TRISOMY 8 MOSAICISM: CELL CYCLE KINETICS AND ...

TRISOMY 8 MOSAICISM: CELL CYCLE KINETICS AND
DISTRIBUTION TRISOMY 8 AND NORMAL CELLS IN
EMBRYONIC AND EXTRA-EMBRYONIC TISSUES
Bonnie J Pettit
Thesis submitted to the
College of Agriculture, Forestry and Consumer Sciences
at West Virginia University
in partial fulfillment of the requirements
for the degree of
Master of Science
In
Genetics and Developmental Biology
Walter Kaczmarczyk, Ph.D., Co-Chair
Sharon L Wenger, Ph.D., Co-Chair
Kenneth Blemings, Ph.D.
Department of Genetics and Developmental Biology
Morgantown, West Virginia
2001
Keywords: Trisomy 8, mosaicism, Fluorescence in situ hybridization,
cell cycle kinetics
Copyright 2001 Bonnie J Pettit

ABSTRACT
TRISOMY 8 MOSAICISM: CELL CYCLE KINETICS AND
DISTRIBUTION OF TRISOMY 8 AND NORMAL CELLS IN
EMBRYONIC AND EXTRA-EMBRYONIC TISSUES
Bonnie J Pettit
Amniocentesis identified a fetus with trisomy 8 in one third of metaphase cells
while remaining cells were normal. The purpose of this project was to determine the
level of trisomy 8 mosaicism within embryonic and extra-embryonic tissues and cell
cycle division rates for the two cell lines. Tissue samples collected at birth included cord
blood, cord tissue, peripheral blood, and placental tissues. The level of trisomy 8 and
normal cells was determined in G-banded metaphases and fluorescence in situ
hybridization of interphase cells. Trisomy 8 metaphase and interphase cell investigations
identified a significant decrease of trisomy 8 cells undergoing mitosis. Cell cycle kinetics
was measured using incorporation of bromodeoxyuridine during 48 hours to determine
the number of cell divisions in metaphase cells. There was no evidence suggesting a
significant difference in growth rates using Chi-squared goodness of fit test.

TABLE OF CONTENTS
iii
Page
Abstract ii
Table of Contents iii
List of Figures and Tables iv
Introduction 1
Methods and Materials 8
Results 17
Discussion 33
Bibliography 39

LIST OF FIGURES AND TABLES
Figure 1. 18
Figure 2. 19
Figure 3. 22
Figure 4. 23
Figure 5. 28
Figure 6. 29
Figure 7. 30
Table 1. 20
Table 2. 24
Table 3. 26
Table 4. 31
iv
Page

Trisomy 8
INTRODUCTION
Trisomy 8 (T8) is a rare genetic condition in which all of an individual’s cells
have 47 chromosomes including 3 chromosome 8s. T8 occurs in 0.1% of all
recognizable pregnancies and 0.7% of spontaneous abortions (Karadima et al. 1998). T8
is associated with clinical manifestations known as Warkany syndrome (WS). It was
first described in 1962 by Warkany et al. and thought to be due to an extra D group
chromosome. Later cytogenetic evaluation proved WS to be caused by an extra
chromosome 8; confusion of the first diagnosis being a D group chromosome was due to
the extra chromosome as a partial 8; deletion of the short arm. Notable abnormalities of
WS include mild to moderate mental retardation, skeletal anomalies, reduced joint
mobility, renal abnormalities, deep palmar and plantar furrows, long narrow face,
absence of corpus callosum, absence of patellae, and congenital heart defects (Riccardi
1977; Berry et al. 1978; Karadima et al. 1998). T8 has been reported to be incompatible
with life and survival may be possible only in the mosaic form (James and Jacobs 1996;
Karadima et al. 1998; Nicolaidis and Petersen 1998).
Trisomy 8 mosaicism (T8m) is a rare genetic condition in which an individual has
two different cell lines including a normal diploid and a trisomy 8 cell line. Over one
hundred cases of T8 have been reported, the majority of which involve mosaicism
(Kurtyka et al. 1988; Miller et al. 1996; Jordan et al. 1998; Karadima et al. 1998;
Nicolaidis and Petersen 1998). T8m occurs in less than 1 in 25,000 liveborn and is found
to be at a higher frequency (5:1) in males than females (Jordan et al. 1998). This
1

