Anna Marsh

Genetic and Histopathologic Evaluation of BRCA1 and BRCA2 DNA Sequence Variants of Unknown Clinical Significance

Classification of rare missense variants as neutral or disease causing is a challenge and has important implications for genetic counseling. A multifactorial likelihood model for classification of unclassified variants in BRCA1 and BRCA2 has previously been developed, which uses data on co-occurrence of the unclassified variant with pathogenic mutations in the same gene, cosegregation of the unclassified variant with affected status, and Grantham analysis of the fit between the missense substitution and the evolutionary range of variation observed at its position in the protein. We have further developed this model to take into account relevant features of BRCA1- and BRCA2-associated tumors, such as the characteristic histopathology and immunochemical profiles associated with pathogenic mutations in BRCA1, and the fact that approximately 80% of tumors from BRCA1 and BRCA2 carriers undergo inactivation of the wild-type allele by loss of heterozygosity. We examined 10 BRCA1 and 15 BRCA2 unclassified variants identified in Australian, multiple-case breast cancer families. By a combination of genetic, in silico, and histopathologic analyses, we were able to classify one BRCA1 variant as pathogenic and six BRCA1 and seven BRCA2 variants as neutral. Five of these neutral variants were also found in at least 1 of 180 healthy controls, suggesting that screening a large number of appropriate controls might be a useful adjunct to other methods for evaluation of unclassified variants.

Subtypes of familial breast tumours revealed by expression and copy number profiling

Extensive expression profiling studies have shown that sporadic breast cancer is composed of five clinically relevant molecular subtypes. However, although BRCA1-related tumours are known to be predominantly basal-like, there are few published data on other classes of familial breast tumours. We analysed a cohort of 75 BRCA1, BRCA2 and non-BRCA1/2 breast tumours by gene expression profiling and found that 74% BRCA1 tumours were basal-like, 73% of BRCA2 tumours were luminal A or B, and 52% non-BRCA1/2 tumours were luminal A. Thirty-four tumours were also analysed by single nucleotide polymorphism–comparative genomic hybridization (SNP-CGH) arrays. Copy number data could predict whether a tumour was basal-like or luminal with high accuracy, but could not predict its mutation class. Basal-like BRCA1 and basal-like non-BRCA1 tumours were very similar, and contained the highest number of chromosome aberrations. We identified regions of frequent gain containing potential driver genes in the basal (8q and 12p) and luminal A tumours (1q and 17q). Regions of homozygous loss associated with decreased expression of potential tumour suppressor genes were also detected, including in basal tumours (5q and 9p), and basal and luminal tumours (10q). This study highlights the heterogeneity of familial tumours and the clinical consequences for treatment and prognosis.

DNA methylome of familial breast cancer identifies distinct profiles defined by mutation status

It is now understood that epigenetic alterations occur frequently in sporadic breast carcinogenesis, but little is known about the epigenetic alterations associated with familial breast tumors. We performed genome-wide DNA-methylation profiling on familial breast cancers (n = 33) to identify patterns of methylation specific to the different mutation groups (BRCA1, BRCA2, and BRCAx) or intrinsic subtypes of breast cancer (basal, luminal A, luminal B, HER2-amplified, and normal-like). We used methylated DNA immunoprecipitation (MeDIP) on Affymetrix promoter chips to interrogate methylation profiles across 25,500 distinct transcripts. Using a support vector machine classification algorithm, we demonstrated that genome-wide methylation profiles predicted tumor mutation status with estimated error rates of 19% (BRCA1), 31% (BRCA2), and 36% (BRCAx) but did not accurately predict the intrinsic subtypes defined by gene expression. Furthermore, using unsupervised hierarchical clustering, we identified a distinct subgroup of BRCAx tumors defined by methylation profiles. We validated these findings in the 33 tumors in the test set, as well as in an independent validation set of 47 formalin-fixed, paraffin-embedded familial breast tumors, by pyrosequencing and Epityper. Finally, gene-expression profiling and SNP CGH array previously performed on the same samples allowed full integration of methylation, gene-expression, and copy-number data sets, revealing frequent hypermethylation of genes that also displayed loss of heterozygosity, as well as of genes that show copy-number gains, providing a potential mechanism for expression dosage compensation. Together, these data show that methylation profiles for familial breast cancers are defined by the mutation status and are distinct from the intrinsic subtypes.

