Novel Inherited Mutations and Variable Expressivity of BRCAI Alleles, Including the Founder Mutation 185Delag in Ashkenazi Jewis

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Am. J. Hum. Genet. 57:1284-1297, 1995 Novel Inherited Mutations and Variable Expressivity of BRCA I Alleles, Including the Founder Mutation 185delAG in Ashkenazi Jewish Families Lori S. Friedman,* Csilla 1. Szabo,* Elizabeth A. Ostermeyer,* Patrick Dowd,t Lesley Butler,* Tari Park,t Ming K. Lee,* Ellen L. Goode,* Sarah E. RowelI,§ and Mary-Claire King* Department of Molecular and Cell Biology and School of Public Health, University of California, Berkeley

Summary Introduction Thirty-seven families with four or more cases ofbreast can- Breast cancer is the most common malignancy among cer or breast and ovarian cancer were analyzed for muta- women, with a cumulative risk by age 85 years of one tions in BRCA1. Twelve different germ-line mutations, four in eight for a female born in 1990 (American Cancer novel and eight previously observed, were detected in 16 Society 1994). The strongest epidemiological risk fac- families. Five families of Ashkenazi Jewish descent carried tor for the disease is a positive family history (Petrakis the 185deIAG mutation and shared the same haplotype at et al. 1982; Kelsey and Gammon 1991). Complex seg- eight polymorphic markers spanning -850 kb at BRCA1. regation analysis of families with high incidence of Expressivity of 185delAG in these families varied, from breast cancer indicated the existence of one or more early-onset bilateral breast cancer and ovarian cancer to autosomal dominant susceptibility genes contributing late-onset breast cancer without ovarian cancer. Mutation to 5%-10% of all breast cancer (Newman et al. 1988; 4184delTCAA occurred independently in two families. In Claus et al. 1991). Approximately 1 of every 200 one family, penetrance was complete, with females devel- women in the general population may carry a predis- oping early-onset breast cancer or ovarian cancer and the posing mutation in one of these genes (Newman et al. male carrier developing prostatic cancer, whereas, in the 1988; King et al. 1993). other family, penetrance was incomplete and only breast In 1990, a breast-ovarian cancer susceptibility gene, cancer occurred, diagnosed at ages 38-81 years. Two novel BRCA1, was mapped to chromosome 17q21 (Hall et al. nonsense mutations led to the loss of mutant BRCA1 tran- 1990). Subsequent studies verified linkage to this region, script in families with 10 and 6 cases of early-onset breast with odds >1026:1 (Narod et al. 1991; Easton et al. cancer and ovarian cancer. A 665-nt segment ofthe BRCA1 1993). Women who inherit a BRCA1 mutation have an 3'-UTR and 1.3 kb ofgenomic sequence induding the puta- 80% lifetime risk of breast cancer and are also at an tive promoter region were invariant by single-strand confor- elevated risk for ovarian cancer (Newman et al. 1988; mation analysis in 13 families without coding-sequence mu- Easton et al. 1993; Ford et al. 1994). BRCA1 was re- tations. Overall in our series, BRCA1 mutations have been cently isolated by positional cloning (Futreal et al. 1994; detected in 26 families: 16 with positive BRCA1 lod scores, Miki et al. 1994) and inherited BRCA1 mutations rap- 7 with negative lod scores (reflecting multiple sporadic idly identified, confirming the identity of this gene (Cas- breast cancers), and 3 not tested for linkage. Three other tilla et al. 1994; Friedman et al. 1994; Futreal et al. families have positive lod scores for linkage to BRCA2, 1994; Miki et al. 1994; Simard et al. 1994). but 13 families without detected BRCA1 mutations have The BRCA1 gene encodes a 7.8-kb transcript com- negative lod scores for both BRCA1 and BRCA2. posed of 22 coding exons that span >80 kb of genomic DNA (gDNA) (Miki et al. 1994). Except for a single C3HC4 RING finger motif (Lovering et al. 1993) located near the amino terminus, the predicted 1,863-amino acid Received June 29, 1995; accepted for publication September 13, protein contains no significant homologies to known 1995. genes. Thus, little insight has been gained into the possible Address for correspondence and reprints: Dr. Mary-Claire King, function of BRCA1. Although 102 germ-line BRCA1 mu- University of Washington, Box 357720, Seattle, WA 98195-7720. E-mail: [email protected] tations have been published to date, the nearly ubiquitous *Present address: Departments of Medicine and Genetics, University distribution of these mutations in the gene (Boyd et al. of Washington, Seattle. 1995; Hogervorst et al. 1995; Shattuck-Eidens et al. tPresent address: Genentech, San Francisco. 1995; Struewing et al. 1995; Takahashi et al. 1995) has tPresent address: Tufts University Medical School, Boston. frustrated not only mutation searching efforts but hopes 5Present address: Kaiser Permanente, Oakland. © 1995 by The American Society of Human Genetics. All rights reserved. that important functional domains would be identified. 