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Introduction Chimerism is a term that describes a condition where a living organism contains two distinct genetic profiles, which is a rare but documented.

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Presentation on theme: "Introduction Chimerism is a term that describes a condition where a living organism contains two distinct genetic profiles, which is a rare but documented."— Presentation transcript:

1 Introduction Chimerism is a term that describes a condition where a living organism contains two distinct genetic profiles, which is a rare but documented event [1]. Although different cell types in the body may not express the same genes, each cell has an identical genome to its neighboring cells, with the exception of common mutations that arise during replication. A chimera is not simply a single mutated cell, but an individual with two populations of cells containing two different cell lines [2]. Three forms of chimerism have been described, gestational, artificial, and tetragametic. Gestation chimerism can happen in one of two ways. Either through blood cells that pass from mother to fetus across the placenta, introducing the mother’s genetically different cells into the fetus’s population of cells, or the distribution of blood and other stem cells between fraternal twins or multiple embryos by means of blood-vessel anastomoses when they share a placenta [1, 3]. Artificial chimerism describes the situation in which foreign cells can be found as a result of a transfusion or transplantation of a second cell line, which occurs when a donor’s cells infiltrates past the transplanted organ and into surrounding tissue. Lastly, tetragametic chimerism, the rarest form, is considered to be the “true” form of chimerism, which occurs as a result of the fusion of non-identical twin embryos shortly after fertilization [3]. The study of chimerism has typically been initiated by a discrepancy or complication within an individual’s record of health and studies initiated to determine the origin of the inconsistency. While a few individuals were determined to be chimeras, no studies published to date have investigated the frequency of chimerism [1, 3, 4]. Therefore, it is unknown how many people within the population contain chimeric properties, which with our new reliance of genetic information may greatly impact a variety of societal issues. Specifically, chimerism may be the underlying cause of certain diseases, affect the development of drug therapies, and maybe most importantly, change the way people are identified within criminal justice system [3]. Paternity testing, missing person’s investigations, mass disasters, and maybe most importantly DNA databases, can all be affected by an individual containing more than one set of DNA. It is for all these reasons that the occurrence of chimerism be better described in a population. The object of the current study was to determine the occurrence of chimerism within a human population, specifically a population of York College students and faculty members, which can then be used to estimate the incidence of chimerism in a greater population. Materials and Methods DNA Isolation: Cheek epithelial cells were isolated from 20 individuals using Chelex, as per manufacturer’s instructions. Briefly, individuals rinsed their mouths with a 0.9% NaCl solution for 10 sec and cells spun down at g for 10 min. DNA was isolated from cell pellet by addition of 500 µl of a 10% Chelex (Carolina) solution and heated to 100ºC for 10 min then spun down at 12,000 RPM for 30 sec. The same 20 individuals provided 5 strands of hair with roots and sheaths. The sheaths were cut off and digested with a mixture of 10% Chelex and proteinase K (100 µg/ml) at 60ºC as per manufacturer’s instructions (Carolina). PCR: Isolated DNA was amplified using 1.5 µl DNA template for cheek and 5 µl for hair using primers specific for the ALU insertion and VNTR polymorphisms. Briefly, for the ALU insertion, 1.5 µl of cheek DNA template was combined with 22.5 µl of supermix (Invitrogen) and 0.5 µl each of forward (´5-GTAAGAGTTCGTAACAGGACAGCT-3´) and reverse (´5-CCCCACCCTAGGAGAACTTCTCTTT-3´) primers and amplified for 30 cycles (94ºC for 1 min, 58ºC for 2 min, and 72ºC for 2 min). For hair DNA, 5 µl of template DNA was combined with 19 µl H2O, 1 µl of forward and reverse primers, into Pure Taq Ready- to-Go PCR bead tubes (Amersham). For the VNTR polymorphism, cheek and hair DNA followed the same protocol as above with primers specific to the VNTR region; forward (´5- GAAACTGGCCTCCAAACACTGCCCGCCG-3´) and reverse (´5- GTCTTGTTGGAGATGCACGTGCCCCTTGC-3´), and amplified for 30 cycles (94ºC for 1 min, 65ºC for 1 min, and 72ºC for 1 min). Agarose Gel: ALU products were resolved on a 2% agarose ETBR gel for 30 min at 100V. VNTR products were run on a 1.5% agarose ETBR gel for 60 min at 100V. Gels were then visualized and photographed on a UV light box.  