Cystic Fibrosis & Genetic Screening

What’s the biggest problem with Cystic Fibrosis

Repeated LRTI (Lower Respiratory Tract Infection) with progressive destruction of lung tissue → bronchiectasis and respiratory failure

What determines life expectancy in CF

CF Pulmonary Infection

Different bacteria cause infection at different stages

Age at which they become permanently colonised/infected with Pseudomonas aeruginosa determines life expectancy

What is a complication seen in 15-20% of infants with CF

Meconium Ileus

Obstruction of the GIT of the infant related to inspissated (thick, dehydrated) material

What determines the risk of developing meconium ileus

Genotype at Cystic Fibrosis Modifier 1 (CFM1) gene on Ch 19

How to confirm CF diagnosis

Define the mutations (targeted mutation panel)

If not a common mutation, then scan exons by PCR amplification and Single Strand Conformation Polymorphism – sequence exons that look different from controls

Explain the CF Mutation Nomenclature

Cystic Fibrosis Carrier = Aa

• A – any CFTR allele that results in a functioning chloride channel (sequence may vary)

• a – any CFTR allele that does not code for a functioning chloride channel

Mutation categories = Class I to V

Explain the difference between CF mutation categories Class I - V

I. Protein production - (no functional protein produced)

II. Protein processing (misfolding)

III. Gating (doesn’t open)

IV. Conduction (faulty channel)

V. Insufficient protein (splice site)

CFTR modulator therapies are designed to do what

Correct the malfunctioning protein made by a mutated CFTR:

Give 3 types of CFTR therapies & what mutations they target

Read-through compounds (non-sense)

Correctors (misfolding)

Potentiators (open channel/increase function)

Give an example of a CF Gating Mutation

G551D (Glycine changed to Aspartic Acid at position 551)

Gating mutations occur in what % of cases of CF

4-5%

What dysfunction does a CF gating mutation cause

The CFTR protein is in place in cell membrane but does not work because the chloride channel does not open

Name a drug used to treat gating mutations

Ivacaftor (Kalydeco)

What type of drug is Ivacaftor (Kalydeco) & what does it do

It is a potentiator - binds to CFTR and allows it to open

Does Ivacaftor (Kalydeco) work for the mutation DF508?

No, but combination drug Orkambi (corrector) does

What is Kaftrio

New Triple Therapy for CF

Comination of Elexacaftor, Tezacaftor (“correctors”) and Ivacaftor (“potentiator”)

Designed to increase the quantity and function of the F508del-CFTR protein at the cell surface

Carrier testing for CF is available to who

Adults over the age of 16 where there is a family history of CF, or where a family member/partner has been found to carry a CF mutation

What kind of diet acts as CF intervention

high energy diet

Newborn Screening for Metabolic Disease is done by what test

Heel prick

Problems associated with IRT (one of the tests done as part of the heel prick)

IRT screening has low specificity

Relatively high false positive rate (better than false negative)

What improves specificity of IRT

Combining the test with mutational analysis

Why is there no CF population carrier screening?

How are adults screenedfor DF508

Testing just for DF508 is straightforward

Amplify relevant sequence from genomic DNA

Assess for wt or DF508 sequence (restriction enzyme / oliognucleotide probe)

Asses for DF508 during amplification using real time approach

What else do we use to screen for common CF mutations

38 mutation panel detects ~93.5% of the CF mutations found in the Irish population

What is NGS used for

Next-generation sequencing (NGS) is making affordable genetic testing based on the identification of variants in extended genomic regions – ~99% detection rate

NGS also being used to design custom CFTR mutation panels for different geographic regions, with around 95% detection rates

The mutations you screen for depend on what about the person

Ethnic background

Prevalence of deltaF508 in non-Hispanic US Caucasians vs US Hispanics

Non-Hispanic US Caucasians ∆F508 = ~70% carriage rate

US Hispanics = ~46%

Most common CF causing mutation in US Ashkenazi Jew (& %)

W1282X (45.92%)

(high due to founder effect)

Haemochromatosis

Clinical condition characterised by accumulation of Iron in liver, skin & other tissues

When do Clinical manifestations of Haemochromatosis develop

in adult life

Symptoms of Haemochromatosis

Hepatic failure

Cardiac failure

Skin pigmentation

Joint Disease

How is hemochromatosis diagnosed

Elevated transferrin saturation

Elevated serum ferritin levels

Haemochromatosis treatmnet

phlebotomy

Classical Haemochromatosis is associated with variant alleles of what gene

HFE gene 6p21.3

Does haemochromatosis have an autosomal dominant/recessive pattern

Autosomal recessive pattern

Role of HFE

HFE regulates iron absorption from the diet and iron storage.

Deficiency = iron overload

What 2 key mutations can occur with the HFE gene

G to A transition at nucleotide 845 (c.845G>A) - Cysteine to Tyrosine (p.C282Y)

C to G at nucleotide 187 (c.187C>G) - Histidine to Aspartic acid 63 (p.H63D)

The mutation C282Y homozygous is found in what % of hemochromatosis cases and leads to what x increase in iron absorption

Found in 80-85% hemochromatosis cases

3x increase iron absorption

We know that C282Y homozygotes result in clinically manifest haemochromatosis.

Do C282Y & H63D Compound Heterozygotes, and Homozygous H63D result in clinically manifesting haemochromatosis?

Heterozygotes do

Homozygous H63D does not result in clinically manifesting  haemochromatosis

Haemochromatosis penetrance

Penetrance may be as low as 1%, even in homozygotes

(It is technically a hereditary disease but outcome is critically dependent on lifestyle factors “exposome”)

Why do we not have a population screening test for Haemochromatosis

Likelihood of discovering undiagnosed patient with HH is <1 in 1000

No evidence of clinical benefit for treatment of asymptomatic carriers

Low positive predictive value – e.g. Haemochromatosis H63D

Low population attributable risk (PAR) – the proportion of total disease risk in the population attributable to the factor being screened for – e.g. G6PDD mutations

Low absolute risk – e.g. FV Leiden thromboembolism relative risk for oral contraceptive users high, but absolute risk low as most of these are young people

No actionable knowledge – no way to improve prognosis

What is CDC’s ACCE framework for Principles of Screening

Analytical validity – how accurate is measurement?

Clinical validity – how accurately does it predict presence/absence of disease?

Clinical utility – how useful are the results (clinical benefit)?

Ethical, legal and social implications?

True/False Screening = diagnosis

False: Screening is not diagnosis and you need to confirm the diagnosis

Genetic drift

the change in allele frequencies in a population from one generation to the next due to chance. These changes are more pronounced in smaller populations than larger populations.