Cystic Fibrosis: Clinical Genetics

Question 1

1) CF has an _________ pattern of inheritance
2) It is commonly a diseasein people of ______ descent (especially ______)
3) Incidence: ~1 in _____ live births (Caucasian)
4) Most common _____ _____ condition causing premature _____ (life-limiting)
5) Carrier frequency: ~1 in ___ (Northern European)
6) H____ (____) selective advantage

Answer 1

1. autosomal recessive
2. caucasian descent, especially northern european
3. ~1 in 2500-3000
4. single genetic condition, premature death
5. ~1 in 25
6. Heterozygote (carrier) selective advantage
   - protection against cholera (?), typhoid fever (?), asthma (?)

Question 2

Heterozygote (carrier) selective advantage

• protection against cholera (?), thyphoid fever (?), asthma (?)

Q) What about CF causes CFTR carriers to have selective advantage against asthma? **potential exam qn

Answer 2

A) ?? think about this

Question 3

Worldwide prevalence of CF

70,000 people affected worldwide

Birth prevalence varies from country to country and ethnic background

Q) There are _____-specific mutations

Answer 3

Ethnic specific mutations

Question 4

Typical pedigree of CF

Example slide

Answer 4

Question 5

Q) Define the term “obligate carriers” of CF

Answer 5

• Generally consider parents to an affected child an “obligate carrier”
• Carrier status can be passed down through multiple generations w/o any evidence of disease

*Partnership with another carrier = 1/4 risk of child affected

Question 6

CFTR mutations are generally _________, “____” mutations are rarely seen

Answer 6

Generally inherited genetically

“de novo” mutations are rarely seen

Question 7

1. Typical for autosomal recessive conditions (M/F affected equally) to have:
2. Most commonly see affected individuals in the same ____
3. With 2 obligate carrier parents, the children have a ____ chance of being affected with CF

Answer 7

1. typical to have no ‘family history’
2. in the same generation
3. 1/4 affected, 3/4 not
   *out of the 3/4, 2/3 chance of being carriers, 1/3 chance of having normal alleles

Question 8

Q) Is there any correlation between class of variant and phenotype?

Q) If so, what?

Answer 8

there is SOME but VARIABLE correlation, especially with pancreatic function (pancreatic insufficiency, PI)

• Classes I, II, III (and probably VI) usually have more severe lung disease and PI
• Classes IV and V usually associated with PS and milder lung disease

Question 9

Some variants have reduced clinical penetrance

One example is R117H - its clinical penetrance also depends on another intragenic polymorphism.

Q) Explain

Answer 9

Intragenic polymorphism i.e. a poly T (5 or 7 or 9 Ts) tract in intron 8 on the same allele (i.e. in cis) as R117H

*Important to establish phase - i.e. which mutations are in cis (through testing parents)

Question 10

R117H clinical penetrance

With one CF-causing variant already present e.g. F508del (pathogenic)

Q) R117H with 5T poly-T tract:

Answer 10

R117H will likely act as disease-causing variant

Most PTs will have elevated sweat chloride and clinical symptoms of CF (variable)

Increased risk for male infertility

Question 11

R117H clinical penetrance

With one CF-causing variant already present e.g. F508del (pathogenic)

Q) R117H with 7T poly-T tract:

Answer 11

R117H unlikely to act as disease-causing variant (particularly for females) but may result in male infertility

May have borderline/elevated sweat chloride + mild clinical symptoms of CF

Question 12

R117H clinical penetrance

With one CF-causing variant already present e.g. F508del (pathogenic)

Q) R117H with 9T poly-T tract:

Answer 12

R117H highly unlikely to act as disease-causing variant

Vast majority will not have CF

Male infertility typically NOT affected by R117H and 9T

Question 13

Environmental and genetic modifiers of CF modify the severity of disease (mendelian disease)

1. Even though CF is a monogenic disease, there is wide ____
2. Even between siblings with same CFTR genotype, there is _____

Answer 13

1. wide variability in clinical features
2. variable phenotype expression - due to environmental and genetic modifiers

Question 14

Environmental and genetic modifiers of CF modify the severity of disease (mendelian disease)

Allelic variation in CFTR correlates with some aspects of CF, but…

Q) Which aspects are well correlated, and which aspects arent?

