Global sequence diversity of BRCA2: analysis of 71 breast cancer families and 95 control individuals of worldwide populations [published erratum appears in Hum Mol Genet 1999 Apr;8(4):717-9]

doi:10.1093/hmg/8.3.413

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Human Molecular Genetics Pages 413-423

Global sequence diversity of BRCA2: analysis of 71 breast cancer families and 95 control individuals of worldwide populations
Introduction
Results
   General sequence variation
   Disease-associated BRCA2 mutations
   Sequence variants in HBC/HBOC families
   Sequence variants detected in the control population
   Frequency and global distribution of continent- and population-specific sequence variants
Discussion
Materials And Methods
   Breast and breast-ovarian cancer families
   Control individuals
   DNA extraction and PCR conditions
   DHPLC
   Direct DNA sequencing
   Cloning of BRCA2 mutations with the TA cloning kit
Acknowledgements
Abbreviations
References

Global sequence diversity of BRCA2: analysis of 71 breast cancer families and 95 control individuals of worldwide populations

Teresa M. U. Wagner¹, Kora Hirtenlehner¹, Peidong Shen⁵, Regina Moeslinger¹, Daniela Muhr¹, Elisabeth Fleischmann^1,2, Hans Concin^7,[dagger], Walter Doeller^8,[dagger], Anton Haid^9,[dagger], Alois Hermann Lang^10,[dagger], Peter Mayer^11,[dagger], Edgar Petru^12,[dagger], Erich Ropp^13,[dagger], Gudrun Langbauer¹, Ernst Kubista^1,2, Otto Scheiner³, Peter Underhill⁵, Joanna Mountain⁶, Michael Stierer¹⁴, Cristoph Zielinski^2,4 and Peter Oefner^5,*

¹Division of Senology, ²Ludwig Boltzmann Institute for Clinical Experimental Oncology, ³Division of Applied Experimental Pathology, Department of General and Experimental Pathology, ⁴Chair for Medical Experimental Oncology, Division of Oncology, Department of Medicine I, University of Vienna, 1090 Vienna, Austria, ⁵Departments of Genetics and Biochemistry and ⁶Department of Anthropological Sciences, Stanford University, Stanford, CA 94305, USA, ⁷Department of Obstetrics and Gynecology, 6900 Bregenz, Austria, ⁸Department of Surgery, 9400 Wolfsberg, Austria, Departments of ⁹Surgery and ¹⁰Internal Medicine, 6800 Feldkirch, Austria, ¹¹Department of Oncology, 5020 Salzburg, Austria, ¹²Department of Obstetrics and Gynecology, University of Graz, 8010 Graz, Austria, ¹³Specialist for Obstetrics and Gynecology, 9020 Klagenfurt, Austria and ¹⁴Department of Surgery, Hanusch Hospital, 1140 Vienna, Austria

Received August 12, 1998; Revised and Accepted November 30, 1998

The aim of this study was to evaluate the prevalence of simple sequence variation in the BRCA2 gene. To this end, 71 breast and breast-ovarian cancer (HBC/HBOC) families along with 95 control individuals from a wide range of ethnicities were analyzed by means of denaturing high-performance liquid chromatography (DHPLC) and direct sequence analysis. In the coding (10 257 bp) and non-coding (2799 bp) sequences of BRCA2, 82 sequence variants were identified. Three different, apparently disease-associated BRCA2 mutations were found in six HBC/HBOC families (8%): two splice site mutations in introns 5 and 21, and one frameshift mutation in exon 11. In the coding region, 53 simple sequence variants were found: 35 missense mutations, one 2 bp deletion (CT) resulting in a stop at codon 3364, one nonsense mutation with a stop at codon 3326, one deletion of a complete codon (AAA) resulting in the loss of leucine, and 15 silent mutations. In the non-coding region, 26 polymorphisms were detected. Of the 79 sequence variants that were not obviously disease-associated, eight were detected only in HBC/HBOC families. The remaining 71 variants were identified in both HBC/HBOC families and control individuals. Sixty three sequence variants (80%) were specific for a continent. Forty two percent (33 out of 79) of the sequence variants were detected exclusively in Africa, though only 13% of the 332 chromosomes screened were of African origin. Our data indicate that, in BRCA2, simple sequence variation is frequent [in the coding region 1 in 194 bp ([thetas] = 2.2 × 10^-4), and in the non-coding region 1 in 108 bp ([thetas] = 4.4 × 10^-4), respectively].

INTRODUCTION

Since the identification of the BRCA2 gene in 1995 (1,2), >468 sequence variants have been detected (3). These sequence variants were identified predominantly in individuals from breast and breast-ovarian cancer (HBC/HBOC) families and primary breast cancer (BC)/ovarian cancer (OC) cases. Approximately 68% were protein truncating mutations, and 32% were polymorphisms or missense mutations of unknown biological relevance, i.e. unclassified variants. Although truncating mutations may be assumed to cause disease, pathogenicity involving unclassified variants is more equivocal because of insufficient information concerning both protein function and genetic variation. Knowledge with regard to potential functional domains as well as the overall function of BRCA2 itself remains limited (4-8). Comparative information regarding BRCA2 genetic diversity in various populations is also limited. The temptation to implicate a missense mutation that occurs at low frequency as a potential functional lesion must be tempered by the possibility that the variant may exist more frequently in another population and thus be considered a mere polymorphism. Schrijver et al. reported such a case for the fibrillin-1 gene (FBN1) (9). Initially, a missense mutation was detected predominantly in Caucasian probands with Marfan’s syndrome (MFS) and consequently classified as likely to be disease associated. Subsequent screening of 416 control individuals from various world populations permitted the classification of the allele as a polymorphism, rather than a mutation, prevalent in Asian and Latin American populations.

The aim of this study was to produce a comprehensive summary of BRCA2 sequence variants in representatives of diverse global populations. DNA from 40 populations was analyzed in conjunction with that of 71 Austrian HBC/HBOC families. Analysis was accomplished by denaturing high-performance liquid chromatography (DHPLC), a novel automated heteroduplex detection method with a proven sensitivity and specificity of >95% (10,11).

RESULTS

General sequence variation

The coding region and intron-exon boundaries (13 056 bp) of BRCA2 of 95 control individuals and 71 HBC/HBOC families were analyzed by means of DHPLC, with heteroduplexes subsequently being identified through direct sequence analysis. A total of 82 different simple sequence variants were detected (66% transitions, 29% transversions and 5% deletions). As of September 1998, only 31 of those variants had been reported previously (Table 1). In the coding regions, 53 out of 10 257 nucleotides (1 in 194) and in the non-coding regions 26 out of 2799 nucleotides (1 in 108) varied.

Table 1. Seventy nine germline sequence variations in BRCA2 found in 332 chromosomes of worldwide populations

Exon/intron Nucleotide^a Codon Nucleotide change Amino acid change^b Global heterozygosity Geographic distribution^c BIC (as of 9/98) citations

