GENETIC TESTINGVolume 1, Number 2, 1997Mary Ann Liebert, Inc.

Genetic Testing for Breast Cancer Susceptibility: Frequency ofBRCA1 and BRCA2 Mutations

ARUPA GANGULY, KEVIN LEAHY, ANDREW M. MARSHALL, ROHINI DHULIPALA,LYNN GODMILOW, and TAPAN GANGULY

ABSTRACT

Genetic testing for breast cancer susceptibility became a reality after two cancer predisposition genes, BRCA1and BRCA1, were identified. Mutations in these two genes were predicted to account for 85% to 90% of hered-itary breast and ovarian cancer syndromes. We present results of mutation analysis of the coding sequenceof these two genes in 110 consecutive non-Jewish breast cancer patients with a positive family history of breastand/or ovarian cancer. The individuals were identified in various cancer risk evaluation centers in the coun-

try. Twenty-two (20%) mutations in the BRCA1 gene and 8 mutations (7%) in the BRCA1 gene were detected.We also analyzed 52 Ashkenazi Jewish breast cancer patients for mutations in the BRCA1 and BRCA1 genes.Eleven Jewish individuals (21%) carried either one of the two common mutations, 185delAG and 5382InsC,in the BRCA1 gene and 4 individuals (8%) had the 6174delT mutation in the BRCA1 gene. The frequency ofmutations in BRCA genes in affected people in this ethnic group was not significantly different from the non-Jewish population. On further analysis, the data demonstrate that neither age of onset nor phenotype of thedisease had any significant predictive value for the frequency of mutations in these genes. These data confirmthe lower prevalence of mutations in either of the BRCA genes in clinical families when compared to high-risk families used for obtaining linkage data in a research setting.

INTRODUCTION

BREAST CANCER IS ONE OF THE MOST COMMON DISEASES af-fecting women. A positive family history is an important

epidemiological risk factor for the disease and accounts forabout 5% of all breast cancer cases. The disease has been linkedto two autosomal dominant genes, BRCA1 and BRCA1 (Hall etal, 1990; Miki et al, 1994; Wooster et al, 1994; 1995). Mu-tations in the BRCA1 gene have been associated with early on-

set breast and/or ovarian cancer, whereas mutations in theBRCA2 gene have been associated with families that includemale breast cancer (Couch andWeber, 1996; Couch etal, 1996;Tavitigian et al, 1996). However, there are many exceptionsto these generalizations. Mutations in the BRCA1 gene havebeen reported in male breast cancer families as well as in lateonset breast cancer individuals. Alterations in either of thesegenes in women have been reported to confer an estimated 85%lifetime risk of developing breast cancer and a significantly el-evated risk of ovarian cancer. These mutations also increase the

risk of prostate, colon, and pancreatic cancer among male car-

riers in these families. To date, hundreds of independent muta-tions have been reported in the BRCA1 (Couch and Weber,1996) and the BRCA2 genes (Breast Cancer Information Coreon the World Wide Web).

This knowledge could be immediately applied to identifi-cation of the mutation in the proband of any high-risk fam-ily followed by the offer of direct testing to additionalpresymptomatic family members. The basis for this signifi-cant clinical application was the prediction that mutations inBRCA1 and BRCA2 genes will account for 85% to 90% offamilial breast and ovarian cancer cases (Easton et al, 1993;Wooster et al, 1994; Rebbeck et al, 1996). Genetic testingcould potentially offer different management options forthese high-risk individuals. Due to the different range of psy-chological and social issues involved in genetic testing ofcancer susceptibility, the American Society of Clinical On-cologists (ASCO) proposed a set of testing guidelines (Offitet al, 1996). These guidelines were aimed at defining the

Department of Genetics, Genetic Diagnostic Laboratory, University of Pennsylvania School of Medicine, Philadelphia, PA 19104.

85

86 GANGULY ET AL.

families with the highest estimated probability of mutation inBRCA1 or BRCA2.

