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European Molecular Genetics Quality Network EMQN Supported by the Standards Measurement and Testing programme of the European Union * * Contract no. SMT4-CT98-7515 Draft Best Practice Guidelines for Molecular Analysis of Hereditary Breast and Ovarian Cancer Mueller C1, Haworth A2. 1 Dept. of Human Genetics, University of Wuerzburg, Wuerzburg, Germany. 2South West Thames Regional Molecular Genetics Laboratory, St Georges Hospital, London, United Kingdom. Draft guidelines prepared by Clemens Mueller ([email protected]) and Andrea Haworth ([email protected] ) following discussions at the EMQN workshop 26th May 2000 in Amsterdam, The Netherlands. Disclaimer These Guidelines are based, in most cases, on the reports drawn up by the chairs of the disease-based workshops run by EMQN and the CMGS. These workshops are generally convened to address specific technical or interpretative problems identified by the QA scheme. In many cases, the authors have gone to considerable trouble to collate useful data and references to supplement their reports. However, the Guidelines are not, and were never intended to be, a complete primer or "how-to" guide for molecular genetic diagnosis of these disorders. The information provided on these pages is intended for chapter authors, QA committee members and other interested persons. All the guidelines are at a draft stage, and must not be used until formally published. Neither the Editor, the European Molecular Genetics Quality Network, the Clinical Molecular Genetics Society, the UK Molecular Genetics EQA Steering Committee nor the British Society for Human Genetics assumes any responsibility for the accuracy of, or for errors or omissions in, these Guidelines. Nomenclature and gene ID See table 1 . Description of the disease Breast cancer is the most common cancer in women with a lifetime risk of up to 1 in 8 depending on the ethnic background. Germline mutations in known and unknown susceptibility genes account for approximately 5-10% of affected women. To date two major genes, BRCA1 and BRCA2 have been identified; mutations in which are strongly associated with predisposition to breast and ovarian cancer (Miki et al 1994, Wooster et al 1995, Tavtigian et al 1996). The majority of mutations in both genes lead to protein truncation and are inherited in an autosomal dominant fashion.Mutations in BRCA1 accounts for approximately 50% of all hereditary breast cancer cases, and in BRCA2 for about 35% (Ellison et al 1998). There are clearly additional genes involved that may act as modifiers, for example the HRAS1 minisatellite (Phelan et al 1996). Other predisposing genes of high to moderate penetrance are known to lead to breast cancer or to syndromes involving breast and/or ovarian cancer, e.g. BRCA3, p53, PTEN (Bishop et al 1994, Easton et al 1997, ). The risk of breast cancer in mutation carriers has first been estimated from the study of selected high-risk families. In this group of patients the risk of developing breast cancer is greater than 80% by age 70. The risk figures given for ovarian cancer vary, with 40% overall risk by age 70 for BRCA1 and 27% for BRCA2 carriers (Easton et al 1993, Narod et al 1995, Ford et al 1998). At present, no penetrance estimates are available for the general population. In a study on an Ashkenazi Jewish population in the USA, unselected for family history, a penetrance of the three common Ashkenazi Jewish mutations of approximately 50-60% has been observed (Struewing et al 1997). There is also evidence that BRCA1 carriers generally have a younger age of onset of breast cancer than BRCA2 carriers (Ford et al 1998). There is little evidence of clear genotype/phenotype correlations for either gene. Most published reports are preliminary or in contradiction to other studies. There is some evidence that indicates that mutation carriers are at increased risk of other cancers, such as prostate cancer for male BRCA1 heterozygotes, and ocular melanoma, prostate and pancreatic cancer for BRCA2 carriers. Gene/Protein structure BRCA1 consists of 22 coding exons, which are transcribed into a 7.8 kb mRNA and encode a 1863 amino acid protein. Most of the exons are small, comprising of 200 or so nucleotides, but one large exon, exon 11, covers 61% of the coding region. Another distinctive feature is that the genomic sequence is rich in Alu repeats (40%). BRCA2 consists of 26 coding exons, which are transcribed into a 11-12 kb mRNA and encode a 3418 amino acid protein. Two large exons, 10 and 11, account for 60% of the coding region; the remaining exons are generally small. The two proteins appear to participate in the same pathways. The following roles have been demonstrated for both genes; both are involved in the maintenance of genomic stability by