2 203 - G->A non-coding 0.260 Eurasia/Oceania BIC,2,12-16

217 - T->G non-coding 0.006 Oceania -

4 550 108 A->C Asn->His (nc) 0.006 Africa -

10 1093 289 A->C Asn->His (nc) 0.120 Global BIC,15,16

1207 327 A->G Lys->Glu (nc) 0.012 Austria -

1342 372 A/C Asn/His (nc) 0.420 Global BIC,2,15-18

1515 429 A->G silent 0.006 Pakistan -

1593 455 A->G silent 0.110 Global BIC,15,18

1972 582 A->C Thr->Pro (nc) 0.006 Central Asia -

11 2347 707 G->T Asp->Tyr (c) 0.006 Africa -

2457 743 T->C silent 0.130 Global 2,15

2578 784 A->G Met->Val (c) 0.006 Asia BIC

2778 850 A->G silent 0.006 Asia -

2885 886 A->T Asn->Ile (nc) 0.006 Africa -

3031 935 G->A Asp->Asn (nc) 0.006 Austria BIC

3111 961 G->A silent 0.006 Austria -

3199 991 A->G Asn->Asp (nc) 0.150 Global 2,15-17

3624 1132 A->G silent 0.340 Global -

4035 1269 T->C silent 0.230 Global BIC,2,15

4086 1286 delAAA del Leu 0.006 Africa -

4097 1290 G->A Cys->Tyr (c) 0.006 Africa -

4194 1322 C->T silent 0.006 Africa -

4469 1414 C->T^d Thr->Met (nc) 0.012 Africa -

4486 1420 G->T Asp->Tyr (c) 0.030 Austria BIC

4765 1513 G->A Asp->Asn (nc) 0.006 Australia -

4791 1521 G->A silent 0.018 Africa -

5007 1593 A->C Glu->Asp (c) 0.006 Pakistan -

5427 1733 C->T^d silent 0.036 Europe/Asia -

5540 1771 G->A Gly->Asp (nc) 0.006 America BIC

5642 1805 A->G Asn->Ser (c) 0.006 Africa -

5868 1880 T->G Asn->Lys (nc) 0.012 Africa BIC,15,17

5932 1902 G->A Asp->Asn (nc) 0.012 Africa BIC

5972 1915 C->T^d Thr->Met (nc) 0.048 Austria/Oceania BIC,13,15-18

6328 2034 C->T^d Arg->Cys (nc) 0.006 Austria BIC,15-17

6448 3074 C->A His->Asn (nc) 0.006 Africa BIC

6640 2138 G->T Val->Phe (c) 0.006 Africa 13

6741 2171 C->G silent 0.018 Africa -

7049 2274 G->T Gly->Val (nc) 0.006 Austria 15,17,19

14 7470 2414 A->G silent 0.250 Global except America BIC,2,12,14-16,18

7625 2466 C->T Ala->Val (c) 0.018 Africa BIC,17,18

15 7666 2480 T->G Leu->Val (c) 0.006 Africa -

7697 2490 T->C Tyr->His (nc) 0.018 America BIC

7772 3015 C->T Thr->Ile (nc) 0.006 Austria BIC

18 8320 2697 G->A^d Arg->His (c) 0.006 Africa -

22 9058 2944 A->T Ile->Phe (c) 0.006 Africa BIC

9079 2951 G->A Ala->Thr (nc) 0.006 America BIC

9133 2969 G->A^d Val->Met (c) 0.006 Asia -

24 9465 3079 T->C silent 0.006 Africa -

25 9642 3138 A->G silent 0.006 Africa -

27 9998 3257 A->G Lys->Arg (c) 0.006 Africa -

10 056 3276 A->T Arg->Ser (nc) 0.006 Africa -

10 152 3308 C->T^d silent 0.006 Africa BIC

10 204 3326 A->T 3326 STOP 0.012 Austria/Sardinia BIC,15,17,20,21

10 319 3364 delCT 3366 STOP 0.006 America -

10 462 3412 A->G Ile->Val (c) 0.024 Africa/Austria/America BIC,15,17,20

2 295+62 T->G non-coding 0.006 Asia 14

3 545-22 C->T non-coding 0.006 Africa -

4 654-47 G->T non-coding 0.006 Austria -

6 744+18 T->C non-coding 0.006 Asia -

745-4 C->G non-coding 0.006 Africa -

8 909+56 C->T non-coding 0.024 Africa/Asia/Oceania -

11 7069+74 T->A non-coding 0.006 Africa -

7069+80 delTTAA non-coding 0.340 Global except America BIC

13 7236-62 A->G non-coding 0.006 Austria -

14 7663+53 C->T^d non-coding 0.130 Global BIC

15 7830-27 T->A non-coding 0.006 Asia -

16 8034-14 T->C non-coding 0.048 Austria BIC

17 8204+12 G->A non-coding 0.012 Africa -

18 8560-66 T->C non-coding 0.006 Africa -

8560-64 A->G non-coding 0.018 Africa -

19 8715+47 C->T non-coding 0.018 Africa -

21 8983-79 G->A non-coding 0.006 Asia -

8983-66 T->C non-coding 0.220 Global -

22 9181+16 C->T non-coding 0.006 Austria -

9181+98 T->C non-coding 0.012 Austria -

25 9730-71 G->T non-coding 0.006 Africa -

26 9876+54 G->A^d non-coding 0.012 Africa -

9876+84 G->A non-coding 0.006 Asia -

9876+106 delT non-coding 0.006 Africa -

^aNucleotide position of variant in reference to cDNA sequence (ref. 2, accession no. U43746).
^bc, conservative change; nc, non-conservative change.
^cGeographic location where variants were detected in the present study; this does not imply that variants may only be found there.
^dSubstitutions associated with methylatable CpG/CpNpG motifs.

Disease-associated BRCA2 mutations

One frameshift mutation with a deletion of 5 bp in exon 11 and two splice site mutations in introns 5 and 21, respectively, were found in six out of 71 HBC/HBOC families (8%). All three are novel mutations and are likely to be disease associated. Pedigrees of the six HBC/HBOC families are shown in Figure 1.

Figure 1. Pedigrees of the six HBC/HBOC families with apparently disease-associated BRCA2 mutations.

The frameshift mutation in exon 11 at nucleotide 6633 delCTTAA results in a stop codon at 2137. In the family with this mutation, three cancer cases could be identified: two BC cases at the age of 35 (carrier of the mutation) and 50 years (not available for testing), and one OC case at the age of 68 years (deceased, no DNA available for testing). The splice site mutation in intron 5 showed an A to G change in the third base pair of the conserved intronic splice donor site. This sequence variant was detected once in 332 chromosomes in a family with three cases of BC (at the ages of 42, 57 and 62 years, all carriers) and one case of BC and OC (at the age of 46 years, deceased, no DNA available for testing). Sequence analysis of the two non-diseased members of the third generation (aged 39 and 30 years) revealed that one inherited the sequence variant. The splice site mutation in intron 21 showed a G to A substitution in the first base of the conserved intronic splice acceptor site. The four families identified with this mutation were A3, G3, G4 and F47. A3 contained three cases of BC at the ages of 38, 48 and 62 years. Only the two younger cases were found to be carriers of this sequence variant. The older case is a sporadic BC. G3 presented three cancer cases, two of which could be tested for the presence of the sequence variant: one case of male BC at the age of 45 years (deceased, no DNA available for testing), one female with BC (at the age of 47 years), melanoma (at the age of 50 years) and OC (at the age of 60 years), and one male with stomach cancer at the age of 88 years. G4 contained three cases of male BC [at the ages of 54 (refused to be tested), 60 and 76 years (deceased, no DNA available for testing)], three cases of female BC [at the ages of 45 (carrier), 54 and 68 years (the latter two are deceased and no DNA has been available for testing)] and one case of prostate cancer (65 years) and stomach cancer (60 years), respectively (both patients are deceased and no DNA has been available for testing). In F47, three BC cases were identified at the ages of 36, 39 and 42 years, with the two younger patients definitely carrying the mutation, while no DNA had been available for testing from the third deceased case. In all four families, the mutation segregated from one generation to the other, and all identified carriers were affected with cancer. Unfortunately, due to the greater number of deceased family members with insufficient pathological specimens for DNA extraction and the refusal of others to be tested because of fear of discrimination, penetrance of the mutations could not be assessed. There was at least one case of sporadic BC in one of the six families (A3).

To resolve the changes caused by these splice site mutations, cDNA was cloned. In three different cloning experiments, a total of 15 plasmids were sequenced. For the intron 5 mutation, four clones showed a complete loss of exon 5. For the intron 21 mutation, three clones showed a complete loss of exon 22 and two clones a loss of exon 22 plus the first 51 bp of exon 23. On the basis of these findings, we deduce that the mutation in the intronic splice donor site of intron 5 causes a complete loss of exon 5, and the mutation in the intronic splice acceptor site of intron 21 can cause both a complete loss of exon 22 and a loss of exon 22 plus the first 51 bp of exon 23.

Sequence variants in HBC/HBOC families

Eight sequence variants were detected only in 11 HBC/HBOC families: five missense mutations, one silent mutation, one nonsense mutation and two sequence variations in introns 4 and 13 (Table 2). Four missense mutations, K327E, D935N, R2034C and T3015I, resulted in non-conservative amino acid changes (Table 2). One family, F54, with two BC cases at the ages of 48 and 49 years, presented with three sequence variants that were only detected in HBC/HBOC families: D935N, G961G and a non-coding sequence change in intron 13.

Table 2. Sequence variants of unknown functional significance detected in HBC/HBOC families only

Exon/intron Nucleotide (codon) Nucleotide change Effect on coding sequence Amino acid change Family No. of BC/OC (age of onset in years)

10 1207 (327) A->G Lys->Glu non-conservative F40 6 BC (34, 37, 42, 42, 46, 47)

M16 2 BC (29, 49)

11 3031 (935) G->A Asp->Asn non-conservative F54 2 BC (48, 49)

11 3111 (961) G->A silent, Gln silent F54 2 BC (48, 49)

11 4486 (1420) G->T Asp->Tyr conservative F35 3 BC (38, 49, 72)

F50 3 BC (39, 52, 60)

M26 3 BC (36, 50, 55 bilateral)

M28 2 BC (39, 47 bilateral)

M41 2 BC (33, 38)

11 6328 (2034) C->T Arg->Cys non-conservative M31 1 BC (29)

15 7772 (3015) C->T Thr->Ile non-conservative M2 2 BC (32, 48)

Intron 4 654-47 G->T non-coding F42 3 BC (43, 53, 59)

Intron 13 7236-62 A->G non-coding F54 2 BC (48, 49)

Table 3. Geographic distribution of observed heterozygosities for common BRCA2 variants

Continent
(no. of individuals) Nucleotide

203 1093 1342 1593 2457 3199 3624 4035 7470 7069+80 7663+53 8983-66

Africa (21) 0.000 0.143 0.190 0.048 0.143 0.238 0.143 0.286 0.190 0.190 0.190 0.381

Austrian controls (18) 0.389 0.167 0.500 0.111 0.111 0.111 0.444 0.167 0.333 0.500 0.111 0.167

Austrian HBOC (71) 0.343 0.029* 0.429 0.029 0.029 0.057 0.429 0.243 0.243 0.400 0.043 0.029*

Other Europeans (12) 0.333 0.167 0.583 0.250 0.250 0.250 0.333 0.333 0.250 0.333 0.250 0.500

Asia (31) 0.258 0.194 0.484 0.194 0.258 0.226 0.290 0.161 0.323 0.323 0.258 0.452

Oceania (7) 0.143 0.286 0.286 0.286 0.286 0.286 0.143 0.143 0.143 0.143 0.429 0.286

America (6) 0.000 0.333 0.500 0.333 0.333 0.333 0.000 0.333 0.000 0.000 0.333 0.167

Total (166) 0.267 0.121 0.424 0.109 0.133 0.152 0.333 0.230 0.248 0.339 0.152 0.218

*P [ap] 0.025.

Sequence variants detected in the control population

In 95 control individuals, a total of 71 different sequence variants were detected (Table 1). Forty six were found in the coding region of BRCA2: 30 missense mutations, one nonsense mutation 10 204 A->T, resulting in a stop at codon 3326, one deletion of 2 bp (CT) with a stop at codon 3364, one deletion of a complete codon (AAA) that results in the loss of leucine, and 14 silent mutations were identified. Of the 30 missense mutations, 17 resulted in non-conservative amino acid changes (Table 1). The non-conservative missense mutation H2074N is located in the eigthth BRC repeat and was detected once in an African control individual. Twenty four changes were identified in the non-coding region, including one deletion of 4 bp, one deletion of 1 bp (T), and two in the 5[prime]-untranslated region (UTR) in exon 2.

