Date of Award

Spring 2001

Document Type


Degree Name

Doctor of Philosophy (PhD)


Biomedical Sciences

Committee Director

Christopher Osgood

Committee Member

Michael Stacey

Committee Member

Wayne Hynes

Committee Member

Frank Castora


Aplastic anemia (AA) is characterized by complete bone marrow failure. Progression to myelodysplastic syndromes (MDS) and acute nonlymphocytic leukemia (ANLL) occurs frequently. At the time of transformation, cytogenetic abnormalities are common. Detection of cytogenetic abnormalities prior to leukemic transformation may indicate future disease progression. Karyotype analysis is the current method of choice to evaluate chromosome aberrations. However, fluorescence in situ hybridization (FISH) is more sensitive in detecting these abnormalities.

hBUB1, a mitotic spindle checkpoint gene, was shown to be mutated in two colorectal cancer cell lines with high levels of aneuploidy (Cahill, et al., 1998). Although theoretically possible, conclusive evidence does not currently exist establishing a link between aneuploidy levels and mutations within a mitotic spindle checkpoint gene. hBUB1 is the most characterized of the mitotic checkpoint genes.

FISH was used to detect cytogenetic abnormalities for chromosomes 7 and 8 in bone marrow samples from patients with AA. In addition, ribonucleic acid (RNA) from all patient samples also underwent hBUB1-specific reverse transcription polymerase chain reaction (RT-PCR), followed by sequencing of the RT-PCR product. Statistical analyses were performed on FISH and sequencing results. Additional samples from patients with a variety of bone marrow disorders also underwent hBUB1-specific RT-PCR and sequencing without FISH analysis.

Seven patient samples out of 46 (15.2%) showed elevated levels of aneuploidy for chromosomes 7, 8, or both. Four of the seven samples showed abnormalities previously undetected by a karyotype analysis. This indicates that FISH analysis is approximately twice as sensitive as a karyotype analysis, and may assist in earlier diagnosis and proper treatment of patients with AA. Statistical analysis showed an increased level of monosomy 8 in African-American males. The age of the patient and responsiveness to treatment did not correlate with the level of aneuploidy. Results from the hBUB1-specific RT-PCR and sequencing were inconclusive due to the high probability of Taq-induced PCR artifact, however there was no apparent correlation between the presence of aneuploidy and the sequencing results. Seventy-eight to 85% of all patient samples analyzed did not amplify any hBUB1-specific RT-PCR product.