Lecture 19: Genetic Diagnostic strategies

Question 1

Central Dogma of Molecular Genomics…?

So once we have the genomic DNA sequence of a
species we have all of the information there is?

Answer 1

James Watson version - 1965

DNA –> RNA –> PROTEIN

2. NO, NOT REALLY

Question 2

Diagram on the increasing complexity of the central dogma of molecular biology

Answer 2

slide 4

Question 3

Genome structure overview:

Answer 3

diagram on slide 5

Question 4

Most detailed model of a human cell to date, obtained

Answer 4

1. “Cellular landscape crosssection through a eukaryotic cell.”
2. obtained using x-ray, NMR and cryoelectron microscopy datasets.
3. Evan Ingersoll and Gael McGill

Question 5

Geographic Information System of a Human Being

Answer 5

diagram on slide 7

Question 6

Overview of Molecular Genetic Diagnosis:

Testing is used for?
Direct mutation analysis?
Molecular techniques?
Clinical utitlity of tests?

Answer 6

1. Molecular genetic testing is used to detect changes in a
   single gene(s), and includes direct DNA sequence analysis & detection of large deletion or duplication.

2 * Direct mutation analysis is possible if the gene causing
the disorder has been identified.

3 * Molecular techniques are powerful tools
– allowing for precise identification of individuals who carry a mutation

4 * Clinical Utility of tests
– Quality of tests used

Question 7

Direct Analysis of Gene Variants

-Types of Mutations

Answer 7

1. Deletions, duplications, CNVs
   – FISH, Microarray, MLPA, PCR, Southern Blot eg FRAXA
   – Smaller deletions may remove an amino acids or result in frame shifts that usually truncate the protein product.
2. • Intragenic rearrangements:
     – Duplications and inversions
     – Point mutations
     – Mutations that affect splicing
3. • Triplet repeat expansion mutations

Question 8

Direct Analysis of Gene Variants
Types of Mutations:

• Deletions, duplications, CNVs = 2

Answer 8

– FISH, Microarray, MLPA, PCR, Southern Blot eg FRAXA

– Smaller deletions may remove an amino acids or result in frame shifts that usually truncate the protein product.

Question 9

Direct Analysis of Gene Variants
* Types of Mutations

Intragenic rearrangements: 3

Answer 9

– Duplications and inversions

– Point mutations

– Mutations that affect splicing

Question 10

Direct Analysis of Gene Variants:

Analysis of Sequence Variations

Answer 10

Assays to Detect Unknown Mutations in a ‘Known Gene’

– Direct DNA sequencing by Sanger or NGS.

Question 11

Sequencing Applications from
Prewomb to Tomb

Answer 11

DIAGRAM ON SLIDE 12

Question 12

Current and emerging application of genomic tests

Answer 12

TABLE OF ‘TYPE OF TEXT’ AND APPLICATION SLIDE 13

Question 13

Types of Genetic Tests =

10 TYPES AND EXPLAIN EACH

Answer 13

1. PRECONCEPTION TESTING
   – Most of us are “carriers” of hidden DNA changes that don’t affect our own health. But if
   you and your partner are both carriers of the same condition, your future child could
   be at risk.
   – RANZCOG recommends carrier screening should be offered to all women planning a pregnancy or in the first trimester of pregnancy.
   * CF, SMA and FXS are most common inherited genetic disorders, causing reduced life expectancy. Medicare funded from 2023. ~1 in 240 couples are at high-risk of their child being born with one of these disorders.
2. PRENATAL TESTING
   – Prenatal testing is used to detect changes in a fetus’s genes or chromosomes before
   birth.
3. NEWBRON SCREENING
   – used just after birth to identify genetic disorders that can be treated early in life eg
   cystic fibrosis, phenylketonuria and congenital hypothyroidism
4. DIAGNOSTIC TESTING
   – genetic testing is used to confirm a diagnosis when a particular condition is suspected based on physical signs and symptoms.
   - Performed at any time during a person’s life, & can influence a person’s choices about health care and the management of the disorder.
5. CARRIER TESTING\
   – used to identify people who carry one copy of a gene mutation that, when present in two copies, causes a genetic disorder. Autosomal recessive or X-linked recessive
   conditions.
   -This type of testing is offered to individuals who have a family history of a
   genetic disorder.
6. PREIMPLANTATION GENETIC DIAGNOSIS
   – PGD a technique that can reduce the risk of having a child with a particular genetic or chromosomal disorder.
   - It is used to detect genetic changes in embryos that were
   created using IVF.
7. PREDICTIVE TESTING
   – used to detect gene mutations associated with disorders that appear after birth, often later in life. For family members with a genetic disorder, but who have no features of the disorder at the time of testing.
   - Predictive testing – if the mutation is present, the
   person has an increased predisposition to develop the disorder in the future.
   - Not everyone with this increased predisposition will develop the condition eg mutations in BRCA1 & 2 (breast cancer) and MLH1 and MSH2 (colon cancer).
8. PRESYMPTOMATIC TESTING
   * if mutation present, the person is almost certain to develop the disorder in the future,
   - eg Huntington disease, familial adenomatous polyposis (FAP) and myotonic dystrophy.
9. FORESNIC TESTING
10. PREDICT DRUG RESPONSE
    – Testing to identify individuals who are less likely to respond or increased risk of adverse
    drug reaction to a medication.

