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Genome-wide search for breast cancer linkage in large Icelandic non-BRCA1/2 familes

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A significant proportion of high-risk breast cancer families are not explained by mutations in known genes. Recent genome-wide searches (GWS) have not revealed any single major locus reminiscent of BRCA1 and BRCA2, indicating that still unidentified genes may explain relatively few families each or interact in a way obscure to linkage analyses. This has drawn attention to possible benefits of studying populations where genetic heterogeneity might be reduced. We thus performed a GWS for linkage on nine Icelandic multiple-case non-BRCA1/2 families of desirable size for mapping highly penetrant loci. To follow up suggestive loci, an additional 13 families from other Nordic countries were genotyped for selected markers. GWS was performed using 811 microsatellite markers providing about five centiMorgan (cM) resolution. Multipoint logarithm of odds (LOD) scores were calculated using parametric and nonparametric methods. For selected markers and cases, tumour tissue was compared to normal tissue to look for allelic loss indicative of a tumour suppressor gene. The three highest signals were located at chromosomes 6q, 2p and 14q. One family contributed suggestive LOD scores (LOD 2.63 to 3.03, dominant model) at all these regions, without consistent evidence of a tumour suppressor gene. Haplotypes in nine affected family members mapped the loci to 2p23.2 to p21, 6q14.2 to q23.2 and 14q21.3 to q24.3. No evidence of a highly penetrant locus was found among the remaining families. The heterogeneity LOD (HLOD) at the 6q, 2p and 14q loci in all families was 3.27, 1.66 and 1.24, respectively. The subset of 13 Nordic families showed supportive HLODs at chromosome 6q (ranging from 0.34 to 1.37 by country subset). The 2p and 14q loci overlap with regions indicated by large families in previous GWS studies of breast cancer. Chromosomes 2p, 6q and 14q are candidate sites for genes contributing together to high breast cancer risk. A polygenic model is supported, suggesting the joint effect of genes in contributing to breast cancer risk to be rather common in non-BRCA1/2 families. For genetic counselling it would seem important to resolve the mode of genetic interaction.
Cosegregation of haplotypes at three chromosomal regions in family 70234. On top, the pedigree of this family is shown with circles denoting females and boxes males, with red filling denoting diagnosis of BC and shaded red also ovarian cancer. The pedigree is somewhat distorted in order to avoid recognition, but preserves the number of male and female meioses. Information about approximate age (in years) at diagnosis of cancer is shown below symbols (Br for breast and Ov for ovarian). One woman inherited a 999del5 BRCA2 mutation from her father (red box), not otherwise blood-related to this family. This woman and one other (denoted by not GWS) were not included in the genome-wide search. One of a pair of identical twins was omitted in linkage calculations. Under the pedigree, genotypes of markers of interest are shown (with chromosomal band position, marker names and genomic distance shown at the left, brackets indicating position between sites of relevant recombination). Colouring of alleles denotes whether they belong to a shared haplotype (allele frequency shown in the rightmost column) or derive from a recombined chromosome (blue). Plain (not coloured) alleles denoted 11 and 3 at D2S367 and D2S2163 respectively, are identical by state, but probably not by descent, to the commonly segregated allele (conclusion supported by fine-genotyping of additional markers, data not shown). The low frequency (0.06) of the shared D6S434 allele was validated by typing in 59 unrelated Icelandic control individuals. This figure was 0.02 in the controls (2/118), which coincides with the published Genethon frequency of the allele.
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RESEARC H ARTIC LE Open Access
Genome-wide search for breast cancer linkage in
large Icelandic non-BRCA1/2 families
Adalgeir Arason
1,2*
, Haukur Gunnarsson
1
, Gudrun Johannesdottir
1
, Kristjan Jonasson
3
, Pär-Ola Bendahl
4
,
Elizabeth M Gillanders
5
, Bjarni A Agnarsson
1,2
, Göran Jönsson
4
, Katri Pylkäs
6
, Aki Mustonen
6
, Tuomas Heikkinen
7
,
Kristiina Aittomäki
8
, Carl Blomqvist
9
, Beatrice Melin
10
, Oskar TH Johannsson
2,11
, Pål Møller
12
, Robert Winqvist
6
,
Heli Nevanlinna
7
, Åke Borg
4
, Rosa B Barkardottir
1,2
Abstract
Introduction: A significant proportion of high-risk breast cancer families are not explained by mutations in known
genes. Recent genome-wide searches (GWS) have not revealed any single major locus reminiscent of BRCA1 and
BRCA2, indicating that still unidentified genes may explain relatively few families each or interact in a way obscure
to linkage analyses. This has drawn attention to possible benefits of studying populations where genetic
heterogeneity might be reduced. We thus performed a GWS for linkage on nine Icelandic multiple-case non-
BRCA1/2 families of desirable size for mapping highly penetrant loci. To follow up suggestive loci, an additional 13
families from other Nordic countries were genotyped for selected markers.
Methods: GWS was performed using 811 microsatellite markers providing about five centiMorgan (cM) resolution.
Multipoint logarithm of odds (LOD) scores were calculated using parametric and nonparametric methods. For
selected markers and cases, tumour tissue was compared to normal tissue to look for allelic loss indicative of a
tumour suppressor gene.
Results: The three highest signals were located at chromosomes 6q, 2p and 14q. One family contributed
suggestive LOD scores (LOD 2.63 to 3.03, dominant model) at all these regions, without consistent evidence of a
tumour suppressor gene. Haplotypes in nine affected family members mapped the loci to 2p23.2 to p21, 6q14.2 to
q23.2 and 14q21.3 to q24.3. No evidence of a highly penetrant locus was found among the remaining families. The
heterogeneity LOD (HLOD) at the 6q, 2p and 14q loci in all families was 3.27, 1.66 and 1.24, respectively. The
subset of 13 Nordic families showed supportive HLODs at chromosome 6q (ranging from 0.34 to 1.37 by country
subset). The 2p and 14q loci overlap with regions indicated by large families in previous GWS studies of breast
cancer.
Conclusions: Chromosomes 2p, 6q and 14q are candidate sites for genes contributing together to high breast
cancer risk. A polygenic model is supported, suggesting the joint effect of genes in contributing to breast cancer
risk to be rather common in non-BRCA1/2 families. For genetic counselling it would seem important to resolve the
mode of genetic interaction.
Introduction
Increased susceptibility to breast cancer (BC) has been
showntobecausedbygermlinesegregationofthree
different classes of alleles: 1) high-penetrance genes with
rare risk variants, 2) moderate-penetrance genes, also
with rare variants and 3) low-penetrance alleles of
common frequency [1]. Hereditary BC, defined by a sig-
nificant familial aggregation of BC and explaining
approximately 5 to 10% of cases diagnosed with BC, is
as yet seen to arise from the first allele class whenever
the causative gene is known. Genetic counselling can
then be provided, based on mutation screening. A sig-
nificant proportion of the families are not associated
with mutations in BRCA1 or BRCA2 or other known
genes [2-5] and may in part be explained by recessive
* Correspondence: adalgeir@landspitali.is
1
Department of Pathology, Landspitali-LSH v/Hringbraut, 101 Reykjavik,
Iceland
Arason et al.Breast Cancer Research 2010, 12:R50
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© 2010 Arason et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
alleles or a polygenic model with risk variants of lower
penetrance jointly affecting risk in miscellaneous combi-
nations [6-8]. However, the gene most recently identi-
fied, RAD51C [9], demonstrates that some proportion
may still have a high-risk-allele cause. RAD51C was
identified using a candidate gene approach, but if more
high-penetrance genes are yet to be identified it might
also be helpful to analyse families from populations
where genetic heterogeneity might be reduced [10,11].
Recent genome-wide searches (GWS) for BC linkage in
families without alterations in known genes (non-
BRCA1/2 families) [10-16], together with earlier sugges-
tions of single loci [17-19], indicate close to 20 candi-
date chromosome regions if accepting LOD scores 1.5
found in a single family or small group of families [16].
Two regions have been independently pinpointed by
more than one study, one on chromosome 2p21 to p22
[11,13] and the other at 6q24 where the ESR1 gene is
located [16,19]. In some studies, notable linkage signals
have been seen at two or three chromosome regions in
the same family [11-13,16], which by chance would have
a low probability [15].
