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Novel de novo BRCA2mutation in a patient with a family history of breast cancer

Novel de novo BRCA2mutation in a patient with a family history of breast cancer Background: BRCA2 germ-line mutations predispose to breast and ovarian cancer. Mutations are widespread and unclassified splice variants are frequently encountered. We describe the parental origin and functional characterization of a novel de novo BRCA2 splice site mutation found in a patient exhibiting a ductal carcinoma at the age of 40. Methods: Variations were identified by denaturing high performance liquid chromatography (dHPLC) and sequencing of the BRCA1 and BRCA2 genes. The effect of the mutation on splicing was examined by exon trapping in COS-7 cells and by RT-PCR on RNA isolated from whole blood. The paternity was determined by single nucleotide polymorphism (SNP) microarray analysis. Parental origin of the de novo mutation was determined by establishing mutation-SNP haplotypes by variant specific PCR, while de novo and mosaic status was investigated by sequencing of DNA from leucocytes and carcinoma tissue. Results: A novel BRCA2 variant in the splice donor site of exon 21 (nucleotide 8982+1 G→A/ c.8754+1 G→A) was identified. Exon trapping showed that the mutation activates a cryptic splice site 46 base pairs 3' of exon 21, resulting in the inclusion of a premature stop codon and synthesis of a truncated BRCA2 protein. The aberrant splicing was verified by RT-PCR analysis on RNA isolated from whole blood of the affected patient. The mutation was not found in any of the patient's parents or in the mother's carcinoma, showing it is a de novo mutation. Variant specific PCR indicates that the mutation arose in the male germ-line. Conclusion: We conclude that the novel BRCA2 splice variant is a de novo mutation introduced in the male spermatozoa that can be classified as a disease causing mutation. Page 1 of 7 (page number not for citation purposes) BMC Medical Genetics 2008, 9:58 http://www.biomedcentral.com/1471-2350/9/58 written consent, blood and tumour samples were col- Background Germ-line BRCA2 (MIM# 600185) mutations in female lected from the proband for mutation screening and after carriers confer a lifetime risk exceeding 80% for breast renewed consent a third blood sample was collected for cancer and 20% for ovarian cancer, and a moderate RNA analysis. Moreover, blood and tumour samples from increased risk of other cancer types [1,2]. Studies have the patient's mother, and blood samples from the indicated that the risk of ovarian cancer is greatest in patient's father and sister were collected. The family his- women with BRCA2 mutations localized in nucleotides tory was verified using the registry of the DBCG (Danish 3035–6629 [3,4]. Several genes are likely to be involved in Breast Cancer Cooperative Group), hospital medical breast cancer predisposition, but no susceptibility gene records and pathology reports, and genetic counselling aside BRCA2 and BRCA1 is likely to account for a large was provided for each family member. Since the study is fraction or a major increase in risk [5]. part of normal diagnostic procedures no ethical approval was obtained. The study was conducted in accordance The BRCA2 gene spans approximately 70 kb and is com- with the Helsinki Declaration. posed of 27 exons, which encodes a protein of 3418 BRCA1 and BRCA2 screening amino acids. BRCA2 is involved in homologous recombi- nation [6,7], but is also suggested to play a role in tran- Genomic DNA was purified from whole blood using the scriptional regulation [8] and cell cycle control [9]. QIAamp DNA mini kit (Qiagen) according to the manu- Mutations are distributed throughout the entire coding facturer's instructions. BRCA1 and BRCA2 were amplified region of BRCA2 and to date numerous deleterious muta- using intronic primer pairs flanking each exon. PCR prod- tions have been reported (Breast Cancer Information Core ucts were pre-screened by dHPLC (denaturing high per- [BIC]; [10]). The majority of patients with BRCA1 or formance liquid chromatography) using the WAWE BRCA2 associated breast and/or ovarian cancer have a system (Transgenomic) and sequenced using an ABI3730 family history, although healthy male carriers may DNA analyzer (Applied Biosystems). Sequence variations obscure the apparent dominant trait. Several BRCA2 were verified in a new blood sample. Moreover genomic founder mutations have been identified, including the DNA was examined by MLPA analysis (MRC-Holland). Ashkenazi Jewish nucleotide 6174delT mutation and the The BRCA1 variant is numbered according to GenBank , in which the A in the AUG Icelandic nucleotide 999del5 mutation [11,12]. In con- accession number U14680 trast, only two mutations in BRCA2 and one in BRCA1 start codon has number 120, whereas the BRCA2 variant have been reported as de novo mutations [13-15], but since is numbered according to GenBank accession number a positive family history is one of the criteria for mutation NC_000059, in which A in the AUG start codon has screening, it is possible that we overlook a number of number 229. Moreover, the BRCA1 and BRCA2 variants patients with these mutations. are numbered according to GenBank accession number NC_000017.9 and NC_000013.9 using the guidelines Here we report the functional characterization of a novel from the Human Genetic Variation Society [16]. de novo BRCA2 splice site mutation located in the inter- vening sequence (IVS) of exon 21 (nucleotide 8982+1 Vector constructs G→A/c.8754+1 G→A) in a Danish breast cancer patient The pSPL3 vector was obtained from Gibco-BRL. pSPL3- with a family history of breast cancer. The parental origin BRCA2-wild-type and pSPL3-BRCA2-mutant plasmids, of the mutation is assigned to the father. containing BRCA2 exon 21 and flanking intron sequences, were constructed by PCR using purified DNA from human blood samples and the following oligonu- Methods Patients cleotides: BRCA2-F, 5'-GATCACGAATTCTTCCT- Family B49363 is a 23 member, three-generation kindred GGAAAACTTATAGCA-3' and BRCA2-R 5'- with two affected subjects. A 40 year old woman was GATCACCTCGAGTTAGGGTAGAGGATTATCAAGTACA- referred to genetic counseling two months after she had 3'. The PCR products were treated with EcoRI