This report provides results of mutation analysis of a groupof individuals identified at various cancer-risk evaluation clin-ics who are affected with breast and/or ovarian cancer and pos-sess a positive family history. We found that 27% of these fam-ilies have mutations in either BRCA1 or BRCA2. The dataindicate that in a significant fraction of familial cases identifiedin the general population, the disease phenotype is not due tomutations in the coding sequence of the BRCA1 and BRCA2genes (Ganguly et al, 1996).

METHODS

Criteria for selection offamiliesThe patient material for this study was collected from mul-

tiple cancers-risk evaluation clinics around the country. The cri-teria for selection of these families followed the recommenda-tions by the ASCO for genetic testing for cancer susceptibility(Offit et al, 1996). These recommendations for eligibility forbreast cancer gene testing were the following: (a) families withmore than 2 breast cancer cases and 1 or more cases of ovar-ian cancer diagnosed at any age; (b) families with more than 3breast cancer cases diagnosed before age 50; (c) sister pairswith the following cancers diagnosed before age 50: 2 breastcancer, 2 ovarian cancers, or 1 breast and 1 ovarian cancer. Ex-ceptions were made in accepting individuals coming from verysmall families where only a mother-daughter pair or a pair ofsiblings are affected with either, (a) very early age at onset(<40) of breast cancer, or (b) bilateral breast of ovarian can-

cer. The protocol was approved by the University of Pennsyl-vania IRB, and informed consent was obtained from each in-dividual tested. Genetic counseling of the individuals, both priorto sampling and after the results of genetic testing became avail-able, was provided by the respective referring centers.

Isolation ofDNA from blood samplesGenomic DNA was isolated from 5 ml of blood using a com-

mercial DNA isolation kit (Qiagen, CA). The DNA was dis-solved in 500 pi of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.Approximately 80 to 100 pg of DNA was obtained. For PCR,the DNA was diluted to 50 nglpl and 1 pi was used in a 50 piPCR reaction. A duplicate sample of DNA was isolated froma second tube of blood and stored separately from the first batchof DNA. This DNA was used for independent verification ofany mutation once it was found in the first sample.

Preparation of PCR products andheteroduplex formation

The primers for genomic DNA amplification oiBRCAl wereinitially downloaded by anonymous FTP (file transfer protocol)from morgan.med.utah.edu in the pub/BRCAl directory. Be-cause Conformation Sensitive Gel Electrophoresis (CSGE) cananalyze fragments larger than SSCP (400-500 bp), primerswere redesigned to generate 10 overlapping fragments for the3.5 kb exon 11 oiBRCAl. Also, for each exon the primers werelocated at least 50 bp away from the exon-intron boundaries.

The sequences of primers for BRCA1 and BRCA2 are obtain-able by email from ganguly@mail.med.upenn.edu. PCR prod-ucts were generated using an automated thermocycler (PE 9600)and commercial PCR kits. The PCR buffer was optimized us-

ing a kit from Stratagene, CA. The composition of the X10PCR buffer was 100 mM Tris-HCl, pH 8.8, 35 mM MgCl2, 250mM KC1; for exons 5 and 11, the MgCL; concentration was 25mM, whereas other components were unchanged. Heteroduplexformation took place during the last two cycles of PCR whichwere 5 min at 94°C followed by 30 min at 68°C.

Validation of conformation sensitive gelelectrophoresis as a method for screening

The first objective of the present study was to evaluate CSGEas a method for scanning the BRCA1 gene for the presence ofsequence differences in PCR products of genomic DNA and itsuse in a routine clinical diagnostic setup. The CSGE methodinvolves heteroduplex analysis of PCR products in a novelmildly denaturing polyacrylamide (PA) gel matrix using a dif-ferent crosslinker, bis-acrolyl-piperazine (BAP, Fluka), insteadof the conventional bis-acrylamide (BIS) (Ganguly etal, 1993;Williams etal, 1995). The enhanced resolution of this methodis due to the novel polyacrylamide matrix with basically largepores (the same as those of a conventional 5% PA-BIS gel) andthe resilience of a 10% PA gel. The molecular basis for the sep-aration is the intrinsic DNA bending in solution and its alter-ation in the presence of mismatched bases (Ganguly et al,1993).For the first part of the evaluation, we obtained a panel of