A deletion mutation in GDF9 in sisters with spontaneous DZ twins

A loss of function mutation in growth differentiation factor 9 (GDF9) in sheep causes increased ovulation rate and infertility in a dosage-sensitive manner. Spontaneous dizygotic (DZ) twinning in the human is under genetic control and women with a history of DZ twinning have an increased incidence of multiple follicle growth and multiple ovulation. We sequenced the GDF9 coding region in DNA samples from 20 women with DZ twins and identified a four-base pair deletion in GDF9 in two sisters with twins from one family. We screened a further 429 families and did not find the loss of function mutation in any other families. We genotyped eight single nucleotide polymorphisms across the GDF9 locus in 379 families with two sisters who have both given birth to spontaneous DZ twins (1527 individuals) and 226 triad families with mothers of twins and their parents (723 individuals). Using case control analysis and the transmission disequilibrium test we found no evidence for association between common variants in GDF9 and twinning in the families. We conclude that rare mutations in GDF9 may influence twinning, but twinning frequency is not associated with common variation in GDF9.

Gene expression profiling of formalin-fixed, paraffin-embedded familial breast tumours using the whole genome-DASL assay.

Tissue sample acquisition is a limiting step in many studies. There are many thousands of formalin‐fixed, paraffin‐embedded archival blocks collected around the world, but in contrast relatively few fresh frozen samples in tumour banks. Once samples are fixed in formalin, the RNA is degraded and traditional methods for gene expression profiling are not suitable. In this study, we have evaluated the ability of the whole genome DASL (cDNA‐mediated Annealing, Selection, extension, and Ligation) assay from Illumina to perform transcriptomic analysis of archived breast tumour tissue in formalin‐fixed, paraffin‐embedded (FFPE) blocks. We profiled 76 familial breast tumours from cases carrying a BRCA1, BRCA2 or ATM mutation, or from non‐BRCA1/2 families. We found that replicate samples correlated well with each other (r2 = 0.9–0.98). In 12/15 cases, the matched formalin‐fixed and frozen samples predicted the same tumour molecular subtypes with confidence. These results demonstrate that the whole genome DASL assay is a valuable tool to profile degraded RNA from archival FFPE material. This assay will enable transcriptomic analysis of a large number of archival samples that are stored in pathology archives around the globe and consequently will have the potential to improve our understanding and characterization of many diseases. Copyright

Sequence variants of DLC1 in colorectal and ovarian tumours

Loss of heterozygosity occurs frequently on the short arm of chromosome 8 in many neoplasms, including colorectal and ovarian cancer. Monochromosome transfer experiments into colorectal tumour cell lines have provided functional evidence for a tumour suppressor gene located at 8p22–23. One of the genes from this region that is expressed by our suppressed hybrids is a candidate tumour suppressor gene, DLC1 (deleted in liver cancer), which has homology to rat RhoGAP. We have delineated the structure of the DLC1 gene and used single‐stranded conformation polymorphism analysis (SSCP) to look for sequence variants in 126 colorectal and 33 ovarian primary tumours and cell lines. One exonic missense mutation and three intronic insertions/deletions were identified in primary colorectal tumours, as well as many polymorphisms present in germline DNAs. The rarity of exonic missense mutations, and the absence of protein‐truncating mutations, indicates that DLC1 is not the target of 8p LOH in colorectal or ovarian tumours. The delineation of the gene structure allows mutation analysis of DLC1 in other tumour types for which it remains a candidate tumour suppressor gene based on its location and homology to rhoGAP. Hum Mutat 15:156–165, 2000.