frequency is questioned because mitotic non-disjunction should occur equally in males
and females, and suggests a possible selection mechanism against T8/T8m in females
(Karadima et al. 1998). T8m may be diagnosed prenatally through amniocentesis or
postnatally by routine cytogenetic analysis of a peripheral blood sample. Patients with
T8m are most commonly diagnosed with WS plus additional anomalies that arise due to
different tissues being affected by the mosaicism (Riccardi 1977; Berry et al. 1978;
Karadima et al. 1998).
Acquired trisomy 8 has been associated with various neoplasms, especially
myeloid leukemias (Zollino et al. 1995; James and Jacobs 1996, Miller et al.1997).
Zollino et al. (1995) have reported on a patient with T8m who developed myelodysplasia.
Due to this association it has been suggested that patients with T8/T8m have an increased
risk for developing cancer (Zollino et al. 1995; James and Jacobs 1996; Karadima et al.
1998). Therefore, long term observation in T8m patients is suggested not only in
accordance with normal routine chromosomal analysis, but with oncological
considerations as well (Miller et al.1997).
The common mechanism for most autosomal trisomy mosaics is meiotic non-
disjunction (James and Jacobs 1996, Nicolaidis and Peterson 1996). However, T8m most
commonly results from a mitotic non-disjunction in a normal diploid developing fetus
(James and Jacobs 1996; Karadima et al. 1998; Nicolaidis and Petersen 1998; Webb et al.
1998; Kalousek 1999). Non-disjunction occurs during cell division when sister
chromatids fail to segregate into opposite daughter cells. The resulting daughter cells are
monosomic and trisomic for that specific chromosome. The monosomic cell lines
involving autosomal chromosomes are not viable, however the trisomic cell line
2

proliferates and thus mosaicism results. Other possible mechanisms for mosaicism
include anaphase lag or spindle fiber attachment failure (Kalousek 1999).
The presence of trisomic cells throughout various tissues, both embryonic and
extra-embryonic, depends upon the timing of the non-disjunction or error event (Webb et
al. 1998). The earlier the error, the more likely both the fetus and placental tissues will
be affected. T8m may occur in the fetus, placenta, or both simultaneously. When only
the placental tissues are affected, it is referred to as Confined Placental Mosaicism
(CPM). CPM occurs in 1-2% of all viable pregnancies analyzed by chorionic villi
sampling. Also CPM is estimated to occur in over 2-5% of spontaneous abortions (Griffin
et al. 1997). CPM has been associated with a broad range of outcomes such as normal
pregnancy, intrauterine growth retardation, and even intrauterine fetal death (Lestou et al.
1998, Kalousek 1999).
CPM can result from both meiotic and mitotic non-disjunction. Meiotic error
results in complete trisomic placental tissue, which can form a normal cell line and
mosaicism by going through trisomic rescue. Trisomic rescue occurs when a
chromosome is lost from either the trisomic trophoblast lineage or true embryonic
progenitors (Kalousek 1999). Whether there is CPM or fetal mosaicism, it has been
documented that chromosomally abnormal placental tissues are correlated with abnormal
fetal development (Farra et al. 2000). Also, as the proportion of trisomic cells in
placental tissue increases, so does the likelihood of intrauterine growth retardation or
fetal death. Compared to chromosomally abnormal placentas, normal placentas enhance
the survival and likelihood of abnormal fetuses going to term (Farra et al. 2000).
3

T8m features are variable, however, some anomalies are more common than
others. The severity of the phenotypes in T8m patients is not directly related to the level
of mosaicism found (Riccardi 1977; Berry et al.1978; Swisshelm et al. 1981; Kurtyka et
al. 1988). Several cases of similar mosaicism levels have been compared, however the
patient phenotypes differ. For example, one patient had 100% trisomy 8 cells in
lymphocytes and over 60% in fibroblasts with a near normal phenotype and IQ (James
and Jacobs 1996). Comparatively, another patient was diagnosed with 94% trisomy 8
cells in lymphocytes and 70% in fibroblasts and had severe mental retardation and
various skeletal anomalies (Jordan et al. 1998). The exact mechanism which causes the
severity of phenotype in T8m patients is unknown, but believed to be associated with
either the “post zygotic origin of the trisomic status” (Miller et al 1997) or dependent
upon the contribution of aneuploid cells in the development of different tissues (Kurtyka
et al. 1988).
T8m is associated with disappearing mosaicism over time. The percentage of
trisomic cells decreases in a patient when tested several months to years after the initial
testing (La Marche 1967; Mark and Bier 1997). Jordan et al. in 1998 described two cases
showing disappearing T8m. Patients were analyzed over an 8 and 9 year period to
evaluate any change in trisomy 8 cell percentages. Both cases were shown to have a
significant decrease of trisomy 8 cells in cultured lymphocytes. Kurtyka et al. (1988) also
reported cases with similar disappearance of trisomy 8 cells in lymphocytes. Mark and
Bier (1997) suggested the trisomic line may disappear completely if enough time has
gone by. The decrease in trisomic cells has been attributed to a selective growth
advantage of the normal cells (La Marche et al. 1967; Mark and Bier 1997).
4

