GENETIC TESTINGVolume 3, Number 2, 1999Mary Ann Liebert, Inc.

BRCA1 Screening in Patients with a Family History ofBreast or Ovarian Cancer

BRITA ARVER,1 ANTONIO CLARO,1 ANITA LANGER0D,2 ANNE-LISE B0RRESEN-DALE,2and ANNIKA LINDBLOM1

ABSTRACT

Several studies have shown that the frequency of BRCA1 mutations in high-risk families differs widely be-tween populations. In a recently published study from the Stockholm region, we found BRCA1 mutations inabout 35% of the breast/ovarian families, but only in 1% of the families with site-specific breast cancer. Todetermine the frequency of BRCA1 mutations in families with a less increased risk for breast or ovarian can-cer, a second study was performed. A total of 94 families with two and six families with only one affectedmember were included. Six mutations were found, all localized in exon 11, and five of them were previouslyknown Swedish founder mutations. The mutation frequency was 6%, similar to the finding in families ful-filling the criteria for hereditary breast cancer (7%) that was disclosed in our first study. All families with a

mutation had at least one individual with ovarian cancer. Thus, our study further implies that for a womanwith breast cancer, a family history of ovarian cancer is far more important than a family history of breastcancer for predicting a BRCA1 mutation. Our results can be used to increase the specificity in selection offamilies for genetic testing.

INTRODUCTION

Breast cancer is the most common malignancy affectingwomen in developed countries. Usually the disease iscaused by genetic alterations in somatic cells of the breast, butit is estimated that 5-10% are due to germline mutations inbreast cancer susceptibility genes, with an autosomal dominantpattern of inheritance (Newman etal, 1988; Claus era/., 1991).Inherited breast tumours have an earlier age of onset than spo-radic tumours (Castilla et al, 1994), and, thus, often affectwomen in reproductive ages. Ovarian cancer is less frequentthan breast cancer but is often diagnosed at a late stage andcauses a large number of deaths among middle-aged women inthe industrial world. The first breast cancer susceptibility geneBRCA1 was located by linkage analysis in site-specific breastcancer families to chromosome 17q21 in 1990 (Hall et al,1990). Linkage to the same region was also obtained in fami-lies with breast-ovarian cancer (Narod et al, 1991) and in fam-ilies with site-specific ovarian cancer (Steichen-Gersdorf et al,1994).The complete sequence of BRCA1 was identified in 1994

(Miki et al, 1994). In 1995, a second breast cancer suscepti-

bility gene, BRCA2, on chromosome 13ql2-13, was identified(Wooster et al, 1995). Of 237 breast cancer families collectedby the Breast Cancer Consortium, 52% showed linkage toBRCA1 and 32% to BRCA2 (Ford et al, 1998). The majorityof the breast-ovarian cancer families were linked to BRCA1,while the majority of families with both male and female breastcancer were linked to BRCA2. Sixteen percent of the familiesdid not show linkage to either of the genes, suggesting otherpredisposing genes (Serova et al, 1997; Ford etal, 1998). Mostmutation screening performed so far have included families athigh risk. The criteria for inclusion in most studies have beenat least three affected individuals, and in some studies inclu-sion has also been restricted to early age of onset cases. In a

recently published study of breast and/or ovarian cancer fami-lies from the Stockholm region, BRCA1 mutations were foundin one-third (7/20) of the breast/ovarian families, but in lessthan 1% (1/106) of the breast cancer families (Zelada-Hedmanet al, 1997). In that study, a family was defined by at leastthree individuals affected by breast or ovarian cancer and therewere no restrictions with respect to age. Many families recruitedto the Cancer Family Clinic, however, are small and includeonly two first-degree relatives affected by breast or ovarian can-

'Department of Clinical Genetics, Karolinska Hospital, S-171 76 Stockholm, Sweden.2Department of Genetics, Institute for Cancer Research, The Norwegian Radium Hospital, N-0310 Oslo, Norway.