homologous recombination and by transcription coupled and double strand break repair (Scully et al 1997, Chen et al 1998). Both play a role in transcriptional regulation (Scully et al 1997 and 4 other refs). BRCA1 is also Guidelines for hereditary breast and ovarian cancer © EMQN 2001 1 Dr. Rob Elles (Co-ordinator) / Dr. Simon Patton (EMQN Administrator) Regional Molecular Genetics Laboratory, St Mary’s Hospital, Hathersage Road, Manchester M13 0JH, United Kingdom Tel: +44 161 276 6129/6741, Fax: +44 161 276 6606 Email: [email protected] / [email protected] European Molecular Genetics Quality Network EMQN Supported by the Standards Measurement and Testing programme of the European Union * * Contract no. SMT4-CT98-7515 involved in ubiquitination of proteins targeted for cellular degradation (Lorrick et al 1999) Ethnic Mutations Among Ashkenazi Jews, three mutations have been found at high prevalence: 185delAG and 5382insC in BRCA1, and 6174delT in BRCA2. Individuals of known Jewish ancestry should be screened for all three mutations. This also applies to those without known Jewish ancestry in whom one of these mutations has previously been identified. Other founder mutations have been described in several populations and it may be worthwhile targeting these common mutations in a patient of relevant origin before initiating more extensive mutation analysis. Selection Criteria and Referrals Throughout Europe referrals are accepted from several types of clinicians, mainly Clinical Geneticists, Oncologists and Obstetricians. The acceptance of referrals is subject to local practices. The mutation detection rate is to a large degree, dependent upon the prior risk of the individual patient. It is extremely useful if this risk, which should be assessed by the referring clinician, is indicated on the referral form along with a copy of the pedigree. Mutation screening can be carried out on either affected individuals, or unaffected individuals with a very strong family history of breast/ovarian cancer. The type of patient accepted for mutation screening is dependent upon local guidelines. Consent should be obtained from all patients prior to storage or analysis of their sample. The tests carried out upon the sample should be within the remit of the referral. It is the responsibility of clinical colleagues who are in personal contact with the patient to obtain written informed consent before the sample is taken. Consequently, it is acceptable for Molecular Genetics laboratories to assume that written informed consent has been obtained if the referral is from an competent source. Strategies Testing should be carried out on the following category of patients using genomic DNA/cDNA Mutation screening in affected individuals. Mutation screening in unaffected individuals with strong family history Mutation screening in unaffected individuals of Jewish ancestry for the three common mutations found in this population. Predictive testing of at risk relatives. Confirmation of the presence of a known mutation. Points to consider Occasionally, mutations in both BRCA1 and BRCA2 have been found in a single patient (one patient in Scotland and by a laboratory participating in a UK Breast and Ovarian Cancer Best Practice meeting unpublished findings). Therefore, it may be worth screening both genes to completion even when one mutation has already been identified, particularly if there is a strong family history on both sides of the pedigree. Similarly, if a mutation is found in an individual, it is advisable where possible, to determine which side of the family it comes from prior to offering predictive testing to the extended family. As breast cancer is a common disease many phenocopies can exist within families, therefore it may be useful to carry out segregation analysis. If this is not possible then it is wise to select the patient with the lowest age of onset available. Whole Gene Screen or Partial Gene Screen? In an ideal world it would be preferable to have the capacity to screen all exons of both genes and, in addition, to perform dosage analysis on BRCA1. However, this may only be practicable for a small cohort of patients, i.e. those at very high risk of being mutation carriers. Also local practices and funding may prevent the full screen of both genes. Thus, it is considered acceptable to carry out a limited screen, which targets areas of the genes where common mutations exist, typically exon 11, 2, 20 and 5 of BRCA1 and exons 10 and 11 of BRCA2, as long as the limits/extent of the analysis is indicated on any subsequent reports. Mutation Detection Many mutation detection techniques are used and thus it is not possible to establish a single recommended technique. The technique used largely depends upon local preferences and facilities. DNA/RNA The source material