Prenatal Testing
Prenatal testing is routine for women with an increased risk for possible fetal
anomalies. Testing is done on women with advanced maternal age, abnormal maternal
triple screen results, or abnormal fetal results from ultrasound. Typical prenatal testing
includes amniocentesis and chorionic villi sampling (CVS). Amniocentesis is usually
performed at 15-17 weeks gestation. Amniotic fluid and cells are removed from the
amniotic sac via a syringe through the abdomen. The amniocytes within the fluid are
grown in culture and harvested for cytogenetic analysis and diagnosis. CVS is performed
during the first trimester and before the amniotic sac is fully expanded in the uterine
cavity (Filkins and Russo 1990). A sampling catheter is inserted into the uterine cavity
vaginally. The catheter is then inserted into the chorion to collect a sample for
cytogenetic testing.
T8m is problematic from the standpoint of genetic counseling due to several T8m
cases diagnosed in amniocytes, with only normal cells found in the aborted fetus. Also,
misdiagnosis can occur when the results obtained from initial diagnosis of amniocytes are
normal, and the pregnancy results in an abnormal fetus (Guichet et al. 1995; Karadima et
al. 1998; Webb et al. 1998). One way to prevent misdiagnosis of T8m when suspected
by abnormal ultrasound findings is to test other cells along with amniocytes, such as
lymphocytes from prenatal umbilical blood (Berry et al. 1978; Guichet et al. 1995; Miller
et al. 1997). The testing of additional tissues is necessary for cases involving mosaicism,
since it will either confirm or negate the original diagnosis. Another cause of concern for
genetic counseling is the lack of correlation between the phenotype and the amount of
trisomy 8 cells present. This makes it very difficult to give a definitive prognosis
5

(Swisshelm et al. 1981; Guichet et al. 1995; Miller et al. 1997; Jordan et al. 1998; Webb
et al. 1998).
Fluorescence In Situ Hybridization
Fluorescence in situ hybridization (FISH) is a molecular technique first
introduced in the late 1970’s (Trask 1991). It can be used for chromosomal analysis and
identification and clinical diagnostic purposes. FISH is widely used due to several
factors including: I.) DNA can be investigated in metaphase spreads and interphase
nuclei II.) The technique is reliable and repeatable due to commercially available probes
and fluorescent reagents III.) A broad range of DNA sizes can be analyzed including
entire genome, chromosome, sub-regions, or single copy sequences (Trask 1991). FISH
involves annealing of DNA probes to subject DNA. Probes are selected based on initial
diagnosis or suspected chromosome abnormalities. Centromeric alpha-satellite probes
can be used for metaphase and interphase analysis (Moore et al. 2000). These
centromeric probes are chosen in cases where patients are suspected to have whole
chromosomal abnormalities, since the probe will only determine if the chromosome is
present and not identify specific chromosomal regions. Probes for chromosome X may
also be used to rule out maternal contamination in placental tissue when the patient is
male, on the basis of males resulting in one X signal and females with two X signals.
Cell Cycle Kinetics
Growth can be easily measured by incorporation of 5’-bromo-2’-deoxyuridine
(BrdU) in tissue culture. BrdU is a base analog that is substituted for thymidine in the
6

DNA undergoing synthesis. Once a sample is subcultured, BrdU is added and will
become incorporated into the genome of the replicating cells (Bonhoeffer 2000). This
newly replicated cell will continue to undergo divisions until the cell culture growth is
terminated. The BrdU is detected in the chromosomes by a fluorescent stain on
metaphase spreads.
Cell divisions are determined by the amount of BrdU incorporation among
chromatids. Equal staining among chromatids represents one division (singly-substituted
double stranded DNA). Two divisions are seen as half the chromatids stained pale
(doubly substituted) and the other half stained dark. Three divisions are recognized by
three-fourths of the chromatids stained pale and one-fourth stained dark. Cell cycle
kinetics analysis allows for detection of growth rate differences between different tissues
and in normal versus abnormal cells.
Purpose
The first purpose of the study is to determine the level of mosaicism among
several embryonic and extra-embryonic tissues from a male patient. Once the
percentages of normal and trisomy 8 cells are determined for each tissue statistical
analysis will be performed. Since a selective growth advantage is known to occur for
normal cells over trisomy 8 cells, the second purpose of the study is to investigate cell
cycle kinetics for each tissue. Cell cycle kinetics will determine the growth rates of the
tissues and statistical analysis performed to denote significant differences in division
times between normal and trisomy 8 cells.
7

Peripheral and Cord Blood preparation
METHODS AND MATERIALS
Chromosomal media was prepared by mixing 100ml RPMI 1640, 20ml Fetal
Bovine Serum, 1.3ml phytohemagglutinin (PHA) (Gibco 10576-015), 1.3ml Penicillin
and Streptomycin (10,000 units/ml Penicillin and sodium and 10,000 µg/ml Streptomycin
sulfate in 0.85% saline), and 1.3ml L-glutamine (29.2 mg/ml in 0.85% NaCl). Media
tubes were pre-prepared in 15ml centrifuge tubes with 9ml aliquots of media and then
frozen until needed. The tube of media was removed and thawed and 0.5ml–1ml of whole
blood was added. The blood was incubated in chromosomal media, with PHA, to
stimulate the proliferation of T-cell lymphocytes. The sample was incubated at 37 o C for
72 hours. Colcemid (10µg/ml) was added at 80µl to each sample and incubated at 37 o C
for 20 minutes. Colcemid, which disrupts spindle fiber formation, was added to prevent
the cells from dividing and therefore suspending them in metaphase for chromosomal
analysis. The sample was centrifuged in an International Equipment Company (IEC)
Centra CL2 centrifuge at 1200 rpm for 8 minutes, followed by aspiration and
resuspension in hypotonic medium. The hypotonic solution consisted of 10ml 0.075M
KCl at 37 o C. The tube was incubated in a 37 o C water bath for 9 minutes. The sample
was preserved by adding 2ml cold fixative (3 parts cold anhydrous methanol and 1 part
glacial acetic acid) and mixed by inverting the tube. The sample was centrifuged in the
IEC Centra CL2 centrifuge at 1200 rpm for 8 minutes. The supernatant was aspirated and
then 10ml room temperature fixative was added to the tube. The sample was allowed to
stand at room temperature for 20 minutes, followed by centrifugation for 8 minutes. The
supernatant was removed and 10ml room temperature fixative was added to the tube. The
8

