Purification of Circulating Cell-Free DNA from Plasma and Urine Using the Automated Large-Volume Extraction on the QIAsymphony® SP Instrument.

Increasing sample numbers for screening and diagnostics using circulating cell-free DNA (ccfDNA) as analyte demands an automated solution for ccfDNA extraction. The efficiency of a new, automated, large volume ccfDNA extraction method was evaluated against a manual reference method. The new kit for automated ccfDNA extraction on the QIAsymphony showed a comparable yield of total ccfDNA from healthy donors as well as a comparable recovery of circulating cancer and fetal DNA. In conclusion, a new kit for automated ccfDNA extraction was established successfully.

Diagnostic accuracy of random massively parallel sequencing for non-invasive prenatal detection of common autosomal aneuploidies: a collaborative study in Europe.

OBJECTIVE: The objective of this study is to validate the diagnostic accuracy of a non-invasive prenatal test for detecting trisomies 13, 18, and 21 for a population in Germany and Switzerland. METHODS: Random massively parallel sequencing was applied using Illumina sequencing platform HiSeq2000. Fetal aneuploidies were identified using a median absolute deviation based z-score equation. A bioinformatics algorithm based on guanine-cytosine normalization was applied after the data were unblinded. Results of massively parallel sequencing and invasive procedures were compared. RESULTS: Overall, 40/42 samples were correctly classified as trisomy 21-positive, including a translocation trisomy 21 [46,XY,der(13;21),+21] and a structural aberration of chromosome 21 [46,XX,rec(21)dup(21q)inv(21)(p12q21.1)] but not including a low percentage mosaic trisomy 21 [47,XY,+21/46,XY], [sensitivity: 95.2%; one-sided lower confidence limit: 85.8%]; 430/430 samples were correctly classified as trisomy 21-negative (specificity: 100%; one-sided lower CL: 99.3%). Using a new bioinformatics algorithm with guanine-cytosine normalization, detection of trisomy 21 was facilitated, and five of five trisomy 13 cases and eight of eight trisomy 18 cases were correctly identified. CONCLUSION: Our newly established non-invasive prenatal test allows detection of fetal trisomies 13, 18, and 21 with high accuracy in a population in Germany and Switzerland.

Noninvasive prenatal detection of chromosomal aneuploidies using different next generation sequencing strategies and algorithms.

OBJECTIVE: Here we describe the successful application of massively parallel sequencing for noninvasive prenatal detection of trisomy 21. In addition, for the detection of a broader spectrum of fetal aneuploidies, a target enrichment approach was successfully tested. METHODS: The circulating cell-free DNA was prepared from 53 maternal blood samples and analysed using Illumina's sequencing systems Genome Analyzer(IIx) and HiSeq2000, respectively. In a first experiment the SureSelect Target Enrichment System was tested. RESULTS: In our initial study analysing 42 samples on the Genome Analyzer(IIx) , all eight samples from women carrying a trisomy 21 fetus were correctly identified. On the basis of our HiSeq2000 sequence data, we discussed new algorithms for detection of fetal trisomy 21. In addition, we successfully used the combination of a target enrichment system followed by sequencing and were able to identify fetal trisomy 13 and fetal trisomy 21. CONCLUSIONS: Our results confirm previous reports that massively parallel sequencing of cell-free fetal DNA allows the reliably noninvasive detection of trisomy 21 from maternal blood with the potential to enhance test selectivity and specificity by bioinformatic means. According to our preliminary results, targeted sequencing might be an alternative strategy to detect chromosomal aneuploidies besides trisomy 21.

Common breast cancer-predisposition alleles are associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers.

