Pathogenic neurofibromatosis type 1 (NF1) RNA splicing resolved by targeted RNAseq.

Neurofibromatosis type 1 (NF1) is caused by loss-of-function variants in the NF1 gene. Approximately 10% of these variants affect RNA splicing and are either missed by conventional DNA diagnostics or are misinterpreted by in silico splicing predictions. Therefore, a targeted RNAseq-based approach was designed to detect pathogenic RNA splicing and associated pathogenic DNA variants. For this method RNA was extracted from lymphocytes, followed by targeted RNAseq. Next, an in-house developed tool (QURNAs) was used to calculate the enrichment score (ERS) for each splicing event. This method was thoroughly tested using two different patient cohorts with known pathogenic splice-variants in NF1. In both cohorts all 56 normal reference transcript exon splice junctions, 24 previously described and 45 novel non-reference splicing events were detected. Additionally, all expected pathogenic splice-variants were detected. Eleven patients with NF1 symptoms were subsequently tested, three of which have a known NF1 DNA variant with a putative effect on RNA splicing. This effect could be confirmed for all 3. The other eight patients were previously without any molecular confirmation of their NF1-diagnosis. A deep-intronic pathogenic splice variant could now be identified for two of them (25%). These results suggest that targeted RNAseq can be successfully used to detect pathogenic RNA splicing variants in NF1.

Evaluation of the XRCC1 gene as a phenotypic modifier in BRCA1/2 mutation carriers. Results from the consortium of investigators of modifiers of BRCA1/BRCA2.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) in genes involved in DNA repair are good candidates to be tested as phenotypic modifiers for carriers of mutations in the high-risk susceptibility genes BRCA1 and BRCA2. The base excision repair (BER) pathway could be particularly interesting given the relation of synthetic lethality that exists between one of the components of the pathway, PARP1, and both BRCA1 and BRCA2. In this study, we have evaluated the XRCC1 gene that participates in the BER pathway, as phenotypic modifier of BRCA1 and BRCA2. METHODS: Three common SNPs in the gene, c.-77C>T (rs3213245) p.Arg280His (rs25489) and p.Gln399Arg (rs25487) were analysed in a series of 701 BRCA1 and 576 BRCA2 mutation carriers. RESULTS: An association was observed between p.Arg280His-rs25489 and breast cancer risk for BRCA2 mutation carriers, with rare homozygotes at increased risk relative to common homozygotes (hazard ratio: 22.3, 95% confidence interval: 14.3-34, P<0.001). This association was further tested in a second series of 4480 BRCA1 and 3016 BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1 and BRCA2. CONCLUSIONS AND INTERPRETATION: No evidence of association was found when the larger series was analysed which lead us to conclude that none of the three SNPs are significant modifiers of breast cancer risk for mutation carriers.

Efficiency of BRCAPRO and Myriad II mutation probability thresholds versus cancer history criteria alone for BRCA1/2 mutation detection.

Considerable differences exist amongst countries in the mutation probability methods and thresholds used to select patients for BRCA1/2 genetic screening. In order to assess the added value of mutation probability methods, we have retrospectively calculated the BRCAPRO and Myriad II probabilities in 306 probands who had previously been selected for DNA-analysis according to criteria based on familial history of cancer. DNA-analysis identified 52 mutations (16.9%) and 11 unclassified variants (UVs, 3.6%). Compared to cancer history, a threshold > or = 10% with BRCAPRO or with Myriad II excluded about 40% of the patients from analysis, including four with a mutation and probabilities <10% with both programs. All four probands had a BRCA2 mutation. BRCAPRO and Myriad II showed similar specificity at 10% threshold, overall BRCAPRO was more sensitive than Myriad II for the detection of mutations. Only two of the probands with an UV had probabilities >20% with BRCAPRO and Myriad II. In summary, BRCAPRO and Myriad II are more efficient than cancer history alone to exclude patients without a mutation. BRCAPRO performs better for the detection of BRCA1 mutations than of BRCA2 mutations. The Myriad II scores provided no additional information than the BRCAPRO scores alone for the detection of patients with a mutation. The use of thresholds excluded from analysis the majority of patients carrying an UV.

Loss of imprinting of IGF2 and not H19 in breast cancer, adjacent normal tissue and derived fibroblast cultures.

Insulin-like growth factors are involved in the paracrine growth regulation of human breast tumor cells. IGF2 is imprinted in most tissues, and shows expression of the paternal allele only. To investigate whether disruption of this monoallelic IGF2 expression is involved in breast cancer development, a series of primary tumors and adjacent, histologically normal, breast tissue samples, as well as matched primary in vitro fibroblast cultures were studied. Biallelic expression (partial) of IGF2 was found in the majority of in vivo samples, and corresponding fibroblast cultures, while monoallelic expression was found in a normal breast sample. In contrast, H19, a closely apposed, but reciprocally imprinted gene, assumed to be regulated by a common control element, showed retention of monoallelic H19 expression in all in vivo and in the majority of in vitro samples. These data indicate that IGF2, but not H19, is prone to loss of imprinting in breast cancer.

The platelet-derived growth factor alpha-receptor 1.5 kb transcript: target for molecular detection of testicular germ cell tumours of adolescents and adults.

