Note: Descriptions are shown in the official language in which they were submitted.
CA 02535506 2006-02-10
WO 2005/013951 PCT/PL2004/000063
Pharmaceutical compositions and methods for the prevention of breast and
ovarian cancer
FIELD OF THE INVENTION
The present invention relates to a composition of selenium (or its salts or
derivatives or any
other selenium compound) as well as methods for the prevention of breast /
ovarian cancer in
females with inherited high risk of cancer, particularly in Garners of BRCA1
gene mutations.
BACKGROUND OF THE INVENTION
BRCA1 (U55654155) and BRCA2 genes are related to high genetic predispositions
to
cancers. BRCA1 and BRCA2 genes have been cloned and, at present, their
abnormalities can be
detected at DNA and RNA levels. Carriers of mutations within above ~ genes
have high risk of
breast and / or ovarian cancers. BRCA1 is the first gene recognized to be
associated with high
risk of breast and ovarian cancer (Mild et al, Science, 266-271, 1994). BRCA1
gene
(GENBANK Accession Numbers: U14680 and 15595) contains 24 exons dispersed
within 100
kbp of genomic DNA and its mRNA is of 7.8 kbp of the length. Intensive studies
of this gene
allowed to identify numerous BRCA1 mutations. For example, US5693473 is
reporting large
registry of such changes. WO 99/29903 is describing the next fifteen BRCA1
mutations.
BRCA2 gene (GENBANK accession Number U43746) contains 27 exons dispersed
within 70
kbp of genomic DNA and its mRNA is of 11-12 kbp of the length. Numerous
mutations of
BRCA2 gene have been reported as associated with cancer predisposition (for
example WO
9928506).
All of described BRCA1 and BRCA2 gene mutations are available on page:
http://www.nchgr.nih.gov/dir/lab-transfer/bic.
BRCA1 and BRCA2 mutations can be located in different exons. Increased
frequency of
selected particular mutations in families with breast / ovarian cancer
aggregation has been
recognized in some ethnic groups / populations such as Ashkenazi Jews among
whom the high
incidence of BRCA1 185delAG and 5382insC and BRCA2 6174de1T are well known. In
Poland,
CA 02535506 2006-02-10
WO 2005/013951 PCT/PL2004/000063
2
increased frequency of the following BRCA1 mutations has been reported: ex 2 -
185delAG, ex
- 300T~ G, ex 5 - 309T~ C, ex 11.15 - 3819de1GTAAA, ex 11.17 - 4153de1A, ex 20
-
5382insC (Polish patent P 335917). Opportunity of DNA testing based on
analysis of limited
number of mutations without significant loss of sensitivity in detection of
abnormalities is
creating a chance, valuable for several countries, of BRCA1BRCA2 examination
with cost-
effectiveness extremely high. In Poland, around 100,000 of women are carriers
of BRCA1
mutation.
Research spanning the last 25 years has established that selenium is effective
in the reduction
of cancer incidence when provided to animals at doses only 5 - to 10 - fold
above nutritional
requirement (El-Bayoumy K., The role of selenium in cancer prevention,
Philadelphia,
Lippincott, 1-15, 1991). Chemoprevention studies with selenium in animal model
systems have
indicated that this element is effective for most, if not all of the organ
systems and is protective
against the carcinogenic effects of a wide variety of insults (El-Bayoumy K.,
The role of
selenium in cancer prevention, Philadelphia, Lippincott, 1-15, 1991). Both
epidemiological
studies and supplementation trails have also supported its efficacy in
lowering the incidence of
cancers of the liver, colon, prostate and lung (Yu S.Y. et al. Protective role
of selenium against
hepatitis B virus and primary liver cancer in Qidong, Biol Trace Elem Res, 56:
117-124, 1997,
Clark L.C. et al. Effects of selenium supplementation for cancer prevention in
patients with
carcinoma of the skin. J Am Med Assoc, 276: 1957-1963, 1996, Yoshizawa K. et
al. Study of
prediagnostic selenium level in toenails and risk of advanced prostate cancer,
J Natl Cancer Inst
(Betsheda), 90: 1219-1224, 1998; Brooks J.D. et al. Plasma selenium level
before diagnosis and
risk of prostate cancer development, J Urol, 166: 2034-2038, 2001). The latest
literature data are,
however, not supporting negative correlation between the level of selenium in
the body of
patients and the risk of breast cancer.