and XhoI breast-conserving surgery at Roskilde County Hospital and cloned into the pSPL3 vector. All constructs were ver- with radical excision of an 8 mm large invasive ductal car- ified by sequencing. cinoma. The tumor was estrogen and progesterone recep- tor positive and had malignancy grade II. The patient Cell culture and transfections received adjuvant radiotherapy followed by seven series of COS-7 cells were cultured in Dulbecco's modified Eagle's chemotherapy with CEF and tamoxifen. The patient's medium (DMEM) containing 4500 mg/l glucose supple- mother had a mastectomy at age 59 with excision of a 22 mented with 10% foetal bovine serum, 100 U of penicil- mm large estrogen and progesterone receptor positive lin per ml, and 100 μg of streptomycin per ml at 5% CO invasive ductal carcinoma, malignancy grade II, and post- and 37°C. One day before transfection, cells were seeded operatively she received tamoxifen. Following verbal and in 6-well culture dishes at a density of 4 × 10 cells/well. Page 2 of 7 (page number not for citation purposes) BMC Medical Genetics 2008, 9:58 http://www.biomedcentral.com/1471-2350/9/58 Cells were transfected with a total of 4 μg plasmid DNA forward: 5'-CAGACCCAGCTTACCTTGAAA-3'), and the using FuGENE 6 transfection reagent (Roche) according two SNP alleles in rs4942485 (rs4942485*A reverse: 5'- to the manufacturer's instructions. Transfection efficien- CTACATTACAGATGGCTAATATCTGATT-3' and cies for each series were determined by cotransfection of rs4942485*G reverse: 5'-CATTACAGATGGCTAATATCT- pEGFP plasmid (Clontech). The next day the media was GAAC-3'. Primer sets were tested on homozygote controls changed and after another 24 h the cells were harvested and unspecific annealing was avoided after introducing and total RNA was isolated using Trizol reagent (Invitro- mismatches (marked in bold). PCR was carried out using gen). standard conditions according to the manufacturer's pro- PLUS tocols using the Expand High Fidelity Taq DNA Exon trapping analysis polymerase (Roche). Allele sizes were: BRCA2 c.8754+1-G First strand cDNA was synthesized using 1 μg RNA, 20 μM and rs942485-G = 2297 bp, BRCA2 c.8754+1-*A and SA2 primer (5'-ATCTCAGTGGTATTTGTGAGC-3') and M- rs942485-G = 2298 bp, BRCA2 c.8754+1-G and MuLV reverse transcriptase (New England Biolabs). The rs942485-A = 2300 bp, BRCA2 c.8754+1-*A and cDNAs were amplified with pSPL3 vector-specific primers rs942485-A = 2301 bp. (5'-TCTGAGTCACCTGGACAACC-3' and 5'-ATCTCAGT- GGTATTTGTGAGC-3') and the PCR products were Results and discussion resolved on a 2% agarose gel. All experiments were The patient was diagnosed with breast cancer at the age of repeated three times. 40. Since her mother had breast cancer at the age of 59 (Fig. 1), the patient was referred to genetic counselling. RNA analysis No other family members had breast or ovarian cancer. A fresh blood sample was obtained from the patient. Total We analysed the entire coding region and the exon-intron cellular RNA was isolated with Trizol (Invitrogen) accord- boundaries of BRCA1 and BRCA2 from genomic DNA by ing to the manufacturer's instructions. For reverse tran- dHPLC and sequencing and for large genomic rearrange- scription-PCR (RT-PCR), cDNA was synthesized using the ments by MLPA analysis. The analysis revealed a polymor- AMV reverse transcriptase (Promega) as described by the phism in exon 11 of BRCA1 (nucleotide 1186 A→G/ supplier. The cDNA were amplified with the BRCA2 spe- c.1067 A→G), which is observed in approximately 5% of cific primers 5'-CGGCCTGCTCGCTGGTAT-3' and 5'- a control group [18], and a nucleotide 8982+1 G→A/ GCCTTCCTAATTTCCAACTGGATCTG-3' resulting in a c.8754+1 G→A mutation in BRCA2 that has not previ- 503 bp fragment. The samples were separated by agarose ously been reported in the BIC database (Fig. 2). The gel electrophoresis and visualized by ethidium bromide mutation occurs at the first base of the conserved GU staining. Finally, the bands were purified, cloned into splice donor site of intron 21. The mutation was verified pCR-Blunt II-TOPO (Invitrogen) and sequenced using an in a second blood sample as well as in breast cancer ABI3730 DNA analyzer (Applied Biosystems). tumour tissue. SNP chip analysis To functionally characterize the BRCA2 nucleotide Genomic DNA was applied to 250K StyI (~238.000 SNPs) 8982+1 G→A/c.8754+1 G→A variant, a fragment con- SNP-microarray chips and processed according to the taining BRCA2 exon 21 (122 bp), 449 bp of intron 20 manufacturer's instructions (Affymetrix, UK). Briefly, 250 (IVS20) and 408 bp of intron 21 (IVS21) containing the ng of genomic DNA was digested with StyI and ligated to wild-type or the nucleotide 8982+1 G→A/c.8754+1 G→A adapters. Adapter-ligated DNA was amplified, purified, variant, respectively, was cloned into the minigene vector fragmented and labeled with biotin and hybridized to the pSPL3 containing exons from HIV-tat under the control of arrays for 18 h. The Affymetrix 450 fluidics station and the the SV40 promoter (Fig. 3A) [19]. Constructs containing Affymetrix 3000 G7 gene scanner were used to wash, either the wild-type or the mutant IVS21 sequence was stain, and scan the arrays. The CEL files were analysed transfected into COS-7 cells. After 48 hours mRNA was using the BRLMM algorithm from Affymetrix Genotyping purified and examined by RT-PCR. The splicing products analysis software (GTYPE). Relationship was inferred by were separated on a 2% agarose gel. The wild-type con- estimating the pairwise identical by descent (IBD) shar- struct yields a product of 299 bp and a product of 177 bp ing, as described [17]. if exon 21 is excluded from the transcript (exon skipping). Moreover a larger product than 299 bp would demon- Assignment of parental origin strate that additional intron sequence is included in the Variant specific primers were designed using the software transcript. The normal wild-type BRCA2 exon 21 (pSPL3- Primer3 in combination with ClustaIW alignments. The BRCA2-wt) generated one transcript comprising the primer sets distinguish the wild-type allele (BRCA2 expected 299 bp, while the BRCA2 nucleotide 8982+1 c.8754+1-G forward: 5'-AGACCCAGCTTACCTTGACG- G→A/c.8754+1 G→A mutant (pSPL3-BRCA2-mut) 3') from the mutation carrying allele (BRCA2 c.8754+1-A yielded a band with