Answer 14

• Well correlated
  
  • pancreatic insufficiency
• NOT well correlated
  
  • lung function, neonatal intestinal obstruction, diabetes, height/weight
    
    • although under strong genetic influence (suspect there are other modifiers)

Question 15

When determining effects of genetic vs environmental modifiers, studies with twins and siblings showed that:

• Monozygotic twins (MZ): share 100% genes
• Dizygotic twins (DZ) and siblings share 50% genes

Answer 15

• When MZ share household, then (to certain extent) can control for environment
• Look for concordance in MZs and DZs for clinical features to estimate genetic control (heritability)

Question 16

Studies in twins and siblings showed that, for example

Lung function in CF is…

Answer 16

E.g. Lung function in CF: 50% genetic, 50% environmental

* Meconium ileus = internal obstruction in neonate due to inpaction of merconium

*DIO presents later in life, usually beyond adolesence
>>clincal presentation entirely on environmental influence

*Diabetes almost entirely due to genetic factors

Question 17

It is important to identify genetic modifers because:

1) Could identify new ______
2) Increases understanding of ______
3) Expect these genes/variants to be minimally penetrant in healthy people, but effects ________ in CF patients

Answer 17

1. new targets for therapies
2. increases understanding of disease variability (within and between families)
3. effects unmasked in CF patients

*These gene modifiers, therefore, may contribute to development or progression of common diseases (e.g. asthma, diabetes) in general population

*Genetic modifiers of CF can teach us about other diseases

Question 18

How do we identify genetic modifiers of CF?

Association studies such as:
1) Linkage

2)

3)

Answer 18

Association studies in CF

1. Linkage
   - track genes/markers associated with specific phenotype in families with CF

Question 19

How do we identify genetic modifiers of CF?

Association studies such as:
1)

2) Candidate gene association

3)

Answer 19

Association studies in CF

2. Candidate gene association
   - Genes with known function (presumably relevant to CF features); correlate variations in gene with presence of features/phenotype in CF patients

Question 20

How do we identify genetic modifiers of CF?

Association studies such as:
1)

2)

3) Genome wide association studies (GWAS)

Answer 20

Association studies in CF

3. Genome wide association studies (GWAS)
   - Examine DNA markers at many positions on multiple chromosomes (i.e. across entire genome) in populations with and without CF phenotype of interest

Question 21

How do we identify genetic modifiers of CF?

Association studies such as:
1)

2)

3)

Answer 21

Association studies in CF

1. Linkage
   - track genes/markers associated with specific phenotype in families with CF
2. Candidate gene association
   - Genes with known function (presumably relevant to CF features); correlate variations in gene with presence of features/phenotype in CF patients
3. Genome wide association studies (GWAS)
   - Examine DNA markers at many positions on multiple chromosomes (i.e. across entire genome) in populations with and without CF phenotype of interest

Question 22

How do we identify genetic modifiers of CF?

Transcriptomic, proteomic, and metabolic analyses in human tissues and animal models (CF vs non-CF controls)

How do these functional studies help with association studies?

Answer 22

Association studies are usually further tested with these functional studies to

• confirm an association
• understand what the mechanism might be for that modifier

Question 23

Genome wide association studies

Look for DNA sequence varaints (Single nucleotide polymorphisms - SNPs) that are shared with much greater frequency among individuals with the same phenotype than among others (control)

*compare CF PTs with specific phenotype vs unaffected or CF PTs without specific phenotype

Q) What are the limitations of GWAS? (3 points)

Answer 23

Limitations:

• When genes/SNPs are less penetrant, need greater sample size
• Need to replicate studies for validation
• Need to demonstrate correlation and cause, not just association, by conducting research showing specific mechanisms

Question 24

Genetic modifiers of different features of CF

Which genes have a probable effect association with lung function (FEV1)? (3 points)

Answer 24

EDNRA

MBL2

TGFB1

Question 25

Genetic modifiers of different features of CF

Which genes have a probable effect association with P. Aeruginosa acquisition/colonisation? (1 point)

Answer 25

MBL2

**This subsequently affects lung function

Question 26

Genetic modifiers of different features of CF

Which genes have a probable effect association with intestinal obstruction? (1 point)

Answer 26

MSRA

Question 27

Genetic modifiers of different features of CF

Summary slide

Answer 27

Question 28

Genetic modifiers of lung disease in CF

1. EDNRA (encodes _____)

2.