An accurate and relatively simple method to detect testicular germ cell tumours of adolescents and adults (TGCTs) could be useful for early detection and may help to avoid unnecessary orchidectomies. We report a highly sensitive reverse-transcription polymerase chain reaction assay for the 1.5 kb transcript of the platelet-derived growth factor-alpha receptor for molecular diagnosis of TGCTs. As a simulation of the clinical application of the assay the approach was applied on through-cut-biopsies from orchidectomy specimens. In a series of 31 specimens, the 1.5 kb transcript was detected in all samples containing either carcinoma in situ, or an invasive TGCT, with mature teratoma as only exception. No expression was detected in normal parenchyma. On the basis of the through-cut-biopsies the assay shows a sensitivity of 0.87 and a specificity of 1.00. The positive and negative predictive values of the test are 1.00 and 1.00. For carcinoma in situ alone these values are 0.86, 1.00, 1.00, and 0.80, respectively. The figures at least equal the results obtained with the most sensitive morphological/enzyme-histochemical study of duplicate biopsies. We conclude that the 1.5 kb transcript of the platelet-derived growth factor-alpha receptor is a useful molecular marker for TGCTs, and therefore of interest in a clinical setting.

Transforming growth factor beta secretion from primary breast cancer fibroblasts.

Transforming growth factor beta (TGF-beta) is a hormonally regulated growth inhibitor with autocrine and/or paracrine functions in human breast cancer. In vivo, enhanced immunohistochemical staining of extracellular TGF-beta 1 has been detected around stromal fibroblasts in response to the antiestrogen treatment. We have investigated the effects of tamoxifen on the production of TGF-beta by primary human breast fibroblast cultures in serum-free medium. Highly variable levels of mainly latent TGF-beta 1 were detected in conditioned media from both tumor and normal tissue derived fibroblasts. Hydroxy-tamoxifen was shown to increase latent TGF-beta 1 secretion in three of the eight tumor tissue-derived fibroblast cultures. Such effect of hydroxy-tamoxifen was not observed in fibroblast cultures established from normal adjacent breast tissue.

Stromal influences on breast cancer cell growth.

Paracrine influences from fibroblasts derived from different sources of breast tissue on epithelial breast cancer cell growth in vitro were investigated. Medium conditioned (CM) by fibroblasts derived from tumours, adjacent normal breast tissue, and normal breast tissue obtained from reduction mammoplasty or from skin tissue significantly stimulated the growth of the steroid-receptor positive cell lines MCF-7 and ZR 75.1. The proliferation index (PI) on MCF-7 cells with CM from fibroblasts derived from breast tumour tissue was significantly higher than that obtained with fibroblasts derived from adjacent normal breast tissue (2p less than 0.05, n = 8). The PI obtained with CM from normal fibroblast cultures from reduction mammoplasty tissue, like normal tissue adjacent to the tumour, fell in the lower range of values. Skin fibroblast, like tumour tissue derived fibroblast, CM caused a high range PI. MDA-MB-231 and Evsa-T, two steroid-receptor negative cell lines, showed only a minor growth stimulatory responses with some of the fibroblast CM's. Evsa-T was occasionally inhibited by CM's. In conclusion, stromal factors play a role in the growth regulation of human breast cancer cells. The effects on cancer cell growth are, however, varying depending on the source of the stroma and the characteristics of the epithelial tumour cells.

Characterization of two Fc receptors for mouse immunoglobulins on human monocytes and cell lines.

We have previously reported a polymorphism in the mitogenic effect of murine (m) IgG1 anti-CD3 monoclonal antibodies. This polymorphism was genetically determined and could be attributed to polymorphism of the Fc receptor (FcR) for mIgG1 present on human monocytes. We have now extended these studies by quantitating FcR expression on monocytes and cell lines by a recently developed EA rosette assay, using the erythrocyte-associated pseudoperoxidase activity. The data show that the polymorphism of the monocyte FcR for mIgG1 is based on a quantitative rather than an absolute difference. Furthermore, this FcR is specific for mIgG1 and does not bind mIgG2a or mIgG2b nor, surprisingly, human IgG. The expression of this FcR on cell lines correlates with their accessory function in IgG1 anti-CD3-induced T cell proliferation. mIgG2a can inhibit the rosetting of monocytes with erythrocytes sensitized with human IgG. The FcR detected by this rosette technique can interact with all four human IgG subclasses but not with mIgG1 or mIgG2b. The expression of this type of FcR on human cell lines correlates well with their ability to support mIgG2a anti-CD3-induced mitogenesis. These direct measurements of FcR expression support the concept that human monocytes have two independent FcR with affinity for mouse IgG: one receptor specific for mIgG2a (which also binds human IgG), and a second specific for mIgG1.

A new photometric method for the quantitation of Fc receptors for murine IgG1 on human monocytes and cell lines.

We have previously shown a polymorphism of human Fc receptors for mouse IgG1 using an EA rosette technique in which human erythrocytes sensitized with a murine IgG1 monoclonal antibody against glycophorin A acted as indicator cells. We now describe a method to quantitate this EA rosetting using the pseudoperoxidase activity present in erythrocytes. This photometric assay allows the sensitive quantitative determination of Fc receptor expression on human monocytes and cell lines. Not only the human Fc receptor for murine IgG1 can be studied in this way, but the method can also be applied to other Fc receptors. An important factor in this type of rosette assay appears to be the amount of negative charge present on the surface of the indicator erythrocytes. Using alcian blue as a probe, we found that this negative charge is higher on human erythrocytes than on sheep erythrocytes, which may contribute to a better signal-to-noise ratio. The method described facilitates the characterization of Fc receptors and permits the rapid screening of monoclonal anti-Fc receptor antibodies.