Two issues helped shape our current GWS study. First,
both BRCA1 and BRCA2 are tumour suppressor genes
and most often involve wild-type loss of heterozygosity
(wt-LOH) in mutation carrierstumours [20,21], resulting
in both parental copies of the gene being damaged in line
with Knudsons two-hit model [22]. Any new loci sugges-
tive by large families to confer high-risk of BC would
predictably gain support from the observation of such a
wt-LOH signature; on the other hand, the lack of it
would leave open the question of different gene func-
tions. Second, in Iceland both BRCA1 and BRCA2 have
been found with recurrent mutations, one in each gene,
with the BRCA2-999del5 mutation occurring in 8.5% of
BC patients and 0.5% of the population [23-25]. Other
mutations in these genes in Iceland have not been pub-
lished and would presumably be very rare [3]. This
accords with the relative geographical isolation of the
Icelandic population which since the settlement of the
island in the ninth century has at times suffered famine-
or epidemic-caused reductions (with population size only
38,000 in the year 1800 compared to the current size of
319,000), which in effect should reduce the complexity of
a gene search. We therefore selected nine large Icelandic
non-BRCA1/2 families for a GWS of high-risk genes
under a parametric dominant linkage model. Regions
considered suggestive (LOD 1.5 per family) were also
subjected to wt-LOH analyses in tumours from putative
gene carriers, and the same regions were genotyped in a
collection of Nordic non-BRCA1/2 families, in order to
estimate the possible proportion of linked families in Ice-
land and other Nordic countries.
Materials and methods
After screening for recurrent Icelandic BRCA1 and
BRCA2 mutations in 438 BC cases diagnosed in Iceland
in the years 1989 to 2001, the history of BC was evalu-
ated in the pedigrees of both the mothersandthe
fathers family side of the non-BRCA1/2 cases. Nine
families were selected and subjected to a GWS of BC
linkage by the selection criteria of (1) at least three
women diagnosed with BC under age 60 years (omitting
bilineal cases), (2) the availability of blood or paraffin-
embedded normal tissue for isolation of DNA of suffi-
cient quality from at least four affected cases (any age),
and (3) evidence against linkage to BRCA1 or BRCA2
according to genotyped microsatellite markers flanking
and within these genes. Each of the nine families con-
sisted of descendents of a single pair of founders. In five
families a DNA sample was available from six or more
BC cases (Additional file 1, Figure S1). In the analyses,
the nine families were treated as 12 because three pedi-
grees (70070, 70228 and 70236) were too large for the
GWS linkage analysis software and were therefore sepa-
rated by branches in two parts each (Additional file 1,
FigureS1).Thetwofamilysideswerecomparedby
inspection of LOD signals (selection of peaks based on
NP-LOD related P-values) in order to find possibly
overlapping positions, which could then be further
examined by manual comparison of haplotypes.
Thirteen additional Nordic families were used in follow-
up studies on suggestive loci on chromosomes 2p, 6q and
14q. They were selected from available non-BRCA1/2
families at other Nordic centres, in line with the above cri-
teria and in such a way that the genotyped affected family
members would not be expected to share by descent more
than approximately 6% of alleles by chance (through at
least six meioses). Written informed consent was obtained
with all blood samples and appropriate Institutional
Review Board approvals were obtained. Characteristics of
the 22 families are summarised in Table 1 and further
details about the families included in the GWS are pro-
vided in Figure S1 (Additional file 1).
DNA was extracted from nuclei of lysed blood sam-
ples according to Miller et al.[26]orbystandardphe-
nol-chloroform extraction, from fresh-frozen tissue
using the Wizard Genomic DNA Purification Kit (Pro-
mega, Madison, Wisconsin, USA) and from paraffin-
embedded tissue using a xylene treatment followed by
proteinase K digestion and phenol/chloroform/isoamyl
alcohol purification. All genotyping was performed at
the same centre; each sample plate contained a blank
well, two duplicate samples and a Centre dEtude du
Polymorphisme Humain (CEPH) control. Samples
included in the GWS were genotyped using the Applied
Biosystems (Foster City, California, USA) HD-5 Linkage
Arason et al.Breast Cancer Research 2010, 12:R50
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Mapping Set, containing 811 fluorescently labelled PCR
primer pairs that define an approximately five centiMor-
gan (cM) resolution human index map. Genotypes were
analysed using an automated ABI PRISM 3130 × l
Genetic Analyzer with GeneMapper software v4.0
(Applied Biosystems, Foster City, California, USA) for
automatic calling of alleles, and then checked manually.
For LOH analysis (eight members of family 70234),
DNA was also isolated from tumour tissue, which was
obtained from paraffin blocks (invasive primary
tumours) after selecting areas rich in tumour cells (>
90%)bymicroscopy(allbythesameinvestigator)and
relative allele intensities were then compared to those of
blood or normal-tissue from the same individual. For six
women, this tumour DNA or DNA from fresh-frozen
tissue was also subjected to array comparative genomic
hybridisation (array-CGH). Arrays were produced at
SCIBLU Genomics, Lund University as previously
described [27] using the 32K tiling BAC clone set from
the CHORI BACPAC resource centre.
Merlin software (Center for Statistical Genetics, Uni-
versity of Michigan, Ann Arbor, Michigan, USA) [28]
was used for the linkage analysis. Four different multi-
point analyses were carried out and associated LOD
scores calculated: (i) parametric dominant and (ii) reces-
sive with age dependent liability classes (14 total) as
defined using the modified Cancer and Steroid Hor-
mone Study (CASH) model [29]; cf. [4,30], (iii) non-
parametric using S-all scoring [31] and (iv) S-pairs scor-
ing [32], in both cases with the exponential model [33].
Only cases with invasive BC were coded as affected; all
other cancers were assigned with unknown status. Two
affected cases in one family (70234) were identical twins
and only one of them was included in linkage calcula-
tions. For parametric linkage heterogeneity LOD scores
(HLOD) are reported. Under the parametric models dis-
ease allele frequencies of 0.0033 (dominant) and 0.08
(recessive) were assumed as in [29]. The Rutgers Map
v.2 [34] (which is based on the deCODE map [35]) was
used to locate markers, and if not present in that map
they were placed with linear interpolation using their
physical position in base pairs relative to flanking mar-
kers. Allele frequencies were estimated separately for
each country by counting in all individuals (the -fa
option of Merlin). LOD scores for individual families
were also calculated, by running each family separately
in Merlin, but using allele frequencies of the total sam-
ple of the relevant country. Genotypes that were incom-
patible with the family relations (inheritance errors), as
well as unlikely genotypes, were eliminated with the
help of Merlin software.
In order to analyse conditional probabilities by family
of being linked under the admixture model, the files
prepared by Merlin software were reformatted to fit
LINKMAP software (National Center for Biotechnology
Information (NCBI), Bethesda, Maryland, USA) for slid-
ing three-point linkage analysis of selected markers.
Eighteen markers were analysed at 6q, 16 at 14q and 7
at 2p, using country-specific allele frequencies. Disease
allele frequency and age dependent liability classes were
as above, under the dominant model [29,30].
The probability of two or three haplotypes from inde-
pendently segregated chromosomal regions (apriori
unknown positions), being simultaneously cosegregated
with a trait through mmeioses, in one out of Nfamilies
with similar or greater total number of meioses (Bonfer-
roni adjustment), was calculated as described (Addi-
tional file 2). Briefly, the resulting P-value for two loci is
Pm
=× ×
N 495 2
0025.(1)
and for three loci
Pm
×
N 1617 125 2
00 0. (2)
Results
Nine Icelandic BC families, unexplained by BRCA1 or
BRCA2 mutations (non-BRCA1/2 families), were geno-
typed for a set of 811 genome-wide distributed
Table 1 Summary of families by group
Number of families
Group Total Number of cases of BC Cases with age at onset < 50 y Number of genotyped
individuals (affected)
456+<4 4+
Iceland* 9 (12) 0 (1) 2 (6) 7 (5) 6 (10) 3 (2) 102 (60)
Lund/Oslo 3 3 1 2 31 (14)
Helsinki 7 1 4 2 5 2 56 (31)
Oulu 3 1 1 1 3 20 (15)
Total 22 2 7 13 15 7 208 (119)
* Except for the last column, numbers within parentheses refer to the number of families as processed in linkage calculations, in which three families were
separated in two parts each due to large size (see Additional file 1, Figure S1).