Frequency and global distribution of continent- and population-specific sequence variants

Twelve sequence variants were detected on at least three continents. The frequency of heterozygotes for these sequence variants ranged from 10.9 to 42.4%. Table 3 shows a comparison of observed heterozygosities for Africa, Europe, Asia, Oceania, America and both affected and unaffected Austrians. A general deficit of heterozygosity, that attains statistical significance (P [ap] 0.025) in nucleotide positions 1093 and 8983-66, respectively, has been observed in HBC/HBOC cases. This deficit of heterozygosity is most probably due to large genomic deletions that are missed by PCR-based mutation screening methods such as DHPLC, using genomic DNA as template. Sixty three sequence variants (80%) were specific for a continent (55 in both the control population and HBC/HBOC families, eight in HBC/HBOC families only). Fifty one sequence variants (65%) were detected only in specific ethnic groups, of which 47 occurred only once. On a regional level, 33 (42%) were detected only in the African control individuals who contributed 13% of the 332 chromosomes. Twelve sequence variants were specific to the Austrian population (eight were detected only in HBC/HBOC families), nine to the Asian, four to the American Indian, two to the Indian/Pakistani, and one each to the Australian and Oceanian populations. One sequence variant was detected only in the Austrian, African and American Indian populations, one only in the Asian, European and Australian populations, one only in the African, Asian and Oceanian populations, one only in the Austrian and Sardinian populations, one only in the Austrian (n = 7) and Australian (n = 1) populations, and one only in the European (n = 5) and Asian (n = 1, Crimean Tartar) populations. Only the latter two (nucleotides 5427 and 5972) were associated with methylatable CpG/CpNpG motifs. Since such motifs are thought to be mutational hotspots, recurrence of those two polymorphisms cannot be excluded.

The fraction of segregating sites for the coding region (1/194) is roughly half that of the non-coding region (1/108). Given the number of segregating sites, heterozygosity, or genetic diversity, is relatively low. As noted elsewhere, 59% of the sequence variants are only found in one out of 332 chromosomes. Our estimates of nucleotide diversity ([thetas]), the number of differences per site between any two randomly chosen sequences, are 22 ± 6.5 × 10^-5 for the coding region and 4.4 ± 1.8 × 10^-4 for the non-coding region. These estimates are based on the assumption that the human groups under consideration constitute, as a whole, a random mating population. To the extent that the human species is subdivided, these values are likely to be underestimates.

DISCUSSION

A disease-causing BRCA2 mutation was identified in 8% of the 71 HBC/HBOC families analyzed. Similar results were determined in 100 Finnish HBC/HBOC families (8% BRCA2 mutations) and in 106 Scandinavian families (11% BRCA2 mutations) (12,20). Though Schubert et al. identified 27% BRCA2 mutations in US families, it is important to note that these were families with at least four cases of BC or OC (13).

All 71 Austrian families had been analyzed previously for BRCA1 mutations (22, unpublished data) and six different BRCA1 mutations were detected. Twelve (17%) out of those 71 families were found to carry a disease-associated mutation in either BRCA gene. The probability of detecting such a mutation was clearly related to the family history, with mutations having been identified in three out of four HBOC families, and two out of three families with male BC. Among the 61 HBC families, 37 and 30% of which presented with two and three cases of BC, respectively, only seven (12%) carried a mutation in either BRCA gene. The latter observation is in accordance with a study of 980 HBC families by T. Bishop (11th Breast Cancer Linkage Consortium Meeting, Pisa, 1997), who was able to show that among the group of families with two cases of BC prior to the age of 50 years, the percentage of both BRCA1 and BRCA2 mutations amounts to ~8% each. In addition, three of the six families with BRCA2 mutations showed one case of OC (one within and two outside the OC cluster region) (23).

Two sequence variants that lead to a stop codon were identified in exon 27. The nonsense mutation 10204 A->T was identified previously with a frequency of 0.02% in a US control population and was not associated with an increase of susceptibility to BC and OC (21). The other variant was a not previously reported CT deletion with a stop at codon 3364 detected once in an American-Indian individual. As this deletion is even closer to the C-terminal end than the 10204 A->T change, it is unlikely to be implicated in disease, but this has not been tested.

Eight sequence variants were detected only in HBC/HBOC families. None were located inside the eight BRC repeats (24,25). Seven could be of functional relevance: four resulted in non-conservative amino acid changes, one in a conservative change and two were silent mutations. The non-conservative missense mutation K327E (nucleotide 1207) is locatedin one of the RAD51-binding domains (residues 982-1066 and 1139-1266) and might therefore interfere with the BRCA2-RAD51 interaction (8). The silent mutation S1733S was detected in four HBC/HBOC families (M24, M28, M37 and M47). In M47, the protein truncating mutation 6633del5 was identified. It seems unlikely, therefore, that S1733S is of clinical relevance. However, recently two examples of silent mutations inducing exon skipping in the fibrillin-1 gene and the calpain gene have been reported (26,27). These findings imply that all sequence variants specific to HBC/HBOC might, at the very least, be of potential, functional relevance, but further tests regarding their effect on RNA stability or splicing are required. One non-conservative missense mutation H2074N, that was identified once in an African control individual and was located in the eighth BRC repeat in exon 11, might also be of functional relevance: the BRC 1, 2, 3, 4, 7 and 8 motifs are highly conserved between species (human, monkey, rat and mouse) (17,28), and Chen et al. (7) demonstrated that BRC repeats are critical for RAD51 binding. Therefore, changes in these motifs might interfere with the binding of RAD51 and lead to a loss of function.

Of particular interest is the observation of a deficit of heterozygosity in the HBC/HBOC cases compared with a group of controls from the same population with no history of BC/OC. This deficit is most probably caused by one or more large genomic deletion that is missed by PCR-based mutation screening such as direct sequencing, DHPLC and the protein truncation test, which was performed on exons 10 and 11 (data not shown), using genomic DNA as a template. No such deficit of heterozygosity had been observed in the same individuals on BRCA1 (unpublished data), and Southern blot analysis did not yield any evidence for the existence of large BRCA1 genomic deletions in Austrian BC/OC patients. However, BRCA1 genomic deletions have been reported to be major founder mutations in Dutch BC patients (29). Southern blot analysis to be carried out on the Austrian HBC/HBOC cases will eventually prove whether large genomic deletions account for the observed deficit of heterozygosity in BRCA2 and are major founder mutations.

The analysis of control individuals from a wide variety of populations, for the purpose of identifying polymorphisms, has particular significance: among the 95 control individuals included in this study, a total of 71 different sequence variants were detected, of which 63 (80%) were specific for a continent and 51 (65%) were detected only in specific populations. The data regarding the prevalence of certain sequence variants in specific populations have a direct bearing on the prevention of inaccurate classification. The control populations commonly consist of unaffected individuals of the same ethnic background as the families. Consequently, a sequence variation with an extremely rare occurrence among the Caucasian population, though more frequent in, for example, the African population, will not turn up in a Caucasian control population either. Vehmanen et al. detected the conservative missense mutation I3412V in exon 27 only in two families but not in the control population (20). In this study, this missense mutation was detected in one out of 89 Austrians, two out of 21 Africans and one out of six American Indians, and can therefore be classified as a polymorphism. Takahashi et al. detected the conservative missense mutation A2466V only once in a patient with OC out of 260 chromosomes (18). In our population sample, it was detected in three out of 21 African control individuals.

In evaluating the sequence variants found in our control populations, those variants occurring with a frequency of >1% in both global and specific populations may be classified reliably as polymorphisms. However, even where only a single occurrence of a sequence variant is detected in the control individuals (frequency 0.6%), this may be considered a strong indication of polymorphism. Additional research into control populations is needed to clarify this issue.

Finally, it is interesting to note that our estimate of nucleotide diversity ([thetas]) of 4.4 × 10^-4 for the non-coding region of BRCA2 is very similar to that reported for 16 725 random human sequence-tagged sites (a total of 1 981 030 bp screened), which was estimated to be 4.58 × 10^-4 (30). The latter estimate was based on 2748 candidate single nucleotide polymorphisms detected in 14 European chromosomes. Given the specificity of oligonucleotide array-based sequencing of 85% (30), their estimate can be considered an overestimate. On the other hand, this may be compensated by the fact that a more ethnically diverse sample is expected to yield a somewhat greater value of nucleotide diversity. A preliminary survey of 31 individuals representing all major racial groups yielded a value of [thetas] that was ~30% larger (30). In contrast, a recent survey of a 9.7 kb region of the human lipoprotein lipase (LPL) gene, that included 998 bp of coding sequence, in 71 individuals of African-American, Finnish and European-American descent reported an estimate of 5.0 ± 5.0 × 10^-4 for the coding and 2.1 ± 1.0 × 10^-3 for the non-coding region of LPL (31). Of the 88 variable sites found, 87 of which were diallelic, 78 had relative allele frequencies >1%, and more than half of the variant sites exceeded a heterozygosity of 10%. In the present study, only 12 out of 79 variable sites (15%) had a heterozygosity >10%, and 46 (58%) of the variable sites were observed only once. Although the nucleotide diversity estimates for LPL are significantly higher than those for BRCA2, the relative numbers of varying sites in coding (1 in every 142 bp) and non-coding (1 in every 108 bp) regions are very similar to those reported here, namely 1 in every 194 bp for the coding and 1 in every 108 bp for the non-coding regions of BRCA2.