0002-9297/95/5706-0005$02.00 The significance of the RING finger motif, however, has 1284 Friedman et al.: Expressivity of Germ-Line BRCA1 Mutations 1285 been underscored by the occurrence of both germ-line probands of breast cancer families. Amplified samples (Castilla et al. 1994; Friedman et al. 1994) and somatic were diluted 1:10 in formamide buffer (98% for- (Merajver et al. 1995) missense mutations resulting in the mamide, 10 mM EDTA pH 8, 0.05% Bromophenol loss of conserved cysteine residues. blue, 0.05% Xylene cyanol), held at 95°C for 5 min, The majority of the 52 distinct BRCA1 mutations pub- then cooled rapidly to 40C and held for 5 min. For each lished so far lead to truncation of the mutant protein sample, S ,l is loaded onto an SSCP gel and run at 6 W (Hogervorst et al. 1995; Shattuck-Eidens et al. 1995; (constant power) for 16 h in 0.6 x Tris-borate EDTA Struewing et al. 1995; Takahashi et al. 1995). These (TBE). (An 80-ml gel solution contains 0.5 x mutation frameshift, nonsense, and splice mutations likely reflect detection enhancement (AT Biochem), 0.6 x TBE, 160 loss of function characteristic of a tumor-suppressor gene. ul 25% ammonium persulfate, and 38 ,l NNN'N' tetra- However, most families in these studies were selected for methylethylenediamine.) Gels are dried on a vacuum gel multiple cases of early-onset breast and ovarian cancer dryer and exposed to film for 12-24 h with an intensi- and may not represent the full spectrum of mutations in fying screen. Variant bands were cut from the gel, rehy- the general population. Further characterization of drated in 100 ,l of water, amplified with appropriate BRCA1 mutations by using several different screening SSCP PCR primers, and directly sequenced with the USB approaches should reveal additional predisposing alleles. PCR product sequencing kit, according to manufactur- Despite the number of BRCA1 mutations identified, er's instructions. Mutations were confirmed in multiple very little information of predictive value can be gleaned members of each family by amplifying gDNA and di- from the existing data, because genotype does not ap- rectly sequencing. SSCP primers for the BRCA1 pro- pear strongly correlated with phenotype (Shattuck- moter region (P-1 through P-8) are given in table 1. Eidens et al. 1995). Evaluation of population-based series of breast cancer patients unselected for family his- Allele-Specific Oligonucleotide (ASO) Hybridization to Dot Blots tory will be necessary to determine accurately the gDNA of breast cancer patients was amplified by nonra- frequency and penetrance of specific BRCA1 mutations dioactive PCR using published primers (Friedman et al. in the general population. Nevertheless, the question of 1994). Five microliters of DNA product from each PCR whether mutations that occur relatively frequently reaction was mixed with 55 p1 of denaturing solution (0.4 among high-risk families arise independently or have a M NaOH, 25 mM EDTA) in a 96-well plate format. Fifty common ancestral origin can begin to be addressed. microliters of each sample was loaded into a 96-well dot- Families with four or more cases of breast or breast blot vacuum apparatus containing a prewetted Genescreen and ovarian cancer were selected for BRCA1 mutation nylon filter (1 x 10 mM Tris-Cl, 1 mM EDTA [TE] for analysis. Linkage to BRCA1 and BRCA2 was tested in 10 min), and vacuum was applied. Wells were washed 34 of the 37 families, but BRCA1 was screened for muta- with 100 ,l 1 x TE, with vacuum. DNA was fixed onto tions in each family, regardless of the magnitude or sign the filter by exposure to UV light for 2 min. Ten picomoles of the lod score. Complementary techniques were used of oligonucleotides specific to each mutation and its wild- to identify germ-line mutations in 16 families. The genotype sequence were end-labeled with 32P by using Gibco mic causes of two "inferred regulatory" mutations were T4 PNK according to manufacturer's instructions. ASOs identified as nonsense substitutions in exons 11 and 13, for known mutations are given in table 1. suggesting a possible mechanism leading to the absence Dot-blot filters carrying amplified DNA were incu- of BRCA1 transcript. Haplotype analysis of kindreds bated with 50 ml of tetramethylammonium chloride that share commonly occurring mutations revealed a (TMAC) hybridization solution (50 mM Tris, pH 8, 3 founder mutation in families of Jewish descent. M TMAC, 2 mM EDTA pH 8, 5 X Denhardts solution, 0.1% SDS, and 100 ,ug/ml salmon sperm DNA) (Wood Material and Methods et al. 1985). Prehybridization was at 56°C for 30-60 min. The ASO probe was then added to hybridization SSCP and Sequencing mix and incubated with the filters at 56°C for 1-3 h. PCR was carried out 50-pl volumes containing 50 ng Filters were washed twice for 10 min in 2 x SSPE, 0.1% cDNA or gDNA; 1 x PCR buffer (Boehringer Mann- SDS, then washed three times for 10-30 min at tempera- heim); 200 gM dATP, dGTP, and dTTP (Boehringer tures ranging from 25°C to 56°C (stringent wash) in a Mannheim); 10 ,uM dCTP (Boehringer Mannheim); 50 solution of 50 mM Tris pH 8, 3 M TMAC, 2 mM EDTA pmol each primer (Operon); 0.5 gCi 32P-dCTP (NEN pH 8, 0.1% SDS. Filters were air dried for 15 min and Dupont); and 1.25 U Taq DNA polymerase (Boehringer were exposed to X-ray film overnight. Mannheim). Amplification conditions used were 35 cy- cles of denaturation at 94°C for 45 s; annealing at the Protein-Truncation Test (PTT) optimal temperature of each primer pair for 30 s; and Three overlapping fragments of exon 11 were ampli- extension at 72°C for 30 s. fied from gDNA (50 ng/PCR reaction) by using primer PCR template was lymphocyte cDNA or gDNA from pairs listed below. In each case, the forward primer con- 1286 Am. J. Hum. Genet. 57:1284-1297, 1995