Arason et al.Breast Cancer Research 2010, 12:R50
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microsatellite markers for subsequent linkage analysis.
As all pedigrees appeared high-risk and dominant, the
main analysis was based on the parametric dominant
model but other models were also considered in order
to see if the LOD scores were sensitive to model mis-
specification. Figure 1a, b shows LOD scores for para-
metric and non-parametric linkage analysis by
chromosomal position for the nine families combined.
For comparison, the position of the main indications of
BC susceptibility according to previously published stu-
dies of non-BRCA1/2 linkage is shown in Figure 1c.
Two of three regions with dominant HLOD > 1.5 over-
lap with previously indicated loci. One of them maps to
2p and contains two separate peaks (peak HLOD 1.72 at
D2S162 and 2.41 at D2S367; both peaks overlapping
with previous indications) and the other to 14q (HLOD
1.68 at D14S63). However, the third region, at 6q
(reaching HLOD 2.44 at D6S300) does not overlap with
previously reported candidates (Figure 1a-c).
When analysing separate families, one (named 70234)
wasseentocontributehighscores(LOD2.63to3.03)
at all three major positions, 2p, 6q and 14q (Figure 1d).
At 6q and 14q haplotypes of 29 and 13 Mb DNA,
respectively, were shared by all nine non-BRCA1/2 BC-
affected women in this family and eight of these women
also shared a haplotype (9 Mb) at 2p (Figure 2). With
reference to the flanking (recombined) markers, the
common haplotypes map to 2p23.2 to p21 (between
D2S165 and D2S2259), 6q14.2 to q23.2 (between
D6S1609 and D6S262) and 14q21.3 to 24.3 (between
D14S288 and D14S1036). A close inspection of the
information in Figure 2 makes it is evident that the 6q
and 14q haplotypes are identical by descent and have
cosegregated through 13 meioses, and that the 2p haplo-
type has cosegregated with the other two through 11
meioses. Of the nine families, a total of five had a com-
parable total number of meioses connecting the affected
cases (ranging from 13 to 18). Therefore a Bonferroni
correction of N= 5 has been chosen. By formula (1) the
probability of observing cosegregation of two loci (6q
and 14q) through 13 meioses in one of five families is
P= 0.006. The corresponding probability for all three
loci and 11 meioses is also P= 0.006 (formula (2)). We
also used Merlin to simulate marker data in order to get
an indication of the frequency of obtaining three sepa-
rate dominant model LOD scores > 2.5. A total of 700
replicates of the per-family runs were generated using
the same allele frequency estimates as in the original
Figure 1 Maximum LOD scores by chromosomal position, and relation to previously suggested candidate loci. Parametric HLOD scores
for the nine Icelandic families are shown in (a) for the dominant (dark teal thick line) and the recessive model (plum). NP-LOD scores are shown
in (b) using different exponential scoring options in Merlin software: S-all (orange thick line) and S-pairs (indigo). The position of previously
published loci is shown in (c), according to GWS studies in red (or black if reported in more than one GWS study) (adapted from Table 5 in
[16]), and according to single locus reports [17-19] in blue. A position at 2p indicated by a subset of relatively early-onset multiple-case families
in one GWS-study [11] is included in (c) and shown in grey. Parametric LOD scores of family 70234 are shown in (d) with line colours as in (b).
Arason et al.Breast Cancer Research 2010, 12:R50
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run. In none of the replicates did we obtain a family
with three peaks of this magnitude, in two instances
there was a family with two peaks, in 75 instances we
obtained a family with one location with dominant LOD
> 2.5 (in six instances a family exceeding LOD 3.0), and
in the remaining simulated cases no dominant LOD >
2.5 was obtained.
Eight breast tumours from family 70234 were assayed
at markers within the shared haplotypes, for loss of het-
erogeneity selective for losing wild-type alleles (wt-LOH)
in harmony with Knudsons model of tumour suppres-
sor genes. At 6q, LOH was seen at all informative mar-
kers in three tumours, with wt-LOH in two but the
third lost all alleles from the risk-related haplotype (data
not shown). Copy-number loss of the region was con-
firmed in the three tumours by array-CGH (data not
shown) which also revealed amplification at 6q21 in a
fourth tumour. At 2p and 14q, signs of allelic losses
were confined to single markers and of inconsistent alle-
lic phase (three tumours each chromosome), and not
supported by array-CGH since intensities were generally
within thresholds.
Family-wise, no other family than 70234 showed para-
metric LOD scores higher than 1.5 (and the highest NP-
LOD for the other families was 2.3). LOD scores of
weaker indication were seen at multiple positions
Figure 2 Cosegregation of haplotypes at three chromosomal regions in family 70234. On top, the pedigree of this family is shown with
circles denoting females and boxes males, with red filling denoting diagnosis of BC and shaded red also ovarian cancer. The pedigree is
somewhat distorted in order to avoid recognition, but preserves the number of male and female meioses. Information about approximate age
(in years) at diagnosis of cancer is shown below symbols (Br for breast and Ov for ovarian). One woman inherited a 999del5 BRCA2 mutation
from her father (red box), not otherwise blood-related to this family. This woman and one other (denoted by not GWS) were not included in the
genome-wide search. One of a pair of identical twins was omitted in linkage calculations. Under the pedigree, genotypes of markers of interest
are shown (with chromosomal band position, marker names and genomic distance shown at the left, brackets indicating position between sites
of relevant recombination). Colouring of alleles denotes whether they belong to a shared haplotype (allele frequency shown in the rightmost
column) or derive from a recombined chromosome (blue). Plain (not coloured) alleles denoted 11 and 3 at D2S367 and D2S2163 respectively,
are identical by state, but probably not by descent, to the commonly segregated allele (conclusion supported by fine-genotyping of additional
markers, data not shown). The low frequency (0.06) of the shared D6S434 allele was validated by typing in 59 unrelated Icelandic control
individuals. This figure was 0.02 in the controls (2/118), which coincides with the published Genethon frequency of the allele.
Arason et al.Breast Cancer Research 2010, 12:R50
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Page 5 of 10
(Additional files 3 and 4, Figures S2 and S3). In order to
see if any position might be indicated by more than one
family, even if too weakly suggestive on a single-family
basis, we listed all peaks that met the criterion of NP-
LOD associated P-values of < 0.005 (Additional file 5,
Table S1). Of 22 peaks, one was found to colocate with
that of family 70234 to the 6q15 to q22 region and two
families shared peaks at 13q32.1 to q33.1. In case of
separate parts of the same pedigree, possibly overlapping
peaks were not observed.
To follow up suggestive linkage signals (HLOD > 1.5
by family), markers at 2p, 6q and 14q were genotyped
in an additional 13 Nordic non-BRCA1/2 families.
Dominant parametric HLOD scores for the 13 families
combined reached 0.92 at D6S434, and were much
lower at 2p and 14q (highest HLOD 0.23 at D2S165).
An analysis of heterogeneity, using LINKMAP was per-
formed on all 22 Nordic families (treated as 25 for the
reasons given above) at the three chromosomal posi-
tions and the results are shown in Table 2. At chromo-
some 6q15 to q22.31, the combined HLOD was 3.27
and a0.40. The data show support for chromosome
6q-linkage among both Icelandic and non-Icelandic
families. HLODs by country subset were in the range
0.34 to 2.02 (aranging between 0.39 and 1). Single
families with a conditional probability of being linked
exceeding the overall a-value of 0.40 (10 families) were
widely distributed among the Nordic subsets (Table 2).
At chromosome 14q21.2 to q24.3 the main support for
linkage lies within the Icelandic subset (Table 2) with
HLOD 1.24 and a0.36, unaltered by the addition of
other Nordic families. At 2p23.2 to p21 most non-Ice-
landic families fail to support the findings since they
indicate a more telomeric position (Table 2) which
does not overlap with the high peak in family 70234.