slightly higher mobility (Fig. 3B). Page 3 of 7 (page number not for citation purposes) BMC Medical Genetics 2008, 9:58 http://www.biomedcentral.com/1471-2350/9/58 products were analysed on a 1% agarose gel (Fig. 4). Two PCR products – one with the expected size of the wild- type (503 bp) and an additional RT-PCR product (549 bp) – were amplified from the patient. Cloning and Acc. 49 yrs sequence analysis of the latter verified the inclusion of 46 bp of intron 21 (data not shown). We examined the patient's mother, who was affected with BRC 59 the same type of breast cancer at the age of 59 years, for BRCA2 c.8754+1 G G G G the same mutation, but she did not have this mutation in rs4 942 485 G G A A either DNA purified from blood or breast cancer tumour samples. Therefore mosaicism was excluded. Moreover, the father and the patient's sister did not carry the muta- tion. To establish the paternity we performed a SNP microarray analysis using 250K Sty gene array (Affyme- trix). The analysis provided the relatedness estimates BRC 40 (Table 1), where k0 is the probability that pairs of individ- BRCA2 c.8754+1 A* G uals at a random loci share no allele identical by descent G G rs4 942 485 G A (IBD), k1 is the probability that pairs of individuals at a G A random loci share one allele IBD, and k2 the probability Fa Figure 1 mily pedigree that pairs of individuals at a random loci share two allele Family pedigree. Breast cancers are indicated as well as IBD. The expected relatedness for full siblings is k0 = 0.25, the age at diagnosis. Acc, accident; BRC, breast cancer. The k1 = 0.5, k2 = 0.25, for parent offspring k0 = 0, k1 = 1, k2 number following the cancer gives the age at diagnosis. = 0, for unrelated k0 = 1, k1 = 0, k2 = 0, which in all cases Moreover, the genotypes from variant specific PCR are indi- fits in this family. We therefore conclude that the identi- cated. Diagonal slash indicates deceased. The proband is indi- fied mutation in the proband is a de novo mutation in cated with an arrow. Proband = individual 1, Sister = BRCA2. In contrast to the previously identified de novo individual 2, Mother = individual 3, Father = individual 4. BRCA2 nucleotide 3034del4 and BRCA1 nucleotide 3888delGA mutations [14,15], the mutation reported in our study resides in a new position not previously Sequencing revealed, that it contained an additional 46 reported in the BIC database. This was also the case with bp from intron 21 generating a transcript of 345 bp (Fig. the BRCA2 nucleotide 7260insA mutation [13]. Several 3C), indicating that it activates a cryptic splice site follow- other variants have been detected in the vicinity of the ing these 46 bp (Fig. 1). To provide direct evidence for the splice donor site in exon 21, including nucleotide 8982 aberrant splicing, RNA was isolated from whole blood of G→A/c.8754 G→A (reported once), nucleotide 8982+1 the affected patient and RT-PCR was performed before the A→G/c.8754+4 A→G (reported seven times), nucleotide Exon 21 Intron 21 G>A Iden Figure 2 tification of the BRCA2 nucleotide 8982+1 G→A/c Identification of the BRCA2 nucleotide 8982+1 G→A/c.8754+1 G→A variant. DNA was purified from whole blood and BRCA2 exon 21 was amplified using the primers 5'-CTTTGGGTGTTTTATGCTTGT-3' and 5'-CTGGCACATCACT- GAAAATC-3' and sequenced. The analysis revealed a nucleotide 8982+1 G→A/c.8754+1 G→A mutation in BRCA2 (sense strand). The nucleotide 8982+1 G→A/c.8754+1 G→A mutation and the cryptic splice site are underlined. Page 4 of 7 (page number not for citation purposes) BMC Medical Genetics 2008, 9:58 http://www.biomedcentral.com/1471-2350/9/58 SD IVS20 IVS21 SA BRCA2 exon 21 pSPL3 pSPL3 449 bp 122 bp 408 bp Exon 21 Exon 21 wild-type mutant 766 bp 500 bp 345 bp 299 bp 200 bp Exon 21 Intron 21 pSPL3 Exon tra Figure 3 pping analysis Exon trapping analysis. (A) Structure of the exon trapping vector pSPL3 containing the BRCA2 exon 21 and 449 bp of intron 20 and 408 bp of intron 21, respectively containing the wild-type or the nucleotide 8982+1 G→A/c.8754+1 G→A vari- ant. (B) COS-7 cells were transfected with pSPL3-BRCA2-exon 21 wild-type or pSPL3-BRCA2-exon 21 mutant plasmids. Total RNA was isolated, RT-PCR analysis was performed and the PCR products (in duplicates) were resolved on a 2% agarose gel. The 299 bp product corresponds to wild-type exon 21 (unaltered splicing), while the 345 bp product corresponds to exon 21 and the inclusion of 46 bp of intron 21. The sizes of the DNA marker are indicated to the right. (C) Sequence of exon 21 (345 bp band). The nucleotide 8982+1 G→A/c.8754+1 G→A mutation and the TAA stop codon are underlined (sense strand). Page 5 of 7 (page number not for citation purposes) BMC Medical Genetics 2008, 9:58 http://www.biomedcentral.com/1471-2350/9/58 Table 1: Relatedness estimates. M 1 Individual Individual k0 k1 k2 Relationship 1000 - 900 - 1 2 0.23 0.49 0.28 full siblings 800 - 1 3 0.01 0.99 0.00 parent offspring 1 4 0.01 0.99 0.00 parent offspring 700 - 2 3 0.01 0.98 0.01 parent offspring 2 4 0.01 0.99 0.01 parent offspring 600 - 3 4 0.98 0.02 0.00 unrelated Exon 21 mutant 500 - Exon 21 wild-type Relationship was infered by estimating the pairwise identical by descent (IBD) sharing as described in [17]. k0 is the probality that pairs of 400 - individuals at a random loci share no allele IBD, k1 the probality that pairs of individuals at a random loci share one allele IBD, and k2 the probality that pairs of individuals at a random loci share two allele IBD. The expected relatedness for full siblings is k0 = 0.25, k1 = 0.5, 300 - k2 = 0.25, for parent offspring k0 = 0, k1 = 1, k2 = 0, and for unrelated k0 = 1, k1 = 0, k2 = 0. The individual number is indicated in figure 1. noteworthy that the age of the probands parents in our study was only 19 and 21 years at the time of birth of their RT-PC from the proband Figure 4 R was performed on RNA purified from whole blood affected daughter. RT-PCR was performed on RNA purified from whole blood from the proband. The cDNA was amplified with Conclusion specific BRCA2 primers. The sample was separated by agar- We conclude that the BRCA2 nucleotide 8982+1 G→A/ ose gel electrophoresis and visualized by ethidium bromide c.8754+1 G→A mutation is a de novo mutation arising staining. Two RT-PCR products (503 bp and 549 