Applications Of Genetic Testing Tools In Medicine PDF

Applications of Genetic Testing Tools in Medicine Dayna Wolff Department of Pathology and Laboratory Medicine [email protected] OBJECTIVES: After studying these lectures you should be able to: 1. Describe the appropriate test(s) to order for common mutations (single nucleotide change, large duplicat...

Applications of Genetic Testing Tools in Medicine Dayna Wolff Department of Pathology and Laboratory Medicine [email protected] OBJECTIVES: After studying these lectures you should be able to: 1. Describe the appropriate test(s) to order for common mutations (single nucleotide change, large duplication/deletion/expanded repeat) 2. Explain the role of genetic testing for diagnostic and carrier purposes in the evaluation of a patient • Explain the differences between a screening and a diagnostic genetic test. • Explain the limitations of genetic testing (ie: a negative test often does not rule out a clinically diagnosed condition). 3. Compare/contrast potentials and pitfalls of genetic testing to be used in providing health care. Molecular Tools for Medicine How can we apply molecular tools/tests to understand genetics of disease? Objectives Students will be able to:   Describe the appropriate test(s) to order for common mutations (single nucleotide change, large duplication/deletion/expanded repeat) Explain the role of genetic testing for diagnostic and carrier purposes in the evaluation of a patient    Explain the differences between a screening and a diagnostic genetic test. Explain the limitations of genetic testing (ie: a negative test often does not rule out a clinically diagnosed condition). Compare/contrast potentials and pitfalls of genetic testing to be used in providing health care. Diagnostic Algorithm for Genomic Medicine Genetic Testing Common Genetic Tests    Cystic Fibrosis Fragile X syndrome Factor V and Factor II (Thrombophilia)            Spinal muscular atrophy Inherited hemochromotosis Prader-Willi/Angelman syndromes Rett syndrome Huntington syndrome Mitochondirial diseases Biochemical disorders Deafness Cardiovascular diseases – Long QT syndromes Inherited cancer – breast, colon, multiple endocrine neoplasia Connective tissue disorders – osteogenesis imperfecta Best resource = http://www.ncbi.nlm.nih.gov/gtr/ Rett syndrome You do not need to know about Rett syndrome – it is being used as something that you might not be familiar with and need info on because you are seeing the patient with Dr Pai in the clinical tomorrow. What will you do? Click here Click here Click here Implications of Results: Context Matters  The informational and counseling needs depend on context and indications for testing  Examples:      Establish or confirm a diagnosis (Gaucher Disease) Reproductive decision-making, carrier testing, fetal testing (cystic fibrosis) Predict risk for future disease (BRCA, Huntington’s Disease) Aid management decisions (e.g., tumor sequencing) Ethical issues vary by context From: TRiG Curriculum: Lecture 4 The context matters in counseling. Regardless of whether a formal informed consent is required, genetic counseling seeks to ensure that patients and families understand the potential results they could learn and what those results may mean. Examples: In diagnostic testing, will a positive result change management? (a) With genetic testing for Gaucher disease, confirmation of this diagnosis may lead to treatment with the enzyme replacement therapy. (b) With genetic testing for cystic fibrosis, results may be used to inform future reproductive decisions for the asymptomatic patient who is either planning a pregnancy or currently pregnant. In such reproductive prenatal genetic testing, what will they do if a fetus is found to be affected? (c) In predictive testing, there may be options for reducing risk or early detection with enhanced surveillance in BRCA mutation carriers. (d) Predictive testing may also be for the purpose of life planning as is often the case in Huntington’s disease. Do they really want to know their risk if the options for risk management are limited or do not exist? (e) Predictive testing may also be used to aid management such as in pharmacogenomic testing or tumor sequencing. There may also be unique ethical issues based on the testing/patient context. These issues and the potential implications of testing should be discussed during pre-test counseling. Overview  Cystic Fibrosis (PCR, ASO and sequencing)    Fragile X (Southern blot)   Screening vs. diagnostic test Bayesian analysis Triplet repeat disorder Prader Willi/Angelman Syndromes (methylation specific PCR)  Imprinted genomic region Clinical scenario  28 year old white female presents for first OB visit at 10 weeks gestation. She reports that her husband’s half brother had cystic fibrosis. She has no known family history of CF. She wants to know her risk for having a child with CF. Risks  