An exception is one Finnish family with LOD 1.03,
matching the position in family 70234. It has a
conditional probability of being linked equal to 0.78
(data not shown), only exceeded by that of family
70234 (0.97).
Discussion
In the present GWS for BC linkage in nine Icelandic
non-BRCA1/2 families, substantial linkage signals were
observed at chromosomes 2p25.1 to p22.1, 6q15 to
q22.31 and 14q21.3 to q31.3, and the strongest contri-
bution to all three regions occurred in the same family
(70234). On a single family basis, all three signals in
the family are exceptionally high compared to previous
studies [10-19], but none of the signals meet the
suggested cut off level of 3.3 for significance in GWS
studies [36]. The regions at 2p and 14q overlap with
those of previous studies of non-BRCA1/2 families.
The position of the more centromeric signal in our 2p
region (2p23.3 to p21) was in fact pinpointed on the
basis of single families by two independent studies
[11,13]. The more telomeric signal in our 2p region
(p25.1 to p24.1) contains a position with relation to
families with a higher number of cases and at a
younger age at diagnosis [11]. The region at 6q15 to
q22.31 does not extend to the 6q24.3 to q25.1/ESR1-
region [16,19] and does therefore not overlap with pre-
vious indications. This region had the strongest signal
(HLOD 3.27 in all families combined) in the current
study and gains support from some other families
besides 70234, both Icelandic and from other Nordic
countries (Table 2).
Recent studies have indicated familial non-BRCA1/2
BC as mainly polygenic with decreasing possibility of
finding new high-risk genes. Our results support this
view in the following way: The current study was pri-
marily designed to find whether dominant mutations of
high penetrance exist in large Icelandic non-BRCA1/2
families with a Mendelian pattern concurrent with such
Table 2 Peak parametric multipoint LOD scores under heterogeneity, at three chromosomal regions as defined by
family-70234 haplotypes
6q15 to q22.31 14q21.2 to q24.3 2p23.2 to p21
Subset Number
of
families*
HLOD acM from
D6S434
Number of
linked
families
HLOD acM from
D14S980
Number of
linked
families
HLOD acM from
D2S367
Number of
linked
families
Icelandic 12 2.02 0.45 -5.4 3 1.24 0.36 30.2 4 2.13 0.61 0.0 7
Lund/
Oslo
3 1.37 0.79 0.3 2 0 0 0.46 1.00 -14.4 0
Helsinki 7 0.34 0.39 12.2 3 0.06 0.34 22.2 0 1.09 1.00 -13.1 2
Oulu 3 1.15 1.00 -1.9 2 0 0 0.75 1.00 -7.5 1
Total 25 3.27 0.40 0.0 10 1.24 0.36 30.2 4 1.66 0.25 0.0 10
*Three Icelandic pedigrees were separated in two parts each in the linkage calculations. Therefore the total number of families in this table is 25.
For each locus, the distance relates to the marker closest to the LOD-peak in family 70234; negative numbers refer to the direction towards the p-telomere.
Linked families are defined as having a conditional probability of linked type (data not shown) exceeding the alpha for all families combined.
Arason et al.Breast Cancer Research 2010, 12:R50
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Page 6 of 10
genetic explanation. Most families failed to reveal evi-
dence of any such locus. Although three chromosomes
provided suggestive linkage signals, their coexistence in
one family hardly supports the idea of a single causative
gene. Some families have previously been reported with
two or three suggestive chromosome regions [11-13,16]
and such an observation was shown to have a low prob-
ability simply by chance [15]. We do not consider chro-
mosomal translocation to be a logical possibility since
the three haplotypes in question segregate independently
to the daughters of affected cases in family 70234
(Figure 2). We estimate the probability to be P=0.006
of seeing any two loci that are not linked to each other
cosegregate by chance with the disease through 13
meiosesashereseenat6qand14q.Thiswouldargue
for the existence of more than one causative gene in
family 70234. Whether the third locus (2p), which also
cosegregates to eight of the nine cases, plays a role in
the risk is a little more ambiguous. Although the ninth
case inherited some alleles identical with the common
haplotype (and therefore contributed to the high LOD
of2.63)theywereoncloserscrutiny(genotypingof
added markers, data not shown) seen to be interrupted
by non-matching alleles and therefore she should be
regarded as a phenocopy with respect to possible 2p-
linkage. By formula (2), the probability of observing
cosegregation of three loci through 11 meioses in one
of five families is P= 0.006. Although this might be
adjusted with respect to different possible ways of
observing a phenocopy, and to the fact that the pheno-
copy did inherit two of the three haplotypes, the
resulting value would still be on the same order as the
compared value for two loci. The 2p-haplotype may
therefore be irrelevant to the BC risk but it gains sup-
port from the two previous reports of a candidate BC
susceptibility locus which overlaps with this 2p-region
[11,13].
Itmaybeaskediftheuniquetriplestrongsignalsin
family 70234 as compared to other families possibly
reflect distinction by genetic linkage models. The
absence of notable linkage peaks in the remaining
families conforms to a polygenic model with frequent
alleles, sometimes cosegregating and sometimes being
replaced by variants of different lineage or of other
genes. Family 70234 seems to differ in this respect,
even if questioning the 2p-linkage, since the two hap-
lotypes at 6q and 14q fully cosegregate with the dis-
ease in nine cases, with maximum LOD ranging from
2.74 to 3.03. Therefore a replacement of one haplotype
by a new variant (if needed) would appear to be a rare
event in this instance, and one might expect a low
population frequency of the gene variants involved.
This would have implications for genetic counselling,
since continued cosegregation of the two (not to men-
tion three) haplotypes would seem improbable for the
descendants of family 70234. It would then also be
important to resolve whether each of the genetic var-
iants contributes an independent proportion of the dis-
ease risk (additive or multiplicative joint effects), or in
an interdependent way, their risk being dependent on
the presence of all cofactors. With such a scenario,
one would view this familys cancer history mainly as a
very rare chance result of cosegregation of limited con-
sequence for later generations of the family. Alterna-
tively, the families studied here may all comply with
the same genetic model, with cooperative alleles of
moderate or high frequency, and the strong linkage
signals in family 70234 to be accounted for by the
absence of phenocopies. We note, in this context, that
by treating the nine families as 12 for linkage calcula-
tions, most were not as highly informative by the num-
ber of cases or meioses as 70234, but nevertheless they
were expected to reveal clues of genomic position by
pairwise comparison of separate parts of the larger
pedigree.
The additional Nordic families support the risk indica-
tion of chromosome 6q, but seem not to support the
2p- and 14q-linkage. One Finnish family may be of the
linked type at the same 2p position as family 70234 but
this is not higher than expected by chance from 13
families, each with up to 6% sharing, by descent, of
genetic material between its affected members. The 6q-
linkage gains some support from other Icelandic families
(for example, 70386 in Additional file 5, Table S1). This
raises the question whether a recurrent mutation may
be involved, but comparison of haplotypes in suggestive
carriers from different families did not support that idea
(data not shown). If present, such a recurrent mutation
would seemingly call upon the genotyping of more den-
sely distributed markers.
At chromosome 2p, a linkage signal in the combined
families, telomeric to the one in family 70234 and con-
current with the reported position of a signal in rela-
tively early-onset multiple-case families [11], also invites
searching for an underlying recurrent mutation in the
Icelandic families. However, although some families con-
tribute to this signal with weakly positive LOD scores
(Additional files 3 and 4, Figures S2, S3), they lack reli-
able indications of which alleles to look for, partly due
to absence of a convincing referencefamily (like 70234
in the case of 6q) and partly due to uncertain recombi-
nation events, and such comparison of haplotypes is
Arason et al.Breast Cancer Research 2010, 12:R50
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Page 7 of 10
therefore meaningless. In short, a sign of a recurrent
mutation (that is, alleles of not too high frequency, seen
in different candidate families) would support a risk
related role of this locus, but it is not seen.
As regards the three chromosome regions most
strongly indicated in our study, we tested by wt-LOH
analysis whether the genes in question might act as clas-
sical tumour suppressors. Three out of eight tumours
from family 70234 showed extensive LOH at 6q but it
affected both wild-type and risk related haplotypes. At
2p and 14q, convincing signs of LOH were absent.
