lecture 9

Question 1

What are the main genetic features of CF?

Answer 1

• autosomal recessive pattern of inheritance
• commonly a disease in people of caucasian descent (especially northern european)
• incidence - ~1 in 2500-3000 live births (caucasian) (In VIC currently ~1 in 3300 - is this a reflection of our ethnic mix?)
• most common single genetic condition causing premature death in children of northern European backgrounds
• carrier frequency - ~1 in 25 (northern European)
• Heterozygote (carrier) selective advantage - protection against cholera (?), typhus (?), asthma (?)

Question 2

What is the worldwide prevalence of CF?

Answer 2

Birth prevalence varies from country to country and ethnic background e.g.:

• 1 in 3000 americans (northern european descent)
• 1 in 4000 Latin Americans
• 1 in 15,000 to 20,000 african americans
• 1 in 350,000 Japanese; also uncommon in Africa and Asie

Ethnic-specific mutations

Question 3

What is are important things to consider in regards to family history of CF?

Answer 3

• often a LACK of family history
• autosomal recessive: need two carriers to get together
• in populations where consanguinity is common you might see a higher carrier rate of CF and therefore there might be some family history; the same for small, isolated populations where there may be some things like intermarriage etc
• don’t often see de novo mutations
• carrier risk for future children - child with affected siblings has a 2/3 carrier risk
• absence of a family history does not mean to say the risk is not there

Question 4

What are some of the genotype-phenotype correlations of CF?

Answer 4

• some but variable correlation between class of mutation and phenotype, especially with pancreatic function (pancreatic insufficiency, PI) - particularly with Classes I, II and III mutations
• classes I, II and II (and probably VI) usually have more severe lung disease and PI (more so with PI than lung disease)
• Classes IV and V usually associated with PS and milder lung disease

Because we aren’t seeing 100% correlation it has to make you wonder whether there are other factors affecting the phenotype. (the answer is yes)

Question 5

What are some of the challenges with mutations of uncertain clinical significance?

Answer 5

• some mutations have reduced clinical penetrance
• one very interesting example is R117H – the clinical penetrance of this mutation also depends on another 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, ie. which mutations are in cis (through testing parents)
• important to consider wider genetic context

Question 6

What are some environmental and genetic modifiers of cystic fibrosis?

Answer 6

• wide variability in clinical features and survival even though CF is a monogenic disease
• variable phenotypic expression even between siblings with the same CFTR genotype
• allelic variation in CFTR correlates with some aspects of CF, but: lung function, neonatal intestinal obstruction, diabetes and anthropometry (e.g. height and weight) NOT well correlated with CFTR mutations although under strong genetic influence.
• Studies with twins and siblings:
• • monozygotic twins (MZ): share 100% genes
• • Dizygotic twins (DZ) and siblings: share 50% genes
• • 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)
• • showed that different symptoms are differentially impacted upon by genes and environment

e.g. lung function in CF: 50% genetic, 50% environmental. Height/weight (to a much lesser extent), diabetes and meconium ileus obstruction are more influenced by genetics while distal intestinal obstruction is almost all environment.

Question 7

How do number of polymorphisms affect penetrance of R117H?

Answer 7

In an individual who is a heterozygote compound where the second mutation is R117H the number of poly Ts.

• those with 5ts: R117H will likely act as a disease- causing mutation. Most patients will have elevated sweat chloride and clinical symptoms of CF – symptoms variable. Increased risk of male infertility
• those with 7T: unlikely to act as a disease-causing mutation (particularly for females), but may result in male infertility. However, a person with this combination may have borderline or elevated sweat chloride and mild clinical symptoms of CF
• those with 9T: highly unlikely to act as a disease-causing mutation. Vast majority of individuals will not have CF. Male fertility is typically not affected by R117H and 9T.

Question 8

Why is it important to identify genetic modifiers of CF?

Answer 8

• identifies new targets for therapies
• increases understanding of disease variability
• expect these genes/variants to be minimally penetrant in healthy people but 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

Question 9

How do you identify genetic modifiers of CF?

Answer 9

Association studies:
- Linkage: track genes/markers associated with specific phenotype in families with CF

• Candidate gene association: genes with known function (presumably relevant to CF features); correlate variations in gene with presence of features/phenotype in CF patients
• 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. GWAS look for DNA sequence variants (single nucleotide polymorphisms: SNPs) that are shared with much greater frequency among individuals with the same phenotype than among others (controls). Moderately successful.

Question 10

What are the limitations of genome wide association studies?

Answer 10

• when genes/SNPS are less penetrant, then 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 11

What are some of the genetic modifiers of different features of CF?