46 patients from the patient population followed by Dr. Bar-baraWeber, Director, Breast Cancer Program at the Universityof Pennsylvania. This panel included 10 patients with knownmutations in the BRCA1 coding sequence found by alternatemethods which were analyzed in a single-blind fashion. The re-

maining 36 individuals were affected with breast/ovarian can-

cer and possessed a strong family history but had unknown mu-tation status for BRCA1. The coding sequence of the BRCA1gene was amplified using genomic DNA and subjected toCSGE, as just described. Nine out of 10 previously known mu-

tations were detected. The tenth mutation-bearing sample failedto display a CSGE shift. This sample was sequenced and foundto be negative for the previously known familial mutation. Theindividual was later found to be an unaffected member of thefamily. Eight new mutations were identified among the 36 in-dividuals who were not previously screened. These mutationsincluded insertions, deletions, missense, and nonsense muta-tions (Couch andWeber, 1996). All reported single-base poly-morphisms in the BRCA1 gene were identified by CSGE as

aberrantly-migrating heteroduplex bands (Ganguly etal, 1995).As a second method of validation, 10 patients were chosen

randomly and the 3.5-kb exon 11 of BRCA1 gene was com-

pletely sequenced and scanned by CSGE. There was a perfectmatch between the results of direct sequencing and CSGE. Forevery CSGE shift, a sequence variation was observed. Allchanges found were shown to be common polymorphisms.

To test for false negatives, two samples of DNA were se-

lected that did not display a single heteroduplex shift by CSGEassay of any exon. The entire coding sequence of BRCA1 in-cluding the exons and the exon-intron boundaries was ampli-

GENETIC TESTING OF BREAST AND OVARIAN CANCER 87

fied and each one of these samples were sequenced using for-ward and reverse primers. No mutation or polymorphism was

detected. These data further suggest that mutation scanning ofthe BRCA1 coding sequence using CSGE detects any nucleotidechange that can be detected by direct sequencing. Mutations inthe coding sequence that would be missed by this method as

well as by direct sequencing of PCR products are complete or

partial deletions, duplications, or rearrangements of exons in-cluding the flanking sequences.

Direct sequencing of PCR products displayingheteroduplex species with unique mobility shifts

Once a subset of PCR products was identified with an aber-rant heteroduplex pattern, PCR products were remade for thesame region, purified from the unincorporated nucleotides andprimers and subjected to dye terminator cycle sequencing (ABI,CA) using one of the PCR primers. The sequencing reactionproducts were purified on a Centrisep spin column (PrincetonSeparations) to eliminate unincorporated nucleotides, denaturedby heating in the standard formamide-containing gel loadingbuffer, and run on an ABI 377 sequencer.

Any sequence change was confirmed either, (a) by se-

quencing with the reverse primer and visualizing the nucleotidechange in both forward and reverse direction, or (b) by selec-

tive restriction enzyme digestion capable of distinguishing thenormal and mutated sequence.

RESULTS

Mutation analysis of the BRCA1-coding sequence

For mutation analysis of the BRCA1 -coding sequences, weadopted CSGE as our preliminary scanning method as describedin the Methods section. The clinical test was offered only to af-fected individuals selected on the basis of criteria set forward byASCO, as described in the Methods, or, with only one affectedfirst-degree relative but one with (a) early age of onset of cancer(<40 y) or (b) bilateral breast or ovarian cancer. However, nolinkage information on these individual families was available.To date, we have completed analysis of the coding sequence ofthe BRCA1 gene of 110 consecutive clinical patients of non-Jew-ish origin. This panel of patients excludes the patients describedin the Methods section who were used for the validation of theCSGE method. We also studied an additional 52 breast cancerpatients from Ashkenazi Jewish families.

Twenty-two disease-causing mutations were detected in theBRCAÍ -coding sequence, including missense, nonsense, or

frameshift mutations (Table 1). Thus, 20% of patients from the

Table 1. Description of Mutations

ID no.