MATERIALS AND METHODS

Breast and breast-ovarian cancer families

All 71 families with BC and/or OC were identified through the genetic counseling program of the Division of Senology, University of Vienna. Selection criteria were: (i) two relatives once removed affected with either BC diagnosed before the age of 50 years, or OC diagnosed at any age; (ii) three or more affected relatives, once or twice removed, at least two of whom hadBC diagnosed before the age of 60 years; (iii) one case of BC diagnosed before the age of 35 years; or (iv) male and femaleBC occurring in the same lineage at any age.

Four families were HBOC families, three families presented with two OC cases (HOC), 61 were HBC families (including four single cases of BC before the age of 30 years), two families with male and female BC, and one family presented with male BC, stomach cancer and one female with BC and OC. All patients were from Austria (Central European origin) and none of the families showed evidence of being related when traced back a minimum of three generations. From all available family members, blood samples and, where feasible, tumor tissue samples were collected. Genomic DNA isolated from lymphocytes was analyzed from 71 family members (43% of 166 screened individuals). One affected individual per family was chosen for screening. Preferably, this was the family member where diagnosis had occurred at the earliest age. All subjects gave their written informed consent regarding their participation in the study.

Control individuals

Ninety five control individuals were screened for sequence variants in the BRCA2 gene. The populations surveyed were as follows. Africa (n = 21): five Biaka (Central African Republic Pygmy), four Mbuti Zaire Pygmy, three Lisongo, two Omega San, four Ethiopian (one Birale, one Hamar, one Berta and one Surma), one Bozo, one Dogon Mali/West Africa and one Tuareg/North Africa; Asia (n = 22): four Japanese, five Han Chinese, four Cambodian, two Taiwanese (Atayal and Ami), two Arab, one Uzbek, one Crimean Tartar, one Turkmen, one Iranian and one Druze; India and Pakistan (n = 9): two Brushaski, one Brauhi, one Balochi, three Sindhi and two Pathan; Pacific (n = 7): three Melanesian, two New Guinean and two Australian; America (n = 6): two Karitiana, one Surui, two Mayan and one Colombian; Europe (n = 30): 18 Austrian, two Sardinian, two Italian, four Basque and four American of European descent.

Control individuals from Austria were women aged 60 years or older with no personal or familial history of cancer. All Austrian control individuals were informed about this research project and signed consent forms. East and West African samples were provided by M. Seielstad (Harvard University, MA) and Central Asian samples by R.S. Wells (Stanford University, CA). All other DNA samples of control individuals were from the Stanford DNA collection (L. Cavalli-Sforza). Samples representing the five continents were collected according to approved human subject protocols.

DNA extraction and PCR conditions

Genomic DNA was isolated from blood samples as described elsewhere (32). PCR was performed using 48 primer pairs in a 50 µl volume containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5-2.5 mM MgCl₂, 50 µM dNTPs, 0.25 µM of each primer and 100 ng of genomic DNA. For all PCRs, AmpliTaq Gold (Perkin Elmer, Foster City, CA) was used. The intron sequences included upstream of the 3[prime] splice site a minimum of 14 nucleotides, average 74 nucleotides, and downstream of the 5[prime] splice site a minimum of two nucleotides, average 75 nucleotides. The PCR cycling regime comprised an initial denaturation step at 95°C for 10 min to activate AmpliTaq Gold. Subsequent denaturing steps were 94°C for 20 s and extension steps of 72°C for 45 s; annealing temperatures typically were decreased from 63°C by 0.5°C increments with each of the initial 14 cycles, followed by 20 cycles at 56°C for 20 s. Sometimes different annealing temperatures were used in spanning the 7°C ‘touchdown’ temperature window (see Table 4 for detailed information on primer sequences, magnesium chloride concentrations and annealing temperatures).

Table 4. PCR and DHPLC conditions for the mutational analysis of BRCA2

Exon Forward (F) and reverse (R) primers (5[prime]->3[prime]) MgCl₂(mM) Annealing
temperature (°C) Size (bp) DHPLC
temperature (°C)