for testing varies; most laboratories extract genomic DNA from blood samples, whereas others also collect RNA from the same patient. Generally RNA is extracted either to store for future analysis and/or to confirm the effect of putative splice mutations. Generally cDNA/RNA is not used as a template for mutation screening, mainly due to technical difficulties such asthe presence of spurious bands and nonsense mediated decay of mRNA. Linkage Guidelines for hereditary breast and ovarian cancer © EMQN 2001 2 Dr. Rob Elles (Co-ordinator) / Dr. Simon Patton (EMQN Administrator) Regional Molecular Genetics Laboratory, St Mary’s Hospital, Hathersage Road, Manchester M13 0JH, United Kingdom Tel: +44 161 276 6129/6741, Fax: +44 161 276 6606 Email: [email protected] / [email protected] European Molecular Genetics Quality Network EMQN Supported by the Standards Measurement and Testing programme of the European Union * * Contract no. SMT4-CT98-7515 Linkage analysis is not offered as a routine service and is only available upon request. Multiplex Heteroduplex analysis. This technique is aimed at detection of small insertions and deletions and not at the detection of single base changes. It can be useful as an initial screen to look for common mutations (Gayther et al 1996) Fluorescent Conformational Sensitive Gel Electrophoresis (F-CSGE) Fluorescent multiplex analysis of fragments on an Applied Biosystems (ABI) 377 DNA sequencer (or equivalent) can be used for the detection of all types of mutations by “automated” heteroduplex analysis (Ganguly et al 1998). The use of various gel conditions may be required to optimise the detection rate. Although not widely used it is reported that the sensitivity is approaching 100%. However, sensitivity is probably lower at the ends of fragments. A possible drawback is that the technique will pick up polymorphic variants and variants of unknown pathological significance as well as truncating mutations. Another problem is that there is a lack of good interpretative software for the anaylsis of results. Single Stranded Conformational Polymorphism (SSCP) SSCP is an easy and low cost procedure which can be automated on an ABI 377. Sensitivity is comparatively low at 70-95% and several gel conditions may need to be used to optimise detection in a single PCR fragment/exon. The optimal fragment size is small at 200-250bp and multiplexing fragments can be difficult. Reproducibility can also be a problem because of the extreme sensitivity of SSCP's to temperature and other gel conditions. As with F-CSGE this technique will pick up all types of mutation/polymorphism. Denaturing Gradient Gel Electrophoresis (DGGE) As a heteroduplex based detection method, DGGE relies upon heteroduplexes having differing melting profiles under denaturing conditions. DGGE requires special primers with a 5’ GC extension (GC clamp). This method has a very high sensitivity once gel conditions have been optimised and multiplexing allows for reasonable throughput capacity. Drawbacks are that the primers are relatively expensive and some special equipment is required. There are programmes available which will calculate the melting profiles of fragments and aid experiment design. However, the technique may be less reliable in GC rich regions. This technique will detect all types of mutation/polymorphism. Denaturing High Performance Liquid Chromatography (DHPLC) Like DGGE, this technique relies upon differential denaturing profiles of heteroduplexes separated on an HPLC column. Melting differences are exploited by the application of a temperature profile specific for each PCR fragment. The technique requires extensive optimisation of conditions for each exon/PCR fragment and a high initial investment for the machine. Although the analyses are done sequentially, throughput is very high and fully automated (96 well format). The running cost per sample is very low and special/ labelled primers are not required. Sensitivity also approaches 100 %. All kinds of sequence variants are detected, though frequent polymorphisms may be recognised by their characteristic elution profile. Protein Truncation Test (PTT) Unlike all other techniques, PTT aims at detecting only those mutations which result in premature termination of the protein product. This technique is particularly suited to analysis of the large exons in BRCA1 and BRCA2 and allows the use of genomic DNA as a test source. The technique is relatively easy to perform and doesn’t require special equipment. A possible technical problem can occur when dividing the large exons into overlapping PCR fragments, as care must be taken to ensure that the primer overlap is large enough that mutations near the end of one fragment, which may be missed, are detectable in the next fragment. If exons are amplified as a single fragment