28 year old white female presents for first OB visit at 10 weeks gestation. She reports that her husband’s half brother had cystic fibrosis. She has no known family history of CF. She wants to know her risk for having a child with CF. What would be a very useful laboratory test result to have? What is the risk that the father is a carrier for CF? Brother’s mutation screen If know brother’s mutations, can screen directly for father’s status I will go over some examples of using specific molecular tools in the context of several diseases. These should be diseases that you already know a little bit about but what is most important is that you understand the concepts of why we are doing certain tests for these diseases and what are the limitations of the testing. Risks  28 year old white female presents for first OB visit at 10 weeks gestation. She reports that her husband’s half brother had cystic fibrosis. She has no known family history of CF. She wants to know her risk for having a child with CF. What is the risk that the father is a carrier for CF? You will learn more about calculating the genetic risk for family members when you do the Small group exercise. However, I expect that you know a bit about how to do a Punnet square and to figure out alleles (like both parents of an autosomal recessive disease must be carriers) What is the risk that the mother is a carrier for CF? Popn risk Testing = risk reduction 1/2 CFTR gene: Cystic Fibrosis Transmembrane Conductance Regulator   Over 2000 mutations have been reported to date The most common mutation is ∆F508 (F508del) that accounts for ~70% of mutations CF Carrier Screening Test    ACOG: guidelines for population based CF carrier screening ACMG: minimal test panel includes 23 mutations R/O carrier status to a confidence of: Chance to be a carrier Accuracy of DNA test Caucasian 1/29 90% Hispanic 1/46 57% Ashkenazi Jew 1/29 97% African American 1/65 75% Asian American 1/90 30% We would do a carrier screening test on the mother. The American College of Obstetrics and Gynecology produced guidelines for population based testing in 2001 and the American College of Medical Genetics determined that the screening test should include the most common 23 mutation as that would allow for 90% of known mutations for the most commonly affected group (Caucasians) to be detected. This test is less sensitive for other ethnic groups. Considerations for Clinical Lab in determining method   How to test for 23+ mutations on a large number of patient samples? Factors to consider     Turn around time; high throughput Fast, efficient method Cost of testing Easily interpretable First need to generate enough DNA to test… Polymerase Chain Reaction PCR PCR 1 PCR 2 PCR 3 Then, need to have post PCR method to detect mutations…     Fragment analysis using capillary gel electophoresis Allele-specific oligonucleotide hybridization (ASO) Sequencing* Important for CF carrier testing, need to be able to detect the mutant and the normal sequence PCR is used to generate tons of the particular DNA sequence of interest, so that you can test this. Prior to PCR, you would have had to clone the DNA of interest into a vector to get enough to study, so the beauty of PCR is that this simple denature, anneal, extend, repeat cycle gives you the DNA that you need. Was only possible when used a heat stable polymerase (Taq) enzyme. Allele-specific oligonucleotide hybridization (ASO)  Multiplex PCR for 36 mutations, followed by reverse dot blot CF ASO Assay For ASO, the patient DNA is PCRed and a label is incorporated into the PCR product. The little nylon strip (shown with green and white stripes) has probes for the normal sequence and the mutant sequences along the entire length (every green line represents a different probe that is complementary to a normal or mutant sequence) The patients’s DNA is hybridized the strips and will only bind to the normal or mutant probes if that DNA is present in the patient. Thus you will only see the probe line light up with a color if that sequence is present in the patient. These are real pictures from a commercially available test: all of the probes on the top half of the strips have probes for mutations and the normal probes are on the bottom half of the strips. Two strips are used because the probes did not fit on a single strip. So the patient sample on the left has no hybridization to mutant and does have hybridization to all of the normal sequences, so no mutation detected. The other samples show one (consistent with being a carrier of a CF mutations) or two mutations (two lines in the top half of each strip) consistent with a diagnosis of CF. Residual Carrier Risk: Bayesian Analysis Carrier Non carrier Prior 1/25 (0.04) 24/25 Conditional 10% (0.1) 1 Joint Posterior 1/250 24/25 1/250 1/250 +240/250 = 1/241 Risks  28 year old white female presents for first OB visit at 10 weeks gestation. She