Question 14

Genetic Testing on Embryos…what are they and examples?

= 5

Answer 14

1 * PGT-A Testing for aneuploidy,
- presence of an extra chromosome

2. • PGT-M Testing for monogenic and single gene disorders.
3. • PGT-SR Testing for structural chromosomal
     rearrangements.
4. • Mitochondrial Donations
5. • HLA matching is used to identify embryos that are HLA
     compatible with a child who needs a bone marrow or cord
     blood transplant.
     - To harvest and use stem cells in the umbilical cord blood of the newly born child.

Question 15

What are some ethical principles? = 10

Answer 15

1 * All medical ethics apply in genomics.

2 * Ethical dilemmas can arise between family members.
3 – Justice – Equity of access to services regardless of residence, ethnicity, gender, religion, age or disability.
4 – Respect for autonomy – Right to privacy and confidentiality.
5 – Beneficence - Taking positive action to do good.
6 – Non-maleficence – Do no harm.

7 * There may be tension when these principles are
considered with respect to the right of an individual to:
8 * know, or not to know, information relevant to their health (autonomy)
9 * disclose, or not to disclose, personal information (privacy)
10 * make an informed decision regarding genetic testing

Question 16

Ethical Dilemmas May Arise…explain them = 3

Answer 16

1 * As a result of genetic testing, an individual’s result may disclose the genetic status of another family member (eg a monozygotic twin) who has not had testing and may not wish to.

2 * An individual refuses to disclose to other family members that they are at risk of a particular disease.

3 * Parents request that their child (<18y) be tested for an
adult-onset condition where there is no health benefit for
the child.

Question 17

Genetic Counselling - what is it? what does it allow for us to do? = 7

Answer 17

1 * Provide information and supportive counselling to members of families with a genetic condition.
To:

2 – Understand the condition, the diagnosis, probable course of condition and available management,
3 – Heredity of the condition and risks to relatives; inheritance pattern,
likelihood that a person who inherits the genetic susceptibility will
develop the condition.
4 – Options for dealing with the risk of recurrence,
5 – Availability of genetic testing – advantages and disadvantages,
6 – Medical and psychological implications for family members and implications for future reproductive choices, employment and insurance,
7 – Issues concerning the privacy and confidentiality of genetic information.

Question 18

Genetics Support Groups and
Organisations: what are they and what do they do? = 3

Answer 18

1 * Support from GP, Genetic Services and professional counselling, patient referral to support groups or organisations can be beneficial.

2 * Provide peer support and practical information about living with a genetic condition.

3 * Families can benefit from contact with other people in similar situations, regardless of their level of coping or need for support.

