MUTATION IN BRIEF

HUMAN MUTATION Mutation in Brief #366 (2000) Online

© 2000 WILEY-LISS, INC.

Received 30 May 2000; Revised manuscript accepted 20 July 2000.

Allelic Imbalance of BRCA1 Transcript in the IVS2012-bp Insertion CarrierPiotr Kozlowski, Krzysztof Sobczak, Anna Jasinska, and Wlodzimierz J. Krzyzosiak

Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego12/14, 61-704 Poznan, Poland

Correspondence to Wlodzimierz J. Krzyzosiak, Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry,Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland; Phone: +48-61 852 85 03 ext. 136;Fax: +48-61 852 05 32; E-mail: wlodkrzy@man.poznan.pl

Contract grant sponsor: Polish Committee for Scientific Research, and Foundation for Polish Science in frame ofprogram “DIAMOL”; Contract grant number: 6P04B00212 and 117/96

Communicated by Mark H. Paalman

One of the unclassified variants of the BRCA1 gene which has drawn considerable attentionin recent years is the 12-bp insertion/duplication in intron 20. In this report, we show that acontribution from one chromosome cannot be detected in the BRCA1 transcript of the 12 bpinsertion carrier. We also demonstrate here that the single transcript variant we observe bycDNA analysis originates from the same BRCA1 allele that harbours the 12-bp insertion.© 2000 Wiley-Liss, Inc.

KEY WORDS: BRCA1; genotyping; transcript analysis; fluorescent SSCP/heteroduplex analysis; capillary electrophoresis

INTRODUCTION

Five years after discovering the first breast cancer susceptibility gene BRCA1 (MIM# 113705), the total numberof its alterations reported to the Breast Cancer Information Core (BIC) Database (www.nhgri.nih.gov/Intramural_research/Lab_transfer/Bic/) reached 3000. There are more than 700 different alterations, whichinclude over 400 protein truncating mutations and 45 polymorphisms. There is also a substantial portion ofunclassified variants, which complicates genetic testing for cancer susceptibility. A good example of these is the12-bp insertion/duplication in intron 20.

Earlier, intronic insertion was reported only in breast or ovarian cancer patients and was considered to be acancer-predisposing mutation (Takahashi et al., 1995; Langston et al., 1996a,b; Stratton et al., 1997). In ourBRCA1 study (Sobczak et al., 1997), the insertion was found in one breast/ovarian cancer patient and in fourhealthy women with varying family histories of cancer. We could not detect any aberrant splicing product in theRNA of the 12 bp insertion carriers (Sobczak et al., 1997). Later, more examples of the insertion were reported(Robledo et al., 1997; Campbell et al., 1997; Malone et al., 1998; Scholl et al., 1999; Dork et al., 1999), includingone male with breast cancer. When genomic DNA of the male carrier, who was heterozygous for the BRCA1coding sequence polymorphism, was compared with his cDNA, only one type of transcript was found (Robledo etal., 1997). The authors concluded that the insertion might be a regulatory, transcript-destabilising mutation. Theydid not show, however, whether the observed transcript derived from the allele with the insertion or from the other

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BRCA1 allele. Here we report that we detected a single type of BRCA1 transcript in the cDNA of another 12 bpinsertion carrier. More interestingly, we provide direct evidence that the single type of transcript we could detectoriginates from the BRCA1 allele containing the 12-bp insertion.

MATERIALS AND METHODS

Genotyping

The study population, PCR primer sequences, and amplification conditions were the same as described in ourprevious study (Sobczak et al., 1997). In all amplifications a protocol for rapid and low volume PCR was used(Sobczak et al., 1995). Genotyping was done by SSCP and heteroduplex analysis of radiolabelled PCR products asdescribed earlier (Kozlowski et al., 1996).

Segregation analysis

Segregation of the 12-bp insertion in intron 20, and exon 11 3667A/G polymorphism was followed by SSCPanalysis of fluorescently labelled PCR products by capillary electrophoresis (Inazukaet al., 1997). The analysis wasperformed on an ABI 310 genetic analyser (Perkin Elmer), using 5% GeneScan polymer and 1x TBE buffercontaining 10% glycerol. Primers used for amplification of the exon 11 fragment (242-bp) and complete exon 20with intron/exon boundaries (401 bp) were respectively: F11: 5’-AGA TTT CTC TCC ATA TCT GAT TTC A-3’;R11: 5’-GGT AAC CCT GAG CCA AAT GT-3’ and F20: 5’-ATA TGA CGT GTC TG CTC CAC-3’; R20: 5’-GGG AAT CCA AAT TAC ACA GC-3’. PCR conditions were: 35 cycles: 94OC-1s, 55OC-1s, 72OC-5s (PerkinElmer 9700 PCR System).