Characterizing mixing in a rotating drum bioreactor for solid-state fermentation

A method, based on the use of wheat bran particles dyed with Rhodamine-WT as tracer particles, was developed to characterize mixing in a 200 l rotating drum bioreactor used for solid state fermentation. The extraction process contributes a 15% relative error in determining Rhodamine concentrations. Extraction efficiency is not affected by autoclaving of the bran and there is no inter-particle transfer of dye during the mixing of bran within the drum. For an unbaffled drum rotated at 5 rpm the axial dispersion coefficient is 9.15 cm2 min−1.

O2 uptake during solid-state fermentation in a rotating drum bioreactor

The maximum O2 uptake by Rhizopus oligosporus grown in a 200 litre rotating drum bioreactor at 0.5 rpm ranged from 6.7 to 7.6 mmol per min per kg initial dry substrate (IDS), for runs done with 4 baffles each 17 cm wide, and 12 baffles each 5 cm wide. Without baffles, the maximum O2 uptake rate at 5 rpm was 6.9 mmol/(min.kg IDS), compared to 5.1 mmol/(min.kg IDS) obtained at 0.5 rpm. Therefore O2 supply is adequate in rotating drum bioreactors as long as slumping flow regimes of the substrate bed are avoided.

BRCA1 and BRCA2 missense variants of high and low clinical significance influence lymphoblastoid cell line post-irradiation gene expression.

The functional consequences of missense variants in disease genes are difficult to predict. We assessed if gene expression profiles could distinguish between BRCA1 or BRCA2 pathogenic truncating and missense mutation carriers and familial breast cancer cases whose disease was not attributable to BRCA1 or BRCA2 mutations (BRCAX cases). 72 cell lines from affected women in high-risk breast ovarian families were assayed after exposure to ionising irradiation, including 23 BRCA1 carriers, 22 BRCA2 carriers, and 27 BRCAX individuals. A subset of 10 BRCAX individuals carried rare BRCA1/2 sequence variants considered to be of low clinical significance (LCS). BRCA1 and BRCA2 mutation carriers had similar expression profiles, with some subclustering of missense mutation carriers. The majority of BRCAX individuals formed a distinct cluster, but BRCAX individuals with LCS variants had expression profiles similar to BRCA1/2 mutation carriers. Gaussian Process Classifier predicted BRCA1, BRCA2 and BRCAX status, with a maximum of 62% accuracy, and prediction accuracy decreased with inclusion of BRCAX samples carrying an LCS variant, and inclusion of pathogenic missense carriers. Similarly, prediction of mutation status with gene lists derived using Support Vector Machines was good for BRCAX samples without an LCS variant (82–94%), poor for BRCAX with an LCS (40–50%), and improved for pathogenic BRCA1/2 mutation carriers when the gene list used for prediction was appropriate to mutation effect being tested (71–100%). This study indicates that mutation effect, and presence of rare variants possibly associated with a low risk of cancer, must be considered in the development of array-based assays of variant pathogenicity.

Mutation analysis of five candidate genes in familial breast cancer

Most of the known breast cancer susceptibility genes (BRCA1, BRCA2, CHEK2 and ATM) are involved in the damage response pathway. Other members of this pathway are therefore good candidates for additional breast cancer susceptibility genes. ATR, along with ATM, plays a central role in DNA damage recognition and Chk1 relays checkpoint signals from both ATR and ATM. PPP2R1B and PPP2R5B code for subunits of protein phosphatase 2A (PP2A), which regulates autophosphorylation of ATM. In addition, EIF2S6/Int-6, which was originally identified as a common integration site for the mouse mammary tumour virus in virally induced mouse mammary tumours, is a candidate breast cancer susceptibility gene because of its putative role in maintaining chromosome stability. To investigate the role of ATR, CHK1, PPP2R1B, PPP2R5B and EIF2S6/Int-6, we carried out mutation analysis of these genes in the index cases from non-BRCA1/BRCA2 breast cancer families. We also screened sporadic breast tumours for somatic mutations in PPP2R1B and PPP2R5B. Although we identified many novel variants, we found no evidence that highly penetrant germline mutations in these five genes contribute to familial breast cancer susceptibility.