Question 19

TECHNOLOGY -Genetic testing workflow

Answer 19

diagram in slide 23

Question 20

slide 24

Answer 20

list of diagnostic mechanisms
types of aberrations
resolution
clinical indication examples

in a table

Question 21

Diagnostic Technologies and
Applications

Answer 21

important look at diagram on slide 25

Question 22

Quality of Tests for Clinical
Work - NEW MOLECULAR TESTS

…what are their characteristics and explain them = 5

Answer 22

1 TRIGGER FOR NEW TESTS
– treating doctor with information from journal articles or meeting.
– information given to doctor by a patient from media.
– an evaluation of new testing based on cost-effectiveness & evidence-based medicine

2. TECHNICAL CHALLENGES
   – variation between different platforms and software to detect the same molecular changes.
3. COST-EFFECTIVENESS
   - important concern in implementing new molecular tests.
   – Patients with metastatic cancer undergo costly drug therapies that have serious adverse reactions. Genomics-based predictive tests can be expensive but may lead to substantial savings by excluding non-responders & the costs of treating side effects.
4. DEVELOPING A NEW TEST IS A COMPLICATED MULTISTEP PROCESS
   – Independent validation is a necessary step plus standardization, test interpretation, the technical
   range and quality control eg prenatal microarray.
5. TEST ACCURACY MAY IMPROVE WITH TIME
   – Re-evaluation of test performance might be necessary. Eg improvement in technical method may improvement in sensitivity and specificity of test.

Question 23

Need to know Clinical Utility of Test - what is it?

Answer 23

Clinical Utility

• the risks and benefits associated with the introduction of a test into practice

Question 24

Clinical Utility Chain of Evidence: 6

Answer 24

1. TECHNICAL EFFICANY - ANALYTIC VALIDITY
2. DIAGNOSTIC ACCURACY EFFICACY - CLINICAL VALIDITY
3. DIAGNOSTIC THINKING EFFICACY - Understanding and refining disease etiology, prognosis and relevant family history
4. THERAPEUTIC EFFICACY - medical management change
5. PATIENT OUTCOME EFFICACY - health-related outcomes
6. SOCIETAL EFFICACY - family and community society impacts

LOOK AT THIER KEY QUESTIONS…

DIAGRAM/FLOW CHART ON SLIDE 29

Question 25

Fryback and Thornby domain and definition …levels?

Answer 25

table on slide 30

with definition and measurement construct and indicator

Question 26

test…

Answer 26

1. There are a range of genetic and genomics
   tests.
   - The right test depends on the type of genetic variant you are trying to find.
2. • Tests also vary in terms of cost, turn -around time and complexity of interpretation.

Question 27

Diagnostic strategies in patients with undiagnosed and rare diseases

…explain and what are some techniques? = 12

Answer 27

1 * Rare diseases (RDs) are very numerous (>6000), with
many being ultra-rare.

2 * ~prevalence of RDs is 3.5%-5.9% which is ~263-446
million individuals worldwide.

3 * RDs are an economic burden,
– medical costs per patient are estimated to be around 3-5 times higher than controls of the same age without RDs.

4 * URDs ~50% of patients with RDs remain undiagnosed even in advanced expert clinical settings where genome sequencing techniques are applied.

5.* ES and GS are recommended by ACMG to be the firstor second-tier tests after CMA or focused gene testing for patients with CA, DD, ID.

6 * Diagnostic yield of 43% for GS and 34% for ES, compared with the diagnostic yield of 21% for standard
genetic testing (CMA, candidate single-gene testing, or large gene panel testing).

7 * Other tests to improve diagnosis

8 * Classical karyotype studies

9 – Multi-omics approachs
10 * Epigenomics
11 * Transcriptomics
12 * Proteomics and metabolomics

Question 28

Diagnostic strategies in patients with undiagnosed and rare diseases

Answer 28

flowchart on slide 36

Question 29

Types of variants missed by ES & recommended diagnostic strategies

Answer 29

slide 42 ..table

Question 30

WA GENOMICS STRATERGY 2022 -2023

= 5 AND EXPLAIN THEM

Answer 30

1. Person and family-centredness
   – ensure consumers, carers, families, and communities are at the heart of how genomic healthcare.
2. Genomic healthcare services
   – integration of genomic knowledge in the WA health system.
3. Workforce, education and training
   – develop a sustainable health workforce that has appropriate capacity, agility and the necessary education and training to deliver genomic healthcare.
4. Digital health and data
   – establish digital health and data solutions, protocols and standards for the delivery of responsible genomic health care.
5. Research and innovation
   – have a health system that values and supports the creation and translation of genomics research and innovation