Therefore no support was found for the hypothesis of a
predisposing tumour suppressor gene similar to the
BRCA1 and BRCA2 genes [20,21]. We note, however,
that the hypothesis is not ruled out because microaltera-
tions could exist that are not seen by our methods.
With respect to the four different ways used to analyse
linkage, we had reasons to choose the parametric domi-
nant analyses as the principal one. Looking at the pedi-
grees none appeared recessive. We also expected low
genetic heterogeneity. That would argue for s-all to be
the primary non-parametric method but for comparison
we also performed s-pairs analyses. By considering all
four analyses (Figures 1 and Additional files 3 and 4,
Figures S2, S3), the parametric dominant analyses did
not appear to be sensitive to model misspecification.
The results of our GWS analysis support previously
reported indications of a polygenicnatureofnon-
BRCA1/2 hereditary BC. Most families in the current
study fail to provide map indications of involved loci
and this may in part be credited to the problem of
phenocopies, which was addressed by simulation
experiments on BRCA1/2 families in the GWS study of
Rosa-Rosa et al. [16]. Finding more families with signals
analogous to those of 70234 in the current study could
provide further clues where to look for interacting risk
loci and a follow-up of more generations could then
help to resolve the significance and mode of possible
genetic interaction.
Conclusions
The results of this study support previous indications
that susceptibility to BC in multiple-case non-BRCA1/2
families seems to be segregated by low- or moderate-
penetrance gene variants jointly contributing to the risk.
A combination of variants at chromosomes 2p, 6q and
14q may in a cooperative or even interdependent way
cause high disease risk in a family. Together with other
such families reported with multiple linkage signals, this
may reflect localised familial clustering of risk alleles
from a pool of many candidate loci. Genetic counselling
would benefit from resolving the mode of interactions
in such families.
Additional material
Additional file 1: Figure S1, pedigrees of the families in the GWS.
This is a jpg file showing pedigrees of the families included in the GWS.
Pedigrees of nine Icelandic non-BRCA1/2 families with each showing BC
cases traced to a single pair of founders but otherwise omitting relatives
if not genotyped. Genotyped family members are marked with an
asterisk. Circles denote females and boxes males, with red filling
denoting diagnosis of BC and shaded red also ovarian cancer. Tan filling
indicates cancer at other sites than breast, or of unknown origin.
Information about the site and approximate age (in years) at diagnosis of
cancer is shown below the symbols (Br for breast, Col colon, Cvx cervix,
Kdn kidney, Lng lung, Ov ovary, Pnc pancreas, Pro prostate, Stm stomach
and Unkn for unknown origin). Dotted vertical lines between family
branches show how the family was separated in two parts for linkage
calculations. Pedigrees are somewhat distorted in order to avoid
recognition.
Additional file 2: Supplementary methods. A Word document
containing a methodological description of intra-family significance
testing for multiple loci.
Additional file 3: Figure S2, parametric LOD scores by family. A jpg
file showing graphs of parametric LOD scores by chromosomal position,
for the families in the GWS (the top graph with all families combined, for
comparison). LOD scores are shown for the dominant (dark teal line) and
the recessive model (plum). Three families (70070, 70228 and 70236)
were separated in smaller units for linkage analysis, as indicated by
adding the letter a or b to the family name.
Additional file 4: Figure S3, NP-LOD scores by family. A jpg file
showing graphs of NP-LOD scores and associated P-values by
chromosomal position in individual families included in GWS, using
different exponential scoring options in Merlin software: S-all (orange
thick line) and S-pairs (indigo). Three families (70070, 70228 and 70236)
were separated in smaller units for linkage analysis, as indicated by
adding the letter a or b to the family name.
Additional file 5: Table S1, Maximum LODs by chromosome and
family, for NP-LODs with P< 0.005. A Word file containing a table of
per-family LOD signals (selected with respect to NP-LOD associated P-
values), for consideration of whether any chromosomal positions may be
indicated by more than one family.
Abbreviations
BC: breast cancer; GWS: genome-wide search/genome-wide scan; HLOD:
heterogeneity LOD (parametric); LOD: logarithm of odds; LOH: loss of
heterozygosity; non-BRCA1/2: not accounted for by mutations in BRCA1 or
BRCA2 or other known genes; NP-LOD: non-parametric LOD; wt-LOH: LOH
with loss from the wild-typechromosome.
Acknowledgements
We thank the patients and their family members whose contribution made
this work possible. We gratefully acknowledge the staff at the Department
of Pathology, Landspitali-University Hospital for providing pathological
information and tissue samples, the Genetic Committee of the University of
Iceland for pedigree information and Valgardur Egilsson, Landspitali-
University Hospital who provided extended pedigree information on family
70234, the Finnish, Icelandic, Norwegian and Swedish Cancer Registry for
information on cancer data, and the staff at Landspitali-University Hospital
and the Service Center at Noatun for help with blood sampling. Financial
support was provided by the Icelandic Research Fund, the Nordic Cancer
Union, the Landspitali University Hospital Research Fund, the Memorial Fund
of Bergthora Magnusdottir and Jakob Bjarnason, the Icelandic association:
Walking for Breast Cancer Research, the Swedish Cancer Society, the
Helsinki University Central Hospital Research Fund, Academy of Finland, the
Finnish Cancer Society, the Sigrid Juselius Foundation, the Finnish Cancer
Foundation, the University of Finland and the Oulu University Hospital.
Arason et al.Breast Cancer Research 2010, 12:R50
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Page 8 of 10
Author details
1
Department of Pathology, Landspitali-LSH v/Hringbraut, 101 Reykjavik,
Iceland.
2
Faculty of Medicine, University of Iceland, Vatnsmyrarvegi 16, 101
Reykjavik, Iceland.
3
Faculty of Engineering and Natural Sciences, University of
Iceland, Hjardarhaga 2-4, 107 Reykjavik, Iceland.
4
Department of Oncology,
Clinical Sciences Lund, Lund University, SE 221 85 Lund, Sweden.
5
Inherited
Disease Research Branch, National Human Genome Research Institute,
National Institutes of Health, 333 Cassell Drive, Suite 1200, Baltimore, MD
21224, USA.
6
Laboratory of Cancer Genetics, Department of Clinical Genetics
and Biocenter Oulu, University of Oulu, Oulu University Hospital, 90220 Oulu,
Finland.
7
Department of Obstetrics and Gynecology, Helsinki University
Central Hospital, P.O. BOX 700, 00029 HUS, Helsinki, Finland.
8
Department of
Clinical Genetics, Helsinki University Central Hospital, P.O. BOX 140, 00029
HUS, Helsinki, Finland.
9
Department of Oncology, Helsinki University Central
Hospital, P.O. BOX 180, 00029 HUS, Helsinki, Finland.
10
Department of
Radiation Sciences, Umeå University, 901 85 Umeå, Sweden.
11
Department of
Oncology, 20A, Landspitali-LSH v/Hringbraut, 101 Reykjavik, Iceland.
12
Section
of Inherited Cancer, Oslo University Hospital, 0310 Oslo, Norway.
Authorscontributions
RBB and AA designed the study and together with EG selected the Icelandic
families after an initial power simulation performed by EG. HG and GJo
carried out the DNA analysis and genotype data collection and AA did the
processing and checking of the data. KJ carried out the linkage analyses and
significance testing, helped with organising and writing statistical parts of
the manuscript, and together with AA, shaped the statistical analysis of
cosegregation of multiple loci. P-OB performed the heterogeneity analyses.
HG and GJo carried out the array-CGH analyses. OThJ, BAA, RBB, AA, HN, RW,
ÅB, PM, BM, KP, AM, TH, KA and CB provided samples and information on
the families included in this study. AA, with the help of RBB, wrote the
manuscript and all co-authors critically read and approved it. RBB conceived
and coordinated the study.
Competing interests
The authors declare that they have no competing interests.
Received: 17 May 2010 Revised: 21 June 2010 Accepted: 16 July 2010
Published: 16 July 2010
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doi:10.1186/bcr2608
Cite this article as: Arason et al.: Genome-wide search for breast cancer
linkage in large Icelandic non-BRCA1/2 families. Breast Cancer Research
2010 12:R50.