Averageage ofonset

Age ofonset inproband

CancerStatus inproband Mutation Exon Gene

96-24096-22196-24196-04796-21196-05696-23896-26496-05796-01796-01696-13696-23196-10596-14496-13096-18396-06196-23296-04996-14196-23596-02096-24596-19596-02796-09996-11796-21496-050

30343739404142424344454546474848484949505050515253555560

44613738

47, 59; 3354, 304346405053464143

54, 3044444934293434

34,4943

31,353849464055

BRBR

BR/OVBR

BR-BL/OVBR-BLBRBRBRBRBRBRBRBR

BR-BLBRBRBRBRBR

BR-BLBR

BR-BLBR

BR/OVBRBRBRBRBR

2034 InsA2458 InsTC554 W4286 del TG2774 Ins C2774 Ins CY42C5232 del 8R1835X4184 del TCAA5946 del CT188 del 11F12Y2800 del AA2774 delC2753 del AA1294 del40C61GE1694XE908X2161 del A1294 del 40Y1563X24811ns T3166 Ins 54232 del GR1835GC61G185 del AGE 1694 X

1011101211113

1824111122

1111111151811111116111112245218

BRCA2BRCA2BRCA2BRCA1BRCA1BRCA1BRCA2BRCA1BRCA1BRCA1BRCA2BRCA1BRCA2BRCA1BRCA1BRCA1BRCA1BRCA1BRCA1BRCA1BRCA2BRCA1BRCA1BRCA2BRCA1BRCA1BRCA1BRCA1BRCA1BRCA1

88 GANGULY ET AL.

Table 2. Frequency of BRCA1 Mutations as a Function of Age of Onset and Nature of Cancer

Average ageof onset

Number ofaffected

individualstested

Breast/ovariansyndrome(nl)

Site-specificbreast

cancer (n2)Ovarian

cancer (n3)

Totalnumber ofmutations

n = nl + n2 + n3

30-4546-5960-66

4260

15 (5, 33%)24 (4, 17%)4 (1, 25%)

27 (3, 11%)34 (9, 27%)4 (0, 0%)

0(0)2(0)0

8 (19%)13 (22%)1 (13%)

families studied had detectable germline mutations in theBRCA1 gene.

eluded 3 families with male breast cancer but only 1 family hada BRCA2 mutation.

Role ofage ofonset on the relative frequency ofBRCAl mu-tations: The panel included 42 families with average age of on-set of breast cancer at s45, including at least one affected first-degree relative with very early age of breast cancer

development, or bilateral breast or ovarian cancer, or multiple-affected first- and second-degree family members. The diseasephenotype was variable and included 15 breast/ovarian cancer

and 27 site-specific breast cancer families. The number and dis-tribution of families positive for BRCA1 mutations is indicatedin Table 2. In total, 8 disease-causing mutations were identi-fied (19%) in this group. In the next group (average age of on-set of cancer 46-60), there were 60 individuals. The distribu-tion among the various phenotypes was as follows: 24breast/ovarian cancer families, 34 families with site-specificbreast cancer, and 2 families with ovarian cancer only. Thir-teen mutations in the BRCA1 -coding sequence were identifiedin this group (22%). The third group (age of onset >60) had 8individuals representing 4 breast/ovarian and 4 site-specificbreast cancer families. One mutation in BRCA1 was detectedin this age group (13%).

Mutation analysis of BRCA2-coding sequence in BRCA1-negative families: To estimate the proportion of BRCA1 -nega-tive families that are positive for BRCA2 mutations, the codingsequences of BRCA2 gene from all BRCA1 -negative individu-als were scanned for mutations following the same protocol asthat for BRCA1. Five frameshift mutations and three missensemutations were identified. Thus, BRCA2 mutations accountedfor roughly 8% of familial cases in this patient pool. Table 3shows the age-specific and phenotype-specific frequency ofBRCA2 mutations. It can be seen that BRCA2 mutations tendto predominate in site-specific breast cancer families. Two ofthe bilateral breast cancer families negative for BRCA1 muta-tions were positive for BRCA2 mutations. This study also in-

Role o/BRCAl/2 mutations in bilateral breast cancer fam-ilies: The patient pool included 20 families with bilateral breastcancer. The number of BRCA1/BRCA2 mutations in patientsdefined by the ages of cancer development are given in Table4. Six mutations (30%) were detected in this subgroup. Onemutation was in an individual with bilateral breast cancer plusearly onset ovarian cancer. A higher proportion of mutationswas expected in this patient group defined by bilateral breastcancer.

