was known that in some pairs of bovine mixed-sex twins, the females were born with both ovaries and testes. This was explained by placental connections between the female and her male co-twin. Not only does an exchange of hematopoietic cells occur, but also primitive gonadal cells can migrate from one twin to the other. In cows, this has been found to be a relatively frequent phenomenon in mixed sex twins, but it has not yet been found in human counterparts. TIMING OF CHIMERISM IN HUMANS When considering the various possible mechanisms of chimerism, it is helpful to think about the timing of the causative event. We should distinguish chimerism that occurs during fetal, childhood, and adult life from that which occurs during fertilization and embryogenesis. Childhood or Adult Life An iatrogenic form of chimerism is produced by bone marrow or other tissue or organ transplantation. There is a large literature related to transplantation immunology, which will not be considered here, except for a description of one instructive case (described below in the section on chimerism in individuals) discovered during preparation for transplantation. Fetal Life Fetomaternal cell trafficking can lead to ‘‘microchimerism:’’ Many normal AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) women in the population develop chimerism during pregnancy, and there is much current interest in the resulting short-term and long-term effects. An example of a short-term effect is the dermatologic condition known as ‘‘pruritic urticarial papules and plaques of pregnancy,’’ or PUPPP. Fetomaternal cell trafficking can lead to ‘‘microchimerism:’’ Many normal women in the population develop chimerism during pregnancy, and there is much current interest in the resulting short-term and long-term effects. An example of a short-term effect is the dermatologic condition known as ‘‘pruritic urticarial papules and plaques of pregnancy’’, or PUPPP. The skin eruption appears during the last trimester of pregnancy and/or early postpartum period, and is due to migration of cells from a male fetus through the placenta and maternal circulation, to the mother’s skin [Aractingi et al., 1998]. A recent study of microchimerism following induced or spontaneous abortion [Sato et al., 2008] showed that 52.8% of women who had male chorions had detectable Y chromosomal DNA in their peripheral blood specimens following the abortion; these cells slowly decreased and became undetectable by 30 days. However, fetal cells can be sequestered and persist in other maternal tissues for considerable time and it has been hypothesized that they may play a role in the development of autoimmune disorders many years after childbirth [Evans et al., 1999]. Trafficking of cells also occurs in the other direction, from mother to fetus. Maloney et al.  discovered that maternal cells can persist in their offspring even into adult life. 149 Embryonic Life We will be focusing on the phenomenon of chimerism that takes place during the earliest times of fertilization and embryogenesis. Examples will be given of chimerism in MZ twins, in DZ twins and even within a single individual. Chimerism in Monozygotic Twins Theoretically, MZ twins cannot have chimerism as they are supposed to have the identical genetic makeup. However, chimerism can occur, but it is probably underdetected, since it is only considered if the twins have dramatically different phenotypes. For instance, there have been several reports of monochorionic (MC) twins who are discordant for sex, including one by Souter et al. , who reported an example of chimerism that occurred during early embryogenesis. In another example, chimerism was recognized because a pair of MZ twins was discordant for Down syndrome [O’Donnell et al., 2004]. In this case report, the MC diamniotic (DA) pregnancy was thought to be suspicious for Down syndrome in one of the twins based on thick nuchal translucency measurements in one twin. A chorionic villous specimen at 13 weeks showed a normal result; amniocentesis from both sacs was suggested but refused by the parents. At delivery, one twin had a normal phenotype and the other showed the dysmorphic features of Down syndrome. Blood from both twins showed an admixture of normal and trisomic cells, indicating hematopoietic chimerism. However, tissue studies of skin and buccal cells of the dysmorphic twin showed only trisomy 21 cells, whereas buccal cells from the normal twin showed only normal cells. Zygosity studies by microsatellite analysis on blood genomic DNA showed that the twins were indeed ‘‘identical,’’ in the sense that they were derived from the same zygote. The extra chromosome 21 was paternally derived. Possible mechanisms include a normal 46, XY conception followed by postzygotic gain of the extra chromosome in one twin, or 150 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) a paternal meiosis II error followed by trisomy rescue (considered less likely). In any event, this case illustrates that zygosity studies should ideally be performed on both blood and fibroblast cells to make it possible to distinguish hematopoietic chimerism from mosaicism. Chimerism in Dizygotic Twins Souter et al.  reported twins who had a MC placenta and were originally assumed to be MZ. In the second trimester, discordance for sex was noted. They found blood chimerism in the twins, that is, a mixture of XX and XY cells, but skin fibroblasts were 46,XX in the female twin and 46,XY in the male twin. Each of the twins carried two populations of lymphocytes, the major subgroup from the male and a minor subgroup from the female. Fluid injection of the placenta showed fine arterial-to-arterial anastomoses on the fetal surface. The pregnancy had been established by in vitro fertilization, which is considered to be one of the risk factors for chimerism. One of the possible