Germline mutations in BRCA1 and BRCA2 confer high risks of breast cancer. However, evidence suggests that these risks are modified by other genetic or environmental factors that cluster in families. A recent genome-wide association study has shown that common alleles at single nucleotide polymorphisms (SNPs) in FGFR2 (rs2981582), TNRC9 (rs3803662), and MAP3K1 (rs889312) are associated with increased breast cancer risks in the general population. To investigate whether these loci are also associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers, we genotyped these SNPs in a sample of 10,358 mutation carriers from 23 studies. The minor alleles of SNP rs2981582 and rs889312 were each associated with increased breast cancer risk in BRCA2 mutation carriers (per-allele hazard ratio [HR] = 1.32, 95% CI: 1.20-1.45, p(trend) = 1.7 x 10(-8) and HR = 1.12, 95% CI: 1.02-1.24, p(trend) = 0.02) but not in BRCA1 carriers. rs3803662 was associated with increased breast cancer risk in both BRCA1 and BRCA2 mutation carriers (per-allele HR = 1.13, 95% CI: 1.06-1.20, p(trend) = 5 x 10(-5) in BRCA1 and BRCA2 combined). These loci appear to interact multiplicatively on breast cancer risk in BRCA2 mutation carriers. The differences in the effects of the FGFR2 and MAP3K1 SNPs between BRCA1 and BRCA2 carriers point to differences in the biology of BRCA1 and BRCA2 breast cancer tumors and confirm the distinct nature of breast cancer in BRCA1 mutation carriers.

Prospects and challenges for the genetic counsellor profession in the German-speaking countries: report of a workshop.

The genetic counsellor profession has not yet been established in the German-speaking countries. In 2019 the Medical University of Innsbruck inaugurated the first German-taught Master's degree programme in Genetic and Genomic Counselling. In order to discuss prospects and challenges of the genetic counsellor profession in Germany, Austria and Switzerland (DACH region), the MSc programme team organized a two-day workshop with international speakers and medical geneticists from the DACH region. Day 1 was dedicated to the history, training and international profile of the genetic counsellor profession. Day 2 focused on four specific topics: (i) professional role, (ii) acceptance and job title, (iii) formal requirements and (iv) remuneration concepts for genetic counsellors in the DACH region. The workshop showed that the key factor for the successful implementation of the genetic counsellor profession is acceptance and trust within the medical genetics team. Genetic counsellors complement patient care in aspects that might be underserved considering the increasing demand of counselling in genomic medicine. Successful establishment of the genetic counsellor profession will entail the development of interprofessional teams under medical supervision and in the team of medical geneticists.

Suppression of polyploidy by the BRCA2 protein.

Mounting evidence implicates BRCA2 not only in maintenance of genome integrity but also in cell-cycle checkpoints. However, the contribution of BRCA2 in the checkpoints is still far from being understood. Here, we demonstrate that breast cancer cells MX-1 are unable to maintain genome integrity, which results in gross polyploidization. We generated MX-1 clones, stably expressing BRCA2, and found that BRCA2 acts to suppress polyploidy. Compared with MX-1, the ectopically BRCA2-expressing cells had different intracellular levels of Aurora A, Aurora B, p21, E2F-1, and pRb, suggesting a BRCA2-mediated suppression of polyploidy via stabilization of the checkpoint proteins levels.

Mutation analysis of BRCA1 and BRCA2 genes in Iranian high risk breast cancer families.