2 F: CCA GGA GAT GGG ACT GAA TTA G 1.5 69-62/62 311 56

R: CTG TGA CGT ACT GGG TTT TTA GC

3 F: TTC CTT ATG ATC TTT AAC TGT TCT G 2.5 63-56/56 406 56

R: GCT AAG ATT TTA ACA CAG GTT TGC

4 F: AGA ATG CAA ATT TAT AAT CCA GAG TA 2.5 63-56/56 249 51, 55

R: AAT CAG ATT CAT CTT TAT AGA ACA AA

5+6 F: TTC CAA CAA TTT ATA TGA ATG AGA ATC 2.5 63-56/56 362 54

R: CTC AGG GCA AAG GTA TAA CGC

7 F: CCT TAA TGA TCA GGG CAT TTC 2.5 63-56/56 214 57

R: CAA CCT CAT CTG CTC TTT CTT G

8 F: GTA GAT GTG CTT TTT GAT GTC TGA C 2.5 63-56/56 315 52

R: GAG AGA CAG CAG AGT TTC ACA GG

9 F: CAG ATA ACT GAA ATC ACC AAA AGT G 2.5 63-56/56 262 54

R: ACA ACA ACA AAA AAA CCT GTA GTT C

10A F: TAT AAA ATA TTA ATG TGC TTC TGT T 2.5 63-56/56 374 54

R: AAA GGG CTT CTG ATT TGC TAC

10B F: ATC TGA AGT GGA ACC AAA TGA TAC 2.5 63-56/56 280 56

R: ACG TGG CAA AGA ATT CTC TGA AGT AA

10C F: TTT CAG AAA AAG ACC TAT TAG ACA 2.5 63-56/56 242 55

R: CTT TTT GAT ACC CTG AAA TGA AGA AG

10D F: TAA AGC AGG CAA TAT CTG GAA CTT CT 2 65-58/58 295 56

R: GTG GAT ATT AAA CCT GCA TTC TTC AA

10E F: TTT AAT TGA TAA TGG AAG CTG G 2.5 63-56/56 268 55

R: TTA CAA AAA AAA AAA GAC AGA GGT

11A F: TTG TCA GAT TTA ACT TTT TTG GAA G 2.5 63-56/56 342 55

R: CAA CTG GGA CAC TTT CTT TCA G

11B F: GCT CAA GAA GCA TGT CAT GG 2.5 63-56/56 394 55

R: TAT GAA AAC CCA ACA GAG TAG GT

11C F: GAA AGA AAG TGT CCC AGT TG 2.5 63-56/56 360 58

R: ACC ACA GTC TCA ATA GAA ACA AGG

11D F: TGA GAC CAT TGA GAT CAC AGC 2.5 63-56/56 709 53

R: TAG TCA CAA GTT CCT CAA CGC A

11E F: TGA TTG ATG GTA CTT TAA TTT TGT CAC 2.5 63-56/56 338 54

R: AGC CAA GAC CTC TTC TTT TAT ATC TG

11F F: AAG CTG ATT CTC TGT CAT GCC TG 2.5 63-56/56 456 55

R: GAT TTG TGT TTT GGT TGA ATT GTA CC

11G F: AAA ATA CAT GAG AGT AGC ATC ACC 2.5 63-56/56 330 55

R: AAA TCT TTT TTA ATT GAC ACT TGG

11H F: CGA ACC CAT TTT CAA GAA CTC TAC CA 2.5 63-56/56 215 54

R: TGT AAT CAT TAT TTT TTT CTG G

11I F: TTG GTT TAT GTT CTT GCA GAG GAG 2.5 63-56/56 487 54

R: CCT TTT GGC TAG GTG TTA AAT TAT GG

11J F: TGG CAT TAG ATA ATC AAA AGA AAC TG 2.5 63-56/56 500 55

R: CCT AAA CCC CAC TTC ATT TTC ATC

11K F: GAA ATT AAA CGG AAG TTT GCT GG 2.5 63-56/56 465 54

R: TGA ATC ACT GCC ATC AAA TTC TAA G

11L F: AAT GAC TAC TGG CAC TTT TGT TG 2.5 63-56/56 401 54

R: CAC TTG CAG TCT GAA AAA ATG TAT C

11M F: GCC AGT ATT GAA GAA TGT TGA AGA TC 1.5 68-61/61 443 55

R: AAA CCT TAT GTG AAT GCG TGC TAC

11N F: AAC GAA AAT TAT GGC AGG TTG TTA C 1.5 68-61/61 536 55

R: GCT TTC CAC TTG CTG TAC TAA ATC C

11O F: CCA GCT CAC AAG AGA AGA AAA TAC TG 1.5 68-61/61 503 54

R: TTA CGT TTT TAG GTG AAG CCT GTT C

11P F: AAA CCC AGA GCA CTG TGT AAA CTC 1.5 68-61/61 487 53

R: TCT CCT CTT CTT TTT CCA ATT CTT G

11Q F: TAC AGA TTC TAA ACT GCC AAG TCA TG 1.5 68-61/61 265 54

R: TAA CCA TAC TCC CCC AAA CTG AC

12 F: AAT TGA CAT TGA AGA CTG ACT TTA CTC 2.5 63-56/56 370 52

R: AGC ACT TTG GAG AGG CAG G

13 F: GCA TCC GTT ACA TTC ACT GAA A 2 65-58/58 310 54

R: ACG GGA AGT GTT AAC TTC TTA ACG

14A F: ACC ATG TAG CAA ATG AGG GTC T 2.5 63-56/56 391 55

R: GCT TTT GTC TGT TTT CCT CCA A

14B F: CAC AGA GTT GAA CAG TGT GTT AGG 1.5 68-61/61 297 55

R: GGG CTT TAA AAT TAC CAC CAC C

15 F: GGC CAG GGG TTG TGC TTT TT 2.5 63-56/56 369 52, 55, 59

R: ATT TCA TTC ATC CAT TCC TGC

16 F: TTT GGT AAA TTC AGT TTT GGT TTG 2.5 63-56/56 396 55

R: AGC CAA CTT TTT AGT TCG AGA G

17 F: CAG AGA ATA GTT GTA GTT GTT GAA 2.5 63-56/56 306 57

R: AGA AAC CTT AAC CCA TAC TGC

18A F: TCA GTT TTT ATT CTC AGT TAT TCA GTG 2.5 63-56/56 298 55

R: GCA TAC CAC CCA TCT GTA AGT TC

18B F: TGT TTC TGA CAT AAT TTC ATT GAG C 2.5 63-56/56 420 53, 57

R: AAA CTT TAA CTG TCT GAA GAA TAT GC

19 F: CTT ATT TAC TGT CTT ACT AAT CTT CCT 2.5 63-56/56 389 52, 56

R: GAC CGA AAC TCC ATC TCA AAC

20 F: GGT GAT CCA CTA ATC TCA GCC TC 2.5 63-56/56 451 54, 57

R: GTC CCT TGT TGC TAT TCT TTG TCT

21 F: GGG TGT TTT ATG CTT GGT TCT 2.5 63-56/56 303 55, 59

R: CAT TTC AAC ATA TTC CTT CCT G

22 F: AAC CAC ACC CTT AAG ATG AGC 2.5 63-56/56 455 53, 56

R: GGG CAT TAG TAG TGG ATT TTG C

23 F: ACT TCT TCC ATT GCA TCT TTC TCA 2.5 66-59/59 290 53

R: AAA ACA AAA CAA AAA TTC AAC ATA

24 F: GCA GCG ACA AAA AAA ACT CA 2.5 63-56/56 365 53, 56

R: ATT TGC CAA CTG GTA GCT CC

25 F: GCT TTC GCC AAA TTC AGC TA 2 65-58/58 427 57

R: TAC CAA AAT GTG TGG TGA TGC

26 F: GTC CCA AAC TTT TCA TTT CTG C 2.5 63-56/56 379 54, 58

R: GGA GCC ACA TAA CAA CCA CA

27A F: CTG TGT GTA ATA TTT GCG TGC T 2.5 63-56/56 495 54, 58

R: GCA AGT TCT TCG TCA GCT ATT G

27B F: GAA TTC TCC TCA GAT GAC TCC A 2.5 63-56/56 417 57

R: TCT TTG CTC ATT GTG CAA CA

DHPLC

DHPLC was carried out on automated HPLC instrumentation (Rainin Instrument, Woburn, MA) equipped with a DNA separation column (Transgenomic, San Jose, CA) (33-35). Crude PCR products-which were subjected to an additional 3 min, 95 denaturing step followed by gradual reannealing from 95 to 65 over a period of 30 min prior to analysis-were eluted with a linear acetonitrile (J.T. Baker, Phillipsburg, NJ) gradient at a flow rate of 0.9 ml/min. The start and endpoints of the gradient were adjusted according to the size of the PCR products. Generally, analysis took <6 min, including column regeneration and re-equilibration to the starting conditions. The temperature required for successful resolution of heteroduplex molecules was determined by use of the DHPLC melting algorithm that can be obtained at http://hardy-weinberg.stanford.edu/dhplc/melt.html . The temperatures at which PCR products were analyzed are given in the last column of Table 4. All 166 samples were analyzed for all BRCA2 exons and intron-exon boundaries, except for exon 1, which is not translated, by DHPLC.

Direct DNA sequencing

PCR products were purified by solid-phase extraction and bidirectionally sequenced with the Applied Biosystems Taq Dye Deoxy terminator cycle sequencing kit (Perkin Elmer) according to the manufacturer’s instructions. Samples were analyzed with an Applied Biosystems 373A sequencer.

Cloning of BRCA2 mutations with the TA cloning kit

For the splice site mutation in intron 5, a cDNA PCR product spanning exons 3-7 was generated according to standard protocols. For the splice site mutation in intron 21, nested PCR primers (first pair exons 20-24, second pair exons 21-23) were used. Both cDNA PCR products were ligated into pCRII and then transformed into One Shot (INVF[prime]) competent cells according to the manufacturer’s instructions (Invitrogen, San Diego, CA). The appropriate white colonies were selected after 24 h and analyzed with an Applied Biosystems 373A sequencer.

ACKNOWLEDGEMENTS

We thank the families that participated in this study. This work was supported by grants of the NIH (R01 HG01707), the European Committee DG-V, the Medizinischer Fond des Bürgermeisters der Stadt Wien, the Wiener Krebshilfe-Krebsgesellschaft, University of Vienna, and the Ludwig-Boltzmann Institut für Klinisch-Experimentelle Onkologie.

ABBREVIATIONS

BC, breast cancer; DHPLC, denaturing high-performance liquid chromatography; FBN1, fibrillin-1; HBC, human breast cancer; HBOC, human breast-ovarian cancer; LPL, lipoprotein lipase; MFS, Marfan syndrome; OC, ovarian cancer.

REFERENCES

1. Wooster, R., Bignell, G., Lancaster, J., Swift, S., Seal, S., Manglon, J., Collins, N., Gregory, S., Gumbs, C., Micklem, G., Barfoot, R., Hamoudi, R., Patel, S., Rice, C., Biggs, P., Hashim, Y., Smith, A., Connor, F., Arason, A., Gudmundsson, J., Ficenec, D., Kelsell, D., Ford, D., Tonin, P., Bishop, D.T. et al). (1995) Identification of the breast cancer susceptibility gene BRCA2. Nature, 378, 789-792. MEDLINE Abstract

2. Tavtigian, S.V., Simard, J., Rommens, J., Couch, F., Shattuck-Eidens, D., Neuhausen, S., Merajver, S., Thorlacius, S., Offit, K., Stoppa-Lyonnet, D., Belanger, C., Bell, R., Berry, S., Bogden, R., Chen, Q., Davis, T., Dumont, M., Frye, C., Hattier, T., Jammulapati, S., Janecki, T., Jiang, P., Kehrer, R., Leblanc, J.-F., Mitchell, J.T. et al). (1996) The complete BRCA2 gene and mutations in chromosome 13q-linked kindreds. Nature Genet., 12, 333-337. MEDLINE Abstract

3. Breast Cancer Information Core-database (http://www.nhgri.nih.gov/Intramural_research/Lab_transfer/Bic/ ).

4. Sharan, S.K., Morimatsu, M., Albrecht, U., Lim, D.-S., Regel, E., Dinh, C., Sands, A., Eichelle, G., Hasty, P. and Bradley, A. (1997) Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking BRCA2. Nature, 386, 804-810. MEDLINE Abstract

5. Wong, A.K.C., Pero, R., Ormonde, P.A., Tavtigian, S.V. and Bartel, P.L. (1997) RAD51 interacts with the evolutionarily conserved BRC motifs in the human breast cancer susceptibility gene BRCA2. J. Biol. Chem., 272, 31941-31944. MEDLINE Abstract

6. Zhang, H., Tombline, G. and Weber, B.L. (1998) BRCA1, BRCA2, and DNA damage response: collision or collusion? Cell, 92, 433-436. MEDLINE Abstract

7. Chen, P.-L., Chen, C.-F., Chen, Y., Xiao, J., Sharp, Z.D. and Lee, W.-H. (1998) The BRC repeats in BRCA2 are critical for RAD51 binding and resistance to methyl methanesulfonate treatment. Proc. Natl Acad. Sci. USA, 95, 5287-5292. MEDLINE Abstract

8. Katagiri, T., Saito, H., Akira, S., Ogawa, H., Kamada, N., Nakamura, Y. and Miki, Y. (1998) Multiple possible sites of BRCA2 interacting with DNA repair protein RAD51. Genes Chromosomes Cancer, 21, 217-222.

9. Schrijver, I., Liu, W. and Francke, U. (1997) The pathogenicity of the Pro1148Ala substitution in the FBN1 gene: causing or predisposing to Marfan syndrome and aortic aneurysm, or clinically innocent? Hum. Genet., 99, 607-611. MEDLINE Abstract

10. Liu, W., Smith, D.I., Rechtzigel, K.J., Thibodeau, S.N. and James, C.D. (1998) Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations. Nucleic Acids Res., 26, 1396-1400.