then it may be necessary to run two gel conditions or to use a gradient gel. Drawbacks are that PTT does not detect putative missense mutations (not considered by many to be a drawback) and also the use of radioactivity (typically 35S-methionine). A chemoluminescent kit for Western blotting is now available. Fluorescent Chemical Cleavage of Mismatch (FCCM) This technique is based upon the cleavage of chemically modified heteroduplex molecules by piperidine. Heteroduplex PCR products are labelled by incorporation of fluorescently labelled dUTP analogues followed by treatment with hydroxylamine and potassium permanganate. After chemical cleavage, PCR products are electrophoresed on a denaturing gel on the ABI 377 (or equivalent) and analysed by Genescan. By multiplexing it is possible to analyse the complete coding sequence of 6 patients for BRCA1, or 3 patients for BRCA2 using a 50 well ABI 377. Advantages of the technique is that it can be automated, sensitivity is reported to approach 100% and large fragments up to 1kb can be analysed. Disadvantages include the toxicity of hydroxylamine Guidelines for hereditary breast and ovarian cancer © EMQN 2001 3 Dr. Rob Elles (Co-ordinator) / Dr. Simon Patton (EMQN Administrator) Regional Molecular Genetics Laboratory, St Mary’s Hospital, Hathersage Road, Manchester M13 0JH, United Kingdom Tel: +44 161 276 6129/6741, Fax: +44 161 276 6606 Email: [email protected] / [email protected] European Molecular Genetics Quality Network EMQN Supported by the Standards Measurement and Testing programme of the European Union * * Contract no. SMT4-CT98-7515 and piperidine; the laborious nature of the analysis of many small exons and the relatively high false positive rate. Another drawback is that when multiple cleavage events are expected in a single fragment, e.g. at 3 common polymorphisms in exon 11 of BRCA1, then it is necessary to introduce a wildtype DNA to allow heteroduplex formation and consistent cleavage. Gene Dosage Gross genomic rearrangements have been reported in BRCA1, presumably due to the presence of a high number of Alu repeats within the genomic sequence of this gene. Detection of such rearrangements has been reported using Southern blot analysis, RT-PCR analysis, fluorescent quantitative PCR and Multiplex Amplification of HYB. So far, rearrangements have been reported in exon 1-2, 8-13, 13-16, 15, 17 and 27of BRCA1. No such rearrangements have been reported in BRCA2 to date. Southern blot analysis is slow, has low sensitivity (especially for duplications), requires large amounts of DNA and frequently uses high energy radioactivity. RT-PCR is technically demanding and the sensitivity is affected by technical problems and decay of mutant mRNAs. Quantitative fluorescent PCR analysis is technically challenging but has been used with considerable success for both, the dystrophin and the BRCA1 genes using labelled PCR primers and incorporation of fluorescent dNTPs (Yau et al 1996, Robinson et al 2000). The advantage of this technique is that it is sensitive, can be automated and has potential high throughput capacity. Disadvantages include the cost of labelled primers and the time taken to optimise the technique. Sequencing Direct sequencing of PCR fragments using dye primers is often quoted as the gold standard with a reported sensitivity of 100 %. It is possible to automate all steps and to have very high throughput capacity. Practically the sensitivity is often not as high as reported, with labs using this technique reporting lower sensitivity due to practical problems and insensitive analysis software. Disadvantages include the laborious nature of the analysis oflarge numbers of small exons, and the expense. Dye terminators can be used instead of Dye Primers, but this reduces the sensitivity of the technique. As with most of the other techniques it will identify all types of mutation/polymorphism, except genomic rearrangements. EMD To be added. Mutation Specific tests. Mutation specific tests such as Allele Specific Oligo’s, ARMS and restriction enzyme digests etc. can be useful when looking for specific common mutations in a patient cohort, e.g. the three common Ashkenazi Jewish mutations. Controls Positive controls should be used on all analyses to ensure that the correct fragment is being analysed and that the technique used is working. For predictive testing a close relative carrying the mutation should be used if possible; if not, another sample containing the relevant mutation should be used. It is advisable to confirm the presence of a particular mutation in the family prior to offering predictive testing. Interpretation and reporting What is a mutation? For the interpretation of an observed sequence variant it is essential to establish the causal role of the variation in the pathogenesis of