CA 02535506 2006-02-10
WO 2005/013951 PCT/PL2004/000063
3
Garland M. et al. concluded that diet supplementation with selenium does not
influence the
risk of female breast cancer (J. Am. Coll Nutr. 1993, Aug; 12(4): 400-11, J
Natl Cancer Inst
1995, Apr S; 87(7) :497-505). Similar conclusions have been reported by
Ghadirian P. et al. (A
case-control study of noenail selenium and cancer of the breast, colon and
prostate, Cancer
Detect Prev, 24: 305-13, 2000).
Further, in EP1003760 relating to selenium-containing compounds and methods
for using
these compounds to protect mammals from toxic insults, it has been stated
that: "Current
selenium supplements rely on inorganic forms, such as sodium selenite
(NaZSe03) or sodium
selenate (Na2Se04). While these forms have some value, they are considered
more toxic than
necessary, and are unlikely to be useful in cancer chemo-prevention".
OBJECTS OF THE INVENTION
It has been recognized, recently, that breast cancer is a heterogeneous
disorder and around
10% of consecutive breast/ovarian cancers occurs as a result of inherited high
genetic
predisposition to cancers. It is, therefore, an object of the invention to
provide pharmaceutical
compositions and methodology of their application allowing efficient lowering
of the risk of
breast / ovarian cancers in individuals with high genetic predisposition to
these tumors,
especially in carriers of BRCA1 gene mutations. Another object of the
invention is to provide
such pharmaceuticals for chemoprevention of breast / ovarian cancers, which
should be offered
to females with constitutional mutations of BRCA1 gene.
SUMMARY OF THE INVENTION
Unexpectedly, the problem described as above has been solved, at least in
large part, due to
this invention.
The present invention is directed to pharmaceutical composition for lowering
the risk of
breast / ovarian cancer in person with inherited high risk of disease,
especially in carrier of
BRCA1 mutation, characterized by contents of therapeutically-effective amount
of selenium
CA 02535506 2006-02-10
WO 2005/013951 PCT/PL2004/000063
4
(e.g. in form of its salt or other derivatives thereof or any other known
selenium comprising
compound) and, if necessary, pharmaceutically acceptable carrier.
According to one embodiment of the invention, selenium is selected among its
organic forms
such as methylselenocysteine, methyloseleninic acid, selenomethinine,
selenocysteine or
inorganic forms such as selenium dioxide, selenonic acid, selenic acid and
their salts. It can be
recommended also that selenium originates from such natural products like Se-
enriched yeast or
broccoli. According to next embodiment of the invention, selenium compound is
a salt selected
among: barium selenite, lithium selenite, calcium selenite and, what is
particularly valuable,
sodium selenite. Preferably, pharmaceutical composition is an isotonic
solution, containing
selenium at concentration 0.1 - 10% w/w. It can be recommended to use as a
dissolvent an
aqueous solution of ethanol at concentration 10 - 96% w/w., distilled water,
physiologic solution
preferably buffered. It can be recommended as well if composition according to
invention
contains selenium salt in aqueous solution of ethanol.
The subject of invention is also the method of prevention of breast / ovarian
cancer in person
with inherited high genetic risk of tumor particularly in female Garner of
BRCA1 characterized
by supplementation of patient with compounds containing selenium (or its salts
or derivatives),
and using compositions as described above. It can be recommended that in
methodology
according to invention patient is supplemented with selenium at doses 50 -
1000 ~,g per day. It is
valuable, in methodology according to invention, to use composition containing
selenium after
detection in patient the constitutional BRCA1 gene mutation.
Unexpectedly, increased sensitivity to bleomycin as measured using cytogenetic
test
according to Hsu was detected in BRCA1 mutation Garners (0.58 vs. 0.39
chromosome breaks
per cell; p < 10~) , that was normalized after 1-month supplementation of diet
with selenium at
doses around 300 pg per day.
CA 02535506 2006-02-10
WO 2005/013951 PCT/PL2004/000063
Additionally, unexpectedly as well, the lowering of breast / ovarian cancer
risk, was
observed (see example 3). For the period of 2 years, occurrence of new breast
/ ovarian cancers
was analyzed in the group of 30, initially healthy, carriers of BRCA1 mutation
supplemented
with ethanol solution of sodium selenite at dose 300 ~g per day and in the
control group of 30
BRCA1 Garners matched for mutation type and year of birth. During above pilot
studies, 1 breast
cancer case was observed in the group supplemented with selenium and 4 breast
cancers plus 1
ovarian cancer in the control group.