11. O'Donovan, M.C., Oefner, P.J., Roberts, S.C., Austin, J., Hoogendoorn, B., Guy, C., Speight, G., Upadhyaya, M., Sommer, S.S. and McGuffin, P. (1998) Blind analysis of denaturing high-performance liquid chromatography as a tool for mutation detection. Genomics, 52, 44-49. MEDLINE Abstract

12. Hakansson, S., Johannsson, O., Johansson, U., Sellberg, G., Loman, N., Gerdes, A.-M., Holmberg, E., Dahl, N., Pandis, N., Kristoffersson, U., Olsson, H. and Borg, A. (1997) Moderate frequency of BRCA1 and BRCA2 germ-line mutations in Scandinavian familial breast cancer. Am. J. Hum. Genet., 60, 1068-1078. MEDLINE Abstract

13. Schubert, E.L., Lee, M.K., Mefford, H.C., Argonza, R.H., Morrow, J.E., Hull, J., Dann, J.L. and King, M.-C. (1997) BRCA2 in American families with four or more cases of breast or ovarian cancer: recurrent and novel mutations, variable expression, penetrance, and the possibility of families whose cancer is not attributable to BRCA1 or BRCA2. Am. J. Hum. Genet., 60, 1031-1040. MEDLINE Abstract

14. Haraldsson, K., Loman, N., Zhang, Q.-X., Johannson, O., Olsson, H. and Borg, A. (1998) BRCA2 germ-line mutations are frequent in male breast cancer patients without a family history of the disease. Cancer Res., 58, 1367-1371. MEDLINE Abstract

15. Teng, D.H.-F., Bogden, R., Mitchell, J., Baumgard, M., Bell, R., Berry, S., Davis, T., Ha, P.C., Kehrer, R., Jammulapati, S., Chen, Q., Offit, K., Skolnick, M.H., Tavtigian, S.V., Jhanwar, S., Swedlund, B., Wong, A.K.C. and Kamb, A. (1996) Low incidence of BRCA2 mutations in breast carcinoma and other cancers. Nature Genet., 13, 241-244. MEDLINE Abstract

16. Couch, F.J., Farid, L.M., DeShano, M.L., Tavtigian, S.V., Calzone, K., Campeau, L., Peng, Y., Bogden, B., Chen, Q., Neuhausen, S., Shattuck-Eidens, D., Godwin, A.K., Daly, M., Radford, D.M., Sedlacek, S., Rommens, J., Simard, J., Garber, J., Merajver, S. and Weber, B.L. (1996) BRCA2 germline mutations in male breast cancer cases and breast cancer families. Nature Genet., 13, 123-125. MEDLINE Abstract

17. McAllister, K.A., Haugen-Strano, A., Hagevik, S., Brownlee, H.A., Collins, N.K., Futreal, P.A., Bennett, L.M. and Wiseman, R.W. (1997) Characterization of the rat and mouse homologues of the BRCA2 breast cancer susceptibility gene. Cancer Res., 57, 3121-3125. MEDLINE Abstract

18. Takahashi, H., Chiu, H.-C., Bandera, C.A., Behbakht, K., Liu, P.C., Couch, F.J., Weber, B.L., LiVolsi, V.A., Furusato, M., Rebane, B.A., Cardonick, A., Benjamin, I., Morgan, M.K., King, S.A., Mikuta, J.J., Rubin, S.C. and Boyd, J. (1996) Mutations of the BRCA2 gene in ovarian carcinomas. Cancer Res., 56, 2738-2741. MEDLINE Abstract

19. Mavraki, E., Gray, I., Bishop, D. and Spurr, N. (1997) Germline BRCA2 mutations in men with breast cancer. Br. J. Cancer, 76, 1428-1431. MEDLINE Abstract

20. Vehmanen, P., Friedman, L.S., Eerola, H., Sarantaus, L., Pyrhönen, S., Ponder, B.A.J., Muhonen, T. and Nevanlinna, H. (1997) A low proportion of BRCA2 mutations in Finnish breast cancer families. Am. J. Hum. Genet., 60, 1050-1058. MEDLINE Abstract

21. Mazoyer, S., Dunning, A.M., Serova, O., Dearden, J., Puget, N., Healey, C.S., Gayther, S.A., Mangion, J., Stratton, M.R., Lynch, H.T., Goldgar, D.E., Ponder, B.A.J. and Lenoir, G.M. (1996) A polymorphic stop codon in BRCA2. Nature Genet., 14, 253-254. MEDLINE Abstract

22. Wagner, T.M.U., Möslinger, R.A., Muhr, D., Langbauer, G., Hirtenlehner, K., Concin, H., Doeller, W., Haid, A., Lang, A.H., Mayer, P., Ropp, E., Kubista, E., Amirimani, B., Helbich, T., Becherer, A., Scheiner, O., Breiteneder, H., Borg, A., Devilee, P., Oefner, P. and Zielinski, C. (1998) BRCA1-related breast cancer in Austrian breast and ovarian cancer families: specific BRCA1 mutations and pathological characteristics. Int. J. Cancer, 77, 354-360. MEDLINE Abstract

23. Gayther, S.A., Mangion, J., Russell, P., Seal, S., Barfoot, R., Ponder, B.A.J., Stratton, M.R. and Easton, D. (1997) Variation of risks of breast and ovarian cancer associated with different germline mutations of the BRCA2 gene. Nature Genet., 15, 103-105. MEDLINE Abstract

24. Koonin, E.V., Altschul, S.F. and Bork, P. (1996) Functional motifs. Nature Genet., 13, 266-267. MEDLINE Abstract

25. Bork, P., Blomberg, N. and Nilges, M. (1996) Internal repeats in the BRCA2 protein sequence. Nature Genet., 13, 22-23. MEDLINE Abstract

26. Liu, W., Qian, C. and Francke, U. (1997) Silent mutation induces exon skipping of fibrillin-1 gene in Marfan syndrome. Nature Genet., 16, 328-329. MEDLINE Abstract

27. Richard, I.A. and Beckmann, J.S. (1995) How neutral are synonymous codon mutations? Nature Genet., 10, 259. MEDLINE Abstract

28. Bignell, G., Micklem, G., Stratton, M.R., Ashworth, A. and Wooster, R. (1997) The BRC repeats are conserved in mammalian BRCA2 proteins. Hum. Mol. Genet., 6, 53-58. MEDLINE Abstract

29. Petrij-Bosch, A., Peelen, T., van Vliet, M., van Eijk, R., Olmer, R., Drüsedau, M., Hogervorst, F.B.L., Hageman, S., Arts, P.J.W., Ligtenberg, M.J.L., Meijers-Heijboer, H., Klijn, J.G.M., Vasen, H.F.A., Cornelisse, C.J., van't Veer, L.J., Bakker, E., van Ommen, G.-J.B. and Devilee, P. (1997) BRCA1 genomic deletions are major founder mutations in Dutch breast cancer cases. Nature Genet., 17, 341-345. MEDLINE Abstract

30. Wang, D.G., Fan, J.-B., Siao, C.-J., Berno, A., Young, P., Sapolsky, R., Ghandour, G., Perkins, N., Winchester, E., Spencer, J., Kruglyak, L., Stein, L., Hsie, L., Topaloglou, T., Hubbell, E., Robinson, E., Mittmann, M., Morris, M.M., Shen, N., Kilburn, D., Rioux, J., Nusbaum, C., Rozen, S., Hudson, T.J., Lipshutz, R., Chee, M. and Lander, E.S. (1998) Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome. Science, 280, 1077-1082. MEDLINE Abstract

31. Nickerson, D.A., Taylor, S.L., Weiss, K.M., Clark, A.G., Hutchinson, R.G., Stengård, J., Salomaa, V., Vartiainen, E., Boerwinkle, E. and Sing, C.F. (1998) DNA sequence diversity in a 9.7-kb region of the human lipoprotein lipase gene. Nature Genet., 19, 233-240. MEDLINE Abstract

32. Godwin, A.K., Vanderveer, L., Schultz, D.C., Lynch, H.A.T., Altomare, D.A., Buetow, K.H. and Daly, M. (1994) A common region of deletion on chromosome 17q in both sporadic and familial epithelial ovarian tumors distal to BRCA1. Am. J. Hum. Genet., 55, 666-677. MEDLINE Abstract

33. Oefner, P.J. and Underhill, P.A. (1995) Comparative DNA sequencing by denaturing high-performance liquid chromatography (DHPLC). Am. J. Hum. Genet., 57, A266.

34. Hayward-Lester, A., Chilton, B.S., Underhill, P.A., Oefner, P.J. and Doris, P.A. (1997) Quantification of specific nucleic acids, regulated RNA processing and genomic polymorphisms using reversed-phase HPLC. In Ferré, F. (ed.), Gene Quantification. Birkhäuser Verlag, Basel, Switzerland, pp. 44-77.

35. Oefner, P.A. and Underhill, P. (1998) DNA mutation detection using denaturing high performance liquid chromatography (DHPLC). Current Protocols in Human Genetics. Wiley & Sons, New York, Supplement 19, 7.10.1-7.10.12.

*To whom correspondence should be addressed. Tel: +1 650 725 6117; Fax: +1 650 725 1534; Email: oefner@genome.stanford.edu
Members of the Austrian Hereditary Breast and Ovarian Cancer Group

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U Hamann, X Liu, S Lange, H U Ulmer, A Benner, and R J Scott
Contribution of BRCA2 germline mutations to hereditary breast/ovarian cancer in Germany
J. Med. Genet., March 1, 2002; 39(3): e12 - 12.
[Full Text] [PDF]

S. Sale, R. Sung, P. Shen, K. Yu, Y. Wang, G. E. Duran, J.-H. Kim, T. Fojo, P. J. Oefner, and B. I. Sikic
Conservation of the Class I {beta}-Tubulin Gene in Human Populations and Lack of Mutations in Lung Cancers and Paclitaxel-resistant Ovarian Cancers
Mol. Cancer Ther., January 1, 2002; 1(3): 215 - 225.
[Abstract] [Full Text] [PDF]

B. J. C. van den Bosch, R. F. M. de Coo, H. R. Scholte, J. G. Nijland, R. van den Bogaard, M. de Visser, C. E. M. de Die-Smulders, and H. J. M. Smeets
Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography
Nucleic Acids Res., October 15, 2000; 28(20): e89 - e89.
[Abstract] [Full Text] [PDF]

J. PLASCHKE, T. COMMER, C. JACOBI, H. K SCHACKERT, and J. CHANG-CLAUDE
BRCA2 germline mutations among early onset breast cancer patients unselected for family history of the disease
J. Med. Genet., September 1, 2000; 37(9): 17e - 17.
[Full Text]