centrifugation, aspiration, and fixative addition were repeated two consecutive times.
Once the final fixative was added, the sample was used for slide making.
Slide Making Procedure
A test tube rack was placed in a 65 o C water bath. A test tube rack was placed in
the water bath and the clean slides were placed on the test tube rack and leaned against
the wall of the water bath at a 45 o angle. Slides were allowed to warm up, then 3-5 drops
of cell suspension were dropped onto the slide. Slides were removed after 30-60 seconds
and the backs were wiped clean with a dry cloth. Slides were placed on a slide rack and
then in the drying oven at 65 o C for 24-48 hours. The slides were G-banded as described
below.
G-banding
Slides were dipped in trypsin, 5ml 0.25% trypsin in 45 ml Hanks Balanced Salt
Solution (HBSS) without Mg +2 and Ca +2 , for 2 minutes. Next, slides were dipped in a
series of two NaCl saline solutions to stop the trypsin action. Finally, slides were stained
in Giemsa, 2ml stock Giemsa (Bio/Medical Specialties, Inc.) and 48ml Gurr’s phosphate
buffer at pH 6.8 (BDH, inc.), for 6 minutes 30 seconds. The slides were rinsed with tap
water and air-dried.
Sister Chromatid Exchange
Blood and tissue subcultures were set up for cell cycle kinetics using routine
cytogenetic techniques. Each subculture was prepared for sister chromatid exchange by
addition of 7.5ug bromodeoxyuridine per ml media. Tubes were wrapped in foil for
9

protection from light, which may increase the exchange rate between chromatids.
Cultures were incubated at 37 o C for 72 hours and then harvested in minimal light. The
slides were prepared and then stained with Wright’s stain (Criterion Sciences, Inc.).
Wright’s stain was made in a small test tube by combining 3ml Gurr’s buffer with 1ml
Wright’s stain. A pipette was used to mix and flood the slide with the solution. Two
slides may be stained from one tube of stain. The slides were rinsed with water after 2
minutes and then air-dried. One hundred banded metaphase cells were scored by
microscope location coordinates and identified as being normal or trisomy 8.
Fluorescence Plus Giemsa (FPG) Technique
Sister chromatid exchange slides were destained by placing them in fresh fixative
for 2 minutes. The slides were then air dried and placed in Hoechst 33258 for 15
minutes. Hoechst stain was made by filling a glass coplin jar with Gurr’s buffer and
adding enough Hoescht powder until the solution was pale yellow. The slides were then
rinsed in distilled water and air-dried. The slides were placed in a square petri dish and
flooded with Gurr’s buffer. The dish was placed 5 inches under a 75 watt sun lamp for
25 minutes. The slides were rinsed in distilled water and placed in a new square petri
dish. Several drops of saline sodium citrate (2XSSC) solution, prepared by adding 4ml
20XSSC (175.3g 3M NaCl and 88.2g 0.3M Na3 citrate dissolved in 1000ml distilled H20)
and 36ml distilled H20, were applied to the slide, which was then covered with a glass
coverslip. The covered petri dish was placed in a 65 o C water bath for 15 minutes. The
slides were rinsed in distilled water and air-dried. The slides were stained in 4% Giemsa
(48ml distilled water and 2ml Giemsa stock) for 10 minutes. Finally, the slides were
10

insed with water and allowed to air dry. Metaphase cells previously located were
evaluated for number of cell divisions. One division was noted when the chromatids were
equally stained. Two divisions were determined by half of the chromatids stained dark
and the other half stained pale. Lastly, three divisions were designated by one-fourth of
the chromatids stained dark and three-fourths of the chromatids stained pale.
Lymphoblast Transformation
Cord blood was transformed with Epstein Barr virus, which stimulated B-cell
proliferation. In a 50ml sterile centrifuge tube containing 5ml–10ml of cord blood, an
equal amount of RPMI 1640, containing 1% antibiotics (10,000units/ml Penicillin and
sodium and 10,000µg/ml Streptomycin sulfate in 0.85% saline), was added to the tube
and mixed by inversion. A 15ml centrifuge tube was prepared with 3ml Histopaque-1077.
The blood mixture(8ml) was carefully overlaid into the Histopaque tube with a pipette.
The tube was centrifuged at 400 g for 35 minutes at room temperature. Mononuclear
cells were in the luminescent band between the serum/media top layer and the
Histopaque bottom layer. The mononuclear cell band was carefully removed and placed
in a clean 15ml centrifuge tube. The volume of the tube was brought up to 15ml with
RPMI 1640 and mixed by inversion. The sample was centrifuged at 400 g for 20
minutes. The supernatant was removed and the cells were resuspended by vortexing.
The tube was filled with 15ml RPMI 1640 medium and mixed well. The sample was
centrifuged for 20 minutes. The supernatant was removed and the cells were resuspended
by vortexing in 10ml RPMI 1640, containing 1% antibiotics, and 15% heat-inactivated
fetal bovine serum. Next, 40ul of 5mg/ml cyclosporin was added. The sample was
distributed into several wells of a 12 well plate. Next, 0.5ml Epstein-Barr virus filtered
11