Genomic DNA/cDNA comparisons

Isolation of genomic DNA and total RNA was performed according to standard procedures (McIndoe et al.,1995; Chomczynski and Sacchi, 1987). Random hexamers and AMV reverse transcriptase (Promega) were used toprepare cDNA from total RNA, according to the manufacturer’s recommendations. Primer sequences and PCRconditions used to amplify BRCA1 fragments from genomic DNA were the same as described above forsegregation analysis or described earlier (Sobczak et al., 1997). Amplifications from cDNA were conducted asfollows. First, a larger fragment of BRCA1 cDNA was amplified from the reverse transcript with primer F11 (asabove) and R16: 5'-CAG CAG TAT CAG TAG TAT GAG C-3’. This 1505-bp fragment spanning exons 11-16could be obtained only from cDNA but not from genomic DNA. The specific PCR product served as a template forseminested amplification of the exon 11 fragment containing the 3667A/G polymorphism. The genomicDNA/cDNA comparisons were performed using combined SSCP/heteroduplex analysis of either radiolabelled orfluorescently labelled RCR products in conditions described above.

RESULTS

Our first experiments were conducted to establish which polymorphic variants of the BRCA1 gene share thesame chromosome with the 12-bp insertion. Five carriers of the insertion, who were probands from the familiesdescribed earlier (Sobczak et al., 1997) and 118 unrelated noncarriers were genotyped at eight BRCA1polymorphic loci (Table 1). In agreement with the earlier results (Malone et al., 1998; Sholl et al., 1999), these

Table 1. Common BRCA1 polymorphisms and genotype frequencies in Polish carriers and noncarriers of the12-bp insertion

int. 8ex.9–57-/insT

ex.112201C/T

ex.112430T/C

ex.113232A/G

ex.113667A/G

ex.134427T/C

int.16ex.17–68

G/A

int.18ex18+66

A/G

int.20 ex20+48

-/ins12

Numberof

genotypes

aa aa aa aa aa aa aa aa -/- 68ab ab ab ab ab ab ab ab -/- 44ab ab ab ab ab ab ab ab -/+ 2bb bb bb bb bb bb bb bb -/- 6bb bb bb bb bb bb bb bb -/+ 3

Positions of the polymorphic nucleotide in cDNA and introns are indicated. The more frequent base is shown to the left, and the less frequent baseto the right of the slash symbol, aa - homozygote for the more frequent polymorphic variant, bb - homozygote for the less frequent variant, ab -heterozygote.

Imbalance of BRCA1 Transcripts 3

polymorphisms are in complete linkage disequilibrium. Interestingly, the 12-bp insertion occurs only in the contextof two genotypes bb and ab. In three cases, the insertion is associated with homozygotes for the less frequentvariant bb. On the other hand, only two out of the 46 heterozygotes and none of the homozygotes aa are 12 bpinsertion carriers. This implies that the 12-bp insertion is most likely linked to the haplotype of the less frequentpolymorphic variants.

To provide direct evidence for the linkage of the 12-bp insertion to the haplotype of polymorphic variants b,also in heterozygous individuals, we performed segregation analysis in the family of case 75 (Figure 1). There isno breast or ovarian cancer in this family. The proband’s mother was diagnosed with uterine cancer and theproband’s father died of lung cancer. The 12-bp insertion was transmitted to the proband from her mother, and oneof the proband’s sons also inherited the insertion. All insertion carriers in this family are heterozygotes 3667A/G.The second proband’s son, who does not carry the insertion, is homozygote A/A for the same intragenicpolymorphism. The observed pattern of inheritance leaves no doubt that the 12-bp insertion/duplication segregatesin this family together with the less frequent polymorphic variant 3667G.

Figure 1. Segregation of the 3667G polymorphic variant with the 12-bp insertion in the family of case 75. In thefamily pedigree, circles and squares indicate females and males, respectively, UtCa – uterine cancer, LuCa – lung

cancer. The individuals whose DNA was analysed are numbered. A - SSCP analysis of BRCA1 fragment spanningthe 3667A/G polymorphism. Primer F11 labelled with TAMRA (black) and primer R11 labelled with JOE (grey).B - SSCP analysis of BRCA1 fragment 20 harbouring the 12-bp insertion. Primer F20 labelled with FAM (grey)

and primer R20 labelled with ROX (black).

Our further experiments were designed to find out whether one type of transcript indeed escapes detection in thecDNA of the insertion carrier. We isolated total RNA from the peripheral blood lymphocytes of three healthywomen. Two of these women, cases 75 and 82, carried the 12-bp insertion in intron 20. Case 82 was homozygoteG/G and case 75 was, as mentioned earlier, heterozygote A/G for BRCA1 polymorphism at nucleotide 3667 inexon 11. The third woman, control 7, who did not carry the insertion, was heterozygote 3667A/G. The results ofthe genomic DNA/cDNA comparisons, using combined SSCP/heteroduplex analysis, are shown in Figure 2. It isapparent that patterns of single strand conformers and duplexes obtained from genomic DNA and cDNA areidentical for the heterozygous control 7 (Figure 2A). This indicates that this woman remains heterozygous also inBRCA1 mRNA. The single strand and duplex patterns of the homozygous case 82, although different from thoseobtained for control 7, are the same for both genomic DNA and cDNA. A clear difference is observed in the SSCPand duplex patterns obtained from the genomic DNA and cDNA of case 75 (Figure 2A, B, C). This result indicatesthat only one type of BRCA1 mRNA gave a significant contribution to the PCR product, which shows ahemizygous SSCP/duplex pattern, identical to that of the G/G homozygote. Reamplification and sequencing of theseparated duplex bands confirmed that the mRNA that was detected by cDNA analysis contained the less frequentpolymorphic variant 3667G. Thus, the observed mRNA was derived from the same BRCA1 allele that alsocontained the 12-bp insertion in intron 20.