Table I BRCAI Primers and ASOs

A. SSCP Primers for Promoter Region Forward Reverse P-.1 .5'-GCCCAGTTATCTGAGAAACCCC-3' 5'-AATCCAGAGCCCCGAGAGACGCTTG-3' P-2. 5'-GCTAAGCAGCAGCCTCTCAGAATAC-3' 5'-ACTGTGACGTAATAAGCCGCAAC-3' P-3. 5'-CGGATGACGTAAAAGGAAAGAGAC-3' 5'-CCCTCCACCTCCCAACAATC-3' P-4. 5'-GATTGTTGGGAGGTGGAGGG-3' 5'-GCTGGATGGGAATCGTAGTCTTC-3' P-5 .5'-CCAGTGGATAGATTGGAGACCTCC-3' 5'-ATGCTGAGGGGCGGAACTAGGAGTG-3' P-6. 5'-CGGGCAAGTAGTCCTCTAAGGTCAG-3' 5'-TGGTGGAGATTGCGTCGATG-3' P-7 .5'-TAGTTCCGCCCCTCAGCATC-3' 5'-ATGCAAGGACCGTCCGCTAC-3' P-8 .5'-TCAATGGCGTGGTCGllT-IlG-3' 5'-TGAACTTCCCCAAACCCTCTTAG-3' B. Allele-Specific Oligonucleotides Normal Mutant

Exon 2 (185AAG) ...... 5' AAA ATC TTA GAG TGT CCC 3' 5' AAA ATC TTA GTG TCC CAT 3' Exon 11 (4184ATCAA) 5' AAA ATA ATC AAG AAG AGC 3' 5' AGA AAA TAA GAA GAG CAA 3' Exon 13 (Arg 1443 ter) 5' AGG ACC TGC GAA ATC CAG 3' 5' AGG ACC TGT GAA ATC CAG 3' Exon 20 (5382 ins C) 5' GAG AAT CCC AGG ACA GAA 3' 5' GAG AAT CCC CAG GAC AGA 3' C. PTT Primers for Exon 11 (HUS14680 nucleotide designations) Forward Reverse

PTTA .792-813 2127-2105 PTTB .1931-1954 3021-2999

PTC ...... PTTC.2916-29372 17e -9o iR exonlilPi Ra

D. SSCP Primers fot 3'-UTR Forward Reverse c12. 5'-TGCCTGGACAGAGGACAATG-3' 5'-CGGTCTTCAGGAAAAGTCC-3' c13 .5'-AGCTCCTCTCACTCTTCAGTCCTTC-3' 5'-AGGTCGGGATTCGGTTGTTGTC-3' c14 .5'-CATTTAAACGCCACCAATTG-3' 5'-GGGTCCAAAGTTCAAAGG-3'