bp) were obtained from the patient (Lane 1). The sizes of the DNA from the male germ line. The mutation is not previously marker are indicated to the left. The PCR products were reported in the BIC database. It leads to the activation of cloned and sequence analysis revealed that the 549 bp band a cryptic splice site 46 base pairs 3' of exon 21 and intro- contained the inclusion of 46 bp of intron 21 (data not duces a premature stop codon and thereby a truncated shown). BRCA2 protein. Therefore, this mutation can be classified as a disease-causing mutation. Analysis of intronic BRCA1 and BRCA2 variants by functional splicing assays can pro- 8982+5 G→A/c.8754+5 G→A (reported once) and nucle- vide information that can be used clinically. otide 8982+5 G→T/c.8754+5 G→T (reported once), sug- gesting that this region could be prone to mutations. Competing interests The authors declare that they have no competing interests. To determine the parental origin of the mutation, variant specific PCR was performed using primers designed to dis- Authors' contributions TVOH designed the study, supervised exon trapping, tinguish between two SNP alleles and the mutation carry- ing allele from the wild-type allele. The father was interpreted the results, and wrote the draft of the paper. homozygote for the haplotype GG, while the mother was MLB was involved in the designing of the study, the vari- homozygote for the haplotype GA (see Fig. 1). PCR anal- ant specific PCR, and the writing of the manuscript. LJ per- ysis of the proband revealed a band using mutant BRCA2 formed the RT-PCR analysis on RNA from whole blood and rs4942485*G primers, indicating that the haplotype and was involved in the writing of the manuscript. AA per- *AG must derive from the father, and that the mutation formed the relationship SNP analysis. BF–B analysed the therefore arose in the testicular germ cells changing carcinoma samples. HE designed and performed the vari- BRCA2 nucleotide 8982+1/c.8754+1 from G to A. This is ant specific PCR. BE selected the patients and participated in agreement with previous findings in BRCA1 [14], in genetic counseling and was involved in the writing of whereas the studies describing de novo mutations in the manuscript. FCN designed the study and was involved BRCA2 were unable to determine parental origin [13,15]. in the writing of the manuscript. All authors have read and Examinations of other cancer families, including MEN2B approved the final manuscript. and retinoblastoma families have indicated that de novo mutations primarily occur in the male germ line [20,21] Acknowledgements Stine Østergaard and Dorthe Petersen are acknowledged for technical and that the fathers age is a major determinant since assistance. Written consent was obtained from the patient or their rela- mutations accumulate during life [22]. It is, however, Page 6 of 7 (page number not for citation purposes) BMC Medical Genetics 2008, 9:58 http://www.biomedcentral.com/1471-2350/9/58 tives for publication of the study. This study was supported by the Neye genomic DNA using exon amplification. Nat Genet 1994, 6(1):98-105. Foundation. 20. Carlson KM, Bracamontes J, Jackson CE, Clark R, Lacroix A, Wells SA Jr., Goodfellow PJ: Parent-of-origin effects in multiple endo- References crine neoplasia type 2B. Am J Hum Genet 1994, 55(6):1076-1082. 21. Kato MV, Ishizaki K, Shimizu T, Ejima Y, Tanooka H, Takayama J, 1. Easton DF, Ford D, Bishop DT: Breast and ovarian cancer inci- dence in BRCA1-mutation carriers. Breast Cancer Linkage Kaneko A, Toguchida J, Sasaki MS: Parental origin of germ-line and somatic mutations in the retinoblastoma gene. Hum Consortium. Am J Hum Genet 1995, 56(1):265-271. 2. Wooster R, Neuhausen SL, Mangion J, Quirk Y, Ford D, Collins N, Genet 1994, 94(1):31-38. 22. Chandley AC: On the parental origin of de novo mutation in Nguyen K, Seal S, Tran T, Averill D, et al.: Localization of a breast cancer susceptibility gene, BRCA2, to chromosome 13q12- man. J Med Genet 1991, 28(4):217-223. 13. Science 1994, 265(5181):2088-2090. 3. Gayther SA, Mangion J, Russell P, Seal S, Barfoot R, Ponder BA, Strat- Pre-publication history ton MR, Easton D: Variation of risks of breast and ovarian can- The pre-publication history for this paper can be accessed cer associated with different germline mutations of the BRCA2 gene. Nat Genet 1997, 15(1):103-105. here: 4. Thompson D, Easton D: The genetic epidemiology of breast cancer genes. J Mammary Gland Biol Neoplasia 2004, 9(3):221-236. 5. 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Publish with Bio Med Central and every 15. van der Luijt RB, van Zon PH, Jansen RP, van der Sijs-Bos CJ, Warlam- scientist can read your work free of charge Rodenhuis CC, Ausems MG: De novo recurrent germline muta- tion of the BRCA2 gene in a patient with early onset breast "BioMed Central will be the most significant development for cancer. J Med Genet 2001, 38(2):102-105. disseminating the results of biomedical researc h in our lifetime." 16. [http://www.hgvs.org/mutnomen]. Sir Paul Nurse, Cancer Research UK 17. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Maller J, Sklar P, de Bakker PI, Daly MJ, Sham PC: PLINK: A Tool Your research papers will be: Set for Whole-Genome Association and Population-Based available free of charge to the entire biomedical community Linkage Analyses. Am J Hum Genet 2007, 81(3):559-575. 18. Hadjisavvas A, Charalambous E, Adamou A, Neuhausen SL, Christo- peer reviewed and published immediately upon acceptance doulou CG, Kyriacou K: Hereditary breast and ovarian cancer cited in PubMed and archived on PubMed Central in Cyprus: identification of a founder BRCA2 mutation. Can- cer Genet Cytogenet 2004, 151(2):152-156. yours — you keep the copyright 19. Church DM, Stotler CJ, Rutter JL, Murrell JR, Trofatter JA, Buckler AJ: BioMedcentral Submit your manuscript here: Isolation of genes from complex sources of mammalian http://www.biomedcentral.com/info/publishing_adv.asp Page 7 of 7 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Medical Genetics Springer Journals

Novel de novo BRCA2mutation in a patient with a family history of breast cancer

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Springer Journals
Copyright
Copyright © 2008 by Hansen et al; licensee BioMed Central Ltd.