supernatant was added to each well while mixing. The plates were incubated in 100%
humidity incubator at 37 o C, and checked one day after initiation for contamination.
Thereafter, cultures were checked at weekly intervals for clumped cells. The cells were
transferred to a 25cm 2 culture flask when a large number of clumps were present, within
3 to 4 weeks. The samples were subcultured by pouring the contents of the flask into a
15ml tube. The cells were pelleted and the supernatant was removed with a sterile pipet.
Double the amount of fresh media was added to the cell suspensions for a 1:2 split. The
flasks, with the cap slightly loose, were stood on end in the incubator. Subculturing was
performed twice a week.
Chromosomal harvest of lymphoblasts
The cells were subcultured by placing the cell pellet into double the media and
then dividing between two 25cm 2 flasks. The flasks were set on their ends and incubated
at 37 o C for 48 hours in 5% CO2 incubator. Two hours before harvest 0.05ml colcemid
(10ug/ml) was added to each flask. The culture was poured into a 15ml tube and
centrifuged at 1500 rpm in IEC Centra CL2 centrifuge for 7 minutes. All except 1-2ml
media was removed and the cells were resuspended by agitation. Once the cells were
mixed, 0.05ml warm 0.075 M KCl was added. The volume of the tube was brought to
10ml with 0.075 M KCl and mixed by inversion. The culture was incubated at 37 o C for 5
minutes and centrifuged for 7 minutes in IEC Centra CL2 centrifuge. The supernatant
was removed and the cells were resuspended by agitation. A small amount of fixative
was slowly added to the tube and the total volume was brought to 5ml with fixative. The
sample was centrifuged and the supernatant removed. Cells were resuspended with 4ml
12

fixative, and the centrifugation and resuspension with 4ml fixative was repeated. Finally,
the sample was centrifuged and all except 0.5 to 1ml of supernatant was removed,
depending on the pellet size. The cells were mixed by gently pipetting. The cells were
prepared for the slide making procedure.
Fibroblast Cultures
The placental tissues were placed on the top half of a sterile 100mm petri dish
containing Amniomax culture medium. The amnion, chorion, and villi were minced into
small pieces, while the blood vessel (fetal tissue) was removed from the umbilical cord
and minced. The pieces were placed into five 35mm culture plates and incubated for 4
hours at 37 o C. Culture media (1ml) was carefully added so as not to dislodge the
explants. Cultures were checked every 48 hours and the media was changed at least once
a week by aspirating off the old media and adding 2ml Amniomax at room temperature.
The cells grew from the explant, usually first as epithelial (round) cells and then as
fibroblasts (elongated). To subculture the cells, 2 to 3 dishes with good fibroblast growth
were selected. The media was removed and the dish was rinsed with HBSS, without
Mg +2 and Ca +2 . To each dish, 0.5ml or less of trypsin/EDTA was added and the sample
was then placed in the 5% CO2 incubator at 37 o C for 5 minutes. The flasks were scanned
under an inverted microscope for the loosening of cells; tapping the bottom of the plate
helped to dislodge the cells. When the cells were detached, 2ml of media were added to
stop the trypsin protease action. A new flask was prepared to receive 1.5 ml of cells, as
0.5ml was replaced into the original dish. The flasks were returned into the incubator.
13

Chromosomal Harvest of Fibroblast Cultures
The newly subcultured flask was checked under the inverted microscope for
proper cell confluence. Two to three flasks, with rounded cells, were selected and 0.1ml
of 10mg/ml colcemid was added, gently rotating the flask to mix. The culture was
incubated at 37 o C for 3 to 4 hours. All of the media was pipetted off and placed in a 15ml
tube. The flasks were rinsed with HBSS without Mg +2 and Ca +2 . The supernatant was
removed and added to the centrifuge tubes. Each flask was filled with 0.5 to 1ml trypsin
EDTA and incubated for 5 minutes. When the cells became detached 2ml HBSS with
Mg and Ca was added to stop the trypsin action and the cell suspension was placed in a
centrifuge tube. The sample was centrifuged at 1000-1200 rpm in IEC Centra CL2
centrifuge for 5 minutes and the supernatant was discarded without disturbing the pellet.
Slowly 10-12ml room temperature 0.075 M KCl was added while mixing the cells. The
sample was placed in the 37 o C incubator for 8 minutes. The cells were centrifuged for 5
minutes and the supernatant was discarded. To the tube, 5ml fresh cold fixative was
added very slowly while mixed into a fine suspension. The suspension was refrigerated
for 1-2 hours. The sample was centrifuged for 5 minutes, the supernatant was discarded,
and the cells were resuspended with new fixative. The sample was centrifuged for 5
minutes and all except 0.5-1ml fixative was removed. The cells were gently resuspended
with a pipette. The cell solution was prepared for slide making procedure. Once made,
the fibroblast slides were dried overnight at 50-55 o C.
14