Question 31

Breast Cancer Gene Testing

Answer 31

Bench Lab NGS

Alamut Visual Software

DIAGRAMS ON SLIDES 45-47

Question 32

5 Tier Classification of Variants

Answer 32

Class… Description …probability of being pathogenic …clinical predictive testing of a at risk relatives

5 - definitely pathogenic …>0.99…yes

4. likely …0.95-0.99 …yes
5. uncertain …0.05-0.949…no
6. likely not …0.001-0.049 ..no
7. not or of no clincial significance …<0.001 …no

table on slide 48

Question 33

Types of Genetic Test Results: 3

Answer 33

1 * Positive – genetic change known cause of disease.

2 * Negative – If variant in family is know –
’true negative’. If not sure which gene causes disease a negative result may not
be definitive.

3 * Uncertain – A variant of uncertain significance (VUS) – not enough information known to classify variant.

Question 34

At Increased Cancer Risk:

If the genetic test result is positive or inconclusive for a gene mutation that could increase your risk of cancer, managing risk is a priority.

= types and explain 5

Answer 34

1 * CHEMOPREVENTION
– eg the drugs tamoxifen and raloxifene can be used to reduce breast cancer risk

2. PROPHYLACTIC (preventive) SURGERY
   – eg some women at high risk for breast / ovarian cancer may decide to have their breasts & ovaries removed.
3. CHANGES IN LIFESTYLE FACTORS
   – eg limiting alcohol intake and exercising to lower the risk of colon or breast
   cancer
4. CANCER DETECTION MONITORING
   – eg early screening for Colon & Breast cancer
5. SHARING RESULTS WITH FAMILY MEMBERS
   – whether to tell other family members who might also be at increased risk

Question 35

NIPT - WHAT IS IT?

Answer 35

1.Non Invasive Prenatal Testing

–Cell Free Fetal DNA (cf DNA) in Maternal Blood

Question 36

Conditions Covered by NIPT:

5 MAIN ONES

5 OTHER

Answer 36

Main Conditions
1. * Down syndrome – an extra copy of chromosome 21 (called trisomy 21 or T21)

2. • Edward’s syndrome – an extra copy of chromosome 18 (called trisomy 18 or T18)
3. • Patau syndrome – an extra copy of chromosome 13 (called trisomy 13 or T13)
4. • Specific sex chromosome number changes
5. • Fetal sex

Others Offered
1. * 22q11 deletion (DiGeorge syndrome)
2. * 15q11 (Angelman/Prader-Willi syndrome)
3. * 1p36
4. 4p (Wolf-Hirschorn syndrome)
5. 5p (Cri-du-chat syndrome)

Question 37

Trisomy 21, 18, 13 screening
- WHAT ARE THEY
- WHAT METHOD IS USED

Answer 37

NON INVASIVE PRENATAL TESTING

• Trisomy 21 (Down syndrome)
• Trisomy 18 (Edwards syndrome)
• Trisomy 13 (Patau syndrome)

Question 38

HOW IS NIPT DONE? =4

Answer 38

1. • cfDNA is released from the FETAL TROPHOBLASTS INTO MATERNAL CIRCULATION and can be detected from ‘10 weeks’
2. • DNA AMPLIFIED by NGS & QUANTIFIED by COMPARING AMOUNTS of DNA from CHROMOSOME OF INTEREST WITH ANOTHER CHROMOSOMES
3. • Results REPORTED AS LOW OR HIGH RISK

4 * SCREENING TEST only

Question 39

Advantages and Problems of NIPT = 9

Answer 39

1. Non-invasive
2. • False positive and inconclusive in 2-6%
3. • Due to:
4. – Low fetal fraction of DNA
   5.– Vanishing twin with aneuploidy
   6.– Placental mosaicism (also for CVS)
5. – Maternal cancer
6. • 20% will be inconclusive on resampling
7. • Cost

Question 40

Ethical Problems of NIPT: 6

Answer 40

1 * Accurate pre-test and post-test counselling required

2 * As a screening test not all implications of test may not have been understood before test

3 * May facilitate sex selective termination by providing accurate information about fetal sex within the first trimester

4 * Maternal chromosome abnormalities can be revealed

5 * Some maternal cancers can be revealed

6 * SCA (Monosomy X, 47,XXY, 47,XYY and 47,XXX)
phenotypes are highly variable.
- Prior to NIPT there had been a decreasing trend of termination for these conditions.