Subject
Biomedicine; Human Genetics; Cytogenetics; Gene Function
eISSN
1471-2350
DOI
10.1186/1471-2350-9-58
pmid
18597679
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Abstract

Background: BRCA2 germ-line mutations predispose to breast and ovarian cancer. Mutations are widespread and unclassified splice variants are frequently encountered. We describe the parental origin and functional characterization of a novel de novo BRCA2 splice site mutation found in a patient exhibiting a ductal carcinoma at the age of 40. Methods: Variations were identified by denaturing high performance liquid chromatography (dHPLC) and sequencing of the BRCA1 and BRCA2 genes. The effect of the mutation on splicing was examined by exon trapping in COS-7 cells and by RT-PCR on RNA isolated from whole blood. The paternity was determined by single nucleotide polymorphism (SNP) microarray analysis. Parental origin of the de novo mutation was determined by establishing mutation-SNP haplotypes by variant specific PCR, while de novo and mosaic status was investigated by sequencing of DNA from leucocytes and carcinoma tissue. Results: A novel BRCA2 variant in the splice donor site of exon 21 (nucleotide 8982+1 G→A/ c.8754+1 G→A) was identified. Exon trapping showed that the mutation activates a cryptic splice site 46 base pairs 3' of exon 21, resulting in the inclusion of a premature stop codon and synthesis of a truncated BRCA2 protein. The aberrant splicing was verified by RT-PCR analysis on RNA isolated from whole blood of the affected patient. The mutation was not found in any of the patient's parents or in the mother's carcinoma, showing it is a de novo mutation. Variant specific PCR indicates that the mutation arose in the male germ-line. Conclusion: We conclude that the novel BRCA2 splice variant is a de novo mutation introduced in the male spermatozoa that can be classified as a disease causing mutation. Page 1 of 7 (page number not for citation purposes) BMC Medical Genetics 2008, 9:58 http://www.biomedcentral.com/1471-2350/9/58 written consent, blood and tumour samples were col- Background Germ-line BRCA2 (MIM# 600185) mutations in female lected from the proband for mutation screening and after carriers confer a lifetime risk exceeding 80% for breast renewed consent a third blood sample was collected for cancer and 20% for ovarian cancer, and a moderate RNA analysis. Moreover, blood and tumour samples from increased risk of other cancer types [1,2]. Studies have the patient's mother, and blood samples from the indicated that the risk of ovarian cancer is greatest in patient's father and sister were collected. The family his- women with BRCA2 mutations localized in nucleotides tory was verified using the registry of the DBCG (Danish 3035–6629 [3,4]. Several genes are likely to be involved in Breast Cancer Cooperative Group), hospital medical breast cancer predisposition, but no susceptibility gene records and pathology reports, and genetic counselling aside BRCA2 and BRCA1 is likely to account for a large was provided for each family member. Since the study is fraction or a major increase in risk [5]. part of normal diagnostic procedures no ethical approval was obtained. The study was conducted in accordance The BRCA2 gene spans approximately 70 kb and is com- with the Helsinki Declaration. posed of 27 exons, which encodes a protein of 3418 BRCA1 and BRCA2 screening amino acids. BRCA2 is involved in homologous recombi- nation [6,7], but is also suggested to play a role in tran- Genomic DNA was purified from whole blood using the scriptional regulation [8] and cell cycle control [9]. QIAamp DNA mini kit (Qiagen) according to the manu- Mutations are distributed throughout the entire coding facturer's instructions. BRCA1 and BRCA2 were amplified region of BRCA2 and to date numerous deleterious muta- using intronic primer pairs flanking each exon. PCR prod- tions have been reported (Breast Cancer Information Core ucts were pre-screened by dHPLC (denaturing high per- [BIC]; [10]). The majority of patients with BRCA1 or formance liquid chromatography) using the WAWE BRCA2 associated breast and/or ovarian cancer have a system (Transgenomic) and sequenced using an ABI3730 family history, although healthy male carriers may DNA analyzer (Applied Biosystems). Sequence variations obscure the apparent dominant trait. Several BRCA2 were verified in a new blood sample. Moreover genomic founder mutations have been identified, including the DNA was examined by MLPA analysis (MRC-Holland). Ashkenazi Jewish nucleotide 6174delT mutation and the The BRCA1 variant is numbered according to GenBank , in which the A in the AUG Icelandic nucleotide 999del5 mutation [11,12]. In con- accession number U14680 trast, only two mutations in BRCA2 and one in BRCA1 start codon has number 120, whereas the BRCA2 variant have been reported as de novo mutations [13-15], but since is numbered according to GenBank accession number a positive family history is one of the criteria for mutation NC_000059, in which A in the AUG start codon has screening, it is possible that we overlook a number of number 229. Moreover, the BRCA1 and BRCA2 variants patients with these mutations. are numbered according to GenBank accession number NC_000017.9 and NC_000013.9 using the guidelines Here we report the functional characterization of a novel from the Human Genetic Variation Society [16]. de novo BRCA2 splice site mutation located in the inter- vening sequence (IVS) of exon 21 (nucleotide 8982+1 Vector constructs G→A/c.8754+1 G→A) in a Danish breast cancer patient The pSPL3 vector was obtained from Gibco-BRL. pSPL3- with a family history of breast cancer. The parental origin BRCA2-wild-type and pSPL3-BRCA2-mutant plasmids, of the mutation is assigned to the father. containing BRCA2 exon 21 and flanking intron sequences, were constructed by PCR using purified DNA from human blood samples and the following oligonu- Methods Patients cleotides: BRCA2-F, 5'-GATCACGAATTCTTCCT- Family B49363 is a 23 member, three-generation kindred GGAAAACTTATAGCA-3' and