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... In a genome-wide search of new chromosomal loci in high-risk BC [4], we earlier reported a pedigree with cosegregation of three haplotypes, located at chromosomes 2p, 6q, and 14q, in nine breast cancer-affected relatives, thus indicating an oligogenic cause of HBC in this family. All three loci had LOD (logarithm of odds) scores close to statistical Genes 2024, 15, 218 2 of 6 significance, which is rarely seen for a locus on a single family basis. ...
... It also remains to be confirmed whether 2p, 6q, and 14q harbor BC risk genes, and if they do, there is a need to identify them. None of these three loci showed, per analysis of their wt-LOH (wild-type loss of heterogeneity), any indications of harboring a tumor suppressor gene [4]. ...
... Our study is based on an extensively traced seven-generation Icelandic pedigree, where detailed clinical and laboratory data are available from the following affected family members: Ten subjects in the main family branch diagnosed with BC (two of them diagnosed with ovarian cancer as well, but one of these was also a carrier of a BRCA2 mutation and is therefore omitted in the count of 6q-haplotype carriers) [4], and a further four cases with BC in a distantly related family branch ( Figure 1). Pedigree and clinical data were obtained from the Genetic Committee of the University of Iceland and the Icelandic Cancer Registry. ...
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Although a number of high-risk breast cancer genes have been identified, including BRCA1 and BRCA2, the risk profile of many high-risk families cannot be explained using known breast cancer genes. Previously, we have shown strong indications of new breast cancer risk loci at chromosomes 2p, 6q, and 14q in a family of six generations including 10 breast cancer cases. In this study, we identified and traced four new family branches descending from siblings of the parents in the top generation of the studied family. One distantly related branch included four breast cancer cases, two of whom were diagnosed at age < 45 years. DNA samples from the cases were typed at selected polymorphic markers from all three chromosome loci, to test identical origin of the haplotypes. All four cases were shown to segregate a common 6q haplotype with a region identical to the previously identified 6q haplotype. The data strongly support a new breast cancer locus at 6q, and narrow it down to a 17 MB interval at 6q15-q21.
... Several studies on linkage analysis in non BRCA1/2 families have been conducted in other populations [20][21][22][23][24][25]. However, the fact that these findings do not replicate in other populations is not surprising given the uniqueness of this Sri Lankan data set. ...
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Objective Although linkage studies have been utilized for the identification of variants associated with cancer in the world, little is known about their role in non BRCA1/2 individuals in the Sri Lankans. Hence we performed linkage analysis to identify susceptibility loci related to the inherited risk of cancer in a cohort of Sri Lankans affected with hereditary breast cancer. The Illumina global screening array having 654,027 single nucleotide polymorphism markers was performed in four families, in which at least three individuals within third degree relatives were affected by breast cancer. Two-point parametric linkage analysis was conducted assuming disease allele frequency of 1%. Penetrance was set at 90% for carriers with a 10% phenocopy rate. Results Thirty-one variants exhibited genome-wide suggestive HLODs. The top overall HLOD score was at rs1856277, an intronic variant in MYO16 on chromosome 13. The two most informative families also suggested several candidate linked loci in genes, including ERAP1 , RPRM , WWOX , CDH1 , EXOC1, HUS1B , STIM1 and TUSC1 . This study provides the first step in identifying germline variants that may be involved in risk of cancer in cancer-aggregated non- BRCA1/2 families from the understudied Sri Lankan population. Several candidate linked regions showed suggestive evidence of linkage to cancer risk.
... The search for "BRCA3" began in earnest in the mid-1990s but the paucity of promising leads suggested that another major cancer predisposing gene explaining the remaining BRCA-negative cancer families was unlikely. Although linkage analyses identified promising chromosomal regions, they were unique to the population in which they were identified [37,38]. These observations suggested that HBC and HBOC syndrome families were more genetically heterogenous than previously expected, suggesting that the carrier frequencies of each high-risk gene candidate would be considerably lower relative to BRCA-carriers. ...
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The French Canadian population of the province of Quebec has been recognized for its contribution to research in medical genetics, especially in defining the role of heritable pathogenic variants in cancer predisposing genes. Multiple carriers of a limited number of pathogenic variants in BRCA1 and BRCA2, the major risk genes for hereditary breast and/or ovarian cancer syndrome families, have been identified in French Canadians, which is in stark contrast to the array of over 2000 different pathogenic variants reported in each of these genes in other populations. As not all such cancer syndrome families are explained by BRCA1 and BRCA2, newly proposed gene candidates identified in other populations have been investigated for their role in conferring risk in French Canadian cancer families. For example, multiple carriers of distinct variants were identified in PALB2 and RAD51D. The unique genetic architecture of French Canadians has been attributed to shared ancestry due to common ancestors of early settlers of this population with origins mainly from France. In this review, we discuss the merits of genetically characterizing cancer predisposing genes in French Canadians of Quebec. We focused on genes that have been implicated in hereditary breast and/or ovarian cancer syndrome families as they have been the most thoroughly characterized cancer syndromes in this population. We describe how genetic analyses of French Canadians have facilitated: (i) the classification of variants in BRCA1 and BRCA2; (ii) the identification and classification of variants in newly proposed breast and/or ovarian cancer predisposing genes; and (iii) the identification of a new breast cancer predisposing gene candidate, RECQL. The genetic architecture of French Canadians provides a unique opportunity to evaluate new candidate cancer predisposing genes regardless of the population in which they were identified.
... DNA was extracted from blood samples and paraffin embedded tissue, as described in previous studies [74]. Quantity of DNA from blood was measured by NanoDrop (Thermo Fisher Scientific), and from paraffin tissue, in the case of LOH analyses, by Qubit Fluorometric Quantification (Thermo Fisher Scientific). ...
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Mutations in BRCA1 result in predisposal to breast and ovarian cancers, but many variants exist with unknown clinical significance (VUS). One is BRCA1 c.4096+3A>G, which affects production of the full-length BRCA1 transcript, while augmenting transcripts lacking most or all of exon 11. Nonetheless, homozygosity of this variant has been reported in a healthy woman. We saw this variant cosegregate with breast and ovarian cancer in several family branches of four Icelandic pedigrees, with instances of phenocopies and a homozygous woman with lung cancer. We found eight heterozygous carriers (0.44%) in 1820 unselected breast cancer cases, and three (0.15%) in 1968 controls (p = 0.13). Seeking conclusive evidence, we studied tumors from carriers in the pedigrees for wild-type-loss of heterozygosity (wtLOH) and BRCA1-characteristic prevalence of estrogen receptor (ER) negativity. Of 15 breast and six ovarian tumors, wtLOH occurred in nine breast and all six ovarian tumours, and six of the nine breast tumors with wtLOH were ER-negative. These data accord with a pathogenic BRCA1-mutation. Our findings add to the current knowledge of BRCA1, and the role of its exon 11 in cancer pathogenicity, and will be of use in clinical genetic counselling.
... It is likely that other genes which mediate cell cycle progression and alter the DDR may also be involved in enhanced susceptibility. Interestingly, studies investigating genes involved in breast cancer susceptibility have identified chromosome 2p, and specifically 2p23.2, as a site which may have genes that contribute to increased breast cancer risk (43)(44)(45). This identified location maps directly to the chromosomal location of the Spy1 gene (SPDYA). ...
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Breast cancer is the most common cancer to affect women and one of the leading causes of cancer related deaths. Maintenance of genomic stability and proper regulation of cell cycle checkpoints play a critical role in preventing the accumulation of deleterious mutations. Perturbations in the expression or activity of mediators of cell cycle progression or checkpoint activation represent important events that may increase susceptibility to the onset of carcinogenesis. The atypical cyclin-like protein Spy1 was isolated in a screen for novel genes that could bypass the DNA damage response. Clinical data demonstrates that protein levels of Spy1 are significantly elevated in ductal and lobular carcinoma of the breast. Using a transgenic mouse driving expression of Spy1 in the mammary epithelium we demonstrate that sustained elevation of Spy1 leads to enhanced proliferation and an increased susceptibility to mammary tumour formation. We find that Spy1 is targeted for degradation by the tumour suppressor p53 to protect checkpoint control. When crossed with p53 deficient mice, elevation of Spy1 leads to an increase in hyperplastic alveolar nodules. Targeting cyclin-like protein activity may therefore represent a mechanism of re-sensitizing cells to important cell cycle checkpoints in a therapeutic setting.