a From Friedman et al. (1994). sisted of the T7 promoter sequence and a consensus D17S1325. Linkage to BRCA2 was tested by genotyping translation initiation sequence (5'-GGA TCC TAA TAC D13S289, D13S171, D13S260, and D13S267 (Wooster GAC TCA CTA TAG GAA CAG ACC ACC ATG-3') et al. 1994). Markers were typed in the manner de- immediately preceding the BRCA1 nucleotides desig- scribed by Anderson et al. (1993). nated according to GenBank sequence HSU14680. The results are given in table 1. Library Screening PCR product (0.5-1 jig) was added to the TnT/T7 A BRCA1 clone containing a portion of the 3'-UTR coupled rabbit reticulocyte lysate system (Promega) ei- was isolated from a placental poly A+ cDNA library ther directly or after purification (Millipore Ultrafree provided by Jeff Boyd (Department of Obstetrics and MC-100 filters). The synthesized 35[S]-Met-labeled pro- Gynecology, University of Pennsylvania School of Medi- teins were seperated by SDS-PAGE (10%-15% acryl- cine) by screening with BRCA1-specific PCR products amide). Autoradiographic exposure of dried gels ranged amplified from lymphoblast cDNA (Friedman et al. from 18 to 48 h. 1994) and radioactively labeled with the Multiprime labeling system (Amersham). Hybridization conditions, Genotyping Markers in the BRCA I and BRCA2 Regions clone isolation, and PCR amplification of the cDNA Linkage to BRCA1 was tested by genotyping insert have been described elsewhere (Friedman et al. D17S1185, D17S1320, D17S1321, D17S855, 1995). SSCP primers for the 3'-UTR (c12-c14) are D17S1322, D17S1323, D17S1327, D17S1326, and listed in table 1. Friedman et al.: Expressivity of Germ-Line BRCA1 Mutations 1287 Human Subjects two sporadic breast cancers, led to a negative lod score This study was approved by the University of Califor- (-1.68) for linkage of breast cancer to 17q21. In con- nia at Berkeley Committee for the Protection of Human trast, in family 2 (pedigree in Hall et al. 1990), all five Subjects. women carrying 4184delTCAA developed breast cancer between ages 23 and 44 years, one developed ovarian Results cancer, as well, and the male carrying 4184delTCAA developed prostatic cancer. Variable Expressivity and Haplotype Sharing of 185deLAG and Five other small deletions or insertions were detected Other Recurrent BRCA I Mutations by SSCP and/or hybridization to ASOs (table 2). Two The entire coding region of BRCA1 was screened from deletions are novel: 230delAA in exon 3 of family 96 probands of 37 families with four or more cases of either and 1048delA in exon 11 of family 34. 230delAA is breast or breast and ovarian cancer (table 2). The most fully penetrant in family 96 and is associated with both frequent mutation was 185delAG in exon 2, which oc- early-onset and later-onset breast cancer (ages 39, 45, curred in five families: 8B, 28, 117, 139 ,and 144 (fig. 56, 60, and 65 years at diagnosis) and ovarian cancer 1). All five families are of Ashkenazi Jewish ancestry and (ages 41 and 42 years at diagnosis). are the only Jewish families in our series with BRCA1 Three other small insertions or deletions were ob- mutations thus far identified. These families share an served previously: 188delll in exon 2 of family 65, extensive portion of the linked BRCA1 chromosome, 2800delAA in exon 11 of family 109, and 5382insC in with allelic identity detected for eight polymorphic exon 20 of family 95. Family 65 is noteworthy because markers spanning -850 kb (table 3). A single exception only two of the five breast cancer patients in the family in family 8B at D17S1327 may reflect a new mutation inherited 188delll (fig. 3), leading to a negative lod at this dinucleotide repeat. The linked haplotype appears score (-0.68) for linkage of breast cancer to 17q21. the same as the partial haplotype reported in four Cana- 1 88dell1 was described elsewhere in a similar large fam- dian families with 185delAG (Simard et al. 1994), which ily with both breast and ovarian cancer (Miki et al. indicates a common origin for the mutation. 1994). Women carrying 185delAG in these families express 2800delAA was previously reported from family 1 of a variety of phenotypes, from early-onset bilateral breast our series (Friedman et al. 1995). The entire 2800delAA- cancer (family 139) and ovarian cancer (families 144 linked haplotype is identical in families 1 and 109 (table and 139) to later-onset breast cancer (age 77 years, fam- 3), which indicates a recent common ancestor, although ily 144; age 62 years, family 117). Nevertheless, pene- the families are not aware of any relationship. This mu- trance of this mutation in known carriers in these five tation has also been reported in two Scottish families high-risk families combined is 38% by age 40 years, who appear to share (with each other) identical alleles 69% by age 50 years, and 85% by age 65 years, close at >4 Mb spanning BRCA1, as indicated by reported to earlier cumulative risk estimates based on segregation homozygosity of THRA1, D17S188, and three interven- analysis of a population-based series of families (New- ing markers in an individual apparently homozygous for man et al. 1988) and evaluation of high-risk families 2800delAA (D. M. Black, unpublished data, cited in (Easton et al. 1993). In family 28, two sporadic cases Boyd et al. 1995). Genotypes of intragenic markers are of early-onset breast cancer effectively obscured linkage not available for the Scottish families, so it is not possible to BRCA1 (lod score = -1.66). to determine whether families 1, 109, and the Scottish 41 84deITCAA in exon 11 was detected in family 1OB families all share 2800delAA by descent. (fig. 2) and previously reported in family 2 (Friedman 5832insC has appeared only in family 95 in our series et al. 1994). Alleles at intragenic and flanking markers but has been frequently reported (Simard et al. 1995; on the 4184delTCAA haplotype differ for families 10B Shattuck-Eidens et al. 1995). Family 95 and the pre- and 2, indicating that the mutation arose independently. viously reported families share the same 5832insC- Family 2 originates from Norway and family 10B from linked haplotype (table 3). 5832insC was detected only England and Germany. 4184delTCAA has been re- by hybridization to the ASO, not by SSCP of either ported in several other families (Shattuck-Eidens et al. gDNA or cDNA. 1995); it will be interesting to determine how frequently Nonsense mutation C2457T was identified in family this mutation has occurred. 7 by using the PTT. Family 7 is American, of Dutch Phenotypes associated with 4184delTCAA in family ancestry. The identical mutation was also detected by 10B varied considerably: two carriers developed early- the PTT in two Dutch families in the Netherlands (Hog- onset breast cancer (bilaterally at ages 38 and 49 years ervorst et al. 1995), so these mutations may also have and unilaterally at age 46 years), another carrier devel- a common ancestor. oped breast cancer at age 78 years, and two other carri- The missense mutation Cys6lGly in the RING finger ers remained cancer-free through ages 73 and 81 years. has been observed in multiple families, including fami- In family 10B, this variation in expressivity, as well as lies 4 and 84 in our series (Friedman et al. 1994). It is Table 2

Germ-Line Mutations in BRCAI and Linkage to BRCAI and BRCA2 in Families with Four or More Breast or Ovarian Cancers

TOTAL CANCERSa LOD SCOREb GERM-LINE BRCA1 MUTATION

FAMILY F Br Ov FT M Br Pr BRCA1 BRCA2 Exon Type Mutation' Codon Predicted Effect

8B ...... 7 2 ... - 2 FS 185 del AG 23 Truncation aa 39 144 ...... 6 3 ... nt nt 2 FS 185 del AG 23 Truncation aa 39 139 ...... 2 4 ... nt nt 2 FS 185 del AG 23 Truncation aa 39 _ 28 ...... 5 ...... 2 FS 185 del AG 23 Truncation aa 39

117 ...... 4 ...... nt 2 FS 185 del AG 23 Truncation aa 39 _ 65 ...... 5 ...... 2 FS 188 del 11 bp 24 Truncation aa 36 5 ...... 9 1 ... + _ 3 SP Exon 3 deleted (54 bp)- truncation aa 27 96 ...... 5 2 ... nt nt 3 FS 230 del AA 33 Truncation aa 39 d _ ...... 10 ...... 5 MS T 300 G Cys 61 Gly Lose zinc-binding motif _ 84d ...... 5 ...... 5 MS T 300 G Cys 61 Gly Lose zinc-binding motif 98 ...... 7 4 ... - Intron 5 SP g Exon 5(+1) a splice donor 22-bp deletion- truncation aa 64

82 ...... 6 2 ...... + Intron 5 SP t Exon 6(-11) g splice acceptor 59-bp insertion-* truncation aa 81

34 ...... S 1 .. . . . + - 11 FS 1048 del A 310 Truncation aa 313

85 ...... 7 2 .. . . . + 11 NS/LOP C 1695 T Glu 526 Stop Loss of transcript

d3 ...... 3 3 .. . . . + 11 FS 2415 del AG 766 Truncation aa 766

7 ...... 7 . . . + 11 NS C 2457 T Gln 780 Stop Truncation aa 780 id...... 16 11 3 + 11 FS 2800 del AA 894 Truncation aa 901