3.

Answer 28

1. EDNRA (encodes endothelin receptor type A)
   
   • particular variants alter smooth muscle tone in airways/vasculature

Question 29

Genetic modifiers of lung disease in CF

1.

2. MBL2 (encodes _____)

3.

Answer 29

1. 2. MBL2 (encodes mannose binding lectin)
      * has role in innate immunity
      * MBL2 deficiency predisposes to early infection with P. Aeruginosa
      * relative impact - only people with CF AND MBL2 gene get sick
      3.

Question 30

Genetic modifiers of lung disease in CF

1.

2.

3. TGFB1 (encodes _____)

Answer 30

1. 2. 3. TGFB1 (encodes transforming growth factor beta)
         * Role in regulating inflammation and tissue remodelling
         * Alleles leading to increased TGFB1 expression cause worse lung function

Question 31

Genetic modifiers of lung disease in CF

1. EDNRA (encodes _____)
2. MBL2 (encodes _____)
3. TGFB1 (encodes _____)

Answer 31

1. EDNRA (encodes endothelin receptor type A)
   
   • particular variants alter smooth muscle tone in airways/vasculature
2. MBL2 (encodes mannose binding lectin)
   
   • has role in innate immunity
   • MBL2 deficiency predisposes to early infection with P. Aeruginosa
   • relative impact - only people with CF AND MBL2 gene get sick
3. TGFB1 (encodes transforming growth factor beta)
   
   • Role in regulating inflammation and tissue remodelling
   • Alleles leading to increased TGFB1 expression cause worse lung function

Question 32

Genetic modifers of Intestinal obstruction (meconium ileus)

1. MSRA (encodes_____)

• Role in modifying _____ such as ____
• certain variants may alter ____, and contribute to formation of ____

Answer 32

MSRA (encodes methionine sulphoide reductase)

• Role in modifying intestinal enzymes such as alpha-1 antitrypsin
• certain variants may alter digestion of intestinal contents, and contribute to formation of viscous meconium

Question 33

Genetic modifiers of Diabetes in CF

1. TCFL2 (encodes _____)

• role in ____ and ____ of ____ of pancreatic islets
• May modify risk for diabetes in CF PTs who have not had recent or prolonged exposure to ____
  
  • ​S.S. _____ _____ risk of diabetes
  • Once someone has been treated w/ ___, then the contribution of this variant to diabetes is ____

Answer 33

TCFL2 (encodes transcription factor 7-like 2)

• role in prolieration and function of beta cells of pancreatic islets
• May modify risk for diabetes in CF PTs who have not had recent or prolonged exposure to systemic steroids
  
  • ​Systemic steroids significantly increases risk of diabetes
  • Once someone has been treated w/ systemic steroids, then the contribution of this variant to diabetes is not significant

Question 34

Environemntal modifiers/determinants of CF

Poorer health outcomes associated with:

1. • may relate to poorer adherence to medical and dietary regimens
2. Lower
3. Exposure to ____ - active and passive
4. ____ exposures
5. Disease _____

Answer 34

Poorer health outcomes associated with:

1. Being female
   
   • may relate to poorer adherence to medical and dietary regimens
2. Lower SES
3. Exposure to tobacco smoke - active and passive
4. Infectious exposures
5. Disease self-management

Question 35

Why test for CF?