Role o/BRCAl and BRCA2 mutations in families ofAshke-nazi Jewish background: In addition, we analyzed 52 Ashke-nazi Jewish affected individuals for the presence of mutationsin the BRCA1 and BRCA2 gene. The average age of onset ofcancer in these families was variable (range 35-64). Six indexcases had bilateral breast cancer and 2 families had only ovar-

ian cancer. We found 8 cases of 185delAG in exon 2 oiBRCAl(16%), 3 cases of 5382InsC in exon 20 oiBRCAl (6%), and 4cases of 6174delT in exon 11 of BRCA2 (8%). The numbersof different mutations in breast/ovarian and breast or ovariancancer only families grouped by age of cancer development aregiven in Table 5. In addition to these common mutations, 1Jewish individual was found to carry a frameshift mutation,2084InsC in exon 10 of BRCA2 leading to a premature stopcodon at amino acid 621. This mutation was not seen in anyother Ashkenazi Jewish individual in this patient subset. Theethnicity of the individual was not verified any further.

DISCUSSION

Clinical testing for the breast cancer susceptibility gene,BRCA1, was started with the assumption that mutations in thisgene accounted for at least 45% of familial breast cancer. The

Table 3. Frequency of BRCA2 Mutations as a Function of Age of Onset and Nature of Cancer

Average ageof onset

Number ofaffected

individualstested

Breast/ovariansyndrome(nl)

Site-specificbreast

cancer (n2)Ovarian

cancer (n3)

Totalnumber ofmutationsnl + n2 + n3

30-4546-5960-66

34477

10 (0, 0%)20 (2, 10%)3 (0, 0%)

24 (5, 21%)25 (1, 4%)4 (0, 0%)

0(0)2(0)0

5 (15%)3 (6%)0

GENETIC TESTING OF BREAST AND OVARIAN CANCER 89

Table 4. Frequency of BRCA1I1 Mutationsin Bilateral Breast Cancer Individuals

Average ageof onset

No. offamilieswith bilateralbreast cancer

Total numberofmutations

30-4546-59

710

2 (29%)4 (40%)

mutation-detection rate in the BRCA1 gene in this clinic-basedpatient pool (22/110; 20%) was far below that expectation. Ofthe 110 individual families represented in this study, 96 fami-lies have a high prior probability of having amutation in BRCA1or BRCA1 according to the ASCO guidelines for testing as de-scribed in the Materials and Methods section. The original pub-lication of the Breast Cancer Consortium suggested that thereis considerable genetic heterogeneity among the breast cancerfamilies (Easton et al, 1993; Rebbeck et al, 1996). The crite-ria for linkage of any family to BRCA1 as set by the consor-

tium were at least 4 members affected with cancer before theage of 60. In our patient population, 60 families met those cri-teria with 31 breast/ovarian and 29 breast-cancer-only families.Only 8 of 31 (26%) of breast/ovarian families and 5 of 29 (17%)site-specific breast cancer families had mutations in the codingsequence of the BRCA1 gene. The total frequency, 13 out of 60(22%), is significantly less than the 61% observed by Rebbecketal (1996) in families with early-onset breast and ovarian can-

cer and multiple-affected relatives. Four BRCA1 mutations (1in breast/ovarian; 3 in breast-cancer-only families) account forthe disease in this patient subgroup (7%). Interestingly, no link-age was observed to the BRCA1 gene on chromosome 13q inany of the families described in Rebbeck et al (1996). In an

additional 36 families with at least 3 members affected withcancer, 7 (20%) had mutations in BRCA1 and 3 (8%) had mu-

tations in BRCA1. In addition, there were 14 individuals in thetotal patient pool who presented very limited family history ofbreast cancer. However, each one of these families had at least1 case of bilateral disease or breast cancer diagnosis at veryearly age of onset (<40 y). One mutation in BRCA2 gene was

detected in this group (ID NO 96-221).Based on published figures, it was expected that 35% of

breast cancer families will be found to segregate with BRCA1mutations (Wooster et al, 1994). In the present report, BRCA2mutations accounted for only 8% of the disease-associated al-íeles. Moreover, the families with BRCA1 mutations had pre-dominantly site-specific breast cancer and not breast/ovariancancer (Table 4).

