Genetic Testing Flashcards
1
Q
Different types of genetic testing
A
- Population carrier screen
- Prenatal test
- Prenatal screen
- New born screen
- Diagnostic test
- Predisposition test
- Predictive test
2
Q
What is purpose of population carrier screen.
A
Identifies heterozygotes – people with one copy of a mutant gene.
3
Q
What is purpose of prenatal test.
A
Detects mutant allele in a foetus for a condition present in a family
4
Q
What is purpose of prenatal screen .
A
Test embryos or foetuses from a population for increased risk of a condition based on family history.
5
Q
What is purpose of new born screen .
A
Population wide testing for several treatable inborn errors of metabolism
6
Q
What is purpose of diagnostic test .
A
Confirms diagnosis based on symptoms
7
Q
What is purpose of predisposition test.
A
Detects allele associated with an illness but not absolutely diagnostic of it.
8
Q
What is purpose of predictive test.
A
Detects highly penetrant mutation with adult onset in an individual at high risk based on family history.
9
Q
How do evaluate the quality of genetic tests.
A
- Analytic validity: is the test result correct?
- Clinical validity: does the result correctly diagnose disease or its absence?
- Clinical utility: does the result guide medical management ?
- Ethical validity: how well does the test meet the expected ethical standards?
10
Q
What are the two key indicators of analytic validity.
A
- Sensitivity: proportion of affected individuals who are correctly identified by testing positive in the test ( if highly sensitive = few false negative)
- Specificity: proportion of unaffected individuals who are correctly identified by testing negative in the test. (If highly specific =few false positives)
11
Q
What is the result of a false diagnosis .
A
Normal individual sent for unnecessary extra test and costs, affected individuals not being treated and may die.
12
Q
What is a false negative or positive.
A
False negative : affected individuals incorrectly give a negative test results.
False positive: affected individuals incorrectly give a negative test result.
13
Q
What is a true positive of negative
A
True positive: affected individuals who tested positive
True negative: unaffected individuals who tested negative
14
Q
Define Sensitivity
A
Fraction of person with disease who have the susceptibility genotype.
+ve present /+ve present +-ve present
15
Q
Define specificity
A
Fraction of persons without disease who do not have the susceptibility genotype.
-ve absent/+ve absent + -ve absent
16
Q
Explain the clinical validity of tests
A
- Primary concern of the clinician: how accurate is a positive and negative test results.
- Use predictive values: directly related to the prevalence of disease= number of cases in a defines population at a specific time.
17
Q
Explain Positive predictive value (PPV)
A
Probability that following a positive test result, that person will truly have the disease.
+ve present/ +ve present + +ve absent
18
Q
Explain negative predictive value (NVP)
A
Probability that following a negative test result, that person will truly not have the disease.
-ve absent / -ve present + -ve absent
19
Q
What is the false positive rate
A
- Proportion of positive tests that are false.
- If a test has significant false positive rates then the predictive value is very low and cannot be used in screening .
- +ve absent/ + ve present + +ve absent
20
Q
Explain what a False negative rates is.
A
- Proportion of negative tests that are false.
- If a test has significant false negative rates then the predictive value is very low and cannot be used in screening
- -ve present/ -ve present + -ve absent
21
Q
What is a direct [DNA] diagnosis of genetic testing.
A
- Used to detect mutations in patients when the disease gene is known
- Disease -caused variants characterised.
22
Q
What is a indirect [DNA] diagnosis of genetic testing.
A
- Method of choice when the disease gene has been localised to a specific chromosomal region, but not necessarily cloned/ characterised.
- Or for very large genes with many exons, difficult -to -find mutations, may be involved in the disease.
- Performed by linkage analysis.
23
Q
List some indirect DNA diagnosis.
A
- Distinguish the two chromosomes in the relevant parents. (Linked marker)
- Determine the phase (which chromosome )
- Work out which chromosomes the person who is being tested inherited.
24
Q
Applications of chromosome analysis.
A
- Prenatal diagnosis
- Pre-implantation diagnosis
- New-born screening
- Clinical diagnosis
- Cancer genetics
- Research
25
We test for the Presence or absence of chromosome abnormalities and CNV before birth.
Due to ….
1. Maternal age(over 35 years)
2. Previous abnormalities (de novo)
3. One of the parents carrier of a structural chromosome abnormalities.
4. Family history (greater family)
5. Abnormal antenatal sonar
6. Screening of maternal serum indication a high risk .
7. Maternal anxiety
26
Explain what human karyotype is.
