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Concordance of the CKIT gene with del(9q) as a cause of Acute Myeloid Leukemia
Kyle Marsh
Abstract
Concordance of the CKIT gene with del(9q) as a cause of
Acute Myeloid Leukemia
Kyle Marsh
Manchester-Essex High School, Manchester-by-the-Sea, MA
Teacher, Dr. Maria Burgess, Manchester-Essex High School
Mentor, Dr. David Sweetser, Mass General Hosp, Boston, MA
Acute Myeloid Leukemia (AML) is caused by a combination of
various classes of genetic mutations, including tumor
suppressor genes, transcription factors/core-binding factors
(CBFs), and chromosomal translocations and deletions. One
specific deletion on chromosome 9, del(9q), and the
translocation t(8;21), have been found together in AML, and it is
likely these two mutations cooperate to cause leukemia. Not all
cases of del(9q) AML have t(8;21) and it appears that other
mutations can cooperate with del(9q). To help better understand
how different mutations cooperate to cause leukemia, AML
samples with del(9q) were screened for mutations in exons 8
and 17 of the CKIT gene, a cytokine receptor. Of 41 samples,
only one mutation was found in exon 8. The low incidence of
mutations suggests further studies are necessary.
Figure 1. The CKIT gene. CKIT is a
cytokine receptor which, when
mutated, has been thought to be
involved in the etiology of acute
myeloid leukemia.
Materials and Methods
•To find mutations in exon 8 and 17, samples were prepared for
sequencing.
•PCR was used to amplify the section of DNA (exon 8 or 17) of
the CKIT gene.
•The PCR reaction “master mix” consisted of Taq (DNA
polymerase), Taq buffer, primers (to flag the exon), dNTP
(nucleotides), and the AML sample.
•The reaction was run at 58oC for 35 cycles.
Figure 2. Pictured is the “master mix”
made for PCR. This mix included
primers for both exons 8 and 17, Taq
(DNA polymerase), Taq buffer, and
dNTP (nucleotides). DNA was then
added to the mix and run at 58 degrees
for 35 cycles.
Figure 3. Pictured is the BioRad
Polymerase Chain Reaction machine
that was utilized for all of our samples.
After the reaction was complete, we
then ran the amplified samples on a gel
in order to see if the DNA sample was
readable and ready for sequencing.
• DNA samples were then size fractioned via electrophoresis
using 1.5% agarose gel.
• Ethidum bromide allowed visualization of the DNA.
• ~4-5 ml of DNA were inserted into each well.
• Pictures of each gel determined if troubleshooting was
necessary.
• If the DNA band was clear, the sample was sent for
sequencing.
Authentic Science Research Program
Manchester-Essex Regional High School, Manchester-by-the-Sea, MA 01944
Results
• In exon 8, we were looking for a deletion in codons 416-420
• In exon 17, we were looking for point mutations.
• We found one mutation for both exons.
• Analysis showed this mutation in sample 267, exon 8 only.
• Found deletion in codon 419, as previously suggested
• Mutations in 2% of the exon 8 samples, compared to 53%, and
0% of the samples had mutations in exon 17, compared to
previous study’s 41%.
Figure 5. Shown is the process of gel electrophoresis. On the left Is a picture
of a gel mid-process. DNA’s natural negative charge forces the DNA to run
down the gel (through electric currents) towards the positive end of the gel.
On the right is a picture of a completed gel – all samples would be strong
enough for sequencing except for sample 6, where troubleshooting would be
necessary.
• DNA isolated via column purification and centrifugation
• Columns have pores that allow excess material, such as dNTP and
Taq, to slide through the membrane and be discarded
• DNA is eluded, and exon sent for sequencing
• After sequencing, analysis for mutations in exon was performed
• MGH “DNA Core” computer software was used
• Sequences were obtained through the DNA Core website represented
via chromatography peaks each peak representing a different base pair
2 colored bands on top of each other for one nucleotide means there
was a possible mutation
• Samples were then aligned to check if this was indeed a mutation on
a word document, which allowed us to compare nucleotides of each
sample.
Figure 6. Once our samples
were sequenced, we would
access the “DNA Core” website to
obtain our samples. Pictured is
the exact sequence of one
particular sample. Each
nucleotide is signified by a
different peak. When there was a
mutation, there would be a twotoned peak, and a pink “N” where
the base pair should be.
• No significant mutations noted.
• Significant polymorphisms in ~5% of the samples at exon 17
• Polymorphism is a synonymous base pair change that is not
expressed.
• Would be significant in the exon, but insignificant in the intronic
sequence, where they were found
Figure 7. Shown in exon 8 of the CKIT gene. In sample 267, we found one
deletion of codon 419, one of the codons highlighted in the green region
that Pollard suggested.
Literature cited
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Conclusions
Acknowledgments
• No mutations found in 41 in exon 17
• 1 mutation in exon 8
I thank Dr. David Sweeter for providing such an excellent opportunity in his
laboratory. He was a wonderful mentor who gave me the perfect balance of
independence and assistance. He was also a wonderful teacher of the genetics behind
AML – I learned a tremendous amount in the laboratory. I would also like to thank
the post-docs in Sweetser’s lab who were supportive and available if I had any
questions.
• Low incidence of these mutations suggest that further studies are
necessary to evaluate the importance of CKIT with concordance to
del(9q).
• Differences between studies supports further studies to characterize
AML samples with the del(9q) for the CKIT gene in order to study its
exact relevance in the etiology of AML.
• Major difference in our work and Pollard’s was our samples. Pollard
solely used pediatric samples, while we used both pediatric and adult
AML samples. Pollard also had ~5x more samples than our lab (203
compared to 41), thus, the sample size and type may have affected our
results.
• Pollard et al. predicted higher incidences of mutations in both exons
AML is a disease controlled by a myriad of both environmental and genetic
factors. In AML, del(9q) is found 30% of the time. High incidence of this
mutation suggests its potential role in leukemogenesis, Thus, the
characterization of each gene associated with del(9q), and of other genes is
warranted.
Figure 8. Pictured is the del(9q) mutation from three different karyotypes.
Supported by the Spaulding Education Fund.
For further information
Please contact [email protected]. More information on this and related projects can
be obtained at MGH’s research website,
http://www.massgeneral.org/research/researchlab.aspx?id=1196.