223

224 ARVER ET AL.

Table 1. BRCAl Mutations Found in 100 Swedish Patientswith a Family History of Breast and/or Ovarian Cancer

Familynumber Exon

Sequencechange

Mutationtype*

Lowest ageat onset

Mean ageat onset

Number ofbreast cancer

Number of ovariancancer

1365000130350546305045

111111111111

2594AC2594AC

3166insTGAGA3166insTGAGA

3745AA3128AITT

FSFSFSFSFSFS

474440494050

565048495750

321I11

aFS, Frameshift.

cer. To define the germline mutation frequency in this risk pop-ulation, a second BRCAl mutation screening was performed.

MATERIALS AND METHODS

FamiliesFamilies with two members affected by breast or ovarian

cancer, families with one woman affected by breast cancer atan early age (<36), or families with one member affected byboth breast and ovarian cancer were included in the study. Onehundred families were willing to participate. The families wererecruited as described in our previous study (Zelada-Hedmanet al, 1997). Fifty-nine of the families were available from a

registry of all existing breast cancer patients in Stockholm1988-1989. The remaining 41 families were recruited throughthe Cancer Family Clinic at the Karolinska Hospital during1990 to 1996. Eighty-nine families were included because oftwo first- or second-degree relatives affected by breast canceronly, four families because of two close relatives with breast

and ovarian cancer, respectively, and one family was recruitedwith two members suffering from ovarian cancer. Six addi-tional families with only one affected woman were included:five because of early onset breast cancer, and, finally, one inwhich a woman had both breast and ovarian cancer. Bloodsamples from one, or occasionally two, members of these fam-ilies were obtained and DNA was extracted according to rou-tine procedures.

Mutation screeningExons 2-6 and exons 14-16, 20 and 24 were screened by

constant denaturing gel electrophoresis (CDGE) as described(Ikdahl Andersen et al, 1998). Exon 11 was screened by theprotein chain truncation test (PTT). SDS Promega kit (TNT TMT7 Coupled Reticulocyte Lysate System) was used in accor-

dance with the manufacturer's instructions. Primers and condi-tions were as described (Hogervorst et al, 1995). For each vari-ant pattern identified by PTT, primers and conditions previouslydescribed were used for manual sequencing of the actual frag-ment (Srivastava et al, 1992).

Family 5045

0—T-0 -0

ji à Jrr&i***ÓÓU^ Ov50/ où

D

Family 1303

25- -ZÍ

Ov55

0 i

Family 630

-Í2T^ Br76/CT~

^ or« Br40 |/¿Moth Ehh 65hIih-r-0 0—^-0E60_J72 I

Co4íT E58 I E30

¿To „fórJi Jr>^Br49 G35S Br51Ov52

Family 136

Ov61 Ov56

4Ov58 7

óñócyBr47Br47SD

/

Family 5000

0-r-fZfOv55

v44|í¿ (TI

FIG. 1. Pedigree of families with a BRCAl mutation. Filled symbols, Affected individuals; open symbols, nonaffected; shadedsymbols, other forms of cancer. Type of cancer and age at diagnosis, if known, is shown below the symbols. Abbreviations: Br,breast; Ov, ovarian; Co, colon; E, endometrial; G, glioma; J, jejunum. Arrows indicate index persons.

BRCAl MUTATION SCREENING 225

RESULTS Normal Patient

Six BRCAl mutations all leading to truncation of the pro-tein and several variants that most likely are polymorphismswere found. Five of the deleterious mutations have previouslybeen reported as Swedish founder mutations. The families werenot aware of any relationship. Two of the mutations were ofthe type 2594AC; In family number 136, initially reported as afamily with two close relatives with breast cancer, an extendedpedigree showed one bilateral breast cancer and three ovariancancers, with the youngest age of onset at 47. Interestingly, thewoman with breast cancer was married to a first cousin, andtheir mothers, who were sisters, both suffered from ovarian can-cer. The woman's husband, however, did not carry the BRCAlmutation.The second family, family 5000, with the mutation 2594AC,

was a small family with a mother and a daughter suffering fromovarian cancer. The mother was adopted at a very young ageand her biological parents were unknown to her.In two families the mutation was of the type 3166 ins

TGAGA. In family 1303, two cases of breast cancer and onecase of cervix cancer were reported. When the mutation was

known, however, an extended family history and check of med-ical records showed one ovarian, instead of cervix cancer. Thesecond family with this type of mutation was number 5054. Inthis family only one woman was affected. She developed bi-lateral breast cancer before 50 years of age and ovarian cancer2 years later. Her mother suffered from endometrial cancer, likeher grandmother and great grandmother, but none of themshared the mutation.Family 630, carried the Swedish founder mutation type