Different purposes:

1. Diagnostic testing of ____ individuals
2. To identify ____ at risk of developing CF
   
   • _____ screening
3. To identify _____ who are ____ of a diagnosed ____ (i.e. for ____ purposes)
   
   • ____ testing (understand their reproductive risk)
4. To identify ____ from the ____ population (i.e. for reproductive purposes)
   
   • _____ _____ screening

Answer 35

Different purposes:

1. Diagnostic testing of symptomatic individuals
2. To identify babies at risk of developing CF
   
   • newborn screening
3. To identify carriers who are relatives of a diagnosed baby (i.e. for reproductive purposes)
   
   • cascade testing (understand their reproductive risk)
4. To identify carriers from the general population (i.e. for reproductive purposes)
   
   • population carrier screening

Question 36

Newborn screening for CF involves:

1. ____ (IRT) on newborn screening (___) __ __ cards
   
   • Heel-prick on all ~2-3 day old neonates
   • Samples with ____ levels (____%) undergo further testing
     ​
2. ___ testing for initial _____ panel - if necessary include ____ panel
   
   • If non-european descent, may want to consider extended panel
3. ___zygotes brought in for ___ test (____ in sweat indicative of CF), and maybe _____ to identify ____
   
   • **Ultimately, definitive diagnostic test is the ____ test

Answer 36

1. Immunoreactive trypsinogen (IRT) on newborn screening (Guthrie) blood spot cards
   
   • Heel-prick on all ~2-3 day old neonates
   • Samples with elevated levels (highest 1%) undergo further testing
2. DNA testing for initial 12 variant panel - if necessary include extended panel
   
   • If non-european descent, may want to consider extended panel
3. Heterozygotes brought in for sweat test (elevated Na+Cl- in sweat indicative of CF), and maybe gene sequencing to identify 2nd variant
   
   • **Ultimately, definitive diagnostic test is the sweat test

Question 37

Newborn screening protocol

Summary slide

Answer 37

**Two CF mutations = will be given a diagnosis of CF

**One CF mutation = child may have CF, brought in for sweat test

Question 38

Sweat test

Done at ~4 weeks of age

Using sweat collecting coil

******ONLY IF CHILD HAS 1 CF MUTATION

Q) Positive result vs Equivocal result vs Normal result

Answer 38

• Positive result (>60 mmol/L)
  
  • Child has CF
    
    • Genetic counselling, carrier testing for 2nd mutation
    • other family members offered cascade testing
• Equivocal result (30-60 mmol/L)
  
  • Child may have CF
    
    • genetic counselling and mutation testing for parents
    • Other family members offered cascade testing
• Normal result (<30 mmol/L)
  
  • Genetic counselling and mutation testing for parents
  • Child does not have CF but is a carrier
  • other family members offered cascade testing

Question 39

Cascade testing statistics

• In a study of NBS in VIC, found only __% of eligible relatives (other than parents who are obligate carriers) underwent genetic testing to find out if they are carriers - even when (at that time) testing was at ___
• Lack of ____ within families regarding ____ is one reason for poor uptake

Answer 39

• In a study of NBS in VIC, found only 12% of eligible relatives (other than parents who are obligate carriers) underwent genetic testing to find out if they are carriers - evem when (at that time) testing was at no charge
• Lack of communication within families regarding relevance of testing is one reason for poor uptake

Question 40

Population carrier screening

• Carrier testing offered through some doctors to people in the general population (obstetricians, to pregnant couples, and GPs) since 2006 (initially __ _____ ± ____ ____ on cheek brush sample)
• Now part of VCCS reproductive carrier screen (CF ___ mutations)

Answer 40

• Carrier testing offered through some doctors to people in the general population (obstetricians, to pregnant couples, and GPs) since 2006 (initially 12 variants ± extended panel on cheek brush sample)
• Now part of VCCS reproductive carrier screen (CF 38 mutations)

Question 41

Reproductive options for carrier couples include:

1. No children, adoption, pregnancy with no testing
2. Egg or sperm (____) donation
3. ___ (and ___) - then ___ (PGD) to…
4. Prenatal testing
   
   • Continue pregnancy, and, if affected, be prepared for baby with CF
   • Terminate affected pregnancy

Answer 41

1. Egg or sperm (non-carriers) donation
2. IVF (and ICSI) - then pre-implantation genetic diagnosis (PGD) - test and implant unaffected embryos
3. Prenatal testing
   
   • Continue pregnancy, and, if affected, be prepared for baby with CF
   • Terminate affected pregnancy