explanations involves fusion of the trophoblasts from the two embryos before implantation. Another theoretical explanation involves double fertilization of the meiotic products of a single oocyte with two sperm, which was ruled out in this case by DNA marker studies. Continued consideration of this mechanism awaited a subsequent report, also by Souter et al.  as described in the next section. In cases of twins with no external phenotypic abnormalities, chimerism can be cryptic. For example, Aoki et al.  described a case of MC twins (again, would have been assumed to be MZ), conceived by induced ovulation, who were found to be discordant for blood types. The twins were also discordant in other tissues, that is, blood lymphoctes and hair roots. Thus, they were discovered to have DZ chimerism. The authors concluded that determination of zygosity in twins conceived via assisted reproduction should be done with caution. Chimerism in Intermediate Types of Twinning Souter et al.  reported a pair of spontaneously conceived twins with 46,XX/46XY karyotypes in lymphocytes, skin fibroblasts and gonads. The chimerism was discovered because one twin had ambiguous genitalia. Molecular genetic studies were very surprising: there was a single maternal contribution, but two paternal genetic contributions. Thus, these twins were more genetically alike than DZ twins, but less alike than MZ twins. Different mechanisms for this intermediate type of twinning were hypothesized, but the cause remains uncertain. Chimerism in a Single Individual A landmark case in chimerism was reported by Yu et al. . This involved a phenotypically normal woman with renal failure who was being evaluated for renal transplantation. Results of the usual histocompatibility testing suggested that she was not the biologic mother of two of her three sons. To explain this surprising result, multiple studies on a variety of tissues were done, including blood grouping, HLA studies, cytogenetic testing, analysis of short tandem-repeat microsatellite markers, mixed-lymphocyte culture and cell-mediated lysis. Blood studies showed only a single cell line in the patient, but her other tissues showed chimerism. The mechanism in this patient is thought to involve separately fertilized XX zygotes, which then went on to fuse together early in development; thus she is a ‘‘tetragametic’’ chimera. The frequency of this condition is unknown and will remain undetected if blood is the only tissue that is studied. The authors note that the increase in twinning associated with in vitro fertilization can also theoretically lead to an increase in tetragametic chimerism, due to either double fertilization of an ovum with two nuclei, or close contact and fusion between the embryos before implantation. WHEN TO SUSPECT CHIMERISM The earliest circumstance in which chimerism should be suspected is when ARTICLE there is apparent mosaicism in chorionic villus sampling (CVS) specimens. Prenatal diagnosis by CVS can sometimes give puzzling results involving apparent mosaicism, with two different cell lines when there is only one fetus present. This can be due to either confined placental mosaicism or to confined placental chimerism. In the case reported by Falik-Borenstein et al. , a CVS specimen had a normal female cell line and also included a cell line with 47,XY þ9. Subsequent amniocentesis and postdelivery peripheral blood specimens were normal. The abnormal cell line most likely came from a vanished twin. Examination of the placenta proved the existence and demise of the karyotypically abnormal co-twin. Postnatally, chimerism can be suspected when MC twins have discordant phenotypes or blood groups, as described above. In addition, other unusual signs, such as peculiar skin pigmentary findings, especially in products of assisted reproductive technology, should raise the possibility of chimerism Postnatally, chimerism can be suspected when MC twins have discordant phenotypes or blood groups, as described above. In addition, other unusual signs, such as peculiar skin pigmentary findings, especially in products of assisted reproductive technology, should raise the possibility of chimerism . In singletons, clinical clues include patchy skin pigmentation, as described by Lipsker et al. , whose patient had bicolored skin pigmentation and another patient described by GerardBlanluet et al. , who also had unusual pigmentary abnormalities, iris heterochromia and hemihypertophy. ARTICLE In addition, a history of a vanishing twin raises suspicion, especially if the surviving twin has any abnormalities. LABORATORY EVALUATION OF CHIMERISM Making a diagnosis of chimerism can be present a challenge, since few clinicians know how to order or interpret testing. Major clinical laboratories that offer chimerism testing focus on evaluation pre- and post-bone marrow transplant rather than twins. While the testing seems complex and impractical to obtain, most molecular genetic laboratories across the country have the technological ability to offer testing and could be requested to do so on a case-by-case basis. As Malan et al.  outline the principle behind the testing is fairly straightforward: to distinguish chimerism from mosaicism by using autosomal markers (DNA testing). If a single maternal and paternal contribution is found for all markers (at all loci), mosaicism can be diagnosed. However, if two or more alleles are demonstrated (at least at one locus), chimerism is likely. At this point, more complex studies can be initiated in attempt to document and elucidate the mechanism of chimeric formation. AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) It is important to emphasize that when blood karyotype does not correlate with the clinical phenotype in twins, tissues in addition to blood should be tested. 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