reports that her husband’s half brother had cystic fibrosis. She has no known family history of CF. She wants to know her risk for having a child with CF. Apriori: ½ x 1/25 x ¼ = 1/200 What is the risk that the father is a carrier for CF? 1/2 1/241 What is the risk that the mother is a carrier for CF? After testing: ½ x 1/241 x ¼ = 1/1928 CF Diagnostic Test 2 day old white male has positive newborn screening test for CF; blood sent to the lab for confirmatory molecular testing    Screen for most common mutations If negative, cannot completely rule out a diagnosis Often additional, diagnostic testing is necessary This calucation is given so that you will have some understanding of how we would calculate residual risk – you do not need to know how do this calculation but you do need to understand that a negative carrier screen for CF does not exclude a person from being a carrier. A negative result from the CF carrier screen will greatly reduce the risk that the person is a carrier however. So in this case, the mom’s risk was reduced from about 1/25 to 1/241 based on her having no mutations detected on the carrier screen. MUSC screen results CF mutation panel results: F508del/Does the patient have CF? Would you suggest additional testing? What? Sequencing Sanger Sequencing More commonly used: massively parallel sequencing Schnekenberg RP, Németh AH; Next-generation sequencing in childhood disorders Archives of Disease in Childhood 2014;99:284-290 Follow Up Testing Results ? CF results: F508del/Y112X CF Testing Review  Carrier Screen    Screen for 23 mutations whites Expanded mutation screen for others Adjust risk based on test results  Make sure patient understands this is screening test so there is residual risk  Diagnostic Testing    Look for most common mutations Reflex to gene sequencing Mutation testing is used to confirm clinical diagnosis – even with gene sequencing, sometimes do not find two mutations Case scenario  3 year old male is referred from general pediatrician to Dr. Champaigne for genetics evaluation due to developmental delay/mental retardation. Child does not have congenital anomalies and appears phenotypically normal. Two mutations were found in the proband with the original diagnosis of CF (the screen for the most common mutations found the F508del mutation, but the other more rare mutation was only detected by full gene sequencing). Now the aunt is curious about CF in one of her children so you would need to be able to do the calculations to detemine risk for the aunt, her husband and then calculate risk for the child Pedigree analysis   3 year old male is referred from general pediatrician to Dr Pai for genetics evaluation due to developmental delay/mental retardation. Child does not have congenital anomalies and appears phenotypically normal. Family history shows that the proband had an uncle on his mother’s side with mental impairment Testing that you might 1. 2. 3. Full mutation >200 consider in child with dev delay Chromosomes Microarray Fragile X test Many expansions>1000 repeats Too large for PCR Remember that these expansions can get very large – very hard to PCR reliably. So we can size the repeats using PCR for those in the normal – premutation range, but not in the full mutation range. Premutation 55-200 Gray zone 45-54 Normal 5-44 Why do we need 2 tests for diagnosis? 5-44 55-200 >200 So you need to be able to do the PCR to size normal, premutation alleles, but need to do a Southern blot to detect the very large full mutation repeats. So with the Southern blot you need to start with a bunch of DNA (remember that we cannot PCR to get a bunch of the sequence we are interested in so we need to isolate a bunch of DNA from the patient directly). Then you use restriction enzymes to cut the DNA into fragments of different sizes, you run this out on a slab gel and separate the fragments by size. Then based on the size, you can determine if the fragment is consistent with a normal, premutation or full mutation size. Detection of the CGG Full Expansion Need for Southern Blotting 2.8kb Methylation sensitive enzyme Normal restriction enzyme CGG repeats Probe You can also use a special enzyme that distinguishes methylated Cs from unmethylated Cs to see if the fragment of interest is methylated and turned off (inactivated gene). So this will let you know if the fragement is expanded in size and whether or not the fragment is methylated (consistent with diagnosis of Fragile X) or not methylated (not Fragile X). * * * * * ** ** * * * CGG repeats 5.2kb Why 4 bands in premutaion female? Remember normal X inactivation process. Thus, you have some cells with a normal sized CGG repeat that is subject to X inactivation (methylated and uncut fragment 5.2 kb) and some cells with a normal sized CGG repeat that is active (unmethylated and cut to give 2.8 fragment). Likewise, you can have a population of cells with an expanded (premutation) sized repeat that is active and cut to give a >2.8 fragment and a population of cells with an expanded premutation X that is uncut, methylated and >5.2 kb fragment. 