3745AT. In this family, two cases of breast cancer and one caseof stomach cancer were reported. The "stomach cancer" wasshown to be ovarian cancer when the death certificate was ob-tained. In family number 5045, a previously unreported non-sense mutation, 3127ATT, was found (Fig. 2). In this family,one woman was affected by breast cancer and her daughter hadovarian cancer, but only the woman with ovarian cancer car-ried the mutation. Her paternal grandmother died of breast can-cer before 50 years of age, but it has not yet been possible toprove that the mutation was inherited from her.

DISCUSSION

In 100 families at risk for breast cancer, 6 mutations inBRCAl were found (Table 1). Three mutations were foundamong 89 (multiple affecteds) and 5 single affected families re-cruited because of breast cancer only and 3 in the 6 familieswith ovarian cancer. However, three of the families with mu-tations that were thought to include only breast cancer cases,harbored members with ovarian cancer, as well. In the 59 fam-ilies recruited from a cancer registry, the family history was

taken through a questionnaire and only the diagnosis of the in-dex person was confirmed by medical records. When the mu-tations were found, the pedigree was extended and all cancerdiagnoses were confirmed by review of medical records. Thefinding of BRCAl mutations solely in families with breast-ovarian cancer, or with ovarian cancer only, strongly empha-sizes the impact that a family history of ovarian cancer has forthe likelihood of finding a BRCAl mutation. The current study

G A T C G A T CNor

G

Mut

I_r\A

T r 3§

FIG. 2. DNA sequence analysis of a germline mutation infamily 5045, done with reverse primers, showing 3127ATT

demonstrates, as did our first investigation (Zelada-Hedman etal, 1997), that the occurrence of breast and ovarian cancer, orovarian cancer only, is far more suggestive of a BRCAl muta-tion than the occurrence of breast cancer only.We think it is likely that these families constitute a repre-

sentative selection of Stockholm families with a history ofbreast or breast-ovarian cancer. However, only 15 BRCAl mu-tations have been found (and two BRCA2 mutations in a sub-set of 167 of the families; Chen et al., 1998), which is a lowfrequency compared to other series published so far.What are the possible reasons for this outcome? Our in-

clusion criteria have been generous with respect to age at on-set, where no restrictions have been made and both first- andsecond-degree relatives were included in our first study. Wedo not think that our choice of methods is biased: Wescreened exon 11, the largest part of the gene, with PTT on

genomic DNA, an easy, stable, method used in many BRCAlstudies and supposed to detect most truncating mutations. Forthe remaining part of the gene, we used single-stranded con-formation polymorphism (SSCP) analysis and CDGE in ourfirst study, and only CDGE in the study described here as thelatter method is supposed to have a higher sensitivity. How-ever, not all exons were screened in this study. The non-

screened exons have sequences not optimal for CDGE analy-ses so any mutations residing in these exons will thereforehave been missed. Previous SSCP of these exons did not,however, detect any mutations, excluding at least foundermutations in these areas. Large deletions will also have beenmissed with the used techniques. Finally, it is known that thebreast cancer gene mutation spectrum varies widely amongpopulations, and that founder mutations are common, partic-ularly in isolated populations like the Icelandic (Thorlaciuset al., 1997), among Ashkenazi Jews (Levy-Lahad et al,1997; Abeliovich et al, 1997), and in our own (Zelada-Hed-man et al., 1997; Johannsson et al, 1996). It is not unlikelythat other breast and ovarian cancer-causing genes, highand/or low penetrant, with or without founder effects, segre-gate in our population. In accordance with our results andthose of others, we now offer clinical BRCAl screening tofamilies with breast and ovarian cancer, to ovarian cancer

226 ARVER ET AL.

families, and to families in which the proband's breast orovarian cancer developed at an early age.

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Address reprint requests to:Dr. Annika Lindblom

Department ofMolecular MedicineKarolinska Hospital

S-171 76 Stockholm, Sweden

Received for publication September 1, 1998; accepted January1, 1999.