FISH slide preparation
Day 1
Slides were made by following the slide making procedure protocol, however
they were air-dried and not oven dried. Once dry, slides were dipped into 37 o C 2XSSC
solution for 30 minutes, removed and rinsed in increasing concentrations (70%, 80%, and
95%) of ethanol for 2 minutes each and air-dried. In a micro-centrifuge tube 1ul of
chromosome 8 alpha-satellite probe, 1ul of chromosome X alpha-satellite probe and 30ul
Hybrisol were mixed. The entire volume was pipetted onto the slide and followed by a
coverslip placed on the slide and sealed with rubber cement. Slides were inserted into the
Hybrite hybridization chamber where the tissue and probe DNA was denatured at 75 o C
for 5 minutes and hybridized at 37 o C overnight.
Day 2
Slides were removed from the chamber and the rubber cement was removed.
Coverslips were carefully lifted straight off the slide, making sure not to disturb the cells.
Slides were dipped in 0.5% SSC solution for 5 minutes at 72 o C. Slides were then placed
in Phosphate Buffer Detergent (PBD) for 2 minutes. Slides were laid flat in a square petri
dish and 60ul of Rhodamine-antidigoxignenin (Ventana Medical Systems) was pipetted
onto the slide. Slides were covered with a coverslip and placed in a 37 o C incubator for 20
minutes. Slides were then rinsed in fresh PBD 3 times for 2 minutes each. Finally, 20ul
DAPI (4’,6-diamidino-2-phenylindole) (Ingen Laboratories), the counterstain, was added
on the slide and covered with a coverslip. Slides were placed in a covered box and kept in
the refrigerator. All steps in the FISH slide preparation were done in minimal light to
ensure maximal brightness of the probe signals. Scoring of the red and green signals was
15

done using a UV-fluorescent microscope with triple band filters. Two red signals and
one green signal designate normal male cells, whereas three red signals and one green
signal indicate trisomy 8 male cells.
16

Case Report
RESULTS
Amniocentesis, performed due to abnormal maternal serum triple screen,
diagnosed the fetus with T8m. Twenty-five metaphase cells were investigated and
resulted in 47,XY+8 [8] and 46,XY [17]. The male infant was delivered naturally at 40
weeks gestation and weighed 3.3kg, which falls in the 50 th percentile. Physical
manifestations included bitemporal narrowing, large fleshy ears, hypoplastic nails, deep
palmar and plantar furrows, first-degree hypospadias, severe cardiac enlargement
secondary to pulmonary hypertension, and feeding difficulties. The infant expired at 8
weeks of age due to pulmonary hypertension.
At the time of birth various fetal tissues were collected including peripheral
blood, cord blood, umbilical cord (blood vessel), and extra-embryonic tissues including
chorion, amnion, and villi. Each sample was investigated separately for metaphase and
interphase analysis, and cell cycle kinetics.
Tissue Mosaicism Determination
Once the tissues were cultured and harvested, the slides were prepared and
G-banded (see figures 1 and 2) for analysis of mosaicism percentage. Trisomy 8 cells
were found in every sample, except for the transformed lymphoblasts (see Table 1).
17

Figure 1 Normal [46, XY] Karyotype
18

Figure 2 Trisomy 8 [46, XY, +8] Karyotype
19

Table 1.
Metaphase Counts
Tissue 46, XY 47, XY+8 Total cells
Cord Blood 45 55 100
Transformed Blood 100 0 100
Peripheral Blood 47 53 100
Umbilical Cord 43 57 100
Amnion 57 43 100
Chorion 92 8 100
Villi 60 6 66*
*insufficient sample size
20

When comparing embryonic tissues, cord blood, transformed blood, peripheral
blood, and umbilical cord, the percentage of trisomy 8 cells were within 4% of each
other, excluding the transformed blood, which resulted in 0% trisomy 8 cells. Since
different cells are stimulated in the peripheral and cord blood (T-cells) than the
transformed blood (B-cells), the data suggests the T-cells are more tolerant of the extra 8
chromosome or perhaps the path of the lymphoblast lineage is involved. Between the
extra-embryonic tissues, the amnion had a higher number of trisomy 8 cells than the
chorion and villi in the metaphase spreads.
The goal for FISH interphase counts was 500 cells in each sample (Table 2). The
interphase cells were scored as a normal male with two red (8) and one green (X) signal
(figure 3) and as trisomy 8 with three red (8) and one green (X) signal (figure 4).
21

Figure 3 FISH Normal [46, XY] Interphase
22

Figure 4 FISH Trisomy 8 [46, XY, +8] Interphase
23

Table 2.
FISH Interphase Counts
Total Maternal
Tissue 45, XY, -8 46, XY 47, XY,+8 Cells Contamination
Cord Blood 8 186 306 500 -
Transformed Blood 7 469 24 500 -
Peripheral Blood 3 127 370 500 -
Umbilical Cord 6 52 442 500 1.96%
Amnion 9 243 248 500 -
Chorion 1 113 36 150† 83.68%
Villi 0 0 0 0‡ -
† low sample size due to high maternal contamination
‡ insufficient sample
24

