THESIS
2014
xii, 129 pages : illustrations (some color) ; 30 cm
Abstract
Genetic testing can provide important clinical benefits. Genetic testing has been
done for diverse purposes, including the diagnostic testing; presymptomatic and
predictive testing and drug-resistance testing. The present study developed
genetic testing for two human diseases. Breast cancer is a complex disease that
causes approximately 23% of new cancer cases annually. Due to the
multifactorial and multiethnicity nature of breast cancer, the identification of the
risk alleles for breast cancer in Asian population greatly lags such identification
amongst American and European populations, and thus takes on special
significance. Concurrently, drug-resistant forms of tuberculosis arising from
mutations in the haploidal genome of Mycobacterium tuberculosis is becoming a
serious c...[
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Genetic testing can provide important clinical benefits. Genetic testing has been
done for diverse purposes, including the diagnostic testing; presymptomatic and
predictive testing and drug-resistance testing. The present study developed
genetic testing for two human diseases. Breast cancer is a complex disease that
causes approximately 23% of new cancer cases annually. Due to the
multifactorial and multiethnicity nature of breast cancer, the identification of the
risk alleles for breast cancer in Asian population greatly lags such identification
amongst American and European populations, and thus takes on special
significance. Concurrently, drug-resistant forms of tuberculosis arising from
mutations in the haploidal genome of Mycobacterium tuberculosis is becoming a
serious cause of deaths worldwide. In view of this, in the present study the novel
One Label Extension (OLE) method developed by our laboratory has been
applied to genotyping the single nucleotide polymorphisms (SNPs) associated
with susceptibility to breast cancer in Asia and the SNPs underlying drug
resistant tuberculosis. The results showed that, even with the use of just a
single-pass OLE genotyping, ≥90% concordance was obtained between the
genotype determined using the OLE method and the genotype determined using
the more expensive direct DNA sequencing method for nine out of twenty of the
breast-cancer associated SNPs, namely No.3 (rs889312), No.5 (rs13281615),
No.9 (rs6504950), No.11 (rs10941679), No.12 (rs2046210), No.13 (rs2725362),
No.14 (rs4784227), No.17 (rs10484919) and No.18 (rs107543). With
drug-resistant tuberculosis, based on phenotyping results obtained from
microbiology-based drug susceptibility tests, the sensitivity, specificity and test
efficiency estimated for One Label Extension assays were respectively 83.9%,
95.5% and 92.4% with the ropB gene in rifampin resistance, 67.3%, 97.1% and
84.3% with rpsL and rrs in streptomycin resistance, 60.0%, 96.0% and 91.4% with embB in ethambutol resistance, 68.4%, 94.9% and 86.3% with inhA and
katG in isoniazid resistance, and 74.1%, 98.9% and 93.2% in multiple drug
resistance defined as resistance to at least both isoniazid and rifampin. Upon
comparison between the genotype of each target site determined by OLE and
the genotype determined by direct DNA sequencing for the PCR amplicons, 100%
agreement between the two sets of determinations was obtained for all of the
121 clinical samples. The results showed that the OLE method yielded a rapid,
accurate and relatively inexpensive method for detecting drug-resistant M.
tuberculosis. These findings suggest that the OLE method can make an
important contribution to the analysis of population-wide screening and
investigation of disease related genes in the diploidal human genome as well as
the haploidal genomes of disease causing microbes.
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