Karyotyping allow visualisation of numerical and large structural chromosome changes during arrested in mitosis.
27
Pros of G-banded karyotype
1. Whole genome analysis
2. Overall impression
3. Can use blindly, no prior knowledge required
4. Can detect balanced chromosome changes
5. Can detect polyploidies and mosaics
28
Cons of G-banded karyotype
1. Low resolution (1 – 10Mb)
2. Culturing needed (needs metaphases)
3. Labour intensive and specialised (very little automation possible)
4. Time consuming, time delay to results (3 – 14 days)
29
Explain what fluorescence in situ hybridisation.
1. Combine Karyotype & Hybridization techniques
2. Chromosomes/Chromatin Immobilized on microscope slide
3. DNA probes anneal to specific target DNA sequence
4. Probes are labelled with fluorescent reporter molecules
5. Presence or absence of a particular genetic aberration can be viewed under fluorescence microscope.
30
Pros of FISH
1. Higher resolution (depend on application / probe design)
2. Can use both inter- and metaphase cells
3. Quick technique to detect abnormal chromosome numbers
4. Many fluorochromes available, can detect multiple probes simultaneously
5. Spectral karyotyping - can evaluate complete karyotype in single experiment
31
Cons of FISH
1. Need prior information (area of interest)
2. Information gained limited to design of probe
3. Not general screening tool
32
Characterise quantitative fluorescence PCR
1. Pre and Postnatal Screening test
2. Short Tandem Repeats (STR) Markers for common aneuploidies
3. Chromosome 13, 18, 21, X and Y
4. Fluorescently labelled primers used for PCR
5. Analysis by Capillary Electrophoresis
33
Material used in QF-PCR
Any DNA containing tissue, uncultured or cultured.
Amniotic cells , chorionic villi, foetal or peripheral blood, baccalaureate cells
34
Explain QF-PCR technique
1. Extract DNA
2. Perform Multiplex PCR using Fluorescently labelled primers.
3. Separate PCR products through Capillary Electrophoresis according to size
4. Analyse using suitable Software e.g. GeneScanTM or GeneMapperTM
35
Pros of QF-PCR
1. Can use any nucleated cells, cultured and uncultured
2. Rapid results, 24 to 48 hours
3. Many SRTs available, can do multiplex PCR
4. Can be use with Non-Invasive Foetal Aneuploidy Screening
5. High accuracy in identifying specific chromosome
6. Largely automated
36
Cons of QF-PCR
1. Only screening tool for common aneuploidy
2. No information gained on structural or other numerical abnormalities
3. Should be used in conjunction with other diagnostic procedure
37
Two types of microarrays
1. Array-based comparative genomic hybridization (aCGH)
2. SNP Microarrays
38
Function of microarray- based genomic copy number changes.
1. Deletions and duplications less than 5-10 Mb of DNA not detected by chromosomal banding techniques
2. Microarray-based DNA hybridization assays scan the DNA of each chromosome for changes in copy number (deletions or duplications)
3. Detect sub-chromosomal changes in DNA copy number
39
Principle array comparative genome hybridisation (aCGH)
1. Patient/test DNA is labeled with one colour (green), reference (normal) DNA is labeled with another colour (red).
2. two samples are mixed, then hybridized competitively to DNA probes immobilized on a slide (chip)
3. ratio’s of red-to-green fluorescence is measured along length of each chromosome
4. amplified DNA in patient will be present in Increase copies, hybridize stronger to normal chromosomes =more green signal
5. deleted DNA in patient will be present in decrease copies, hybridize less to normal DNA = more red signal
40
What types of arrays can you do during array comparative genome hybridisation. (ACGH)
1. Whole genome arrays
- Cover the entire human genome
2. Targeted arrays
-Microdeletion syndromes
- Sub-telomeric rearrangements
-Genomic abnormalities in cancer
- Sub-microscopic aberrations
- Prenatal Genetic Diagnosis
41
Application of aCGH
1. Detection chromosomal imbalances and large- scale changes in cancer
-Tumour samples against a control of lymphocyte DNA from the same individual
2. Identifying suspected gene imbalances in children with developmental disabilities or congenital anomalies
42
Characterise SNP microarray hybridisation
1. Single nucleotide polymorphism microarrays – panels of oligonucleotides shorter in length than those in aCGH
2. Hybridized only to individual alleles
3. Compares the dosage of the individual being tested at any locus to equivalent values in a database of SNP array results from control individuals
4. Detect gains and losses of sequences across the genome as well as absence of heterozygosity
43
When is copy number variation clinically significant. (Abnormal (Pathogenic))
1. If described in well established syndromes
2. De novo (different from parents)
3. Identical to an affected parent
4. Large changes – gene rich
5. The CNV is a deletion (homozygous)
6. The CNV is greater than 1 copy gain (amplification)
44
When is copy number variation clinically significant.(likely benign)
1. Not previously reported but inherited from a healthy parent
2. Identical to a healthy parent
3. Described in CNV database of healthy individuals
4. Duplication with no known dosage-sensitive genes
5. Devoid of known regulatory elements
45
WHen is conventional karyotyping still indicated?