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Exon/intron	Nucleotide^a	Codon	Nucleotide change	Amino acid change^b	Global heterozygosity	Geographic distribution^c	BIC (as of 9/98) citations
2	203	-	G->A	non-coding	0.260	Eurasia/Oceania	BIC,2,12-16
2	217	-	T->G	non-coding	0.006	Oceania	-
4	550	108	A->C	Asn->His (nc)	0.006	Africa	-
10	1093	289	A->C	Asn->His (nc)	0.120	Global	BIC,15,16
	1207	327	A->G	Lys->Glu (nc)	0.012	Austria	-
	1342	372	A/C	Asn/His (nc)	0.420	Global	BIC,2,15-18
	1515	429	A->G	silent	0.006	Pakistan	-
	1593	455	A->G	silent	0.110	Global	BIC,15,18
	1972	582	A->C	Thr->Pro (nc)	0.006	Central Asia	-
11	2347	707	G->T	Asp->Tyr (c)	0.006	Africa	-
	2457	743	T->C	silent	0.130	Global	2,15
	2578	784	A->G	Met->Val (c)	0.006	Asia	BIC
	2778	850	A->G	silent	0.006	Asia	-
	2885	886	A->T	Asn->Ile (nc)	0.006	Africa	-
	3031	935	G->A	Asp->Asn (nc)	0.006	Austria	BIC
	3111	961	G->A	silent	0.006	Austria	-
	3199	991	A->G	Asn->Asp (nc)	0.150	Global	2,15-17
	3624	1132	A->G	silent	0.340	Global	-
	4035	1269	T->C	silent	0.230	Global	BIC,2,15
	4086	1286	delAAA	del Leu	0.006	Africa	-
	4097	1290	G->A	Cys->Tyr (c)	0.006	Africa	-
	4194	1322	C->T	silent	0.006	Africa	-
	4469	1414	C->T^d	Thr->Met (nc)	0.012	Africa	-
	4486	1420	G->T	Asp->Tyr (c)	0.030	Austria	BIC
	4765	1513	G->A	Asp->Asn (nc)	0.006	Australia	-
	4791	1521	G->A	silent	0.018	Africa	-
	5007	1593	A->C	Glu->Asp (c)	0.006	Pakistan	-
	5427	1733	C->T^d	silent	0.036	Europe/Asia	-
	5540	1771	G->A	Gly->Asp (nc)	0.006	America	BIC
	5642	1805	A->G	Asn->Ser (c)	0.006	Africa	-
	5868	1880	T->G	Asn->Lys (nc)	0.012	Africa	BIC,15,17
	5932	1902	G->A	Asp->Asn (nc)	0.012	Africa	BIC
	5972	1915	C->T^d	Thr->Met (nc)	0.048	Austria/Oceania	BIC,13,15-18
	6328	2034	C->T^d	Arg->Cys (nc)	0.006	Austria	BIC,15-17
	6448	3074	C->A	His->Asn (nc)	0.006	Africa	BIC
	6640	2138	G->T	Val->Phe (c)	0.006	Africa	13
	6741	2171	C->G	silent	0.018	Africa	-
	7049	2274	G->T	Gly->Val (nc)	0.006	Austria	15,17,19
14	7470	2414	A->G	silent	0.250	Global except America	BIC,2,12,14-16,18
14	7625	2466	C->T	Ala->Val (c)	0.018	Africa	BIC,17,18
15	7666	2480	T->G	Leu->Val (c)	0.006	Africa	-
	7697	2490	T->C	Tyr->His (nc)	0.018	America	BIC
	7772	3015	C->T	Thr->Ile (nc)	0.006	Austria	BIC
18	8320	2697	G->A^d	Arg->His (c)	0.006	Africa	-
22	9058	2944	A->T	Ile->Phe (c)	0.006	Africa	BIC
	9079	2951	G->A	Ala->Thr (nc)	0.006	America	BIC
	9133	2969	G->A^d	Val->Met (c)	0.006	Asia	-
24	9465	3079	T->C	silent	0.006	Africa	-
25	9642	3138	A->G	silent	0.006	Africa	-
27	9998	3257	A->G	Lys->Arg (c)	0.006	Africa	-
	10 056	3276	A->T	Arg->Ser (nc)	0.006	Africa	-
	10 152	3308	C->T^d	silent	0.006	Africa	BIC
	10 204	3326	A->T	3326 STOP	0.012	Austria/Sardinia	BIC,15,17,20,21
	10 319	3364	delCT	3366 STOP	0.006	America	-
	10 462	3412	A->G	Ile->Val (c)	0.024	Africa/Austria/America	BIC,15,17,20
2	295+62		T->G	non-coding	0.006	Asia	14
3	545-22		C->T	non-coding	0.006	Africa	-
4	654-47		G->T	non-coding	0.006	Austria	-
6	744+18		T->C	non-coding	0.006	Asia	-
6	745-4		C->G	non-coding	0.006	Africa	-
8	909+56		C->T	non-coding	0.024	Africa/Asia/Oceania	-
11	7069+74		T->A	non-coding	0.006	Africa	-
11	7069+80		delTTAA	non-coding	0.340	Global except America	BIC
13	7236-62		A->G	non-coding	0.006	Austria	-
14	7663+53		C->T^d	non-coding	0.130	Global	BIC
15	7830-27		T->A	non-coding	0.006	Asia	-
16	8034-14		T->C	non-coding	0.048	Austria	BIC
17	8204+12		G->A	non-coding	0.012	Africa	-
18	8560-66		T->C	non-coding	0.006	Africa	-
18	8560-64		A->G	non-coding	0.018	Africa	-
19	8715+47		C->T	non-coding	0.018	Africa	-
21	8983-79		G->A	non-coding	0.006	Asia	-
21	8983-66		T->C	non-coding	0.220	Global	-
22	9181+16		C->T	non-coding	0.006	Austria	-
22	9181+98		T->C	non-coding	0.012	Austria	-
25	9730-71		G->T	non-coding	0.006	Africa	-
26	9876+54		G->A^d	non-coding	0.012	Africa	-
	9876+84		G->A	non-coding	0.006	Asia	-
	9876+106		delT	non-coding	0.006	Africa	-

Exon/intron	Nucleotide (codon)	Nucleotide change	Effect on coding sequence	Amino acid change	Family	No. of BC/OC (age of onset in years)
10	1207 (327)	A->G	Lys->Glu	non-conservative	F40	6 BC (34, 37, 42, 42, 46, 47)
10	1207 (327)	A->G	Lys->Glu	non-conservative	M16	2 BC (29, 49)
11	3031 (935)	G->A	Asp->Asn	non-conservative	F54	2 BC (48, 49)
11	3111 (961)	G->A	silent, Gln	silent	F54	2 BC (48, 49)
11	4486 (1420)	G->T	Asp->Tyr	conservative	F35	3 BC (38, 49, 72)
					F50	3 BC (39, 52, 60)
					M26	3 BC (36, 50, 55 bilateral)
					M28	2 BC (39, 47 bilateral)
					M41	2 BC (33, 38)
11	6328 (2034)	C->T	Arg->Cys	non-conservative	M31	1 BC (29)
15	7772 (3015)	C->T	Thr->Ile	non-conservative	M2	2 BC (32, 48)
Intron 4	654-47	G->T	non-coding		F42	3 BC (43, 53, 59)
Intron 13	7236-62	A->G	non-coding		F54	2 BC (48, 49)

Continent (no. of individuals)	Nucleotide
Continent (no. of individuals)	203	1093	1342	1593	2457	3199	3624	4035	7470	7069+80	7663+53	8983-66
Africa (21)	0.000	0.143	0.190	0.048	0.143	0.238	0.143	0.286	0.190	0.190	0.190	0.381
Austrian controls (18)	0.389	0.167	0.500	0.111	0.111	0.111	0.444	0.167	0.333	0.500	0.111	0.167
Austrian HBOC (71)	0.343	0.029*	0.429	0.029	0.029	0.057	0.429	0.243	0.243	0.400	0.043	0.029*
Other Europeans (12)	0.333	0.167	0.583	0.250	0.250	0.250	0.333	0.333	0.250	0.333	0.250	0.500
Asia (31)	0.258	0.194	0.484	0.194	0.258	0.226	0.290	0.161	0.323	0.323	0.258	0.452
Oceania (7)	0.143	0.286	0.286	0.286	0.286	0.286	0.143	0.143	0.143	0.143	0.429	0.286
America (6)	0.000	0.333	0.500	0.333	0.333	0.333	0.000	0.333	0.000	0.000	0.333	0.167
Total (166)	0.267	0.121	0.424	0.109	0.133	0.152	0.333	0.230	0.248	0.339	0.152	0.218

