Abstract:
The tumor suppressor gene TP53 encodes a nuclear protein that prevents the cells from
dividing before DNA damage is repaired. Mutations in TP53 gene have effects on its
biological activities. The objectives of present study aims at determining the frequency TP53
mutations in sporadic, genetic lineage and analysis of the data i.e. questionnaire collected
from breast cancer patients from Pakistan, during the study.
Female breast cancer patients were recruited at Shaukat Khanum Memorial Cancer Hospital
& Research Centre and Mayo Hospital, Lahore Pakistan, from January 2005-December 2008.
A total of 150 sporadic breast cancer patients and three families with breast cancer cases
were included in the study. From all study participants, a blood sample and a piece of tissue
of normal and tumor both were collected. DNA was extracted and exons 5-8 (central region)
of TP53 gene were PCR amplified. Each sample was heteroduplexed with a normal control
sample (confirmed by sequencing). To screen TP53 mutations Temporal Temperature
Gradient Gel Electrophoresis (TTGE) was performed. The mutations were confirmed by
sequencing. Restriction Fragment Length Polymorphism (RFLP) was used for understanding
the status of codon 72, exon 4 of TP53 gene polymorphism (arg/arg) in Pakistan. The data
was analyzed using the R15 programme, provided by International Agency for Research on
Cancer. Three deleterious mutations were detected in the sporadic breast cancer patients, viz.,
codon 238 where TGT is mutated to TAT (cys to tyr), codon 248 where CGG is mutated to
CAG ( arg to glu), and codon 278 where CCT is mutated to TCT (pro to ser). These
mutations were not detected in normal breast tissue and blood samples of these patients. R15
analysis (IARC, 2011) of TP53 gene mutations showed that the mutations detected in
Pakistani breast cancer patients are reported most prevalent somatic mutations (codon 238 =
79 tumors, codon 248 = 779 tumors and codon 278 = 74 tumors) in breast cancer patients of
the world. Three-dimensional structures were predicted by 3D Viewer (software given on
IARC website) and found that all these three mutations are in DNA binding region of TP53
and could change the structure of protein and, therefore, affect its function. TP53 mutation
has not been observed in normal persons and breast cancer families blood samples. One
family was detected with Li-Fraumeni syndrome characters but TP53 mutations are not
found in it.
Although the polymorphism arg/arg, codon 72, exon 4 of TP53 gene is reported as a
functional relevant polymorphism that contributes to breast cancer development yet in the
vpresent study, genotype arg/pro and pro/pro, both polymorphisms were found more
significant in Pakistani breast cancer patients as compared to arg/arg with corresponding ratio
of arg/pro (53.3): pro/pro (34.6): arg/arg (12). Normal controls showed about the same
difference in ratio of arg/pro: pro/pro: arg/arg, (50:40:10).
Correlation of TP53 mutations with clinicopathological parameters (data collected by
questionnaire) was observed. Patients were divided into two groups; group 1 (TP53 non
mutated) and group 2 (TP53 mutated). As both groups have not shown any difference so no
prominent correlation between TP53 mutations and clinicopathological parameters was
found.
It is concluded that the frequency of TP53 gene mutations in DNA coding region (5-8 exon )
is low in Pakistani breast cancer patients. However, present study is in favor of the fact that
the frequency of TP53 gene mutations is different in different geographical areas. Genotype
arg/arg is less prevalent in the female breast cancer patients and normal population of
Pakistan. There was no significant correlation between TP53 mutation and tumor
aggressiveness e.g. nodal status, size, ER/PR, histopathology etc. Epidemiologically, no
carcinogen was found important as a causative factor of TP53 gene mutations in Pakistani
breast cancer patients.