Answer 11

Research has been able to classify the level of association of a gene with a particular of CF, e.g. lung function or P. Aeruginosa acquisition/colonisation.

Genes are determined as to have either a probable effect, possible effect, likely no effect, or no effect.

Some that have been determined to have probably effects include:

• EDNRA: lung function (FEV1)
• MBL2: lung function, and P. Aeruginosa acquisition/colonisation
• TGFB1: lung function
• MSRA: intestinal obstruction
• TCF7L2: CF related diabetes

EDNRA (encodes endothelin receptor type A): variants alter smooth muscle tone in airways and/or vasculature and therefore may modify CF lung disease

MBL2 (encodes mannose binding lectin):

• has role in innate immunity
• MBL2 deficiency predisposes to early infection with P. Aeruginosa and therefore leads to more severe lung disease

TGFB1 (encodes transforming growth factor beta):

• role in regulating inflammation and tissue remodelling
• Alleles leading to increased TGFB1 expression cause worse lung function in CF patients

MSRA (encodes methionine sulphoxide reductase)

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

TCF7L2 (encodes transcription factor 7-like 2)

• may have role in proliferation and function of beta cells of pancreatic islets
• may modify risk for diabetes in CF patients who have not had recent or prolonged exposure to systemic steroids

Question 12

What are some of the environmental modifiers/determinants of CF?

Answer 12

Poorer health outcomes associated with:

• Being female – may relate to poorer adherence to medical and dietary regimens
• lower socio-economic status
• exposure to tobacco smoke - active and passive
• infectious exposures
• disease self-management

Question 13

What are different purposes for screening for CF?

Answer 13

• To identify babies at risk of developing CF (i.e. for diagnostic purposes) – newborn screening
• To identify carriers who are relatives of a diagnosed baby (i.e. reproductive purposes) – cascade testing
• To identify carriers from the general population (i.e. reproductive purposes) – population carrier screening

Question 14

How is newborn screening for CF carried out? Why do we do it?

Answer 14

CF screening for neonates was introduced in 1989
In Vic we screen for PKU, and for congenital hypothyroidism both of which have very clear treatments that can certainly reduce the effects of the condition. Although we do not have a cure for CF early treatments etc can improve the quality of life. In 2002 about 20 other metabolic conditions were introduced into newborns in Vic.

Picks up most people with CF but not 100%

1. Immunoreactive trypsinogen (IRT) on newborn screening (Guthrie) cards
   - heel-prick on all ~2-3 day old neonates
   - samples with elevated levels undergo further testing
   - if you are in top 1% of IRT levels you go on to next stage
2. DNA testing for initial 12 mutation panel – if necessary include extended panel
   - two mutations: i.e. homozygotes and compound heterozygotes (two different mutations) have CF.
   - if they only have one mutation showing here they will undergo step 3.
3. Heterozygotes brought in for sweat test (elevated NaCl in sweat indicative of CF), and maybe gene sequencing
   - usually wait until 6 weeks because they need a particular volume of sweat in order to perform the test properly
   - there is not the same urgency with CF as there is with other metabolic conditions
   - non- invasive for the baby

Question 15

How often does cascade testing occur?

Answer 15

• in a recent study, found that only 12% of eligible relatives (other than parents who are obligate carriers) underwent genetic testing to find out if they are carriers – even when (then) testing was at no charge
• lack of communication within families regarding relevance of testing one reason for poor uptake

Question 16

When does population carrier screening occur?

Answer 16

• Carrier testing offered through some doctors to people in the general population (obstetricians, to pregnant couples, and GPs) since 2006 (initially 12 mutations + extended panel on cheek brush sample)
• Fee for service ($200 per person)
• no such thing as zero risk - potentially you have a mutation that has not yet been identified
• Now part of VCCS reproductibe carrier screen (CF (38 mutations), SMA, & FXS - $385) on blood sample; other pathology providers

lots of ethical issues:

• who should pay for population carrier?
• • only people from northern european background only?
• • anyone who wants it?
• some companies/labs (overseas) now offering panels of 100 mutations - many are ‘mild’ mutations
• • what is the clinical utility of known about ‘mild’ mutation?
• • what happens when whole genome sequence becomes available? How do we interpret this information?

Question 17

What are the options for carrier couples?

Answer 17

Reproductive options for carrier couples:

• no children; adoption; pregnancy with no testing
• pre-implantation genetic diagnosis (PGF) – test and implant unaffected embryos
• prenatal testing
• • continue pregnancy and, if affected, be prepared for birth of baby with CF
• • terminate affected pregnancy