April Journal-2009.p65 - Association of Biotechnology and Pharmacy

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