1. Array-CGH can’t detect balanced chromosomal rearrangements – no gain or loss of DNA
2. Inversions
3. Balanced chromosomal translocations
46
Pros of aCGH/SNP array
1. Whole genome analysis
2. Resolution high (Array - depending on design)
3. Can use blindly
4. Detect all gains & losses simultaneously
5. SNP Array can detect Uniparental disomy
47
Cons of aCGH/SNP array
1. Needs Chip (Array) - expensive
2. Difficult to interpret results
3. Cannot detect balanced abnormalities
4. Cannot distinguish between duplication, insertion, marker chromosome, unbalanced translocation.
5. Chromosomal localization not possible
6. Cannot detect mosaic state
48
Analysing a SNP-array
1. Log R Ratio (LRR) – normalized measure of the total signal intensity for 2 alleles of a SNP
2. B allele Frequency (BAF) – normalized measure of the allelic intensity ratio of 2 alleles
3. Using LRR and BAF – used to infer copy number changes in the genome
4. deletion – decrease in LRR values and a lack of heterozygote values in BAF
5. Duplication – increase in LRR values and splitting of heterozygote clusters in 2
49
Describe Multiplex ligation-dependent probe amplification (MLPA)
1. PCR-based method to detect copy number changes
2. Scan intragenic deletions and duplications by monitoring copy number of exons within genes
3. Duchenne muscular dystrophy due to high number of deletions/duplications
4. Complements DNA sequencing to scan for copy number changes
50
What is direct DNA testing.
1. Mutation scanning / screening: detect and localise unknown mutations.
2. Mutation testing/ detection: detection of known or common mutations.
51
What are the different types of frequency and nature of mutations.
1. Point mutations, small insertion/deletions . (Account for most mutations)- found in APC, BRCA1 and BRCA2.
2. Large insertion/deletion-found in dystrophin(deletion) and PMP (duplication)
3. Gross arrangements - found in haemophilia A
4. Expansion of triplet repeat-found in friedrich Ataxia.
52
Criteria for designation of mutation as being of pathogenic significance.
1. In coding regions causing truncation (Frameshift or nonsense) - likely to be phenotype modifying.
2. If missense- more likely to be pathogenic when: Located in functionally important part of protein.
4. Evolutionary conserved amino acid- greater functional importance.
5. Non-conservative amino acid change- greater effect.
6. If mutation segregates with disease may be disease-causing.
7. Mutation = rare<% prevalent
53
Variation in types of mutation and location/ extent of genetic heterogeneity .
1. Specific mutation in a specific gene in all affected individuals. Eg sickle cell or achondroplasia
2. Multiple potential mutation sites in a specific gene in affected individuals. Eg cystic fibrosis and beta thalassemia
3. Multiple potential mutation sites in several genes in affected individuals eg. Hereditary deafness + cardiomyopathy
54
When screening a disease with specific mutation in a specific gene in all affected individual.
You will pick up a specific mutation in all affected individuals
55
When screening for a disease with multiple potential mutation sites in a specific gene in affected individuals. What do you look for ?
1. Affected individuals will show a limited range /set of mutations .
2. Could be founder mutation
3. So you screen for common mutations such as repeat expansions, missense, detection of an amino acid.
56
When screening for a disease with multiple potential mutation sites in several gene in affected individuals. What do you look for ?