There was a notable difference between the normal and trisomy 8 cells in the cord
and peripheral blood (T-cells) versus the transformed blood (B-cells) in the interphase
counts also (Table 2). Once again the number of trisomy 8 cells is higher in the cord and
peripheral blood and low in the transformed blood. The amnion was also elevated in
trisomy 8 interphase cells than in the chorion, the villi could not be tested for comparison
due to tissue growth failure.
When recording the interphase FISH counts maternal contamination was a factor.
The chorion was only scored for 150 cells due to high maternal contamination of 83.68%.
The umbilical cord resulted in 1.96% maternal contaminant, while the remaining tissues
were not contaminated.
25

Table 3.
Trisomy 8 Cells in G-banded Metaphases vs. FISH Interphases
Tissue Metaphase Interphase* Chi Squared
Cord Blood 55% 62% p

For each tissue the metaphase and interphase percentages of trisomy 8 cells were
statistically compared by the Chi-squared goodness of fit test (Table 3). The trisomy 8
cell percentages of the cord blood and amnion were found not to be significantly
different, while the transformed blood, peripheral blood, umbilical cord, and chorion
were found to have a significant difference of at least p

Figure 5. First Division – Cell Cycle Kinetics
28

Figure 6 Second Division – Cell Cycle Kinetics
29

Figure 7 Third Division – Cell Cycle Kinetics
30

Table 4.
Division Rates for Normal vs. Trisomy 8 Cells
Number 1 st 2 nd 3 rd Chi
Tissue Metaphase Cells Division Division Division Squared
Cord Blood 46,XY 39 10% 36% 54% p

Cell cycle kinetics (Table 4) was performed to analyze possible growth rate
differences between the trisomy 8 and normal cells. Cord blood, transformed blood, and
amnion were the only tissues that resulted in successful cell cycle kinetics information.
Statistical investigation was done by contingency tables and the Chi-squared goodness of
fit test, which in all three cases showed no evidence of a significant difference in cell
growth rates between normal and trisomy 8 cell lines.
32

Origin of Trisomy 8 cells
DISCUSSION
T8m occurs due to mitotic nondisjunction within a normal fetus, therefore
introducing a second cell line (James and Jacobs 1996; Karadima et al. 1998; Nicolaidis
and Petersen 1998; Webb et al. 1998; Kalousek 1999). The presence of trisomic cells
throughout fetal tissues and placental tissues depends directly on the timing of the
nondisjunctional event (Webb et al. 1998). The earlier the event in embryonic
development, the more tissues will be affected. This includes CPM, which results in a
normal fetus and abnormal placenta, due to the nondisjunction occurring in a placental
lineage cell. Abnormal placental tissues are correlated to abnormal fetal development,
whereas normal to low placental mosaicism supports growth and intrauterine survival of
abnormal fetuses (Farra et al. 2000). T8m anomalies, however, are not correlated to the
level of mosaicism present within fetal tissues. A similar mosaic constitution within the
tissues has been reported for patients, who have had drastic differences in their
phenotypes, some being phenotypically normal, where others have severe mental
retardation and multiple skeletal anomalies (James and Jacobs 1996; Jordan et al. 1998).
The investigation of tissues collected from our patient lead to the determination of
an early nondisjunction event since both embryonic and extra-embryonic tissues were
mosaic. The embryonic samples, which included umbilical blood, peripheral blood
(T-lymphocytes), and umbilical cord each were scored for over 50% trisomy 8 cells in
G-banded metaphases, while no trisomic cells were found in the transformed blood
(B-lymphoblasts) (Table 1). This difference seen in the level of mosaicism between the
33

T-lymphocytes and B-lymphoblasts in G-banded metaphases may be due to cell lineage
separation or perhaps T-lymphocytes are more tolerant of an extra 8 chromosome.
The extra-embryonic samples, which included amnion, chorion, and villi, each
contained fewer than 50% trisomy 8 cells, with chorion and villi having less than 10%
trisomy 8 cells (Table 1). The presence of trisomic cells in the embryonic and extra-
embryonic tissues confirms the early timing of the non-disjunction mitotic event; before
the embryonic and placental lineages separated. Had the non-disjunction occurred later,
after late blastocyst, the resulting levels of mosaicism would have been either confined to
the fetus, therefore having a normal placenta, or CPM with a normal fetus. The lower
level of trisomy 8 cells in the placental tissues could provide an explanation for the
pregnancy progressing to full term, with normal birth weight, despite the severity of the
patient’s phenotype. Once again this supports the concept of a normal placenta
supporting the growth and development of an aneuploid fetus to full term (Farra et al.
2000).
Growth disadvantage of Trisomy 8 cells
Selective growth advantage of normal cells in mosaic tissues was described by La
Marche et al. in 1967, while studying mosaic trisomy 18. La Marche et al. found an
individual’s mosaicism significantly changed from 90% trisomy cells/10% normal cells
at birth to 100% normal cells at ten months of age. They described this event as
disappearing mosaicism and concluded, “the normal population of cells exercised a
growth advantage, with eventual replacement of the abnormal cells” (La Marche et al.
1967). More recently, Mark and Bier (1997) and Jordan (1998) each concurred with the
34