BRCA2-R 5'- with two affected subjects. A 40 year old woman was GATCACCTCGAGTTAGGGTAGAGGATTATCAAGTACA- referred to genetic counseling two months after she had 3'. The PCR products were treated with EcoRI and XhoI breast-conserving surgery at Roskilde County Hospital and cloned into the pSPL3 vector. All constructs were ver- with radical excision of an 8 mm large invasive ductal car- ified by sequencing. cinoma. The tumor was estrogen and progesterone recep- tor positive and had malignancy grade II. The patient Cell culture and transfections received adjuvant radiotherapy followed by seven series of COS-7 cells were cultured in Dulbecco's modified Eagle's chemotherapy with CEF and tamoxifen. The patient's medium (DMEM) containing 4500 mg/l glucose supple- mother had a mastectomy at age 59 with excision of a 22 mented with 10% foetal bovine serum, 100 U of penicil- mm large estrogen and progesterone receptor positive lin per ml, and 100 μg of streptomycin per ml at 5% CO invasive ductal carcinoma, malignancy grade II, and post- and 37°C. One day before transfection, cells were seeded operatively she received tamoxifen. Following verbal and in 6-well culture dishes at a density of 4 × 10 cells/well. Page 2 of 7 (page number not for citation purposes) BMC Medical Genetics 2008, 9:58 http://www.biomedcentral.com/1471-2350/9/58 Cells were transfected with a total of 4 μg plasmid DNA forward: 5'-CAGACCCAGCTTACCTTGAAA-3'), and the using FuGENE 6 transfection reagent (Roche) according two SNP alleles in rs4942485 (rs4942485*A reverse: 5'- to the manufacturer's instructions. Transfection efficien- CTACATTACAGATGGCTAATATCTGATT-3' and cies for each series were determined by cotransfection of rs4942485*G reverse: 5'-CATTACAGATGGCTAATATCT- pEGFP plasmid (Clontech). The next day the media was GAAC-3'. Primer sets were tested on homozygote controls changed and after another 24 h the cells were harvested and unspecific annealing was avoided after introducing and total RNA was isolated using Trizol reagent (Invitro- mismatches (marked in bold). PCR was carried out using gen). standard conditions according to the manufacturer's pro- PLUS tocols using the Expand High Fidelity Taq DNA Exon trapping analysis polymerase (Roche). Allele sizes were: BRCA2 c.8754+1-G First strand cDNA was synthesized using 1 μg RNA, 20 μM and rs942485-G = 2297 bp, BRCA2 c.8754+1-*A and SA2 primer (5'-ATCTCAGTGGTATTTGTGAGC-3') and M- rs942485-G = 2298 bp, BRCA2 c.8754+1-G and MuLV reverse transcriptase (New England Biolabs). The rs942485-A = 2300 bp, BRCA2 c.8754+1-*A and cDNAs were amplified with pSPL3 vector-specific primers rs942485-A = 2301 bp. (5'-TCTGAGTCACCTGGACAACC-3' and 5'-ATCTCAGT- GGTATTTGTGAGC-3') and the PCR products were Results and discussion resolved on a 2% agarose gel. All experiments were The patient was diagnosed with breast cancer at the age of repeated three times. 40. Since her mother had breast cancer at the age of 59 (Fig. 1), the patient was referred to genetic counselling. RNA analysis No other family members had breast or ovarian cancer. A fresh blood sample was obtained from the patient. Total We analysed the entire coding region and the exon-intron cellular RNA was isolated with Trizol (Invitrogen) accord- boundaries of BRCA1 and BRCA2 from genomic DNA by ing to the manufacturer's instructions. For reverse tran- dHPLC and sequencing and for large genomic rearrange- scription-PCR (RT-PCR), cDNA was synthesized using the ments by MLPA analysis. The analysis revealed a polymor- AMV reverse transcriptase (Promega) as described by the phism in exon 11 of BRCA1 (nucleotide 1186 A→G/ supplier. The cDNA were amplified with the BRCA2 spe- c.1067 A→G), which is observed in approximately 5% of cific primers 5'-CGGCCTGCTCGCTGGTAT-3' and 5'- a control group [18], and a nucleotide 8982+1 G→A/ GCCTTCCTAATTTCCAACTGGATCTG-3' resulting in a c.8754+1 G→A mutation in BRCA2 that has not previ- 503 bp fragment. The samples were separated by agarose ously been reported in the BIC database (Fig. 2). The gel electrophoresis and visualized by ethidium bromide mutation occurs at the first base of the conserved GU staining. Finally, the bands were purified, cloned into splice donor site of intron 21. The mutation was verified pCR-Blunt II-TOPO (Invitrogen) and sequenced using an in a second blood sample as well as in breast cancer ABI3730 DNA analyzer (Applied Biosystems). tumour tissue. SNP chip analysis To functionally characterize the BRCA2 nucleotide Genomic DNA was applied to 250K StyI (~238.000 SNPs) 8982+1 G→A/c.8754+1 G→A variant, a fragment con- SNP-microarray chips and processed according to the taining BRCA2 exon 21 (122 bp), 449 bp of intron 20 manufacturer's instructions (Affymetrix, UK). Briefly, 250 (IVS20) and 408 bp of intron 21 (IVS21) containing the ng of genomic DNA was digested with StyI and ligated to wild-type or the nucleotide 8982+1 G→A/c.8754+1 G→A adapters. Adapter-ligated DNA was amplified, purified, variant, respectively, was cloned into the minigene vector fragmented and labeled with biotin and hybridized to the pSPL3 containing exons from HIV-tat under the control of arrays for 18 h. The Affymetrix 450 fluidics station and the the SV40 promoter (Fig. 3A) [19]. Constructs containing Affymetrix 3000 G7 gene scanner were used to wash, either the wild-type or the mutant IVS21 sequence was stain, and scan the arrays. The CEL files were analysed transfected into COS-7 cells. After 48 hours mRNA was using the BRLMM algorithm from Affymetrix Genotyping purified and examined by RT-PCR. The splicing products analysis software (GTYPE). Relationship was inferred by were separated on a 2% agarose gel. The wild-type con- estimating the pairwise identical by descent (IBD) shar- struct yields a product of 299 bp and a product of 177 bp ing, as described [17]. if exon 21 is excluded from the transcript (exon skipping). Moreover a larger product than 299 bp would demon- Assignment of parental origin strate that additional intron sequence is included in the Variant specific primers were