the disease. Given the vast heterogeneity of sequence changes in both genes this constitutes a major challenge which requires extensive biological assays and/or family studies. Usually, this is not offered as part of a routine diagnostic service. Published data are available for a limited number of (recurrent) mutations only. In the absence of experimental evidence, the pathological significance of an observed sequence change has to rely on plausibility considerations. The following mutation types have most likely pathological consequences for the protein function: • • • • Mutations which formally interfere with proper protein synthesis [nonsense mutations (= stop codons), frame shifting mutations, Mutations which are likely to lead to altered splicing of the mRNA (splice site mutations). This may be checked by mRNA studies, Other mutations with experimental proof (published or own data) of their impairment of the protein’s function. Sequence variants which have been shown to strictly co-segregate with the disease in several unrelated pedigrees and which are not found in a large number of control samples. All other sequence variants must be considered as “unclassified” until functional evidence becomes available. Reports should be made to a Clinical Geneticist or other acceptable source. The exact content of the report is very much dependant on the extent of the genetic knowledge of the referring Guidelines for hereditary breast and ovarian cancer © EMQN 2001 4 Dr. Rob Elles (Co-ordinator) / Dr. Simon Patton (EMQN Administrator) Regional Molecular Genetics Laboratory, St Mary’s Hospital, Hathersage Road, Manchester M13 0JH, United Kingdom Tel: +44 161 276 6129/6741, Fax: +44 161 276 6606 Email: [email protected] / [email protected] European Molecular Genetics Quality Network EMQN Supported by the Standards Measurement and Testing programme of the European Union * * Contract no. SMT4-CT98-7515 clinician. Many have specialised in cancer genetics and should thus be aware of the full clinical spectrum of each gene. Reports should include a statement of which genes were tested and why, the extent and the limits of the analysis and the methods used (refer to the UK Clinical Molecular Genetics Society (CMGS) guidelines on reporting - http://www.emqn.org/reports.htm) Positive result in an affected index case. The report should state that this result is consistent with affection status and that other family members are at risk. It should also state that the mutation is likely to be the cause of the breast/ovarian cancer in the family. It is generally felt inappropriate at this time to point out the increased risk of other cancers in mutation carriers i.e. the increased risk of prostate cancer in male BRCA1 heterozygotes. Also interim reports on PTT or other positive results (such as a SSCP band shift) should only be issued if absolutely necessary, and reporting should wait until the causative mutation has been characterised. Negative result in an affected index case The risk of a pathological mutation segregating in the family has not been changed. This also applies to a negative result for an unaffected individual with a strong family history or tested for common ethnic mutations. No comment should be made regarding the percentage of mutations excluded, as ascertainment is incomplete, even when every exon has been screened and RNA expression examined. As mentioned above it is necessary to include details of the extent of the analysis. Predictive testing There is a large amount of discussion on the type of sample required for predictive testing for a known mutation. The methods used at the moment include the following: • • • • A single test on a single sample Two identical tests on the same sample Two different tests on the same sample One identical test carried out on duplicate blood samples taken at the same time or on different days. It is likely that other variations also occur. Generally it is felt that this must be left to local practice or local legislation. The report should state that the risk of breast/ovarian cancer has been reduced to that of the general population in females. In males the test result should be reported and mentioned that the result has implications for any of his offspring, in particular his daughters. Positive predictive test The report should state that this results in a high lifetime risk of breast/ovarian cancer. It is not felt appropriate to mention other BRCA related cancers at this time. It is advisable not to report a specific risk, as there is potential bias in the calculated risk figures, as families with very high incidence of cancer were used in their calculation (Easton et al 1997). There is clear evidence that familial factors may influence risk and that risk may be different for those unselected for family