Question 41

Diagnostic Performance of a Test

Answer 41

• The accuracy of a test to discriminate diseased cases from normal cases is evaluated using ROC curve analysis.
• ROC curves can also be used to compare the diagnostic performance of two or more tests

BELL CURVE … TEST RESULTS AND CRITERION VALUE
DIAGRAM WITH DISEASE AND WITHOUT DISEASE ON SLIDE 65

Question 42

The Two-by-two Table

Answer 42

DRAW AND UNDERSTAND THE TABLE

Question 43

The Two-by-two Table

ACCURACY =

Answer 43

ACCURACY = (TP +TN)/ TP + TN + FP + FN

Question 44

The Two-by-two Table…TEST CHARACTERISTICS 4

Answer 44

1 * Sensitivity: TP/(TP + FN)
– True positive test results in patients with disease

2 * Specificity: TN/(TN + FP)
– True negative results in patients without disease

3 * Positive predictive value: TP/(TP + FP)
– Predictive value of a positive (abnormal) result OR post-test probability of disease, given positive test

4 * Negative predictive value: TN/(TN + FN)
– Predictive value of a negative (normal) result OR post-test probability of non disease, given negative test

Question 45

What Affects Sensitivity & Specificity? = 6

Answer 45

1. Choice of cutoff value
2. Quality of test
   – Equipment, technique, reagents, questionnaire
3. Quality of interpretation of data
4. Spectrum of disease (severity distribution)
   – for sensitivity
5. Spectrum of non-disease other diseases may cause false positives
   – for specificity
6. NOT prevalence

Question 46

The two-by-two table…PRESENTATION OF RESULTS = 3

Answer 46

1 * PPV tells you how likely that you actually have a disease if you test positive for it.
– The number of true positives / by the total number of people who test positive.

2 * It varies with test sensitivity, test specificity &
disease prevalence.

3 * NPV tells you how likely that you do not have a
disease if you test negative for it.

Question 47

COMPLETE A TWO-BY-TWO TABLE WITH THE FOLLOWING SCENARIO…

A genetic condition affects 5% of population in a city of
1,000,000 ie 50,000 people will have bad gene. Genetic
test gives 1% false positive and 1% false negative
results.

Answer 47

Disease Non-disease
Test
Positive
49,500 9,500 59,000
PPV=83.9%
Test
Negative
500 940,500 941,000
NPV=99.9%
50,000
Sensitivity= 99%
950,000
Specificity= 99% 1,000,000

Question 48

UNDERSTANDING

Receiver-Operating Characteristic (ROC) Curves = 4

Answer 48

1. ROC curves estimate sensitivity & specificity at
   various cutoffs.
2. • In a ROC curve the true positive rate (Sensitivity) is plotted in function of the false positive rate (100- Specificity) for different cut -off points.
3. • Each point on the ROC Curve represents a sensitivity/specificity pair corresponding to a particular decision threshold.
4. • The area under the ROC curve (AUC) is a measure of how well a parameter can distinguish between two diagnostic groups (diseased/normal)

Question 49

(ROC) Curves FEATURES… 5

Answer 49

1. In a ROC curve the true
   positive rate (Sensitivity)
   is plotted in function of the
   false positive rate (100
   - Specificity) for different cut-off points.
2. Each point on the ROC curve represents a sensitivity/specificity pair corresponding to a particular decision threshold.
3. A test with perfect
   discrimination has a ROC
   curve that passes through
   the upper left corner
   (100% sensitivity, 100% specificity).
4. The closer the ROC curve
   is to the upper left corner,
   the higher the overall
   accuracy of the test.
5. DIAGRAM/GRAPH - ON SLIDE 78

Question 50

Conclusions

Answer 50

1 * Types of genetic tests

2 * Types of genetic tests and Clinical Utility.

3 * Techniques used ROC analysis,
Sensitivity, Specificity, Positive and
Negative Predictive Values apply to all
diagnostic tests