109 ...... 3 2 ...... + nt 11 FS 2800 del AA 894 Truncation aa 901

1 02d ...... S 3 .. . . . - - ~~~11 FS 2863 del TC 915 Truncation aa 915 . . . 14dlOB ...... 6 - - ~~~11 MS G 3238 A Ser 1040 Asnf . . . 74d ...... 6 i + 11 NS C 3726 T Arg 1203 Stop Truncation aa 1203 . . . 1 2d ...... S + 11 FS 4184 del TCAA 1355 Truncation aa 1364 . . . 10B ...... 11 . - - 11 FS 4184 del TCAA 1355 Truncation aa 1364 . . . 81 ...... 11 . . . + - 13 NS/LOr C 4446 T Arg 1443 Stop Loss of transcript . . . 95 ...... 6 . . . + 20 FS 5382 ins C 1756 Truncation aa 1829 . . . 77d ...... 7 . . . + 24 FS 5677 ins A 1853 Truncation aa 1853 6...... 6 ...... + 94 S ...... + 17 ...... 4 1 .. . . . 99 ...... 6 1 + . . . 9...... 13 . . . 16 ...... 3 . . . 1 . . . 87 ...... S 33 ...... S ...... 19 ...... 9 ...... 90 ...... 9 ...... 22 8 ...... 1 91 ...... 7 ...... 11 ...... 7 ...... 13 ...... 7 . . .

20 ...... 6 ......

18 ...... S ......

10 ...... 23 ......

68 ......

67 ...... 4 . . .

72 ...... 4 ni ni ..

' Cancer sites: F Br = female breast; Ov = ovary; FT = fallopian tube; M Br = male breast; and Pr = prostate. b Lod scores: A plus sign (+) indicates >1.0; a minus sign (-) indicates < -1.0 (-0.7 for family 65); nt = not tested; and ni = not informative (too few cases available for genotyping). c Intronic nucleotides are designated by lowercase letters. d BRCA1 mutation previously reported by Friedman et al. (1994). 'LOT = loss of transcript. f Missense reported as a rare polymorphism by Castilla et al. (1994).

1288 > . 00 o -o

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Cl)>~~~~~~~C

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NIn~~~~~~~~~~~~~C 0~~~~~

N > kU-

a) 0 <0 0

co~~~O

m co ~~~~~~~~~~~~~~~~~I I0

1289 n ) t t r O O C T 0

N oC oC> C>000C) NNeq I-'t cqI rn

\-O N \ 0 N N NN CN N

No N (f

N N

------' - 0 d r- N : N 0 N

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0 c0 (A el . - r-- .-*r-t-1*0 .2

E 3 3 4, en} I- o- - 0r. c 0t u 0- 00 0 41 0t L L. ._ *_O *_ *_ *_ CU * 0 -, -0xC -0C C C C ' c CU CU 0o

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0 ON aq 00 pq -- rnI ,t o o t

0f IV . .~ (A z 0-00 "0N 0 . ~ . aL. . . . .- . .

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Family B1 0

Br3 6 79 74 Br3 5 98 95 Br48 84 wt wt V wt wt wt wt

Br57 73 69 Br46 Br38,49 wt V wt V V V

47 wt wt wt Figure 2 Variable expressivity of the 4184de1TCAA BRCA1 mutation. Phenotypes associated with this BRCA1 mutation (indicated by "V") range from early-onset bilateral breast cancer (Br), in individual IV:5, to late age of breast cancer onset (11:2), and, in the extreme, a female carrier is unaffected by the disease at age 81 years (III:11).

now clear that a partial haplotype of -400 kb is com- exon 5. Analysis of intronic sequence from gDNA sur- mon to these two families (table 3). Divergence of the rounding exon 5 revealed a G-+A substitution in the first markers flanking BRCA1 through recombination of the base of the conserved intronic splice donor site. This ancestral chromosome in families 4 and 84 indicates substitution activates a cryptic splice site within exon 5 that the original mutation is relatively old. Family 4 is at the closest upstream GT dinucleotide (fig. 4). The of German and Russian ancestry, whereas family 84 is junction of the truncated exon 5 with exon 6 encodes of Polish descent. In families 4 and 84, Cys6lGly is an immediate stop codon at amino acid 64. associated only with breast cancer, not ovarian cancer, A different splice variant in the proband of family 5, but ovarian cancer has been observed in at least one described elsewhere (Friedman et al. 1994), was simi- woman with this mutation (Takahashi et al. 1995). larly detectable only by analysis of cDNA. Mutant transcripts precisely splice around exon 3, and juxtaposition Novel Splicing Errors Leading to Protein Truncation of exon 2 with exon 5 creates a stop codon. (BRCA1 In family 98, a variant cDNA SSCP band was se- does not have an exon 4, because the genomic sequence quenced and revealed a deletion of the last 22 bp of so named [Miki et al. 1994] is an intronic Alu element.)

