BRCA-1/2

A genotype-phenotype study in a
Newfoundland population

MED:6393
Dec 8th 2010
 BRCA1/2 ‘s
BRC first linked to chrm 17 in 1990 4
BRCA1 (17q.21)5 and BRCA2 (13q12.3)6 were
discovered in 94 and 95 respectively (Wooster et al., 1995).

 www.genetests.org: copyright University of Washington, Seattle

 www.geneclinics.org/profiles/BRCA1/
 BRCA1/2

BRCA1/2
◦ Interactions..

Deficiencies 
< gen. integrity
 Oncogenic var. 
Tumor proliferation

www.genetests.org: copyright University of Washington, Seattle

Risk
27.8% of all Canadian female cancer was
attributed to breast cancer (BRC)1.
Rate of BRC is twice the 2nd leading
cancer(lung) 1.
Women 20-59
◦ 37% of all cancer incidence 1
◦ 22% of cancer related death 1
Early onset
Earlyonset partly due to allelic variations
in BRCA1 and BRCA2
◦ 60%~80% lifetime risk of developing BRC2
◦ account for 5~10% of all BRC3

BRCA
Sporadic/Oth
er
 Different Variations Infer Different Phenotypes

Cancer risk
◦ age
Cancer location
◦ ovarian, prostate
Tumor Expression
◦ ER receptors, lymphatic invasion
Progression
◦ 5 year survival
Prognosis (Debate)

◦ Norway and the UK collaboration

 73% vs. 92% 5 year survival (BRCA1+/-)7
 62% vs. 86% 5 year survival (BRCA1+/-)8
◦ Another study on Israeli women
 concluded equivalent results between carriers and
non carriers9
◦ Two other studies also produce equivalent
results (Tamer et al,/Brekelmans et al.)10 11
Prevalence and Penetrance
“The contribution of [pathogenic variations] to
breast cancer risk within any specific
population is a function of both their
prevalence and their penetrance. Mutation
prevalence varies among ethnic groups and
may be influenced by founder mutations.
Penetrance can be influenced by mutation-
specific phenotypes and the potential
modifying effects of the patient's own genetic
and environmental background.”
 Nat Rev Cancer 2007;7:937
Founder populations
clear phenotypic description if you have many
affected with the same variation
◦ Ashkenazi Jewish families
 BRCA1:185delAG, 5382insC; BRCA2: 6174delT
◦ Sweden
 BRCA1:3171ins5
◦ Finland
 BRCA2: 4088insA
◦ Québec Canada
 BRCA1: 2953delGTA + C, 3875delGTCT, 4446C --> T
 BRCA2: 2816insA, 3398delAAAAG, 6085G --> T,
6503delTT, 8765delAG)
Newfoundland
Unique genetic environment
◦ much of the population share a common
ancestry which has remained genetically
isolated over time
Recently studies
Bardet-Biedl Syndrome
Colorectal cancer
Blindness
BRC???
Hypothesis

We hypothesize that Newfoundland

families, with high incidences of breast
cancer, may present a distinct BRCA
genotype-phenotype relationship thus far
unreported.
 Objectives

1) To evaluate the risk and tumor characteristics

seen within high incidence BRC families in
Newfoundland.

2) To identify the BRCA genotype of these

families through sequencing screening.

3) To examine associations between BRCA

variations and cancer risk, tumor presentation and
prognosis.
Rationale

The etiology of High incidence BRC

families has yet to be determined in
Newfoundland1
There is still much debate with regards to
prognosis of BRCA patients7,8,9,10,11
Is there a BRC founder variation in
Newfoundland?
Overview
Referred Families

Inclusion
Excluded/Declin
Critereia/Consen
ed
ted

FHQ/Data
Blood Sample
Exttraction

DNA Time to event

Sequencing analysis

Genotype Phenotype

Genotype -
Phenotype/
Follow Up
Recruitment

~100 families
Retroactively contact probands
Inclusion Criteria
◦ Adult
◦ US Preventive Task Force for BRCA screening15
 2 first-degree relatives with BRC, 1 of whom received the diagnosis at age 50 years
or younger
 a combination of 3 or more first- or second degree relatives with BRC regardless of
age at diagnosis;
 a combination of both breast and ovarian cancer among first- and second- degree
relatives
 a first-degree relative with bilateral BRC
 a combination of 2 or more first- or second-degree relatives with ovarian cancer
regardless of age at diagnosis
 a first- or second-degree relative with both breast and ovarian cancer at any age
 a history of breast cancer in a male relative
Recruitment
Informed consent
Blood samples for DNA analysis.
Data extraction of medical records
Family History Questionnaire (FHQ)
◦ Pedigrees
Phenotype- Risk Profile
FHQ
◦ Determine Severity of Disease
 number of affected
 mean age of diagnosis
 mean age of death
 types of cancer

