April Journal-2009.p65 - Association of Biotechnology and Pharmacy
April Journal-2009.p65 - Association of Biotechnology and Pharmacy
April Journal-2009.p65 - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong><br />
Vol. 3 (2) 181-187, <strong>April</strong> 2009. ISSN 0973-8916<br />
182<br />
represents at least 50% <strong>of</strong> the total hepatic<br />
CYP3A content in people polymorphically<br />
expressing CYP3A5, it might be the most<br />
important genetic contributor to interindividual <strong>and</strong><br />
interracial differences in CYP3A-dependent drug<br />
clearance <strong>and</strong> in responses to many medicines.<br />
There are substantial interindividual differences<br />
in CYP3A expression, exceeding 30-fold in some<br />
populations like African Americans, Southeast<br />
Asians, Pacific Isl<strong>and</strong>ers <strong>and</strong> Southwestern<br />
American Indians. The higher prevalence <strong>of</strong><br />
CYP3A5 expression in non-Caucasians indicated<br />
that they are more likely to experience higher<br />
clearance <strong>of</strong> drugs principally inactivated by<br />
CYP3A so less likely to experience dose-limiting<br />
toxicities, <strong>and</strong> have different risks <strong>of</strong> diseases that<br />
are associated with the CYP3A5 expressor<br />
phenotype. The relatively low levels <strong>of</strong> metabolic<br />
activity <strong>of</strong> CYP3A5 protein as observed in Korean<br />
population suggested that they would be at greater<br />
risk for drug toxicity even at conventional doses<br />
(5).<br />
Similar frequencies <strong>of</strong> CYP3A5*3 were<br />
observed in the leukemic patients <strong>and</strong> normal<br />
controls. Consequently, the finding suggested that<br />
the CYP3A5 polymorphism was not associated<br />
with the risk <strong>of</strong> myeloid leukemia (6). CYP3A5*6<br />
was not found in Asian population.<br />
Materials <strong>and</strong> Methods<br />
A group <strong>of</strong> 250 breast cancer patients were<br />
selected for the present study. 250 healthy <strong>and</strong><br />
age matched women without family history <strong>of</strong><br />
breast cancer or any other cancers were selected<br />
to serve as control group. Cases were chosen<br />
from Nizam’s Institute <strong>of</strong> Medical Sciences after<br />
confirmed diagnosis <strong>and</strong> controls included healthy<br />
volunteers. The diagnosis <strong>of</strong> breast cancer was<br />
established by pathological examination,<br />
mammography, Fine needle aspiration (FNAC)<br />
<strong>and</strong> biopsy. Epidemiological history such as age<br />
at onset <strong>of</strong> breast caner, diet, socioeconomic<br />
status, occupation, reproductive history, family<br />
Surekha et al<br />
history <strong>and</strong> consanguinity was taken through<br />
personal interview with breast cancer patients<br />
using specific pr<strong>of</strong>orma. The patients were<br />
screened for receptor status <strong>of</strong> estrogen,<br />
progesterone <strong>and</strong> HER-2/neu by immunohisto<br />
chemical assay. Clinical history such as size <strong>of</strong><br />
the tumor, presence <strong>of</strong> auxiliary nodes, extent <strong>of</strong><br />
metastasis, stage <strong>and</strong> type <strong>of</strong> the breast cancer,<br />
chemotherapeutic drugs used <strong>and</strong> prognosis <strong>of</strong><br />
the disease was collected with the help <strong>of</strong><br />
oncologist. Informed consent was taken from all<br />
patients <strong>and</strong> controls included in the study. The<br />
approval <strong>of</strong> ethical committee was taken before<br />
initiation <strong>of</strong> the work.<br />
Five milliliters <strong>of</strong> blood was collected in an<br />
EDTA vaccutainer from patients as well as<br />
controls. DNA was isolated (7) <strong>and</strong> used for<br />
amplification <strong>of</strong> CYP3A5*3 <strong>and</strong> CYP3A5*6 by<br />
PCR-RFLP (8). CYP3A5*3 polymorphism<br />
This polymorphism was detected by using<br />
modified primers. The polymorphism <strong>of</strong> the<br />
CYP3A5*3 (nt 22893 G) with the mutagenic base<br />
C at nt 22889 was introduced as the fourth base<br />
at 3’ end <strong>of</strong> the forward primer created the Dde<br />
I site after PCR (Fig 1) The 155 bp product was<br />
digested into 121 <strong>and</strong> 34 bp fragments for<br />
CYP3A5*1 <strong>and</strong> 97, 34 & 24 bp fragments for<br />
CYP3A5*3 allele. The heterozygote is identified<br />
by the presences <strong>of</strong> 121, 97, 34 & 24 bp fragments.<br />
CYP3A5*6 polymorphism<br />
PCR-RFLP was done for identification <strong>of</strong><br />
CYP3A5*6 polymorphism using specific primers.<br />
The amplified product (268 bp) was digested with<br />
1 unit <strong>of</strong> Dde I enzyme (New Engl<strong>and</strong> biolabs) at<br />
37 0 C for overnight <strong>and</strong> electrophoresed on 14%<br />
PAGE (Fig 2) <strong>and</strong> noticed DNA fragments <strong>of</strong><br />
size 120, 103, 25 &20 for CYP3A5*1 <strong>and</strong> 128,<br />
120 & 20 for CYP3A5*6 allele.<br />
Statistical analysis<br />
The results were analyzed using appropriate<br />
statistical tests by SPSS Version 14. Odds ratio<br />
was estimated to calculate the relative risk for