台灣乳癌病理成因之研究-乳癌檢體中含變異之動情激素接受體 與臨床上之關聯性(第三年)
Date Issued
1999-07-31
Date
1999-07-31
Author(s)
張金堅
DOI
882314B002364
Abstract
To investigate the correlation
between the presence of ERa variants
and prognosis of breast tumors from
Taiwan, we detailed examined the coding
region of ERa gene. One sense point
mutation and 3 polymorphisms have been
found. The point mutation, P324S, is
located at the end of the beginning helix
of ER ligand binding domian. The 3
polymorphisms showed strong
disequilibrium in allele distribution and
one of the polymorphisms showed allele
preference in tumor. However, the allele
preference is not significantly associated
with tumor recurrence. To elucidate the
possible reason of disequilibrium in
allele distribution, we compared
polymorphisms of paired tumor-normal
counterpart tissues, and found the
disequlibrium may due to a loss
expression of one allele phenomenon. In
searching for exon deletion variants,
alternative spliced variants were found.
Moreover, a deletion of 32 nucleotide 3’
to the exon 1 only (d32nt) or together
with the whole exon 2 (d32nt-D2)
through aberrant splicing, which has
never been reported, was found to exist
extensively in tumor tissues in our study.
In addition, types and amount of deletion
variants were more dominant in tumor
tissue than normal tissues. However, the
elevated expression of deleted variant forms did not showed significant
association with tumor recurrence.
Introduction
Estrogen exposure is one of the
major risk factors of breast cancer
development. Many epidemiological
surveys support this relationship
(reviewed in Pike et al. 1993). The
biological mechanism is poorly
understood. One hypothesis is the
estrogen metabolic derivatives play a
genotoxic role in the carcinogenesis of
breast tissue. The other possible
mechanism is the cell proliferationpromoting
ability through estrogen
receptor(ER) pathway. There are two
known estrogen receptors-- ERa and
ERb. ERa protein plays a major role in
the development and normal physiology
of the breast tissue and is important for
the regulation of cell growth and
differentiation. To investigate the
correlation between the presence of ERa
variants and prognosis of breast tumors
from Taiwan, we detailed examined the
coding region of ERa gene from 98 out
of 199 primary breast cancers, 19 normal
counterparts and 8 benign masses.
Results
Detection of Nucleotide Changes by
PCR-SSCP Analysis PCR-SSCP
analysis was used to detect small
insertions/deletions and point mutations
present in ERa gene. Nine overlapped
amplification regions (about 250 base
pairs each in size) were carefully
examined and only 1 point mutation and
was found from these 38 putative ERa
misfunctioning cases. The point mutation,
which changed a proline residue (CCC)
to serine (TCC) at codon 324, is located
in the end of the beginning helix (H1,
numbered based on the canonical
structure of NR LBD, Wurtz et al., 1996)
of the ligand binding domain. Since a
proline residue may play a role in
determination and orientation of local
secondary structure, this mutant is
possibly with altered function. However,
functional assay of this mutant has not
been conducted yet.
Detection of Polymorphisms by PCRSSCP
Analysis 3 neutral
polymorphisms were found in the 38
putative ERa-misfunctioning cases by
PCR-SSCP analysis. These polymorphiic
sites are located in codon 10
[TCT®TCC (Ser), exon 1], codon 325
[CCC®CCG (Pro), exon 4], and codon
594 [ACA®ACG (Thr), exon 8],
respectively. Polymorphism patterns of
ERa-PR consistent and normal group
were also analyzed in order to compare
with those of the ERa-PR inconsistent
group. When the alleleic distribution
pattern of these polymorphic regions
being examined, all of the 3
polymorphisms in the ERa-PR
inconsistent group showed linkage
disequilibriums but only polymorphism
in codon 594 showed a slight allele
preference (Table 1).
Detection of Exon Deletion Var iants by
PCR-Southern Blotting Analysis In the
PCR-Southern blotting based detection of
truncated transcripts, truncated
transcripts (such as clone 4) or longer
exon deletion variants (such as D2-3-4)
can not be detected by our strategy. We
could not also discriminate transcripts
with single exon deletion or combine
exon deletion ( for instance, exon 4 and
exon 7 deletions were found in the same
transcript). But these transcripts present
in relatively low level and the majority of
variant transcripts bearing shorter
deletions. In our study, the alternative
spliced transcripts, D2, D3, D4, D5, D7,
D2-3, D3-4, D4-5, reported by previous
studies of other groups were also detected.
We also found a truncated transcript
comprising 32 nucleotides 3’ to the exon
1 (denoted as d32nt) which has never
been reported in previous publishes. It
might because the shorter PCR spanning
region in our study is favored finding out small deletions. The 32 nt bears a
sequence character with a beginning of
GT and an ending of AG, which is
seemly the feather of most introns. Thus
the d32nt may owe to be mistaken as an
intron and spliced out. A deletion of this
32 nt together with the whole exon 2
(denoted as d32nt-D2) was also found in
our studies. Protein translated from d32nt
and d32nt-D2 variants will terminated
prematurely just after Thr140 in addition
to 5 or 35 more extra amino acids and
bearing only an incomplete A/B domain
possibly without any biological activity.
Compared with the basal level by
quantifying the relative ratio of variant to
wildtype bands, tumor groups beard
significant increase in D4, D5, D7, and
D2-3 expression (Table 2).
Discussion
In the present study for the search
of ERa variants, we used a preliminary
IHC analysis to raise the possibility of
finding out ERa variants with altered
function. However, we only found 1
sense point mutation out of 38 primary
breast cancer cases, much fewer from the
expected number, 2~10 mutations based
on multiply 1~5% reported mutation rate
and 199 cases. It is possible that some
mutations with unaltered function may
exist in the 161 cases we did not detailed
screened. 2 variants comprising deletion
of 32 nucleotides 3_ to exon 1 only
(d32nt) or together with the whole exon 2
(d32nt-D2) were first reported in this
study. They were probably generated
from aberrant splicing mechanism since
the 2 sequences are flanked with genuine
or cryptic splice sites. Aberrant splicing
mechanism is reported important in
altering normal function of some cellular
proteins but aberrant splicing variant of
ERa was never identified except a 69-
nucleotide insertion between exon 5 and
6. The variant was resulted from a new
splice donor site generated by a point
mutation in the intron 5 (Wang et al.,
1997). However, it is the first time that
aberrant splicing is found to occur within
the ERa coding sequences. It is also the
first time to suggest that aberrant splicing
may play some role in altering the
signaling pathway of ERa gene. Since
these variants were only reported in
Taiwan population, more extensive
studies must be undertaken in different
populations to determine whether they
are ethnic-specific variants or they were
found in the advantage of shorter PCR
amplified region comparing with those
used by other groups. By using
polymorphisms as genetic markers, we
also found significantly strong
disequilibrium in allele distribution
comparing with the result by an U.S.
group (Roodi et al., 1995) Only 2 out of 6
polymorphisms showed relatively slighter
significance. Furthermore, the
polymorphisms they analyzed did not
reveal the loss expression of one allele
phenomenon we observed in our cases. It
is thus strongly suggested that changes in
ERa signaling may play a different role
in breast cancer tumoriogenesis in
Taiwan.
Subjects
Breast Cancer
Estrogen Receptor
SDGs
Publisher
臺北市：國立臺灣大學醫學院外科
Type
report
File(s)![Thumbnail Image]()
Loading...
Name
882314B002364.pdf
Size
43.45 KB
Format
Adobe PDF
Checksum
(MD5):6060fc54a28606e28e785283372978b4