Family 65

wt wt wt wt

51 47 Br3 3 wt wt wt wt wt wt wt wt

Br3 3 Br3 2 V V V

18 V Figure 3 Genetic heterogeneity of breast cancer in family 65. Frameshift mutation 188delll occurs in individuals indicated by "V" (variant). Three sporadic cases of breast cancer in women wild type at this site, designated "wt," contributed to the negative lod score (-0.68) for linkage of breast cancer to D17S855 in this family. 1292 Am. J. Hum. Genet. 57:1284-1297, 1995

wt wt wt wt V wt V wt

v V V

* Exon 5 Intron 5 Exon 6

I I

GT CCT TTA TGT AAG AAT GAT ATA ACC AAA AG . ;AG CCT ACIA,

Wild Type Splice Junction

Exon 5 Intron 5 Exon 6

GT CCT TTA ,CCT ACA

Mutant Splice Junction Results in TGA: Premature Stop Codon Figure 4 Splice variant in family 98. BRCAI status and cancer designations are as in the previous figures. Prophylactic mastectomy is indicated as "pm." The wild-type GT consensus splice donor sequence is present at the 5' end of intron 5 (upper sequence). The substitution of the conserved G to A (marked with asterisk [-], lower sequence) activates a cryptic splice site, utilizing the next available GT dinucleotide 22 bases upstream within exon 5. The resultant aberrant splice junction forms an immediate stop codon. Nucleotides deleted from the mutant transcript are underlined.

It is now clear that this altered transcript is present in tive for two-allele polymorphisms within the BRCA1 cDNA from lymphoblastoid cells of all linked family coding sequence were identified, and heterozygosity of members but has not been observed in 24 lymphoblast gDNA was compared to heterozygosity of cDNA. In cDNA samples from unlinked family 5 members or un- families 81 and 85, the BRCA1 transcripts on the chro- related individuals. Sequence of the intron/exon bound- mosome linked to the disease was not expressed (fig Sa). aries surrounding exon 3 appears normal, as does the Exclusive expression of the wild-type transcript strongly splice donor site of exon 2 and the splice acceptor of suggested either that mutant BRCA1 was not tran- exon 5. The genomic alteration causing this aberrant scribed or that the mutant transcript was unstable. splice variant remains unknown. Analysis of 1.3 kb of the putative promoter region (Brown et al. 1994) in families 81 and 85 failed to reveal Nonsense Mutations Leading to Loss of Mutant Transcript any variants. This portion of the promoter was also For families with breast cancer linked to 17q21 but analyzed by SSCP in 11 other 17q21-linked breast-ovar- without known BRCA1 mutations, individuals informa- ian cancer families without detectable coding sequence Friedman et al.: Expressivity of Germ-Line BRCA1 Mutations 1293 a

gDNA cDNA 1 2 3 4 5 1 2 3 4 5

gDNA 1 gDNA 2 cDNA 1 G A T C G ATC G ATC

Figure 5 Loss of transcript mutations. In a, SSCP analysis of gDNA shows that probands from family 81 (lane 1) and from family 85 (lane 5) are heterozygous for an informative polymorphism in exon 11. However, only the wild-type SSCP band is detectable in cDNA from these same individuals, indicating loss of the mutant transcript. Lanes 2-4 are heterozygous controls. In b, the C4446T (Argl443ter) substitution in exon 13 is heterozygous in gDNA from the family 81 proband (gDNA1). However, only the wild-type sequence is observed from the cDNA of this same individual (cDNA1). gDNA2 is wild-type genomic sequence from a control individual.