... Compared with sporadic cancer, the prognosis of BRCA-positive cancer has been reported to be worse [3,4] not different [5,6] or better [7]. In about 70% of families with aggregation of breast and ovarian cancers and a pedigree strongly suggesting hereditary breast cancer, the BRCA test was negative and no pathogenic mutation was identified [8][9][10][11][12]. This subgroup of familial non-BRCA mutated breast cancers are very heterogeneous and not well defined in terms of histopathological features, clinical presentation and prognosis. ...
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Background Approximately 5–10% of breast cancers are hereditary and their biology and prognosis appear to differ from those of sporadic breast cancers. In this study we compared the biological features and clinical characteristics of non metastatic breast cancer in patients with BRCA mutations versus patients with a family history suggesting hereditary breast cancer but without BRCA mutations (BRCA wild type) versus patients with sporadic disease, and correlated these findings with clinical outcome. Methods We retrieved the clinical and biological data of 33 BRCA-positive, 66 BRCA-wild type and 1826 sporadic breast cancer patients contained in a single institution clinical database between 1980 and 2012. Specifically, we recorded age, tumor size, nodal status, treatment type, pattern of relapse, second primary incidence, outcome (disease-free survival and overall survival), and biological features (estrogen receptor [ER], progesterone receptor [PgR], tumor grade, proliferation and c-erbB2 status). Median follow-up was 70 months. ResultsBRCA-positive patients were significantly younger than sporadic breast cancer patients, and less likely to be ER-, PgR- or c-erbB2-positive than women with BRCA-wild type or sporadic breast cancer. Tumor size and grade, nodal status and proliferation did not differ among the three groups. Rates of radical mastectomy were 58, 42 and 37%, and those of conservative surgery were 42, 58 and 63% in women with BRCA-positive, BRCA-wild type and sporadic breast cancer (p = 0.03), respectively. The incidence of contralateral breast cancer was 12, 14 and 0% (p <0.0001) and the incidence of second primary tumors (non breast) was 9, 1 and 2% (p <0.0001) in BRCA-positive, BRCA-wild type and sporadic breast cancer, respectively. Median disease-free survival in years was 29 in BRCA-wild type, 19 in BRCA-positive and 14 in sporadic breast cancer patients (log-rank = 0.007). Median overall survival in years was not reached for BRCA-wild type, 19 for BRCA-positive and 13 for sporadic breast cancer patients (log-rank <0.0001). At multivariate analyses only BRCA-wild type status was related to a significant improvement in overall survival versus the sporadic breast cancer group (HR = 0,51; 95% CI (0,28–0,93) p = 0.028). Conclusions The biology and outcome of breast cancer differ between patients with BRCA mutations, patients with a family history but no BRCA mutations and patients with sporadic breast cancer.
... Mutation-positive cases. Testing took place in two genetics laboratories in Reykjavik, Iceland, in the context of several previous research projects that are described elsewhere, (Tulinius et al, 2002;Arason et al, 2010;Tryggvadottir et al, 2013) and the results have been combined to generate a cohort of mutation-positive breast cancer patients. The research projects were originally based on the Family Collection of the Icelandic Cancer Society that was founded in 1972 and consists of 995 breast cancer probands who were selected for genealogical tracing by defined periods of diagnosis and year of birth and thus unselected with respect to family history (Tulinius et al, 2002). ...
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Background: The impact of an inherited BRCA2 mutation on the prognosis of women with breast cancer has not been well documented. We studied the effects of oestrogen receptor (ER) status, other prognostic factors and treatments on survival in a large cohort of BRCA2 mutation carriers. Methods: We identified 285 breast cancer patients with a 999del5 BRCA2 mutation and matched them with 570 non-carrier patients. Clinical information was abstracted from patient charts and pathology records and supplemented by evaluation of tumour grade and ER status using archived tissue specimens. Univariate and multivariate hazard ratios (HR) were estimated for breast cancer-specific survival using Cox regression. The effects of various therapies were studied in patients treated from 1980 to 2012. Results: Among mutation carriers, positive ER status was associated with higher risk of death than negative ER status (HR=1.94; 95% CI=1.22-3.07, P=0.005). The reverse association was seen for non-carriers (HR=0.71; 95% CI: 0.51-0.97; P=0.03). Conclusions: Among BRCA2 carriers, ER-positive status is an adverse prognostic factor. BRCA2 carrier status should be known at the time when treatment decisions are made.British Journal of Cancer advance online publication, 18 August 2016; doi:10.1038/bjc.2016.249 www.bjcancer.com.
Chapter
Breast cancer is the most commonly diagnosed cancer in women and the leading cause of cancer death. About one million new cases are diagnosed each year in China [1]. According to the latest data released by the World Health Organization in 2017, the number of breast cancer deaths in China reached 49,011, accounting for 0.52% of the total deaths. The age-adjusted mortality rate is 5.76 deaths per 100,000 people, ranking China 179 globally. Worldwide, there will be an estimated 2.1 million newly diagnosed breast cancer cases for women in 2018, accounting for a quarter of all cancers in women. And there will be approximately 0.63 million breast cancer death in 2018 [2]. Recently, the incidence of this cancer is increasing in developing countries. Breast cancer is the most frequently diagnosed cancer in most countries (154 of 185 countries) and is also the leading cause of cancer deaths in more than 100 countries; the main exceptions are Australia/New Zealand, Northern Europe, North America (where lung cancer is the top one), and many countries in sub-Saharan Africa (due to the increased incidence of cervical cancer [2]). The incidence of breast cancer is the highest in countries such as Australia/New Zealand, Northern Europe (e.g., UK, Sweden, Finland, and Denmark), Western Europe (Belgium, Netherlands, and France), Southern Europe (Italy), and North America [2].
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Background: Breast cancer is the most common cancer to affect women and one of the leading causes of cancer-related deaths. Proper regulation of cell cycle checkpoints plays a critical role in preventing the accumulation of deleterious mutations. Perturbations in the expression or activity of mediators of cell cycle progression or checkpoint activation represent important events that may increase susceptibility to the onset of carcinogenesis. The atypical cyclin-like protein Spy1 was isolated in a screen for novel genes that could bypass the DNA damage response. Clinical data demonstrates that protein levels of Spy1 are significantly elevated in ductal and lobular carcinoma of the breast. We hypothesized that elevated Spy1 would override protective cell cycle checkpoints and support the onset of mammary tumourigenesis. Methods: We generated a transgenic mouse model driving expression of Spy1 in the mammary epithelium. Mammary development, growth characteristics and susceptibility to tumourigenesis were studied. In vitro studies were conducted to investigate the relationship between Spy1 and p53. Results: We found that in the presence of wild-type p53, Spy1 protein is held 'in check' via protein degradation, representing a novel endogenous mechanism to ensure protected checkpoint control. Regulation of Spy1 by p53 is at the protein level and is mediated in part by Nedd4. Mutation or abrogation of p53 is sufficient to allow for accumulation of Spy1 levels resulting in mammary hyperplasia. Sustained elevation of Spy1 results in elevated proliferation of the mammary gland and susceptibility to tumourigenesis. Conclusions: This mouse model demonstrates for the first time that degradation of the cyclin-like protein Spy1 is an essential component of p53-mediated tumour suppression. Targeting cyclin-like protein activity may therefore represent a mechanism of re-sensitizing cells to important cell cycle checkpoints in a therapeutic setting.
Chapter
Pathology is at the cornerstone of cancer diagnosis, prognosis, prevention, and treatment as well as the molecular mechanisms of disease, while epidemiology provides insights into the burden of cancer, its causes and opportunities for prevention. Pathology and epidemiology often work together by incorporating pathological and tumor biomarker data for individuals in epidemiologic studies, or by enriching pathological studies with the principles of epidemiologic methods such as study design and population selection. These fields can also work more synergistically toward the common goal of elucidating disease etiology and progression, as we will highlight in this chapter. Within, we feature studies from the intersection of patho-epidemiology of cancer pathogenesis to illustrate our inspiration for this textbook. By integrating these two disciplines, we provide a unique and comprehensive overview that helps the reader to understand neoplastic disease processes.
