Download Figure 1: Representative gels of cheek and hair samples for ALU.

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
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.
Figure 4: Illustration tetragametic chimerism.
Non-identical twins fuse to form one embryo with
two genetic profiles [5].
Data
CHEEK ALU
A
Results
HAIR ALU
B
100
BP 3 15 20 7 16 17 8 2 10 14 13 19
6
100
BP 12
4
13 14 15
16 17
18
19
 The PCR reaction for the ALU insertion and
VNTR polymorphism was replicated numerous
times.
20
ALU: cheek 19/20; hair 17/20 yielded bands
VNTR: cheek 16/20; hair 6/20 yielded bands
400
400
100
100
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 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.
CHEEK VNTR
100
BP 5 12 18 1
3 15 20 7 16 17 8
2 10 14
800
600
400
Based on the population of York County (381,751),
the projected number of chimeras is 22,456
individuals.
200
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 500-1000 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).
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 Readyto-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
(´5GAAACTGGCCTCCAAACACTGCCCGCCG-3´)
and
reverse
(´5GTCTTGTTGGAGATGCACGTGCCCCTTGC-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.
 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.
Conclusions
Materials and Methods
PCR:
 VNTR was not as successful as ALU and
exhibited random priming, un-amplified DNA,
and unclear results.
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.
A
100
100BP
BP
ALU
C\H
13
C\H
14
C\H
19
VNTR
B
C\H
20
100
BP
C\H
13
C\H
14
C\H
19
C\H
20
800
600
400
400
200
100
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.
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.
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. 2002. Disputed
maternity leading to identification of tetragemtic chimerism. The New England
Jounral of Medicine. 346:20:1545-1552.
2. Bowen, R. 1998 August 5. Mosaicism and Chimerism. Available from:
http://arbl.cvmbs.colostate.edu/hbooks/genetics/medgen/chromo/mosaics.html.
Accessed 2003 September 30.
3. Pearson, H. 2002. Dual Identities. Nature. 417:6884:10-11.
4. Amor, D., Delatycki, M.B., Susman, M., Casey, E. et al. 1999. 46, XX/ 46, XY
at amniocentesis in a fetus with true hemaphroditism. Journal of Medical
Genetics. 36:11:866-869.
5. Ainsworth, C. 2003. The Stranger Within. New Scientist. 180:2421:34-38.
Acknowledgements
Ronald Kaltreider Ph.D., Biology Faculty Mentor
Deborah Ricker Ph.D., Biology Chair