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Figure 2. The SSCP/heteroduplex analysis of the 3667A/G polymorphism in genomic DNA and cDNA. Threeprobands from families: 7, 82 and 75, were selected, as specified in panel A, according to their different carrier

status for the 12-bp insertion, and exon 11 polymorphism. A - analysis of radiolabelled PCR products, inset - DNAsequencing across the region of nucleotide 3667 for the allele detected in the cDNA of case 75 (left) and for the

other allele (right), B - SSCP and C - duplex analysis of fluorescently labelled fragment of the exon 11 of case 75,spanning the 3667A/G polymorphism. The primer sequences and labels used were the same as described in the

legend to Figure 1.

DISCUSSION

In this study we have found the BRCA1 intragenic haplotype to which the 12-bp insertion in intron 20 waslinked. We have also shown that only the transcript originating from the BRCA1 allele with the insertion wasdetected in the Polish carrier, who is heterozygous for the intragenic polymorphisms.

Imbalance of BRCA1 Transcripts 5

The linkage of the 12-bp insertion to the haplotype of the less frequent BRCA1 polymorphic variants, which isdemonstrated in this paper, has been postulated also by other authors recently (Sholl et al., 1999). Those authors, aswell as others (Dork et al., 1999), did not find any imbalance of BRCA1 transcripts in the blood cells of two breastcancer patients who carried the insertion. However, the transcript imbalance is clearly shown in this study, as wellas in the earlier report of the Spanish workers (Robledo et al., 1997). Therefore, two questions need to be asked:Why is a transcript not detected from the BRCA1 allele lacking the 12-bp insertion in the presence of a transcriptfrom allele carrying the insertion? and: Why has this happened in only two out of four insertion carriersinvestigated thus far?

The observed transcript imbalance could result, on the one hand, from a normal expression of the allelecontaining the insertion and a strongly reduced expression of the other allele in both the Polish and Spanishcarriers. The possibility (i) - that an undetected deleterious LOT (loss of transcript) mutation occurred in each ofthese two carriers in combination with the 12-bp insertion in the second BRCA1 allele seems, however, ratherunlikely. The entire BRCA1 coding sequence of case 75, together with exon/intron boundaries, was scanned formutations by a combined SSCP/heteroduplex analysis and no mutation was found (Sobczak et al., 1997).Similarly, the Spanish workers did not find any other mutation in their sample (Robledo et al., 1997).

On the other hand, the observed transcript imbalance could result from strongly enhanced expression of allelewith the 12-bp insertion and normal expression of the other BRCA1 allele. In accordance with possibility (ii) - aregulatory GOT (gain of transcript) mutation linked to the insertion, but located outside the region scanned formutations, could be responsible for over-expression of this allele in the Polish and Spanish carriers. Alternatively,according to possibility (iii) - the duplicated 12 bp sequence itself could be a GOT mutation involved in positiveregulation of BRCA1 transcription or splicing. Mutations that occur in introns and up-regulate transcript are notunusual (Cohen et al., 1988; Malkinson et al., 1994). In this case, the specificity of the effect observed in two outof four investigated carriers could only be explained by the different abilities of different carriers to exert thisspecific expression-activating mechanism.

Explanations (i) and (ii) imply that the 12-bp insertion/duplication in BRCA1 intron 20 is a rare neutralpolymorphism, accompanying some undetected causative mutations, which either down-regulate or up-regulate theBRCA1 transcript. According to alternative (iii), the insertion itself is a causative factor that facilitates expressionof its own allele. The GOT mutation in a tumour-suppressor gene, proposed by alternatives (ii) and (iii), shouldplay rather a protective role in breast and ovarian cancer tissue by compensating for the effect of LOH (loss ofheterozygosity) at the BRCA1 locus, if the insertion allele remains. On the other hand, the loss of insertion allele inbreast tumours with LOH would contribute to cancer development. The latter scenario found support in the resultsof British workers who analysed tumour tissue from three breast cancer patients, and in each case the insertionallele was lost (Campbell et al., 1997).

In summary, our study presents new results that contribute to a better understanding of the role played by theBRCA1 variant of controversial clinical significance. Although interpretation of our data in terms of the neutral orpositive role of the insertion in the regulation of BRCA1 expression is inconclusive, it appears more certain nowthat the intronic insertion is not a cancer-predisposing mutation.

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