Exon	Forward (F) and reverse (R) primers (5[prime]->3[prime])	MgCl₂(mM)	Annealing temperature (°C)	Size (bp)	DHPLC temperature (°C)
2	F: CCA GGA GAT GGG ACT GAA TTA G	1.5	69-62/62	311	56
	R: CTG TGA CGT ACT GGG TTT TTA GC
3	F: TTC CTT ATG ATC TTT AAC TGT TCT G	2.5	63-56/56	406	56
	R: GCT AAG ATT TTA ACA CAG GTT TGC
4	F: AGA ATG CAA ATT TAT AAT CCA GAG TA	2.5	63-56/56	249	51, 55
	R: AAT CAG ATT CAT CTT TAT AGA ACA AA
5+6	F: TTC CAA CAA TTT ATA TGA ATG AGA ATC	2.5	63-56/56	362	54
	R: CTC AGG GCA AAG GTA TAA CGC
7	F: CCT TAA TGA TCA GGG CAT TTC	2.5	63-56/56	214	57
	R: CAA CCT CAT CTG CTC TTT CTT G
8	F: GTA GAT GTG CTT TTT GAT GTC TGA C	2.5	63-56/56	315	52
	R: GAG AGA CAG CAG AGT TTC ACA GG
9	F: CAG ATA ACT GAA ATC ACC AAA AGT G	2.5	63-56/56	262	54
	R: ACA ACA ACA AAA AAA CCT GTA GTT C
10A	F: TAT AAA ATA TTA ATG TGC TTC TGT T	2.5	63-56/56	374	54
	R: AAA GGG CTT CTG ATT TGC TAC
10B	F: ATC TGA AGT GGA ACC AAA TGA TAC	2.5	63-56/56	280	56
	R: ACG TGG CAA AGA ATT CTC TGA AGT AA
10C	F: TTT CAG AAA AAG ACC TAT TAG ACA	2.5	63-56/56	242	55
	R: CTT TTT GAT ACC CTG AAA TGA AGA AG
10D	F: TAA AGC AGG CAA TAT CTG GAA CTT CT	2	65-58/58	295	56
	R: GTG GAT ATT AAA CCT GCA TTC TTC AA
10E	F: TTT AAT TGA TAA TGG AAG CTG G	2.5	63-56/56	268	55
	R: TTA CAA AAA AAA AAA GAC AGA GGT
11A	F: TTG TCA GAT TTA ACT TTT TTG GAA G	2.5	63-56/56	342	55
	R: CAA CTG GGA CAC TTT CTT TCA G
11B	F: GCT CAA GAA GCA TGT CAT GG	2.5	63-56/56	394	55
	R: TAT GAA AAC CCA ACA GAG TAG GT
11C	F: GAA AGA AAG TGT CCC AGT TG	2.5	63-56/56	360	58
	R: ACC ACA GTC TCA ATA GAA ACA AGG
11D	F: TGA GAC CAT TGA GAT CAC AGC	2.5	63-56/56	709	53
	R: TAG TCA CAA GTT CCT CAA CGC A
11E	F: TGA TTG ATG GTA CTT TAA TTT TGT CAC	2.5	63-56/56	338	54
	R: AGC CAA GAC CTC TTC TTT TAT ATC TG
11F	F: AAG CTG ATT CTC TGT CAT GCC TG	2.5	63-56/56	456	55
	R: GAT TTG TGT TTT GGT TGA ATT GTA CC
11G	F: AAA ATA CAT GAG AGT AGC ATC ACC	2.5	63-56/56	330	55
	R: AAA TCT TTT TTA ATT GAC ACT TGG
11H	F: CGA ACC CAT TTT CAA GAA CTC TAC CA	2.5	63-56/56	215	54
	R: TGT AAT CAT TAT TTT TTT CTG G
11I	F: TTG GTT TAT GTT CTT GCA GAG GAG	2.5	63-56/56	487	54
	R: CCT TTT GGC TAG GTG TTA AAT TAT GG
11J	F: TGG CAT TAG ATA ATC AAA AGA AAC TG	2.5	63-56/56	500	55
	R: CCT AAA CCC CAC TTC ATT TTC ATC
11K	F: GAA ATT AAA CGG AAG TTT GCT GG	2.5	63-56/56	465	54
	R: TGA ATC ACT GCC ATC AAA TTC TAA G
11L	F: AAT GAC TAC TGG CAC TTT TGT TG	2.5	63-56/56	401	54
	R: CAC TTG CAG TCT GAA AAA ATG TAT C
11M	F: GCC AGT ATT GAA GAA TGT TGA AGA TC	1.5	68-61/61	443	55
	R: AAA CCT TAT GTG AAT GCG TGC TAC
11N	F: AAC GAA AAT TAT GGC AGG TTG TTA C	1.5	68-61/61	536	55
	R: GCT TTC CAC TTG CTG TAC TAA ATC C
11O	F: CCA GCT CAC AAG AGA AGA AAA TAC TG	1.5	68-61/61	503	54
	R: TTA CGT TTT TAG GTG AAG CCT GTT C
11P	F: AAA CCC AGA GCA CTG TGT AAA CTC	1.5	68-61/61	487	53
	R: TCT CCT CTT CTT TTT CCA ATT CTT G
11Q	F: TAC AGA TTC TAA ACT GCC AAG TCA TG	1.5	68-61/61	265	54
	R: TAA CCA TAC TCC CCC AAA CTG AC
12	F: AAT TGA CAT TGA AGA CTG ACT TTA CTC	2.5	63-56/56	370	52
	R: AGC ACT TTG GAG AGG CAG G
13	F: GCA TCC GTT ACA TTC ACT GAA A	2	65-58/58	310	54
	R: ACG GGA AGT GTT AAC TTC TTA ACG
14A	F: ACC ATG TAG CAA ATG AGG GTC T	2.5	63-56/56	391	55
	R: GCT TTT GTC TGT TTT CCT CCA A
14B	F: CAC AGA GTT GAA CAG TGT GTT AGG	1.5	68-61/61	297	55
	R: GGG CTT TAA AAT TAC CAC CAC C
15	F: GGC CAG GGG TTG TGC TTT TT	2.5	63-56/56	369	52, 55, 59
	R: ATT TCA TTC ATC CAT TCC TGC
16	F: TTT GGT AAA TTC AGT TTT GGT TTG	2.5	63-56/56	396	55
	R: AGC CAA CTT TTT AGT TCG AGA G
17	F: CAG AGA ATA GTT GTA GTT GTT GAA	2.5	63-56/56	306	57
	R: AGA AAC CTT AAC CCA TAC TGC
18A	F: TCA GTT TTT ATT CTC AGT TAT TCA GTG	2.5	63-56/56	298	55
	R: GCA TAC CAC CCA TCT GTA AGT TC
18B	F: TGT TTC TGA CAT AAT TTC ATT GAG C	2.5	63-56/56	420	53, 57
	R: AAA CTT TAA CTG TCT GAA GAA TAT GC
19	F: CTT ATT TAC TGT CTT ACT AAT CTT CCT	2.5	63-56/56	389	52, 56
	R: GAC CGA AAC TCC ATC TCA AAC
20	F: GGT GAT CCA CTA ATC TCA GCC TC	2.5	63-56/56	451	54, 57
	R: GTC CCT TGT TGC TAT TCT TTG TCT
21	F: GGG TGT TTT ATG CTT GGT TCT	2.5	63-56/56	303	55, 59
	R: CAT TTC AAC ATA TTC CTT CCT G
22	F: AAC CAC ACC CTT AAG ATG AGC	2.5	63-56/56	455	53, 56
	R: GGG CAT TAG TAG TGG ATT TTG C
23	F: ACT TCT TCC ATT GCA TCT TTC TCA	2.5	66-59/59	290	53
	R: AAA ACA AAA CAA AAA TTC AAC ATA
24	F: GCA GCG ACA AAA AAA ACT CA	2.5	63-56/56	365	53, 56
	R: ATT TGC CAA CTG GTA GCT CC
25	F: GCT TTC GCC AAA TTC AGC TA	2	65-58/58	427	57
	R: TAC CAA AAT GTG TGG TGA TGC
26	F: GTC CCA AAC TTT TCA TTT CTG C	2.5	63-56/56	379	54, 58
	R: GGA GCC ACA TAA CAA CCA CA
27A	F: CTG TGT GTA ATA TTT GCG TGC T	2.5	63-56/56	495	54, 58
	R: GCA AGT TCT TCG TCA GCT ATT G
27B	F: GAA TTC TCC TCA GAT GAC TCC A	2.5	63-56/56	417	57
	R: TCT TTG CTC ATT GTG CAA CA

				N. M. Suter, R. M. Ray, Y. W. Hu, M. G. Lin, P. Porter, D. L. Gao, R. E. Zaucha, L. M. Iwasaki, L. P. Sabacan, M. C. Langlois, et al. BRCA1 and BRCA2 Mutations in Women from Shanghai China Cancer Epidemiol. Biomarkers Prev., February 1, 2004; 13(2): 181 - 189. [Abstract] [Full Text] [PDF]

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				G. Palmieri, G. Palomba, A. Cossu, M. Pisano, M. F. Dedola, M. G. Sarobba, A. Farris, N. Olmeo, A. Contu, A. Pasca, et al. BRCA1 and BRCA2 germline mutations in Sardinian breast cancer families and their implications for genetic counseling Ann. Onc., December 1, 2002; 13(12): 1899 - 1907. [Abstract] [Full Text] [PDF]

				D. Trikka, Z. Fang, A. Renwick, S. H. Jones, R. Chakraborty, M. Kimmel, and D. L. Nelson Complex SNP-Based Haplotypes in Three Human Helicases: Implications for Cancer Association Studies Genome Res., April 1, 2002; 12(4): 627 - 639. [Abstract] [Full Text] [PDF]

				U Hamann, X Liu, S Lange, H U Ulmer, A Benner, and R J Scott Contribution of BRCA2 germline mutations to hereditary breast/ovarian cancer in Germany J. Med. Genet., March 1, 2002; 39(3): e12 - 12. [Full Text] [PDF]

				S. Sale, R. Sung, P. Shen, K. Yu, Y. Wang, G. E. Duran, J.-H. Kim, T. Fojo, P. J. Oefner, and B. I. Sikic Conservation of the Class I {beta}-Tubulin Gene in Human Populations and Lack of Mutations in Lung Cancers and Paclitaxel-resistant Ovarian Cancers Mol. Cancer Ther., January 1, 2002; 1(3): 215 - 225. [Abstract] [Full Text] [PDF]

				B. J. C. van den Bosch, R. F. M. de Coo, H. R. Scholte, J. G. Nijland, R. van den Bogaard, M. de Visser, C. E. M. de Die-Smulders, and H. J. M. Smeets Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography Nucleic Acids Res., October 15, 2000; 28(20): e89 - e89. [Abstract] [Full Text] [PDF]

				J. PLASCHKE, T. COMMER, C. JACOBI, H. K SCHACKERT, and J. CHANG-CLAUDE BRCA2 germline mutations among early onset breast cancer patients unselected for family history of the disease J. Med. Genet., September 1, 2000; 37(9): 17e - 17. [Full Text]