Additionally, for the period of 1 year, occurrence of new breast/ovarian
cancers was analysed
in the group of 200, initially unaffected, Garners of BRCA1 mutations
supplemented with
ethanol solution of sodium selenite at dose 300 ~g per day and in the control
group of 200
BRCA1 Garners matched for mutation type and age/average age 38.6 yrs). During
above pilot
studies 1 breast and 1 peritoneal cancers were observed in the group
supplemented with
selenium, whereas 2 breast and 2 ovarian cancers were diagnosed in the control
group.
DETAILED DESCRIPTION OF THIS INVENTION
Invention provides pharmaceutical compositions and methodology of their
application
allowing efficient lowering of the risk of breast / ovarian cancers in
individuals with high genetic
predisposition to these tumors, especially in carriers of BRCA1 gene
mutations. Further, the
invention provides such pharmaceuticals for chemoprevention of breast /
ovarian cancers, which
should be offered to females with constitutional mutations of BRCA1 gene.
Invention provides the pharmaceutical composition for lowering the risk of
breast / ovarian
cancer in person with inherited high risk of disease, especially in carrier of
BRCA1 mutation,
which composition contains selenium (or its salt or derivatives or any other
selenium comprising
compound) and, if necessary, pharmaceutically acceptable Garner.
The term "selenium or its salt or derivatives" as used herein, unless
otherwise specified,
refers to any known selenium comprising compound. The selenium can be present
in elemental
CA 02535506 2006-02-10
WO 2005/013951 PCT/PL2004/000063
6
form and/or as all other organic or inorganic selenium compounds. In such
compounds selenium
can have -2, 0, +2, +4, and +6 oxidation states. Usually, the body detoxifies
the higher oxidation
state forms by reducing them to -2 and methylating to form dimethyl selenide.
Dimethyl selenide
is preferred and it can be used in conjunction with other selenium containing
compounds.
Particularly, as inorganic selenium compounds may be used such compounds as
selenium
dioxide, selenonic acid, selenic acid and their salts, especially metal
selenides or selenites (e.g.
potassium selenide, sodium selenide, sodium selenite, zinc selenide, barium
selenite, lithium
selenite, calcium selenite and other metal selenides or selenites). Moreover,
selenium can be
used in its organic forms such as seleno-amino acid e.g. methylselenocysteine,
methyloseleninic
acid, selenomethinine, selenocysteine. Examples of other useful organic
selenium compounds
are dimethyl selenide, all other organic selenides, selenoglutathione, and the
organo-selenium
complex Factor 3. It can be recommended also that selenium originates from
such natural
products like Se-enriched yeast or broccoli. Selenium occurs naturally in
varying amounts in a
wide variety of foods and also is present as an impurity in the natural form
of the sulfur-
containing amino acids, e.g., with methionine as the compound
selenomethionine. This latter is
due to the fact that the chemical behavior and reactivity of sulfur and
selenium are very similar.
Thus, in food grade sulfur-containing amino acids, the corresponding seleno-
amino acid is
normally present and thus contains selenium in significant amounts. Examples
of such useful
edible sulfur-containing amino acids are: cysteine, methionine, cystine,
cystathionine,
pencillamine cysteine disulfide, penicillamine, 2-amino-4,4-dimethyl-
mercaptobutyric acid,
vitamin U, brasinine, djenkolic acid, 2-amino-4-isopropyl-mercaptobutyric
acid, 2-amino-4-butyl
mercaptobutyric acid, 2-amino-4,4-diethyl-mercaptobutyric acid, dibenzo-
yldjenkolic acid, the
monohydrochloride of djenkolic acid, the hydrochloride of cysteine, 2-amino-2-
ethyl-3-
mercapto-propanoic acid, 2-thiolhistidine, thiomalic acid, the hydrochloride
of cysteine,
homocysteine, pantetheine, panthethine, Coenzyme A, and cysteic acid. All
isomeric forms can
CA 02535506 2006-02-10
WO 2005/013951 PCT/PL2004/000063
7
be used. The sulfur-containing amino acids, proteins, and peptides are
normally used in the
hydrochloride form or in weak acid or base salt form because they are more
readily water
soluble. Examples of useful natural sulfur-containing peptides are:
glutathione (a tripeptide of
glutamic acid, cystein and glycine, also termed gamma-glutamyl-cysteinyl-
glycine), cysteinyl-
glycine, and gamma-cysteinyl-methionyl-glycine. Examples of useful sulfur-
containing proteins
are: keratin, insulin, albumin, ribonuclease, fibroin, collagen and elastin.