designed using the software transcript. The normal wild-type BRCA2 exon 21 (pSPL3- Primer3 in combination with ClustaIW alignments. The BRCA2-wt) generated one transcript comprising the primer sets distinguish the wild-type allele (BRCA2 expected 299 bp, while the BRCA2 nucleotide 8982+1 c.8754+1-G forward: 5'-AGACCCAGCTTACCTTGACG- G→A/c.8754+1 G→A mutant (pSPL3-BRCA2-mut) 3') from the mutation carrying allele (BRCA2 c.8754+1-A yielded a band with slightly higher mobility (Fig. 3B). Page 3 of 7 (page number not for citation purposes) BMC Medical Genetics 2008, 9:58 http://www.biomedcentral.com/1471-2350/9/58 products were analysed on a 1% agarose gel (Fig. 4). Two PCR products – one with the expected size of the wild- type (503 bp) and an additional RT-PCR product (549 bp) – were amplified from the patient. Cloning and Acc. 49 yrs sequence analysis of the latter verified the inclusion of 46 bp of intron 21 (data not shown). We examined the patient's mother, who was affected with BRC 59 the same type of breast cancer at the age of 59 years, for BRCA2 c.8754+1 G G G G the same mutation, but she did not have this mutation in rs4 942 485 G G A A either DNA purified from blood or breast cancer tumour samples. Therefore mosaicism was excluded. Moreover, the father and the patient's sister did not carry the muta- tion. To establish the paternity we performed a SNP microarray analysis using 250K Sty gene array (Affyme- trix). The analysis provided the relatedness estimates BRC 40 (Table 1), where k0 is the probability that pairs of individ- BRCA2 c.8754+1 A* G uals at a random loci share no allele identical by descent G G rs4 942 485 G A (IBD), k1 is the probability that pairs of individuals at a G A random loci share one allele IBD, and k2 the probability Fa Figure 1 mily pedigree that pairs of individuals at a random loci share two allele Family pedigree. Breast cancers are indicated as well as IBD. The expected relatedness for full siblings is k0 = 0.25, the age at diagnosis. Acc, accident; BRC, breast cancer. The k1 = 0.5, k2 = 0.25, for parent offspring k0 = 0, k1 = 1, k2 number following the cancer gives the age at diagnosis. = 0, for unrelated k0 = 1, k1 = 0, k2 = 0, which in all cases Moreover, the genotypes from variant specific PCR are indi- fits in this family. We therefore conclude that the identi- cated. Diagonal slash indicates deceased. The proband is indi- fied mutation in the proband is a de novo mutation in cated with an arrow. Proband = individual 1, Sister = BRCA2. In contrast to the previously identified de novo individual 2, Mother = individual 3, Father = individual 4. BRCA2 nucleotide 3034del4 and BRCA1 nucleotide 3888delGA mutations [14,15], the mutation reported in our study resides in a new position not previously Sequencing revealed, that it contained an additional 46 reported in the BIC database. This was also the case with bp from intron 21 generating a transcript of 345 bp (Fig. the BRCA2 nucleotide 7260insA mutation [13]. Several 3C), indicating that it activates a cryptic splice site follow- other variants have been detected in the vicinity of the ing these 46 bp (Fig. 1). To provide direct evidence for the splice donor site in exon 21, including nucleotide 8982 aberrant splicing, RNA was isolated from whole blood of G→A/c.8754 G→A (reported once), nucleotide 8982+1 the affected patient and RT-PCR was performed before the A→G/c.8754+4 A→G (reported seven times), nucleotide Exon 21 Intron 21 G>A Iden Figure 2 tification of the BRCA2 nucleotide 8982+1 G→A/c Identification of the BRCA2 nucleotide 8982+1 G→A/c.8754+1 G→A variant. DNA was purified from whole blood and BRCA2 exon 21 was amplified using the primers 5'-CTTTGGGTGTTTTATGCTTGT-3' and 5'-CTGGCACATCACT- GAAAATC-3' and sequenced. The analysis revealed a nucleotide 8982+1 G→A/c.8754+1 G→A mutation in BRCA2 (sense strand). The nucleotide 8982+1 G→A/c.8754+1 G→A mutation and the cryptic splice site are underlined. Page 4 of 7 (page number not for citation purposes) BMC Medical Genetics 2008, 9:58 http://www.biomedcentral.com/1471-2350/9/58 SD IVS20 IVS21 SA BRCA2 exon 21 pSPL3 pSPL3 449 bp 122 bp 408 bp Exon 21 Exon 21 wild-type mutant 766 bp 500 bp 345 bp 299 bp 200 bp Exon 21 Intron 21 pSPL3 Exon tra Figure 3 pping analysis Exon trapping analysis. (A) Structure of the exon trapping vector pSPL3 containing the BRCA2 exon 21 and 449 bp of intron 20 and 408 bp of intron 21, respectively containing the wild-type or the nucleotide 8982+1 G→A/c.8754+1 G→A vari- ant. (B) COS-7 cells were transfected with pSPL3-BRCA2-exon 21 wild-type or pSPL3-BRCA2-exon 21 mutant plasmids. Total RNA was isolated, RT-PCR analysis was performed and the PCR products (in duplicates) were resolved on a 2% agarose gel. The 299 bp product corresponds to wild-type exon 21 (unaltered splicing), while the 345 bp product corresponds to exon 21 and the inclusion of 46 bp of intron 21. The sizes of the DNA marker are indicated to the right. (C) Sequence of exon 21 (345 bp band). The nucleotide 8982+1 G→A/c.8754+1 G→A mutation and the TAA stop codon are underlined (sense strand). Page 5 of 7 (page number not for citation purposes) BMC Medical Genetics 2008, 9:58 http://www.biomedcentral.com/1471-2350/9/58 Table 1: Relatedness estimates. M 1 Individual Individual k0 k1 k2 Relationship 1000 - 900 - 1 2 0.23 0.49 0.28 full siblings 800 - 1 3 0.01 0.99 0.00 parent offspring 1 4 0.01 0.99 0.00 parent offspring 700 - 2 3 0.01 0.98 0.01 parent offspring 2 4 0.01 0.99 0.01 parent offspring 600 - 3 4 0.98 0.02 0.00 unrelated Exon 21 mutant 500 - Exon 21 wild-type Relationship was infered by estimating the pairwise identical by descent (IBD) sharing as described in [17]. k0 is the probality that pairs of 400 - individuals at a random loci share no allele IBD, k1 the probality that pairs of individuals at a random loci share one allele IBD, and k2 the probality that pairs of individuals at a random loci share two allele IBD. The expected relatedness for full siblings is k0 = 0.25, k1 = 0.5, 300 - k2 = 0.25, for parent offspring k0 = 0, k1 = 