The PCR reaction for the ALU insertion and VNTR polymorphism was replicated numerous times.  ALU: cheek 19 / 20 ; hair 17 / 20 yielded bands  VNTR: cheek 16 / 20 ; hair 6 / 20 yielded bands  VNTR was not as successful as ALU and exhibited random priming, un-amplified DNA, and unclear results.  Consistently, sample 20 suggests a possible chimera using the ALU insertion with cheek tissue yielding a band at 400 bp and hair yielding bands at 100 and 400 bp. Results  A possible human chimera was observed in a small population (N=20) of York College students and faculty. Using this as a representative sample, chimerism has an occurrence rate of 5.88% within a population.  Based on the population of York County (381,751), the projected number of chimeras is 22,456 individuals.  Forensic cases rely heavily on DNA and the comparison of an evidentiary sample to a donated sample from a suspect. Typically an analysis uses 7-9 loci to match DNA samples, which may possibly increase the detection of a chimera. Our data suggests that there may be significant implications regarding the use of DNA for identification and comparison within the criminal justice system. Conclusions Acknowledgements Ronald Kaltreider Ph.D., Biology Faculty Mentor Deborah Ricker Ph.D., Biology Chair Literature Cited 1. Yu, N., Krushkall, M.S., Yunis, J.J., Knoll, J., Uhl, L., Alosco, S., Ohashi, M., Clavijo, O., Husain, Z., Yunis, E.J., Yunis, J.J., and Yunis, E.J Disputed maternity leading to identification of tetragemtic chimerism. The New England Jounral of Medicine. 346:20: Bowen, R August 5. Mosaicism and Chimerism. Available from: Accessed 2003 September Pearson, H Dual Identities. Nature. 417:6884: Amor, D., Delatycki, M.B., Susman, M., Casey, E. et al , XX/ 46, XY at amniocentesis in a fetus with true hemaphroditism. Journal of Medical Genetics. 36:11: Ainsworth, C The Stranger Within. New Scientist. 180:2421: Identification of a Possible Chimera in a Small Sample Size of 20 Individuals from York College Samantha Ciacco Department of Biological Sciences, York College of Pennsylvania Figure 4: Illustration tetragametic chimerism. Non-identical twins fuse to form one embryo with two genetic profiles [5]. 100 BP Figure 2: Representative gel of cheek cells for VNTR. Cheek samples were obtained from 20 individuals and DNA was isolated, then amplified using primers specific to the VNTR region. A 100 BP ladder was used. PCR products were run on a 1.5% agarose ETBR gel for 60 min and photographed CHEEK VNTR BP Figure 1: Representative gels of cheek and hair samples for ALU. Cheek and hair samples were obtained from 20 individuals and DNA was isolated, then amplified using primers specific to the ALU region. Figure 1A: Cheek samples. Figure 1B: Hair samples. A 100 BP ladder was used and PCR products were run on a 2% agarose ETBR gel for 30 min and photographed. Samples were either homo- or heterozygous yielding bands at 100 and 400 BP. Above gels indicate hair and cheek samples from most individuals are genetically consistent and do not contain chimeric properties. A CHEEK ALU 100 BP B HAIR ALU 100 BP Data Figure 3: Representative gels of side by side comparison of cheek and hair samples for ALU and VNTR primers. Figure 3A: ALU cheek and hair samples run side by side. Samples 13 and 14 are tissues that match. Samples 20 is a possible chimera with homozygous (400) cheek cells and heterozygous (100/400) hair cells. Figure 3B: VNTR side by side cheek and hair samples. Samples 13 and 14 are easily identifiable, where 19 and the possible chimera 20 are unclear. B VNTR C\H 13 C\H 14 C\H 19 C\H BP A C\H 13 C\H 14 C\H 19 C\H 20 ALU 100 BP


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