1. Panel of genes are nowadays used as alternative to sequencing gene by gene
2. Also WES or WGS if the phenotype is not clear.
57
Methods of testing for specific mutations [mostly small scale change such as point mutation]
1. ARMS (amplification refractory mutation system)
2. Oligonucleotide ligation assay (OLA)
3. Pyrosequencing
58
Purpose of identifying genetic disease through newborn screening.
1. Management of disease or its complications in a more effective manner than would otherwise be possible.
2. Screening for inborn errors of metabolism such as phenylketonuria in newborns so that it can be treated with a different diet.
3. Screening for congenital hypothyroidism for hormone replacement.
59
Purpose of identifying individuals at increased risk of having affected children through carrier screening.
1. To take advantage of reproductive options that may prevent birth of affected children.
2. Detection of heterozygous carriers for Tay-Sachs disease among Ashkenazi Jews populations.
3. Detection of heterozygous carries for Thalassemia in Sardinia and Cyprus.
60
Purpose for testing for adult onset diseases, individuals with a genetic predisposition to a disease (at risk), through adult screening/ genetic risk assessment.
1. To enable them to institute measures that will prevent or delay development of the disease.
2. This can be done through predictive testing/predisposition testing
3. Predisposition testing
61
Distinguish between predictive and predisposition tests.
1. Predictive tests/ pre-symptomatic: identify Whether an individual has inherited a gene mutation that may lead to a genetic disorder- adult onset.= is predictive of the actual risk of developing the disorder.
2. Predisposition tests: identify an individual that may be at increased risk of a complex disorder. = is not predictive of the actual risk of developing the disease.
62
Goal of prenatal screening/ diagnosis
1. Give parents a range of informed choices
2. Provide reassurance and reduce anxiety
3. Allows couples to begin a pregnancy with the knowledge that the presence or absence of the disorder can be confirmed
4. Give the option of appropriate management for the impeding birth of a child with a genetic disorder
5. Enable prenatal treatment of the affected fetus
63
Indications for prenatal diagnosis
1. Advanced maternal age (AMA) – older than 35 years
2. Previous child with a de novo chromosome abnormality.
3. Presence of a structural chromosome abnormality (balanced chromosome translocation) in one of the parents
4. Family history of a genetic disorder that may be ruled out by biochemical or DNA analys
5.Family history of an X-linked disorder for which there is no specific prenatal diagnosis (sex selection)
6. Increased risk for a neural tube defect 7.Abnormal maternal serum screening and
ultrasound examination
8. Abnormalities in the fetus suspected
64
Traditional prenatal screening
1. Combination of biochemical testing of substances in the mother blood and ultrasound to detect trisomies and other abnormalities.
2. Screening tests- only gives a risk (1 in 100/1 in 250)
65
Prenatal screening is carried out
1. First trimester (11-13 weeks)
2. Second trimester (15-20 weeks)
66
First trimester screening (ultra sound)
1.1 Measure nuchal translucency by ultrasound.
1.2 We Subcutaneous oedema of the neck of the foetus, this could indicate increased in trisomies.
67
1. Increase in trisomy 21
2. Decrease in other trisomies
2.1 Measurement of certain substances in maternal serum
2.2 Pregnancy-associated plasma protein A (PAPP-A) = Decreased in trisomies 2.1 Measurement of certain substances in maternal serum.
3.1 human chorionic gonadotropin (beta-hCG)
3.2 Increase in trisomy 21
3.3 decrease in other trisomies
68
Second trimester screening (AFP)
1.1 Maternal serum alpha- fetoprotein (AFP) can be used to Diagnose defects in the fetus body wall such as Open neural tube defect (spina bifida)
1.2 Measured at +- 16 weeks if there is an increase in AFP in spina bifida.
1.3 Combined with ultrasound most defects detected
1.4 if trisomy present AFP ↓ (not useful on its own)
69
Second trimester screening (triplet and quadruple test)
1. Triplet tests
1.1 Measures 3 markers in maternal blood serum
1.2 Alpha-fetoprotein (AFP)
1.3 Unconjugated estriol (uE3)
1.4 Human chorionic gonadotropin (β-hCG)
1.5 Detection rate of 70% at a 5% false positive rate
2. Quadruple test
2.1 Add Inhibin A
2.2 Increase the detection rate to 80%
70
Trisomy test for trisomy 21
1. AFP: decrease
2. UE3: decrease
3. B-hCG: increase
71
Triple test for trisomy 13 and 18
1. AFP: decrease
2. UE3: decrease
3. B-hCG: decrease
72
Triple test for NTD
1. AFP: large increase
2. UE3: N/A
3. B-hCG: N/A
73
Why do cytogenetic in prenatal diagnosis, what are some indications for the test to be done.