mutations or informative polymorphisms: no variants thus confirming our previous observation of mutant were detected. Nor were any variants detected in 665 BRCA1 transcript loss (fig. Sb). bp of the 3'-UTR by using SSCP primers to amplify Family 85 was independently ascertained by our gDNA samples. group and by the International Agency for Research on Nonsense mutations were soon revealed in both fam- Cancer (IARC) (Torchard et al. 1994). In parallel with ily 81 and family 85 by other investigators. Family 81 our observation of the loss of the mutant BRCA1 tran- was independently ascertained by the Berkeley group script, the nonsense mutation C1695T was identified in (Bowcock et al. 1993) and by the Cancer Research Cam- exon 11 by 0. Serova (personal communication). The paign (CRC) Human Cancer Genetics Research Group, PTT detects C1695T. However, the biologically im- Cambridge (family 88 in Smith et al. 1993). In Cam- portant consequence of BRCA1 transcript loss would bridge family 88, a nonsense mutation at codon 1443 have been missed if PTT had been the only method used. was identified by heteroduplex analysis by Gayther et al. (in press). We verified C4446T in Berkeley family 81 Discussion by ASO analysis and direct sequencing of exon 13 from 1 85delAG in the Ashkenazi Jewish Population gDNA. Furthermore, we only detected the wild-type se- Five families in our series carry the 185delAG muta- quence in lymphoblast cDNA from these individuals, tion in exon 2. These families share a common ancestral 1294 Am. J. Hum. Genet. 57:1284-1297, 1995 haplotype spanning -850 kb (table 3) and the same ulation genetics of 5382insC are difficult to evaluate intragenic marker alleles as the four Canadian families because this mutation has been reported multiple times carrying this mutation (Simard et al. 1994). All five fami- and haplotype analysis is still underway for some families are of Ashkenazi descent, with ancestors immigrat- lies. In contrast, 4184delTCAA probably occurred inde- ing to America from central and eastern Europe. This pendently in families 2 and 10B, in which alleles are mutation has been identified in 18 Ashkenazi Jewish shared at only D17S1323 and D17S1327. These shared families thus far (Simard et al. 1994; Shattuck-Eidens et alleles are the most common at each marker so may not al. 1995; Takahashi et al. 1995; Tonin et al. 1995). reflect identity by descent. A more complete analysis of Because the specificity of 185delAG appears to be the the most common recurrent mutations is in progress (D. result of a founder effect in the Ashkenazi Jewish popu- Goldgar, unpublished data). lation, it is possible that inherited breast cancer repre- sents a higher proportion of breast cancer in this popula- Polymerase Slippage as a Possible Mutational Mechanism for tion than others. If so, the prevalence of 185delAG might - or 2-bp Deletions and Insertions partially explain the increased risk of breast cancer Of the 19 distinct mutations identified in 26 families among Jewish women (Helmrich et al. 1983). It thus from our series, 10 (59%) are protein-truncation muta- becomes important to determine the penetrance of tions caused by short insertions or deletions within the 185delAG among carriers not selected for family history coding sequence (table 2). Most 1- and 2-bp insertion of breast cancer and to identify genetic (Dietrich et al. and deletion mutations published thus far occur within 1993) or environmental factors that may influence vari- short stretches of mono- or dinucleotide repeats. For able expressivity or penetrance of this allele. example, 185delAG is flanked by TTAGAGTG, 2415delAG is flanked by CTGTAGAGAGTAG, Founder Effects of Recurrent Mutations 2800delAA is flanked by AAAGAAACAAA, and On the basis of the first published survey of BRCA1 5382insC is flanked by GAATCCCCA. These mutations mutations (Shattuck-Eidens et al. 1995), 30% of muta- suggest polymerase slippage as a possible mutational tions have been observed more than once. Five of these mechanism (Streisinger et al. 1966; Kunkel 1986; Tautz 11 recurrent mutations have appeared in families in our 1989; Schlotterer and Tautz 1992). The mutation series (table 3). Comparison of haplotypes among fami- 3875delGTCT may also occur by this mechanism, be- lies carrying the same mutation can distinguish whether cause it removes a tandemly repeated tetranucleotide frequently occurring alleles originated from a single an- (Castilla et al. 1994). cient mutational event, a single more recent mutational event, or arose independently more than once. Single Nonsense Mutations Explain Two "Inferred Regulatory" events will be reflected in shared alleles at polymor- Mutations in BRCA I phisms flanking the mutation, with more recent muta- The first BRCA1 mutations (Miki et al. 1994) in- tions sharing alleles over a longer region, because fewer cluded one "inferred regulatory" mutation, so named recombination events would have occurred to disrupt because of the apparent absence of BRCA1 mRNA from the haplotype. Of course, the association between allele the disease-linked allele. The genomic source of this mu- sharing and time of mutation is also influenced by size tation apparently remains unknown. Families 81 and 85 and endogamy of the population in which the mutation from our series yielded the same observation; only the occurred and by mutation rate at the marker loci and wild-type BRCA1 allele was expressed. Hence, mutant at BRCA1. BRCA1 was not transcribed at all, or the mutant tran- The most ancient mutation appearing in families in script was unstable. Mutation of the promoter sequence our series is probably the RING finger missense muta- could lead to transcription failure, but no promoter mu- tion Cys61Gly. The two families in our series with this tations were found. Absence of BRCA1 transcript could mutation share alleles at the five markers most closely also result from mRNA destabilizing mutations. One flanking BRCA1, but differ at four more distant markers major determinant of rapid mRNA decay is the presence (table 3). These families are Polish and German/Russian. of AU-rich cis-acting elements located in 3'-UTRs (Roy The next-oldest mutation is probably 185delAG, for et al. 1992). Lability of transcripts containing these ele- which the five Ashkenazi Jewish families in our series ments appears to be modulated through sequence-spe- share alleles at eight of the nine flanking markers (with cific binding of cytoplasmic factors (Amara et al. 1993; the exception of the D17S1327 mutation in family 8B) Henics et al. 1994; Wang et al. 1995). Introduction of and carry one of two alleles at the most distant marker, an mRNA-destabilizing sequence into the BRCA1 3'- D17S1185. 2800delAA appears recent, because the two UTR through a fortuitous mutation could result in rapid English and "western European" families with this mu- degradation of the mutant transcripts. However, no 3'- tation share alleles at all flanking markers. 