Family history Score (FHS)

 Determine overall risk by comparing observed and
expect rates of cancer
Tumor Phenotype
Data extraction from medical records
 cancer stage
 lymphatic vascular invasion
 mucinous Y/N
 margins
 estrogen/progesterone receptor status
 IHC HER-2 status
 number of lymph nodes examined and status invasive ductal or
lobular carcinoma
 lobular carcinoma insitu status
 tumor grade and differentiation
 Tumor characteristics and risk information will give
a well defined phenotype within each family.
Genotype Analysis
Prediction model 17

◦ screen families most likely to harbor a BRCA

variation first

Oij =Observed BRC status, Pij= Prob. Of genotype given

parental genotype, nij, nij= number of affected individuals
Genotype Contd……..
Sequencing
◦ ABI 377 DNA Sequencer
 all exons and intron/exon boundaries
 coding regions derived from RefSeq
 NM_007294.2 (BRCA1)
 NM_012772.1 (BRCA2).

◦ Primer sets
 previous studies 5, 7, 8
 Primer 3.
Genotype Contd……..
Analysis of sequencing results
◦ Seqeuncher
◦ Mutation Surveyor
◦ ABI Sequencing Analysis
Deletions
◦ detected by multiplex ligation–dependent
probe amplification (MLPA)
Methylation status
◦ methylation-specific PCR
Genotype Contd……..
Predictions of Allele variation
pathogenicity and conservation
◦ Polyphen, SIFT, PANTHER and Clustral.

PredictedBRCA variations will be

confirmed in affected family members

Multiple families?
Genotype-Phenotype Analysis
Assign Phenotype to BRCA1/2
◦ Tumor Presentation
◦ Cancer Risk
 FHS
 Average age of onset
 Risk of BRC and other related Cancers vs. other
genotypes
Prognosis
 Survival analysis Kaplan-Meir
 BRCA1/2 vs. Sporadic
 BRCA1 vs. BRCA2 outcomes
Difficulties
Samples
◦ Retroactive
 Probands
 Confirmation
May not find identical pathogenic
variations
◦ Homozygous nature of Newfoundland may
work against us
Conclusion
We will have genotyped high risk BRC
families in Newfounland
Evaluated each family with regards to
risk, pathology, and prognosis
Add to the prognosis debate
Regardless of findings, high risk families
will have a clear phenotypical work up
that will lower the burden of disease and
possible aid in novel gene discovery
 References
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implications for genetic testing. JAMA. 1997;278:1242-1250.
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3. Rennert G, Dishon S, Rennert HS, Fares F. Differences in the characteristics of families with BRCA1 and BRCA2 mutations in israel. Eur J Cancer Prev.
2005;14:357-361.
4. Antoniou A, Pharoah PD, Narod S, et al. Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case series
unselected for family history: A combined analysis of 22 studies. Am J Hum Genet. 2003;72:1117-1130.
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6. Miki Y, Swensen J, Shattuck-Eidens D, et al. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science. 1994;266:66-71.
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2002;8:3776-3781.
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genotype-phenotype
Higherrisk of BRC and ovarian cancer when
compared to BRCA2
BRCA1
 < risk of BRC risk with variations in the central
gene region BRCA1 allele{{331 Thompson,D.
2002}}
◦ Variations 3’ to nucleotide 4,191
 < risk ovarian cancer {{331 Thompson,D. 2002}}
genotype-phenotype
BRCA2
Variations exon 11
◦ effect its ability to bind RAD51
 when compared to other BRCA2 deleterious
variations
 > the risk of ovarian cancer
 < the risk of BRC {{330 Thompson,D. 2001}}.
6174delT
 > ovarian , < prostate cancer risk
6503delTT
 < ovarian cancer ,> male breast cancer