original finding of La Marche in their patients with T8m. The growth advantage of
normal cells over the trisomic cells resulted in the decrease of trisomic cells and lead to
the complete disappearance of trisomic cells over time (Mark and Bier 1997).
Support for selective growth advantage of normal cells is evident from the
decrease of trisomy 8 cells from interphase to metaphase found in the transformed blood,
peripheral blood, umbilical cord, and chorion (Table 3). The significant difference
suggests there is a mechanism resulting in the decrease of trisomic cells in metaphase.
Alpha-satellite probes used in the FISH interphase study are known to give a large signal
and hence have a greater chance of overlap within the cell (Moore et al. 2000). The
sensitivity of the DNA probes has been reported as 80-90% efficiency for detecting the
specific region if present on the chromosome (Moore et al. 2000 and Ruangvutilert et al.
2000). Therefore, the efficiency of FISH probes would explain a decrease in trisomic
interphases compared to metaphase, but would not explain the reverse.
Mechanisms
Mechanisms that may be responsible for the significant decrease in trisomy 8
metaphases include varying cellular growth rates, apoptosis or cell death, and entry into
the GO phase. First we suggested a growth rate difference between the normal and
trisomy 8 cells. Trisomy 8 cells may have a more difficult time undergoing mitosis due
to the extra chromosome and therefore genetic information imbalance, while the normal
cells would divide as usual. This would possibly slow down the division rate since
replication, chromosomal alignment, and division times must be in synchrony for proper
cellular division. Growth disadvantage has been reported in a comparison of 45,X and
35

46,XX cells (Verp et al.1988). Cell generation times were significantly different between
the two cell lines, resulting in abnormal cells with a longer turnover time (Verp et
al.1988). However, the results from our cell cycle kinetics analysis (Table 4) show
normal and trisomy 8 cells in the cord blood, transformed blood, and amnion do not have
a significant difference in growth rates. Therefore, these results exclude the growth rate
difference as a causal mechanism for the decrease in trisomy 8 cells.
Another mechanism proposed for the loss of trisomy 8 cells is apoptosis or
programmed cell death. Apoptosis occurs when a cell has sustained DNA damage or
been subjected to an injury or viral infection. This ingrained defense mechanism
recognizes errors and deters cellular division, resulting in cellular arrest. Chromosome 8
houses an oncogene, c-myc, directly related to checkpoint cyclins and the cell cycle.
Overexpression of c-myc has been known to result in the arresting of cells at the G2 phase
checkpoint, and therefore causing cells to undergo apoptosis before reaching metaphase
(Felsher et al. 2000). The apoptotic mechanism is assumed possible due to the additional
8 chromosome in the aneuploid cells, and may be involved in the complete disappearance
of trisomic cells over time. Studies on our patient were not performed to examine cell
death.
Finally, we suggested the entry of trisomy 8 cells into the GO phase of the cell
cycle would cause a decrease in trisomic metaphases. The GO phase, usually associated
with non-dividing cells and resting cells, occurs after mitosis and during the G1 phase of
interphase. This phase may last a short length of time, extended period of time, or
indefinitely. A passage into the GO phase would inhibit the procession into mitosis,
therefore resulting in a decrease of metaphase cells. This has been seen in mosaic
36

Pallister Killian patients with the absence or reduced level of i(12p) cells in metaphase
(Wenger et al. 1990; Thornberg Reeser and Wenger 1992). Cells exiting the cell cycle
and entering the GO phase is more likely a mechanism for our patient than apoptosis,
because cell death should not change the ratio of interphase to metaphase trisomy 8 cells,
whereas cells entering the GO phase would cease to continue with cell division and
therefore result in a decline of mitotic cells.
Conclusion
The trisomy 8 cell line as a whole is not delayed in going through the cell cycle,
which is evident in the statistical comparison shown in Table 4. However, a portion of
the cells are apparently exiting out of the cell cycle and no longer dividing, but not
undergoing apoptosis. This provides a selective growth advantage for the normal cells.
Exiting of trisomy 8 cells from the cell cycle will result in a decrease of the cell line over
time, which is supported in the literature (La Marche 1967; Mark and Bier 1997; Jordan
1998).
Further Studies
Further investigative measures would have to occur to determine the exact
mechanism causing the decrease of trisomy 8 cells in metaphase. Follow-up testing in
this case could not be performed to investigate each mechanism due to the patient
expiring and exhausting cell lines. However, in other cases, tissues could be examined
by tests including flow cytometry for apoptotic body identification (Felsher et al. 2000).
Also, DNA content measuring by fluorescence-activated cell analysis would identify
37

interphase cycle status to determine if cells were ascertained in the GO phase (Felsher et
al. 2000). These tests as well as others could pinpoint the mechanism involved, resulting
in other cases of T8m patients with decreased trisomic cells undergoing mitosis, making a
distinction between cells in GO and apoptosis.
38

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