1. Invasive testing to confirm screening results
2. Specific indication such as a known cytogenetic defect in the family
74
Explain what chorionic villus sampling (CVS) is.
1. Done at 11-12 weeks gestation through cervix or trans- abdominally under ultrasound guidance
2. Sample of the placenta (cytotrophoblast cells and extra-embryonic mesoderm)
3. Risk for fetal loss due to CVS ±1%
4. Problems with interpretation – chromosomal mosaicism, maternal contamination
75
Explain what amniocentesis is.
1. Done at 16-18 weeks gestation with needle inserted trans abdominally under ultrasound guidance
2. Sample of amniotic fluid
3. Largely derived from fetal urine – skin and bladder cells
4.Risk for fetal loss 0.5%
76
Methods to clarify mosaicism
1. Uncultured CVS material must be cultured, If confined to the cytotrophoblast much less likely to be clinically significant.
2. Amniotic fluid samples – grown in multiple culture flasks .Analysis of colonies derived from individuals cells,If true mosaic = will be present in multiple colonies from several cultures
3. Pseudo-mosaicism – non-disjunction in culture (artifact)? If Present in a single colony, not clinically significant
77
Explain Percutaneous umbilical blood sampling (PUBS) - chordocentesis
1. After 18 weeks of pregnancy
2. Indications: Late in pregnancy Failure of culture of amniotic cells
3. Biochemical tests
4. Risk of foetal loss 2%
78
Tests performed on prenatal tissue
1. Standard chromosomal analysis
2. FISH or QF-PCR for aneuploidy syndromes
3. Chromosome microarray analysis
4. Biochemical tests
5. DNA tests for single gene disorders
79
Explain Non-invasive prenatal testing (NIPT)/ diagnosis (NIPD) and what they test for.
1. Isolation of fetal DNA rom maternal blood
2. Maternal circulation contains fetal DNA in very low abundance
3. Test for:
3.1 Single gene disorders
3.2 Chromosomal abnormalities (Down syndrome etc.)
3.3 Sensitivity and specificity of 99% (NGS)
80
Explain Pre-implantation genetic diagnosis and what tests are performed during.
1. During in vitro fertilization – 8 cell stage
2. Single cell (blastomere) removed for analysis
3. Unaffected embryos implanted
4. No detrimental effects to the
embryo
5. Tests done:
5.1 FISH – trisomy or chromosome
rearrangement
5.2 DNA analysis – specific mutation in the family
81
Genetic counselling - definition
1. The process by which patients or relatives at risk of a disorder that may be hereditary are advised of the consequences of the disorder, the probability of developing or transmitting it and of the ways in which this may be prevented, avoided or ameliorated. Harper 1981
2. An educational process that seeks to assist affected and/or at risk individuals to understand the nature of the genetic disorder, its transmission and the options open to them in the management
and family planning.
Kelly 1986
82
Counselling deals with:
1.The risk that the fetus is affected
2. Nature and possible consequences of the specific problems
3. Risk and limitations of the procedure to be used
4. The time required before a report can be issued
5. Possible need for a repeated procedure in the event of a failed attempt
6. Results might be difficult to interpret 7.Further testing and consultation might be
required
8. Results may not necessarily be definitive
83
Procedure of counseling
1. Obtain history (family, etc.) Which Determine the recurrence risk
2. Advise the parents to the genetic risk to them and other family members
3. Offer genetic testing or prenatal diagnosis 4. Outline the various treatment or management options to reduce the risks
5. NB: Non-directive counselling
84
Ethical issues
1. Autonomy
2. Beneficence
3. Non-maleficence
4. Justice
85
Define Autonomy
incorporating respect for the persons, their privacy, the importance of informed consent and confidentiality
86
Define beneficence
the principle of seeking to do good and therefor acting in the best interest of the patient
87
Define non-maleficence
the principle of seeking, overall, not to do harm, i.e. not to leave the patient in a worse condition than before the treatment
88
Define justice
incorporating fairness for the patient in the context of the resources available, equity of access and opportunity
89
Challenges in prenatal diagnosis
1. Availability of tests
2. Different indications for testing
3. Subtleties of interpretation of test results
4. Personal, ethical and religious issues into reproductive decision making