2.8kb Southern Blot Results CGG repeats Probe * * * * * ** ** * * * CGG repeats 5.2kb 5.2kb 2.8kb Relative would like to know her risk of being a carrier Size Markers ?? F Premutation F Premutation F Normal M Premutation M For Carrier Testing: Use PCR to size her triplet repeat 100 repeats 50 repeats 30 repeats 20 repeats Testing algorithm for FMR1-related disorders (www.ncbi.nlm.nih.gov/books/nbk1384) Full mutation individuals Premutation Carriers POF Premutation Carriers FXTAS = Fragile X-associated temor/ataxia syndrome Fragile X Testing Review  Diagnostic testing indicated in ANY individual with intellectual disability   Southern blot indicated Carrier testing indicated in ANY female with family history of intel disability  PCR testing; possible reflex to SB Case scenario  Two year old male presents to the endocrinologist due to morbid obesity. Child is short, obese, has hypogonadism and has development delays. There is a history of hypotonia in the newborn period and failure to thrive as an infant. Family history was non-contributory. Premutation carriers do not have Fragile X, but are at risk for developing premature ovarian failures in carrier (premutation) females or FTAS in premutation males. Read over this algorithm carefully – there is lots of info in this and it is very important to understand. Mechanism for Uniparental Disomy Trisomy Rescue 1. 2. Maternal meiotic nondisjunction Mitotic nondisjunction Genomic Imprinting    Different gene expression dependent on parent Mediated by methylation DNA sequence identical So how can we do a molecular test for this? Testing that you might consider      Chromosomes? FISH? Microarray? DNA sequencing? Special type of PCR that can distinguish methylation of cytosines? Testing Algorithm for PWS/AS  Methylation-Sensitive PCR:   Distinguishes maternal and paternal alleles Detects PWS/AS due to deletion, uniparental disomy or imprinting error    >99% PWS ~ 75-85% AS Positive result confirms diagnosis of PWS or AS Methylation sensitive testing paternal maternal 174 bp 100 bp For this assay, you expose the patient’s DNA to a treatment with bisulfite, this changes the unmethylated Cytosines to Urosils (C to U) – now you have a difference in the DNA from the different parent. For example, on the dad’s chromosome the UBE3A gene, the gene responsible for Angelman syndrome, is methylated and inactive, but on the mom’s chromosome, the gene will be unmethylated and active. The opposite is true of the SNRPN gene that is shown above. So in the gel, you have designed the assay so that after bisulfite treatment, if the cytosines are methylated (inactive – and the SNRPN gene is inactive on the mom’s chromosome), then you get a larger PCR fragment; and you will get a smaller sized fragment (100 bps) from the PCR reaction with primers for the unmethylated and active SNRPN gene from the dad’s chromosome. So if you see both sized alleles, it is normal (having both mom and dad fragments), but if only one is present, you have to interpret which band is there and figure out what disease this is associated with. (Do this and you will be glad you did!) Prader-Willi Testing Review  Best diagnostic method = methylation sensitive PCR   Detects almost all cases of PWS (deletion and uniparental disomy Only detects ~80% of Angelman because single gene mutations account for about 15% of cases  If negative by methylation sensitive PCR, full gene sequencing is performed. Summary  Lots of genetic molecular tests   Need to have some idea of molecular testing to order appropriate tests Selection of testing platform depends on what type of aberration being tested Question  1. A woman comes for counseling about her risk for breast cancer. Both her mother and her sister were diagnosed with breast and ovarian cancers at an early age. Her sister had sequencing of her BRCA1 and BRCA2 genes and no mutations were detected. You suggest that additional testing of these genes may be needed to detect what type of aberration?     Chromosome analysis to look for a balanced rearrangement RFLP analysis for creation of a new restriction cut site Southern blot analysis to look for large deletions Realtime PCR analysis of introns Question  1. A woman comes for counseling about her risk for breast cancer. Both her mother and her sister were diagnosed with breast and ovarian cancers at an early age. Her sister had sequencing of her BRCA1 and BRCA2 genes and no mutations were detected. You suggest that additional testing of these genes may be needed to detect what type of aberration?     Chromosome analysis to look for a balanced rearrangement RFLP analysis for creation of a new restriction cut site Southern blot analysis to look for large deletions Realtime PCR analysis of introns