history. Prenatal diagnosis Prenatal diagnosis is to be carried out under the same criteria as with every other late onset disease. It should always be referred from a clinical geneticist after extensive genetic counselling. Testing of minors As there is no evidence that any symptoms will manifest in childhood the testing of minors is not recommended. What constitutes a minor will however, differ depending upon national considerations. Unknown variants As extensive mutation analyses become common practice many rare sequence variants are being discovered in both genes. A major challenge for the near future will be in how to interpret these variants. Some may be pathogenic with high/medium/low penetrance and may act as modifying factors. Until functional analyses are obtained and proven it is prudent to report them as “variants of unknown pathological significance”, for all but the most common variants which have been shown to segregate with the disease in large pedigrees. Breast cancer Information Core (BIC) Although considered to be a useful resource the data submitted to the Breast Cancer Information Core is not validated and therefore care should be taken when referring to it. Also submission is not widespread from the diagnostic community and therefore the mutations presented will not truly represent the full mutational spectrum of either gene for many populations. Negative predictive test Guidelines for hereditary breast and ovarian cancer © EMQN 2001 5 Dr. Rob Elles (Co-ordinator) / Dr. Simon Patton (EMQN Administrator) Regional Molecular Genetics Laboratory, St Mary’s Hospital, Hathersage Road, Manchester M13 0JH, United Kingdom Tel: +44 161 276 6129/6741, Fax: +44 161 276 6606 Email: [email protected] / [email protected] European Molecular Genetics Quality Network EMQN Supported by the Standards Measurement and Testing programme of the European Union * * Contract no. SMT4-CT98-7515 Future Developments Apart from the obvious development in mutation screening technology there are also exciting developments in diagnosis. In particular the potential use of microarrays to look at gene expression within tumours and the ability to tie that in with family history information. Likewise the improved capacity of pathologists to recognise BRCA1-like tumours in patients may offer further valuable information to clinicians in assessing risk within families. • • • • • • References • • • • • • • Miki Y et al (1994) A stong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266:66-71 Wooster R et al (1995) Identification of the breast cancer susceptibility gene BRCA2. Nature 378:789-792 Tavtigian S et al (1996) The complete BRCA2 gene and mutations in chromosome 13q-linked kindreds. Nature Genet 12:333-337 Ellison et al (1998) Phelan CM et al (1996a) Ovarian cancer risk in BRCA1 carriers is modified by the HRAS1 variable number of tandem repeat (VNTR) locus. Nature Genet 12:309-311 Phelan CM et al (1996b) Mutation analysis of the BRCA2 gene in 49 site-specific breast cancer families. Nature Genet 13:120128 Bishop DT (1994) BRCA1, BRCA2, BRCA3 ... a myriad of breast cancer genes. Eur J Cancer 30A:1738-1739 • • • • • • Easton D (1997) Breast cancer genes - what are the real risks. Nature Genet 16:210-211 Easton et al (1993) Narod SA et al (1995) Risk modifiers in carriers of BRCA1 mutations. Int J Cancer 64:6 394-398 Ford D et al (1998) Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. The Breast Cancer Linkage Consortium. Am J Hum Genet 62:3 676-689 Struewing JP et al (1997) The risk of cancer associated with specific mutations of BRCA1 and BRCA2 among Ashkenazi Jews. N Engl J Med 336:20 1401-1408 Scully R et al (1997) Dynamic changes of BRCA1 subnuclear location and phosphorylation state are initiated by DNA damages. Cell 90:3 425-435 Chen et al (1998) Lorrick et al (1999) Gayther SA et al (1996) Rapid detection of regionally clustered germ-line BRCA1 mutations by multiplex heteroduplex analysis Am J Hum Genet. 58(3): 451-456 Ganguly T et al (1998) High throughput fluorescence-based conformation-sensitive gel electrophoresis (F-CSGE) identifies six unique BRCA2 mutations and an overall low incidence of BRCA2 mutations in high-risk BRCA1-negative breast cancer families Hum Genet 102:5 549-556 Yan et al (1996) Robinson et al (2000) Table 1. Nomenclature and gene ID Gene BRCA1 BRCA2 Useful links OMIM# 113705 600185 Breast Cancer Information Core Breast cancer linkage consortium Human Gene Mutation Database Myriad Genetics Rosgen Guidelines for hereditary breast and ovarian cancer © EMQN 2001 6 Dr. Rob Elles (Co-ordinator) / Dr. Simon Patton (EMQN Administrator) Regional Molecular Genetics Laboratory, St Mary’s Hospital, Hathersage Road, Manchester M13 0JH, United Kingdom Tel: +44 161 276 6129/6741, Fax: +44 161 276 6606 Email: [email protected] / [email protected]