All of the scleroproteins
(albuminoids), some of which are mentioned above, may be also useful as
natural source of
selenium.
The term "pharmaceutically acceptable carrier" as used herein refers to term
very well know
for the person skilled in the art. Methods and pharmaceutical Garners for
preparation of
pharmaceutical compositions are well known in the art, as set out in textbooks
such as
Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company,
Easton,
Pennsylvania, USA. Further information on preferred formulations is also
provided in the more
detailed description of the invention set out below (see example 1).
The term "therapeutically-effective amount" means an amount of a selenium
comprising
compound, as defined above, to yield a desired therapeutic response, i.e.
allowing efficient
lowering of the risk of breast / ovarian cancers in individuals with high
genetic predisposition to
these tumors, especially in Garners of BRCA1 gene mutations. The specific
"therapeutically-
effective amount" will, obviously, vary with such factors as the physical
condition of the subject,
the specific formulations employed and the structure of the used "selenium or
its salt or
derivatives". Particularly, it can be recommended that in methodology
according to invention
patient is supplemented with selenium at doses 50 - 1000 ~g per day or
appropriate dose of
selenium comprising compounds which dose includes such amount of selenium. The
compound
of the invention may be administered in conjunction with one or more other
known anti-
CA 02535506 2006-02-10
WO 2005/013951 PCT/PL2004/000063
8
neoplastic and/or cancer preventive agents. The compound of the invention and
the second agent
may be administered together or sequentially.
In order to present the sense of invention, the description of invention is
expanded by
examples 1-3. However, it is not our intention to introduce claims limited to
embodiments
described in examples, because basing on presented sense of invention combined
with
knowledge generally available, experts will be able to prepare other variants
comprised in
defined claims.
Example 1. Compositions containing selenium.
Basing on information available from pharmakopoeia, experts are able to
prepare different
variants of compositions containing selenium, especially selenium (IV). For
example, selenium
can be chosen from organic forms such as methylcysteine, methylseleninic acid,
selenomethionine, selenocysteine or from inorganic forms such as selenium
dioxide, selenic
acid, (HZSe03) or its salts. For example selenium (IV) salt valuable for
preparation of
compositions according to invention is sodium selenite. Natural products with
high concentration
of selenium such as selenium-enriched yeast or broccoli are attractive
compounds as well.
Particularly valuable, although not the only one, composition according to
invention are
solutions of selenium at concentration 0.1 - 10% w/w, especially isotonic
solutions. Such
solutions can be made, for example, using such dissolvents as aqueous solution
of ethanol at
concentration 10 - 96% w/w, distilled water, physiologic solution especially
if buffered.
It has been shown that the optimum of pharmacological effect can be achieved
if daily dose
is 50 - 1000 ~g of selenium. The above values are modifiable depending on
biological access of
selenium applied to preparation of composition, as well depending on influence
of associated
compounds, pharmaceutical Garners.
Selected, although not the only ones, examples of such compositions are
presented below:
A) Ethanol solution of sodium selenite.
CA 02535506 2006-02-10
WO 2005/013951 PCT/PL2004/000063
9
In order to prepare 1000 g of composition - 2.03 g of sodium selenite
(Na2Se03) is dissolved
in 334.32 g of HZO. To aqueous solution of sodium selenite obtained as above
663.65 g of 96%
aqueous solution of ethanol is added, and the solution is carefully mixed.
Composition prepared
in such way can be used for oral supplementation of patient, 2 times 10 drops
per day, what
corresponds to daily dose around 300 ~g of pure selenium. It can be
recommended to divide
composition into bottles containing 11-12 g, because such amount covers one-
month
supplementation. Described composition is stable, maintains its form and
activity at least 18
months if stored at 4° C. It can be used by patients without giving
side effects.
B) Aqueous solution.
Composition based on aqua can be prepared by replacement of ethanol by
distilled water or
physiologic solution (preferably buffered) in recipe as in A.
Example 2.Increased rates of chromosome breakage in BRCAl carriers are reduced
by
oral selenium supplementation.
Study subjects were recruited from among the attendees of a single familial
cancer clinic of
the Hereditary Cancer Centre of the Pomeranian Academy of Medicine in
Szczecin, Poland.