PURPOSE: Germline mutations in either BRCA1 or BRCA2 genes are responsible for the majority of hereditary breast and ovarian cancers. At present, over thousand distinct BRCA1 and BRCA2 mutations have been identified. Specific mutations are found to be common within particular populations, resulting from genetic founder effects. To investigate the contribution of germline mutations in these two genes to inherited breast cancer in Iran, we performed BRCA1/BRCA2 mutation analyses in ten Iranian high risk breast cancer families. This is the first study analysing the complete coding sequences of both genes that concerns the Iranian population. METHODS: BRCA1/BRCA2 mutation detection included sequencing of the coding and the 3' and 5' untranslated regions. To detect large genomic rearrangements in the BRCA1 gene semi-quantitative multiplex PCR was performed. RESULTS: Two pathogenic mutations in the BRCA2 gene were detected: a novel deletion c.4415_4418delAGAA and a previously described insertion c.6033_6034insGT. In addition, one intronic variation g.5075-53C > T and a deletion/insertion g.*381_389del9ins29 in the 3' untranslated region of BRCA1 were found in two of the investigated families. Both sequence alterations were absent in an age matched Iranian control group. The BRCA2 homozygous variation p.N372H, previously associated with an increased risk for developing breast cancer, was not identified in this study. We did not detect large genomic rearrangements in BRCA1 in patients tested negatively for disease causing mutations in both genes by standard sequencing. CONCLUSIONS: At present, the BRCA2 mutations c.4415_4418delAGAA and c.6033_6034insGT have not been identified in any investigated population except the Iranian. Whether both mutations are specific for the Iranian population or a special subgroup remains to be investigated in larger studies. The absence of BRCA1 mutations in the analysed families may suggest that penetrance or prevalence of BRCA1 mutations may be lower in Iran.

Evaluation of BRCA1/2 mutational status among German and Austrian women with triple-negative breast cancer.

PURPOSE: Testing for BRCA1 and BRCA2 mutations in breast cancer patients is used to identify the risk of second primary cancers and the risk of cancer in the patients' family. Women with triple-negative breast cancer (TNBC) are thought to be more likely to be BRCA1/2 mutation carriers, but most national guidelines for genetic testing, including those used in Germany and Austria, do not consider receptor triple negativity. METHODS: We determined the prevalence of BRCA1 and BRCA2 mutations within a cohort of 100 unselected TNBC cases, including patients from Germany and Austria to identify those BRCA-positive patients with a masked family history and who would have been missed due to respective current national guidelines. Double-stranded Sanger sequencing of all exons of BRCA1 and BRCA2, respectively, was performed. RESULTS: We identified a total of 13 deleterious mutations in BRCA1 and a total of four deleterious mutations in BRCA2. The total rate of deleterious BRCA1/2 mutation carriers was 21 % in our cohort. Six novel mutations, including two deleterious mutations, have been identified, which have not been described in public mutation databases so far. According to current German and Austrian national guidelines for genetic testing, 38.1 and 52.4 %, respectively, of BRCA1/2 mutation carriers would have been overlooked. CONCLUSIONS: We conclude that the prevalence of BRCA1 and BRCA2 mutations is high in TNBC patients and that BRCA1/2 mutations are not restricted to young women or patients with a positive family history. Receptor triple negativity should therefore be considered in BRCA1/2 genetic testing guidelines.

Screening for large rearrangements of the BRCA1 gene in German breast or ovarian cancer families using semi-quantitative multiplex PCR method.

Since the identification of the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2, a large number of different germline mutations in both genes have been found by conventional PCR-based mutation detection methods. Complex germline rearrangements such as those reported in the BRCA1 gene are often not detectable by these standard diagnostic techniques. To detect large deletions or duplications encompassing one or more exons of the BRCA1 gene and in order to estimate the frequency of BRCA1 rearrangements in German breast or ovarian cancer families, a semi-quantitative multiplex PCR method was developed and applied to DNA samples of patients from families negatively tested for disease causing mutations in the BRCA1 and BRCA2 coding regions by direct sequencing. Out of 59 families analysed, one family was found to carry a rearrangement in the BRCA1 gene (duplication of exon 13). The results indicate that the semi-quantitative multiplex PCR method is useful for the detection of large rearrangements in the BRCA1 gene and therefore represents an additional valuable tool for mutation analysis of BRCA1 and BRCA2.

Large BRCA1 gene deletions are found in 3% of German high-risk breast cancer families.