1, k2 = 0, and for unrelated k0 = 1, k1 = 0, k2 = 0. The individual number is indicated in figure 1. noteworthy that the age of the probands parents in our study was only 19 and 21 years at the time of birth of their RT-PC from the proband Figure 4 R was performed on RNA purified from whole blood affected daughter. RT-PCR was performed on RNA purified from whole blood from the proband. The cDNA was amplified with Conclusion specific BRCA2 primers. The sample was separated by agar- We conclude that the BRCA2 nucleotide 8982+1 G→A/ ose gel electrophoresis and visualized by ethidium bromide c.8754+1 G→A mutation is a de novo mutation arising staining. Two RT-PCR products (503 bp and 549 bp) were obtained from the patient (Lane 1). The sizes of the DNA from the male germ line. The mutation is not previously marker are indicated to the left. The PCR products were reported in the BIC database. It leads to the activation of cloned and sequence analysis revealed that the 549 bp band a cryptic splice site 46 base pairs 3' of exon 21 and intro- contained the inclusion of 46 bp of intron 21 (data not duces a premature stop codon and thereby a truncated shown). BRCA2 protein. Therefore, this mutation can be classified as a disease-causing mutation. Analysis of intronic BRCA1 and BRCA2 variants by functional splicing assays can pro- 8982+5 G→A/c.8754+5 G→A (reported once) and nucle- vide information that can be used clinically. otide 8982+5 G→T/c.8754+5 G→T (reported once), sug- gesting that this region could be prone to mutations. Competing interests The authors declare that they have no competing interests. To determine the parental origin of the mutation, variant specific PCR was performed using primers designed to dis- Authors' contributions TVOH designed the study, supervised exon trapping, tinguish between two SNP alleles and the mutation carry- ing allele from the wild-type allele. The father was interpreted the results, and wrote the draft of the paper. homozygote for the haplotype GG, while the mother was MLB was involved in the designing of the study, the vari- homozygote for the haplotype GA (see Fig. 1). PCR anal- ant specific PCR, and the writing of the manuscript. LJ per- ysis of the proband revealed a band using mutant BRCA2 formed the RT-PCR analysis on RNA from whole blood and rs4942485*G primers, indicating that the haplotype and was involved in the writing of the manuscript. AA per- *AG must derive from the father, and that the mutation formed the relationship SNP analysis. BF–B analysed the therefore arose in the testicular germ cells changing carcinoma samples. HE designed and performed the vari- BRCA2 nucleotide 8982+1/c.8754+1 from G to A. This is ant specific PCR. BE selected the patients and participated in agreement with previous findings in BRCA1 [14], in genetic counseling and was involved in the writing of whereas the studies describing de novo mutations in the manuscript. FCN designed the study and was involved BRCA2 were unable to determine parental origin [13,15]. in the writing of the manuscript. All authors have read and Examinations of other cancer families, including MEN2B approved the final manuscript. and retinoblastoma families have indicated that de novo mutations primarily occur in the male germ line [20,21] Acknowledgements Stine Østergaard and Dorthe Petersen are acknowledged for technical and that the fathers age is a major determinant since assistance. Written consent was obtained from the patient or their rela- mutations accumulate during life [22]. It is, however, Page 6 of 7 (page number not for citation purposes) BMC Medical Genetics 2008, 9:58 http://www.biomedcentral.com/1471-2350/9/58 tives for publication of the study. This study was supported by the Neye genomic DNA using exon amplification. Nat Genet 1994, 6(1):98-105. Foundation. 20. Carlson KM, Bracamontes J, Jackson CE, Clark R, Lacroix A, Wells SA Jr., Goodfellow PJ: Parent-of-origin effects in multiple endo- References crine neoplasia type 2B. Am J Hum Genet 1994, 55(6):1076-1082. 21. Kato MV, Ishizaki K, Shimizu T, Ejima Y, Tanooka H, Takayama J, 1. Easton DF, Ford D, Bishop DT: Breast and ovarian cancer inci- dence in BRCA1-mutation carriers. Breast Cancer Linkage Kaneko A, Toguchida J, Sasaki MS: Parental origin of germ-line and somatic mutations in the retinoblastoma gene. Hum Consortium. Am J Hum Genet 1995, 56(1):265-271. 2. Wooster R, Neuhausen SL, Mangion J, Quirk Y, Ford D, Collins N, Genet 1994, 94(1):31-38. 22. 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Publish with Bio Med Central and every 15. van der Luijt RB, van Zon PH, Jansen RP, van der Sijs-Bos CJ, Warlam- scientist can read your work free of charge Rodenhuis CC, Ausems MG: De novo recurrent germline muta- tion of the BRCA2 gene in a patient with early onset breast "BioMed Central will be the most significant development for cancer. J Med Genet 2001, 38(2):102-105. disseminating the results of biomedical researc h in our lifetime." 16. [http://www.hgvs.org/mutnomen]. Sir Paul Nurse, Cancer Research UK 17. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, Maller J, Sklar P, de Bakker PI, Daly MJ, Sham PC: PLINK: A Tool Your research papers will be: Set for Whole-Genome Association and Population-Based available free of charge to the entire biomedical community Linkage Analyses. Am J Hum Genet 2007, 81(3):559-575. 18. Hadjisavvas A, Charalambous E, Adamou A, Neuhausen SL, Christo- peer reviewed and published immediately upon acceptance doulou CG, Kyriacou K: Hereditary breast and ovarian cancer cited in PubMed and archived on PubMed Central in Cyprus: identification of a founder BRCA2 mutation. Can- cer Genet Cytogenet 2004, 151(2):152-156. yours — you keep the copyright 19. Church DM, Stotler CJ, Rutter JL, Murrell JR, Trofatter JA, Buckler AJ: BioMedcentral Submit your manuscript here: Isolation of genes from complex sources of mammalian http://www.biomedcentral.com/info/publishing_adv.asp Page 7 of 7 (page number not for citation purposes)

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