The total frequency of mutations detected in BRCA1 andBRCA1 genes in this non-Jewish clinical population was only27%. Although a major class of mutations involving the non-

coding regions of the BRCA genes, such as promoter or intronmutations have not been ruled out, the data suggest the exis-tence of other as yet unidentified genetic loci causing cancer

susceptibility.One criticism of this study could be that the mutation scan-

ning assay used fails to detect 100% of mutations. The resultswe describe indicate that scanning the coding sequences ofBRCA1 gene by CSGE is specific and sensitive in detecting a

very high proportion of mutations, including single-base sub-stitutions. Also, CSGE has now been successfully applied tothe analysis of a number of large genes such as BRCA2,COL2A1, and COL7A1 (Couch et al, 1996; Christiano et al,1996a; 1996b). In a recent report, two different gene-scanningmethods, Denaturing Gradient Gel Electrophoresis (DGGE) andCSGE, were compared for detection efficiency of single-basesubstitutions in two different collagen genes, COLIXA2 andCOLXIA1. Each and every change detected by DGGE was alsopicked up by CSGE (Korkko et al, 1996).

The possibility exists that an excess of sporadic breast andovarian cancer patients in our patient pool reduced the fractionof patients positive for mutations in the BRCA genes. This im-plied that more than 1 affected individual from any familyshould be tested when the preliminary screen is negative. How-ever, the cost of such clinical testing would be prohibitive.

These results are significant for discussions of the clinicalsignificance of the breast cancer susceptibility genes. In con-

trast to previous predictions, this study confirms that a smallerfraction of breast cancer families can be accounted for by mu-

tations in the coding sequences of BRCA1 or BRCA2 genes.Similar results have recently been obtained by others (Couchet al, 1997) for different patient pools. The earlier predictionswere based on linkage analyses of extremely high-risk familiesthat included multiple-affected members in multiple genera-tions. Family size and the number of additional affected mem-

bers tend to be much smaller in the case of the average clini-cal patient who presents for genetic testing. It should beemphasized in the pre-test counseling sessions, that, in the ab-sence of linkage information, there is a significant probabilitythat nomutation will be found in either of the two genes, BRCA1or BRCA2.It is also true that the patients with breast and/or ovarian can-

cer syndrome in our series lacked any specific phenotype thatsuggested which BRCA gene was a good candidate for analy-sis. Thus, patients can be trapped in an endless cycle of testsas each new cancer susceptibility gene is found. As these tests

Table 5. Frequency of BRCA1I2 Mutations in Ashkenazi Jewish Breast Cancer Individuals

Mutation

185delAG5382InsC6174delT

Total numberof

mutations

Average age of onset, 30-45Breast/ovariansyndrome

300

Site-specificbreast Ovarian

cancer

010

Average age of onset, 46-64Breast/ovariansyndrome

411

Site-specificbreast Ovarian

cancer

000

90 GANGULY ET AL.

are labor-intensive and expensive, the situation could becomedifficult for all concerned. Therefore, from a clinical perspec-tive, the direct genetic test should be only presented to indi-viduals who have been counseled and have a realistic under-standing of the possible benefits and limitations of the test. Inthe end, in deciding whether or not to be tested, a woman mustweigh the potential benefits of knowing her BRCA mutationstatus, with the significant negatives associated with the test-

ing—including that the results may prove to be emotionally bur-densome to herself and her family, and that they may be am-

biguous or inexact as predictors of cancer risk.

ACKNOWLEDGMENTS

This work was supported partially by a grant from NIH,1R21CA66179 through the Cancer Center at University ofPennsylvania. We acknowledge the support of this work byHaig Kazazian, Jr, MD, and the participation of numerous can-

cer-risk evaluation programs that sent us clinical samples. Wealso thank Barbara Weber, MD, for providing us with the ini-tial samples for validation of the assay system.

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Address reprint requests to:Arupa Ganguly, Ph.D.Department of Genetics

University of Pennsylvania415 Curie Boulevard

Philadelphia, PA 19104-6145