Women were referred to this clinic because of a family history of breast or
ovarian cancer. The
women who participated in this study had previously been offered, and had
consented to, genetic
testing. Case women were recruited from among those who had been found to be
Garners of a
deleterious mutation in the BRCA1 gene. The most common mutation was the
Polish founder
mutation 5382 ins C (16 carriers) but there were 10 women with other BRCA1
mutations.
Control subjects were recruited from among the family members of the Garners
(cases), but who
had been determined not to carry the deleterious mutation. It was possible to
generate 26 case-
control pairs. In 20 instances the control was the sister of the case and in
six instances she was a
more distant relative. Only healthy women were invited to participate in this
study; women with
a past history of breast, ovary or other form of cancer were excluded. Each
woman provided a
blood sample at some time during one year study.
Chromosome sensitivity to bleomycin was measured according to the method of
Hsu et al.
(Sensitivity to the genotoxic effects of bleomycin in humans; possible
relationship to
environmental carcinogenesis, 1nt J Cancer, 1989, 43, 403-409). Bleomycin
(Nippon-Kayuka)
CA 02535506 2006-02-10
WO 2005/013951 PCT/PL2004/000063
was added to the cell culture five hours before the end of the culture at a
concentration of 0.03
ICT/ml. Conventional harvesting and Giemsa staining were made. For each
subject, 100
consecutive euploid cells in metaphase were read (cells with overlapping
chromosomes were
excluded). Each chromatid aberration (excluding gaps) was scored as one
breakpoint and each
exchange-type aberration was scored as two breakpoints. The total number of
breakpoints per
100 cells was recorded for each subject. Reading of coded slides was performed
blindly by one
of us. For twenty randomly selected cases the counts were determined
independently by two
cytogenetic technicians with good agreement. The mean number of breaks per
cell was
calculated after inspection of 100 cells. The mean level of chromosome breaks
was compared in
the 26 case-control pairs using the paired t-test.
The mean number of chromosome breaks per cell was 0.58 for the BRCA1 mutatin
carriers
(range 0.34 to 0.73) and was 0.39 for the controls (range 0.28 to 0.62) (table
1). For 23 of the 26
pairs, the value for the Garner exceeded that for the related control, for two
pairs the control
value was the higher of the two and for one pair the two values were equal (p
< 0.0001; sign
test). The mean difference between the two groups was highly significant (p <
0.0001 ).
CA 02535506 2006-02-10
WO 2005/013951 PCT/PL2004/000063
11
Table 1. Comparison of chromosomes breaks per cell in BRCA1 carriers and
matched controls.
Case carrier Control non-carrier
Case ID Chromosome Chromosome
age breaks er cell age breaks er
cell
1. 22 0.58 22 0.41
2. 43 0.61 54 0.55
3. 29 0.34 33 0.44
4. 39 0.51 36 0.62
5. 21 0.48 31 0.31
6. 24 0.57 20 0.49
7. 45 0.60 51 0.40
8. 47 0.52 55 0.52
9. 51 0.61 29 0.44
10. 24 0.66 22 0.42
11. 48 0.57 28 0.35
12. 24 0.65 29 0.28
13. 23 0.65 22 0.42
14. 31 0.73 29 0.46
15. 31 0.52 34 0.32
16. 23 0.52 38 0.36
17. 44 0.59 35 0.36
18. 59 0.48 58 0.30
19. 53 0.54 61 0.29
20. 58 0.59 50 0.34
21. 44 0.51 46 0.38
22. 22 0.67 27 0.45
23. 34 0.60 30 0.28
24. 34 0.71 36 0.41
25. 18 0.59 21 0.28
26. 27 0.64 31 0.35
mean 35.3 0.58 35.7 0.39
A second group of BRCA1 carriers was recruited from this clinic for the
purpose of studying
the effect of selenium supplementation on frequencies of chromosome breaks.
Thirty-five
women with BRCA1 mutations agreed to participate in this phase of the study.
Blood samples
were taken prior to the onset of selenium supplementation, and again at a time
from one month
to three months following the start of daily selenium supplementation. Prior
to selenium
supplementation, the mean number of induced chromosome breaks per cell was
0.63 (range 0.42
to 0.81). An oral selenium solution was provided to the study subjects which
contained 690 pg of
pure selenium, in the form of sodium selenite (Na2Se03) per ml of 70% ethanol.
Subjects were
requested to consume 0.2 ml of the solution twice daily. A second blood sample
was taken at a
time from one month to three months after commencement of selenium
supplementation and the
CA 02535506 2006-02-10
WO 2005/013951 PCT/PL2004/000063
12
bleomycin test was repeated. After a mean of 1.5 months of selenium
supplementation, the mean
number of chromosome breaks per cell was reduced to 0.40 (range 0.27 to 0.60).