We have tested for large BRCA1 gene rearrangements in German high-risk breast and ovarian cancer families previously screened negative for point mutations by dHPLC and sequencing. Using the novel MLPA method, two deletions of exons 1A, 1B and 2 and exon 17, respectively, were detected in four out of 75 families investigated in Southern Germany. An identical exon 17 deletion with the same breakpoints and a deletion of exons 1A, 1B and 2 were found by fluorescent multiplex PCR in two out of 30 families investigated in Northern Germany. Combining both populations, genomic rearrangements were found in 6% of the mutation-negative families and 3% of all high-risk families and account for 8% of all BRCA1 mutations. Our data indicate that the exon 17 deletion may be a founder mutation in the German population. The prevalence of BRCA1 gene deletions or duplications in our patients is similar to previous reports from Germany and France. Genomic quantification by MLPA is a useful method for molecular diagnostics in high-risk breast cancer families.

Fetal Aneuploidy Detection by Cell-Free DNA Sequencing for Multiple Pregnancies and Quality Issues with Vanishing Twins.

Non-invasive prenatal testing (NIPT) by random massively parallel sequencing of maternal plasma DNA for multiple pregnancies is a promising new option for prenatal care since conventional non-invasive screening for fetal trisomies 21, 18 and 13 has limitations and invasive diagnostic methods bear a higher risk for procedure related fetal losses in the case of multiple gestations compared to singletons. In this study, in a retrospective blinded analysis of stored twin samples, all 16 samples have been determined correctly, with four trisomy 21 positive and 12 trisomy negative samples. In the prospective part of the study, 40 blood samples from women with multiple pregnancies have been analyzed (two triplets and 38 twins), with two correctly identified trisomy 21 cases, confirmed by karyotyping. The remaining 38 samples, including the two triplet pregnancies, had trisomy negative results. However, NIPT is also prone to quality issues in case of multiple gestations: the minimum total amount of cell-free fetal DNA must be higher to reach a comparable sensitivity and vanishing twins may cause results that do not represent the genetics of the living sibling, as described in two case reports.

Genetic testing: new challenges in the healthcare of women. Interview by Hannah Branch.

Dr Wera Hofmann is an expert in biochemistry and has over 12 years of human genetic diagnostics experience. Until 2006, she supervised a diagnostic unit for BRCA gene testing at the Interdisciplinary Center for Hereditary Breast Cancer (Max Delbrück Center, Berlin, Germany). She has also been the Managing Director of the Professional Association of German Human Geneticists, BVDH, which is a trade association. In 2008, Hofmann became a Medical Director at LifeCodexx (Konstanz, Germany), where she has worked on the development of a noninvasive prenatal diagnostic test that detects chromosomal aneuploidies in fetuses.

Limited relevance of the CHEK2 gene in hereditary breast cancer.

To establish the importance of CHEK2 mutations for familial breast cancer incidence in the German population, we have screened all 14 of the coding exons in 516 families negative for mutations in both the BRCA1 and BRCA2 genes. We found 12 distinct variants in 30 unrelated patients (5.81%), including 5 that are novel and an additional 4 found for the first time in breast cancer. These aberrations were evaluated in 500 healthy women aged over 50 years and in the case of the 2 exon 10 mutations, 1100delC and 1214del4bp, in 1315 randomized healthy controls. According to our results, a statistically significant association for the exon 10 mutations was observed (p = 0.006). The prevalence of the 1100delC mutation in the German population, however, is significantly lower than those reported for other Caucasian populations both in familial breast cancer patients (1.6%) and controls (0.5%), and shows independent segregation with breast cancer in 2 of 4 families analyzed. The remaining 10 variants were more abundant in patients (21) compared to the controls (12) although the difference was not statistically significant. Interestingly, we found no increased breast cancer risk associated with the splice site mutation IVS2+1G-->A or the most common missense mutation I157T, which account for more than half (12/21) of the variants observed in patients. The low prevalence and penetrance of the exon 10 deletion mutations together with no, or an uncertain elevation in risk for other CHEK2 mutations suggests a limited relevance for CHEK2 mutations in familial breast cancer. Further evaluation of the unique variants observed in breast cancer is required to determine if they may play a role in a polygenic model of familial breast cancer. Nevertheless, it seems premature to include CHEK2 screening in genetic testing.