5382insC UTR variants were observed, either. also appears recent, on the basis of family 95 and those Another possible mechanism leading to transcript in- reported from Canada (Simard et al. 1994), but the pop- stability is nonsense-mediated mRNA degradation (Los- Friedman et al.: Expressivity of Germ-Line BRCA1 Mutations 1295 son and Lacroute 1979). This "surveillance" pathway (Hogervorst et al. 1995). The relative ease of this PTT is well characterized in several species (Leeds et al. 1991; is offset by its inability to detect disease-associated mis- Pulak and Anderson 1993; Cui et al. 1995) and may sense alterations and the requirement for cDNA tem- play a role in the reduced mRNA expression in human plate, since RNA is often unavailable from patient sam- disease-related nonsense mutations (Dunn et al. 1989; ples. Nevertheless, the unusually large exon 11 of Lim et al. 1992; Menon and Neufeld 1994). The non- BRCA1 enables -60% of the BRCA1 coding sequence sense mutations revealed in families 81 and 85 strongly to be screened by PTT in three fragments amplified from suggest degradation of the mutant transcript by the non- gDNA. Three of four nonsense mutations in families in sense-mediated pathway. our series were not identified by SSCP of either gDNA Thus, neither "inferred regulatory" mutation is due or cDNA, but two were detectable by PTT. However, to an alteration in the putative promoter region. Rather, it is important that transcript instability caused by two the underlying genetic defect in both cases is a nonsense of these nonsense mutations would not be revealed by mutation. It is unclear why these particular nonsense PTT but was only detected by comparison of genomic mutations should lead to mRNA degradation whereas and cDNA for heterozygosity at polymorphisms in the others do not. The extent of mRNA destabilization in coding sequence. the nonsense-mediated degradation pathway appears to Analysis of cDNA by SSCP is critical for detection of depend on position, with nonsense mutations in the 3' some mutations. Mutations in five families in our series portion of the transcript having a reduced effect or no affect splicing (families 5, 98, and 82) or expression or effect on mRNA stability, relative to rapid degradation stability of the BRCA1 transcript (families 85 and 81). of transcripts with early nonsense mutations (Losson These mutations were initially detected only by analysis and Lacroute 1979). However, other BRCA1 translation of cDNA. Together, these mutations represent 17% terminating mutations in the 5' portions of the gene, of all mutations identified in our series, which indicates and termination mutations between GluS26ter and that a significant proportion of BRCA1 mutations fall Argl443ter (e.g., Gln780ter in family 7 and Argl203ter into these categories. Patients not screened for these in family 74) do not lead to loss of the mutant transcript. types of mutations may receive false-negative results. Alternatively, it is possible, albeit unlikely, that a second Somatic point mutations leading to splice alterations BRCA1 mutation in family 81 or 85 abolishes transcrip- are prevalent in the p53 (Hartmann et al. 1995) and tion and that such a mutation lies in an unscreened part retinoblastoma (Dunn et al. 1989) tumor-suppressor of the promoter or enhancer. genes. It is therefore conceivable that the puzzling pau- Detection of the nonsense mutation in family 85 also city of inactivating somatic BRCA1 mutations in breast completes the story of coinheritance of breast and ovar- and ovarian tumors (Futreal et al. 1994; Hosking et al. ian cancer and palmoplantar keratoderma in this French 1995; Merajver et al. 1995; Takahashi et al. 1995) re- family (Blanchet-Bardon et al. 1987). Cosegregation of flects a failure of current mutation-screening methods these phenotypes has proved to be a phantom example to detect alterations in the promoter, regulatory regions, of a contiguous-gene syndrome. The mutation causing and intronic sequences affecting RNA expression and PPK was found in the keratin 9 gene proximal to BRCA1 processing. This possibility is illustrated by the absence (Torchard et al. 1994). In parallel with our observation of a detectable variant in either the coding sequence or of the loss of the mutant BRCA1 transcript, the nonsense the intron/exon boundaries of the splicing mutation in mutation C1695T was identified in exon 11 (0. Serova, family 5. Similarly, the specific "inferred regulatory" personal communication). Independent mutations in mutation causing loss of BRCA1 expression in family keratin 9 and in BRCA1 occurred by chance on the same 2035 (Miki et al. 1994) has yet to be defined. The recent ancestral chromosome 17 in this family. observation that expression of BRCA mRNA is frequently down-regulated in sporadic breast carcinomas Advantages and Limitations of Various Mutation-Detection (Thompson et al. 1995) further indicates that somatic Methods inactivation of BRCA1 may occur by mechanisms other The simplicity and efficiency of SSCP makes this mu- than mutation of the BRCA1 coding sequence. tation-detection method appealing for rapid analysis. However, analysis of other genes suggested that only Linkage versus Inherited Mutation: Implications for Counseling 80% of single-bp substitutions were detected by this Among 41 families in our series with four or more technique (Vidal-Puig et al. 1994). The success rate for cases of breast or breast and ovarian cancer who were insertions and deletions is higher but not 100% (e.g., tested for linkage to BRCA1 and BRCA2, 19 families 5382insC is missed in our hands). Alternatively, the PTT had lod scores >1.0 for BRCA1, and only 3 had lod reveals premature protein-translation mutations (Roest scores >1.0 for BRCA2, a higher proportion of BRCA1 et al. 1993). PTT has been successfully applied to detec- families than identified in most series. Seven families with tion of mutations in DMD (Roest et al. 1993), APC negative BRCA1 lod scores nevertheless carry inherited (van der Luijt et al. 1994), and, most recently, BRCA1 mutations in this gene: lod scores were negative because 1296 Am. J. Hum. Genet. 57:1284-1297, 199S women without inherited mutations also developed Dietrich W. Lander E, Smith J, Moser A, Gould K, Luongo breast cancer. The frequent occurrence of BRCA1 muta- C, Borenstein N, et al (1993) Genetic identification of Mom- tions in families with negative lod scores has important 1, a major modifier locus affecting Min-induced intestinal implications for genetic counseling. Families cannot be neoplasia in the mouse. Cell 75:631-639 assumed to be free of mutations at BRCA1 simply be- Dunn JM, Phillips RA, Zhu X, Becker A, Gallie BL (1989) Mutations in the RB1 gene and their effects on transcription. cause the lod score with 17q21 markers is negative. Mol Cell Biol 9:4596-4604 Easton D, Ford D, Peto J (1993) Inherited susceptibility to breast cancer. Cancer Surv 18:95-113 Acknowledgments Easton DF, Bishop DT, Ford D, Crockford GP, Breast Cancer We thank J. Boyd for providing the polyA' placental cDNA Linkage Consortium (1993) Genetic linkage analysis in fa- library. We gratefully acknowledge B. A. J. Ponder and S. milial breast and ovarian cancer: results from 214 families. Gayther, at the CRC Human Cancer Genetics Research Am J Hum Genet 52:678-701 Group, and G. Lenoir, J. Fenuteun, and 0. Serova, at the Ford D, Easton DF, Bishop DT, Narod SA, Goldgar DE, Breast International Agency for Research on Cancer, for sharing data Cancer Linkage Consortium (1994) Risks of cancer in before publication. This work was supported by National In- BRCA1-mutation carriers. Lancet 343:692-695 stitutes of Health (NIH) grant RO1 CA27632. L.S.F. is a Ko- Friedman LS Ostermeyer EA, Lynch ED, Welcsh P. Szabo CI, men Postdoctoral Fellow and C.I.S. is supported by NIH Post- Meza JE, Anderson LA, et al (1995) Twenty-two Genes doctoral National Research Service Award F32 CA66293. 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