In every case,
the post-supplementation level showed a decline from the baseline level (table
2) and the mean
difference was highly significant (p < 10-'°).
Table 2. Comparison of chromosome breaks per cell in BRCA1 carriers before
and after selenium supplementation.
Case ID Before selenium su lementationAfter selenium su lementation
12 0.65 0.60
15 0.52 0.40
18 0.48 0.36
24 0.71 0.49
25 0.59 0.30
26 0.64 0.39
27 0.63 0.29
28 0.66 0.44
29 0.66 0.32
30 0.42 0.33
31 0.55 0.32
32 0.65 0.38
33 0.67 0.37
34 0.65 0.30
35 0.55 0.32
36 0.60 0.32
37 0.74 0.43
38 0.55 0.43
39 0.61 0.27
40 0.57 0.36
41 0.59 0.27
42 0.65 0.44
43 0.62 0.34
44 0.67 0.52
45 0.67 0.50
46 0.63 0.58
47 0.77 0.42
48 0.57 0.32
49 0.62 0.31
50 0.58 0.46
51 0.70 0.52
52 0.59 0.34
53 0.71 0.47
54 0.81 0.51
55 0.65 0.45
mean 0.63 0.40
CA 02535506 2006-02-10
WO 2005/013951 PCT/PL2004/000063
13
Summarising, oral selenium is a good candidate for chemoprevention in women
who carry a
mutation in the BRCA1 gene.
Example 3. Supplementation of female BRCAl mutation carriers with selenium is
reducing significantly the risk of breast / ovarian cancers.
Occurrence of breast / ovarian cancers in the group of 30, initially healthy,
carriers of
BRCA1 mutations taking orally ethanol solution of sodium selenite at dose 300
~g daily and in
the control group of 30 BRCA1 carriers matched for mutation type and year of
birth, was
observed during two years. Average age in both groups was 46.5 years. During
the period of
observation 1 breast cancer case was diagnosed in the group supplemented with
selenium and 4
breast cancers plus 1 ovarian cancer have been diagnosed in control group.
(Tab. 3).
Additionally, for the period of 1 year, occurrence of new breast/ovarian
cancers was analysed
in the group of 200, initially unaffected, Garners of BRCA1 mutations
supplemented with
ethanol solution of sodium selenite at dose 300 ~g per day and in the control
group of 200
BRCA1 Garners matched for mutation type and age/average age 38.6 yrs). During
above pilot
studies 1 breast and 1 peritoneal cancers were observed in the group
supplemented with
selenium, whereas 2 breast and 2 ovarian cancers were diagnosed in the control
group.
CA 02535506 2006-02-10
WO 2005/013951 PCT/PL2004/000063
14
Table 3. Occurence of breast / ovarian cancers In initially healthy carriers
of
BRCA1 mutations after supplementation with selenium and in matched controls.
Studi ed rou Con trol rou
L . Mutation Year of Occurrence Year of Occurrence of
birth of birth cancer
cancer
1 5382insC 1929 - 1933 -
2 5382insC 1947 - 1944 -
3 5382insC 1947 - 1946 breast cancer
4 5382insC 1947 - 1946 -
S 5382insC 1950 - 1949 -
6 5382insC 1951 - 1951 -
7 C61G 1952 - 1951 -
8 C61G 1955 - 1952 -
9 5382insC 1954 - 1953 -
5382insC 1954 - 1954 breast cancer
11 4153de1A 1956 - 1957 ovarian cancer
12 5382insC 1956 - 1957 -
13 4153de1A 1966 - 1963 -
14 4153de1A 1959 - 1960 breast cancer
5382insC 1942 - 1948 breast cancer
16 5382insC 1960 breast cancer1960 -
17 C61 G 1962 - 1962 -
18 4153de1A 1963 - 1962 -
19 C61 G 1963 - 1963 -
C61 G 1963 - 1963 -
21 C61 G 1964 - 1964 -
22 5382insC 1965 - 1965 -
23 5382insC 1966 - 1964 -
24 5382insC 1966 - 1966 -
5382insC 1967 - 1967 -
26 5382insC 1956 - 1956 -
27 5382insC 1943 - 1943 -
28 5382insC 1963 - 1965 -
29 C61G 1968 - 1968_ -
5382insC 1955 - 1956 ~ -
