LETTER TO THE EDITOR

Caution should be taken in the methodology used to confirmc.156_157insAlu BRCA2 mutation

Patricia Machado Æ Fatima Vaz

Received: 1 July 2008 / Accepted: 1 July 2008 / Published online: 17 July 2008

� Springer Science+Business Media, LLC. 2008

To the Editor

We read with great interest the article by Peixoto et al. [1]

about the screening of the c.156_157insAlu BRCA2

mutation in Northern Portugal. As we previously reported,

[2] this BRCA2 rearrangement first described by Teugels

et al. [3] in a Portuguese family living in Belgium, will

surely be the explanation for breast and ovarian cancer risk

in several families of Portuguese ancestry. Most of these

families have been, as reported by Peixoto et al. [1], con-

sidered negative by the usual screening methods.

We are writing this letter to comment about the method-

ology that must be used to confirm the splicing effect of the

c.156_157insAlu mutation. Peixoto et al. report RT–PCR

data in their paper, that corroborates our own data concerning

the segregation of c.156_157insAlu BRCA2 mutation and

cancer in individuals from positive families, but this should

not be taken as the most appropriate methodology for the

confirmation of this rearrangement. The primers used for

their RT–PCR reactions amplify not only the pathogenic

Alu-mediated splicing product, lacking exon 3, but also a

physiologic, ubiquitous splicing product, also exon 3-defi-

cient, observed in individuals with sporadic cancers and

healthy persons [4–8]. Although both splicing products lack

exon 3, only the one associated with c.156_157insAlu

mutation includes at least the first part of BRCA2 exon 10 and

this distinguishes them [9]. Primers like the ones used by

Peixoto et al., located in exons 1 and 6 of BRCA2 cDNA,

amplify (as shown in Fig. 1a of their report) three fragments

in all samples, both c.156_157insAlu carriers and wild type

carriers. As a confirmation step, RT–PCR is expected to

amplify only true positive samples, originating from really

positive c.156_157insAlu carriers. Although the authors

explain the three bands observed, as a rule, for diagnosis, we

should avoid relying on subjective observations like band

intensity.

For reasons of length, we did not include the reference

to the ubiquitous exon 3-defficient transcript in our original

paper [2] but we did discuss it when, very pertinently, Diez

et al. [4] wrote a letter raising doubts about our method-

ology to confirm c.156_157insAlu as a pathogenic BRCA2

mutation. They were very well aware of previous data

showing a physiological BRCA2 transcript lacking exon 3

[5–8]. Their questions allowed us, in a reply [9], to explain

that indeed, we had tried several primers for our RT–PCR

confirmation step and eventually chose primers 1FcDNA/

10RcDNA, first described by Nordling et al. [10], because

these never amplify the ubiquitous splicing transcript. As in

the case of our families, and in the case of the BRCA2

family described by Nordling et al., they only amplify a

BRCA2-mutated associated transcript.

Our three-step-PCR (patent pending) was optimized and

every step decided after several studies. The first two steps

described by Peixoto et al., are so similar to our own that

we believe that no false positive was reported: from our

experience, with a total of 29 c.156_156insAlu families

already diagnosed, if the first two steps are positive the

third always confirms it. But BRCA1 and BRCA2 molecular

diagnosis has implications for individuals and their fami-

lies at clinical, emotional, legal and ethical levels. No

doubt must remain in the methodologies for screening,

P. Machado � F. Vaz (&)

Molecular Biology Department, Instituto Portugues

de Oncologia de Lisboa, Francisco Gentil, Lisbon, Portugal

e-mail: fvaz@ipolisboa.min-saude.pt

P. Machado

e-mail: pmachado@ipolisboa.min-saude.pt

F. Vaz

Breast Cancer Risk Evaluation Clinic, Instituto Portugues

de Oncologia de Lisboa, Francisco Gentil, Lisbon, Portugal

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Breast Cancer Res Treat (2009) 116:617–618

DOI 10.1007/s10549-008-0124-0

most specially if one refers to methods screening a specific

mutation, that is, so far, the most frequent BRCA2 rear-

rangement described to date and the first BRCA founder

mutation described in the Portuguese population.

References

1. Peixoto A, Santos C, Rocha P et al (2008) The c.156_157insAlu

BRCA2 rearrangement accounts for more than one-fourth of

deleterious BRCA mutations in northern/central Portugal. Breast

Cancer Res Treat (Mar):25 (Epub ahead of print)

2. Machado PM, Brandao RD, Cavaco BM et al (2007) Screening

for a BRCA2 rearrangement in high-risk rearrangement in high-

risk breast/ovarian cancer families: evidence for a founder effect

and analysis of the associated phenotypes. J Clin Oncol 25:2027–

2034. doi:10.1200/JCO.2006.06.9443

3. Teugels E, De Brakeleer S, Goelen G et al (2005) De novo Alu

element insertions targeted to a sequence common to the BRCA1and BRCA2 genes. Hum Mutat 26:284. doi:10.1002/humu.9366

4. Dıez O, Gutierrez-Enrıquez S, Ramon y Cajal T et al (2007)

Caution should be used when interpreting alterations affecting the

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Clin Oncol 25:5035–5036. doi:10.1200/JCO.2007.13.4346

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the transactivation domain of the BRCA2 gene and mutations in

the splice acceptor site of intron 2. Genes Chromosomes Cancer

26:381–382. doi:10.1002/(SICI)1098-2264(199912)26:4\381::

AID-GCC14[3.0.CO;2-N

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7. Speevak MD, Young SS, Feilotter H et al (2003) Alternatively

spliced, truncated human BRCA2 isoforms contain a novel coding

exon. Eur J Hum Genet 11:951–954. doi:10.1038/sj.ejhg.5201063

8. Koul A, Nilbert M, Borg A (1999) A somatic mutation in RER

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24:207–212. doi:10.1002/(SICI)1098-2264(199903)24:3<207::

AID-GCC5>3.0.CO;2-3

9. Machado PM, Cavaco BM, Brandao RD et al (2007) In reply to:

Dıez O, Gutierrez-Enrıquez S, Ramon y Cajal T et al. Caution

should be used when interpreting alterations affecting the exon 3

of the BRCA2 gene in breast/ovarian cancer families. J Clin

Oncol 25:5036–5038. doi:10.1200/JCO.2007.13.5442

10. Nordling M, Karlsson P, Wahlstrom J et al (1998) A large

deletion disrupts the exon 3 transcription activation domain of the

BRCA2 gene in a breast/ovarian cancer family. Cancer Res

58:1372–1375

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