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The contribution of BRCA1 and BRCA2 to inherited breast cancer was assessed by linkage and mutation analysis in 237 families, each with at least four cases of breast cancer, collected by the Breast Cancer Linkage Consortium. Families were included without regard to the occurrence of ovarian or other cancers, Overall, disease was linked to BRCA1 in an estimated 52% of families, to BRCA2 in 32% of families, and to neither gene in 16% (95% confidence interval [CI] 6%-28%), suggesting other predisposition genes, The majority (81%) of the breast-ovarian cancer families were due to BRCA1, with most others (14%) due To BRCA2. Conversely, the majority of families with male and female breast cancer were due to BRCA2 (76%). The largest proportion (67%) of families due to other genes tvas found in families with four or five cases of female breast cancer only, These estimates were not substantially affected either by changing the assumed penetrance model for BRCA1 or by including or excluding BRCA1 mutation data. Among those families with disease due to BRCA1 that were tested by one of the standard screening methods, mutations were detected in the coding sequence or splice sites in an estimated 63% (95% CI 51%-77%). The estimated sensitivity was identical for direct sequencing and other techniques. The penetrance of BRCA2 was estimated bg maximizing the LOD score in BRCA2-mutation families, over all possible penetrance functions, The estimated cumulative risk of breast cancer reached 28% (95% CI 9%-44%) by age 50 years and 84% (95% CI 43%-95%) by age 70 pears. The corresponding ovarian cancer risks were 0.4% (95% CI 0%-1%) by age 50 years and 27% (95% CI 0%-47%) by age 70 years, The lifetime risk of breast cancer appears similar to the risk in BRCA1 carriers, but there was some suggestion of a lower risk in BRCA2 carriers <50 years of age.
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Efforts to find disease genes using high-density single-nucleotide polymorphism (SNP) maps will produce data sets that exceed the limitations of current computational tools. Here we describe a new, efficient method for the analysis of dense genetic maps in pedigree data that provides extremely fast solutions to common problems such as allele-sharing analyses and haplotyping. We show that sparse binary trees represent patterns of gene flow in general pedigrees in a parsimonious manner, and derive a family of related algorithms for pedigree traversal. With these trees, exact likelihood calculations can be carried out efficiently for single markers or for multiple linked markers. Using an approximate multipoint calculation that ignores the unlikely possibility of a large number of recombinants further improves speed and provides accurate solutions in dense maps with thousands of markers. Our multi-point engine for rapid likelihood inference (Merlin) is a computer program that uses sparse inheritance trees for pedigree analysis; it performs rapid haplotyping, genotype error detection and affected pair linkage analyses and can handle more markers than other pedigree analysis packages.
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We investigate the contribution of the Iberian bat fauna to the cryptic diversity in Europe using mitochondrial (cytb and ND1) and nuclear (RAG2) DNA sequences. For each of the 28 bat species known for Iberia, samples covering a wide geographic range within Spain were compared to samples from the rest of Europe. In this general screening, almost 20% of the Iberian species showed important mitochondrial discontinuities (K2P distance values > 5%) either within the Iberian or between Iberian and other European samples. Within Eptesicus serotinus and Myotis nattereri, levels of genetic divergence between lineages exceeded 16%, indicating that these taxa represent a complex of several biological species. Other well-differentiated lineages (K2P distances between 5–10%) appeared within Hypsugo savii, Pipistrellus kuhlii and Plecotus auritus, suggesting the existence of further cryptic diversity. Most unsuspected lineages seem restricted to Iberia, although two have crossed the Pyrenees to reach, at leas...
Conference Paper
Accurate and comprehensive linkage maps are critical for the success of positional cloning projects and other genetic studies. We have constructed a second generation high-resolution genetic map that includes a much larger set of polymorphic markers (N=28,219) than the first version and is consistent with the latest genome assembly (NCBI Build 36). To build the new combined linkage-physical map, a set of SNP genotype data including 5,172 SNPs from the Affymetrix Human Mapping 10K Array, 3,506 SNPs from the Applied Biosystems SNPlex Human Linkage Mapping Set 4k, and 4,782 SNPs from the Illumina Linkage IV Panel were collected and integrated into our existing data set (N=14,759). In the initial mapping stage, only markers with known physical positions were used and were added to the map only if the results of linkage analysis were consistent with physical position. Markers with no physical position were added in later steps using only linkage information. The data were also cleaned by removal of genotypes that are likely to be erroneous, identified as those that lead to close double-recombination events. Then markers whose linkage positions did not originally match physical position were re-evaluated against the more complete map. Finally, Those markers which still had discordant positions were assigned to map intervals determined by linkage analysis. This map represents the most comprehensive linkage map ever constructed. The average map resolution is 0.17 cM (127kb) between neighboring markers, with 58 percent of neighboring markers showing no recombination events. This map is a valuable tool for obtaining map distances for linkage studies to identify genetic loci. It is disseminated via our MAP-O-MAT website (http://compgen.rutgers.edu/mapomat).
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This paper reports the results of a collaborative linkage study involving 214 breast cancer families, including 57 breast-ovarian cancer families; this represents almost all the known families with 17q linkage data. Six markers on 17q, spanning approximately 30 cM, were typed in the families. The aims of the study were to define more precisely the localization of the disease gene, the extent of genetic heterogeneity and the characteristics of linked families and to estimate the penetrance of the 17q gene. Under the assumption of no genetic heterogeneity, the strongest linkage evidence was obtained with D17S588. Multipoint linkage analysis allowing for genetic heterogeneity provided evidence that the predisposing gene lies between the markers D17S588 and D17S250, an interval whose genetic length is estimated to be 8.3 cM in males and 18.0 cM in females. This position was supported over other intervals by odds of 66:1. The location of the gene with respect to D17S579 could not be determined unequivocally. Under the genetic model used in the analysis, the best estimate of the proportion of linked breast-ovarian cancer families was 1.0 (lower LOD -- 1 limit 0.79). In contrast, there was significant evidence of genetic heterogeneity among the families without ovarian cancer, with an estimated 45% being linked. These results suggest that a gene(s) on chromosome 17q accounts for the majority of families in which both early-onset breast cancer and ovarian cancer occur but that other genes predisposing to breast cancer exist. By examining the fit of the linkage data to different penetrance functions, the cumulative risk associated with the 17q gene was estimated to be 59% by age 50 years and 82% by age 70 years. The corresponding estimates for the breast-ovary families were 67% and 76%, and those for the families without ovarian cancer were 49% and 90%; these penetrance functions did not differ significantly from one another. 42 refs., 5 figs., 2 tabs.
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Mutations in BRCA1 and BRCA2 that cause a dominantly inherited high risk of female breast cancer seem to explain only a small proportion of the aggregation of the disease. To study the possible additional genetic components, we conducted single-locus and two-locus segregation analyses, with and without a polygenic background, using three-generation families ascertained through 858 women with breast cancer diagnosed at age <40 years, ascertained through population cancer registries in Melbourne and Sydney, Australia. Extensive testing for deleterious mutations in BRCA1 and BRCA2, to date, has identified 34 carriers. Our analysis suggested that, after other possible unmeasured familial factors are adjusted for and the known BRCA1 and BRCA2 mutation carriers are excluded, there appears to be a residual dominantly inherited risk of female breast cancer in addition to that derived from mutations in BRCA1 and BRCA2. This study also suggests that there is a substantial recessively inherited risk of early-onset breast cancer. According to the best-fitting model, after excluding known carriers of mutations in BRCA1 and BRCA2, about 1/250 (95% confidence interval [CI] 1/500 to 1/125) women have a recessive risk of 86% (95% CI 69%-100%) by age 50 years and of almost 100% by age 60 years. Possible reasons that our study has implicated a novel strong recessive effect include our inclusion of data on lineal aunts and grandmothers, study of families ascertained through women with early-onset breast cancer, allowance for multiple familial factors in the analysis, and removal of families for whom the cause (i.e., BRCA1 or BRCA2) is known. Our findings may have implications for attempts to identify new breast cancer-susceptibility genes.