Genetics lecture 9.PDF

Genetics Lecture 9 – Cystic Fibrosis& Genetic Screening Case – Joe - 2nd child - Older brother aged 4 big for age and healthy - Infancy – he is slow to gain weight - Coughs a lot - Hospital admission at 5 months LRTI (lower respiratory tract infection) - Bulky offensive stool - Below 5th Centile for...

Genetics Lecture 9 – Cystic Fibrosis& Genetic Screening Case – Joe - 2nd child - Older brother aged 4 big for age and healthy - Infancy – he is slow to gain weight - Coughs a lot - Hospital admission at 5 months LRTI (lower respiratory tract infection) - Bulky offensive stool - Below 5th Centile for Height and Weight - Sweat Test: Na+ 87mmol/L (upper limit of normal is 60mmol/L) (Sweat test for cystic fibrosis) - What are the implications for cystic fibrosis? Mucus build up, respiratory distress, infertility (95%) - Cystic fibrosis – life impact - Marked reduction in life expectancy - Duration and Quality of Life critically dependent on quality of services - The Biggest Problem: Repeated LRTI with progressive destruction of lung tissue (bronchiectasis and respiratory failure) CF Pulmonary Infection - Major determinant of life expectancy - Different bacteria cause infection at different stages - Age at which they become permanently colonised/infected with Pseudomonas aeruginosa is a critical issue - Where does the bacteria come from? Everywhere – environmental – difficult to prevent exposure Meconium Ileus - Obstruction of the GIT of the infant related to inspissated (thick, dehydrated) material - Occurs in 15 to 20% of infants with CF - Genotype at Cystic Fibrosis Modifier 1 (CFM1) gene on Ch 19 may determine risk of developing meconium ileus Question: You are pretty sure that Joe has CF, how should you confirm the diagnosis? - Define the mutations (targeted mutation panel) - If not a common mutation, what then? Scan exons by PCR amplification and Single Strand Conformation Polymorphism – sequence exons that look different from controls 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 - (> 2000 mutated CFTR alleles have been described) Mutation categories (Class I to V) include: 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) Why is diagnosing the mutations so important for Joe? Treatment CFTR Pharmacogenetics - CFTR modulator therapies are designed to correct the malfunctioning protein made by a mutated CFTR: o Read-through compounds (non-sense) o Correctors (misfolding) o Potentiators (open channel/increase function) Some Less Common CF Mutations - Gating Mutations - e.g. G551D (Glycine changed to Aspartic Acid at position 551) - 4-5% of Cases of CF (depends on population) - The CFTR protein is in place in cell membrane but does not work because the chloride channel does not open - Ivacaftor (Kalydeco) (potentiator) binds to CFTR and allows it to open - Does not work for DF508 but combination drug Orkambi (corrector) does New Triple Therapy approved 2020- Kaftrio - Available for patients 12 + with at least one (or two) DF508 and one “minimal unction” mutation since Oct 2020 - Elexacaftor, Tezacaftor (“correctors”) and Ivacaftor (“potentiator”) - Designed to increase the quantity and function of the F508del-CFTR protein at the cell surface - Since March 2023 extended to patients aged 6-11, possibly to 2-5-year-olds in 2024 (pending EMA approval) - Ireland is among the first 4 countries in Europe to secure full access to Kaftrio. (UK, Germany and Denmark) Question: What are the implications for Joe’s extended family? - Carrier analysis may be requested - “Cascade screening/cascade testing” - Carrier testing is available to 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 - Testing is available through the Molecular Genetics Department at the National Centre for Medical Genetics (NCMG), Our Lady's Children’s Hospital Crumlin Cystic Fibrosis Pedigree (doesn’t show carriers) Purposes of Genetic Screening for Cystic Fibrosis - Carrier screening - to plan pregnancy - Testing of embryos - embryo selection - Prenatal testing on amniotic fluid/CVS - termination of pregnancy - Newborn screening - early detection and intervention: o high energy diet, physio, medicines for lung function & gut absorption Newborn Screening for Metabolic Disease Established in 1966 - Guthrie test, “Heel prick test” - CF 1: 1,500 [2011] - Phenylketonuria 1: 4,500 [1966] - Homocystinuria 1: 65,000 [1971] - Classical Galactosaemia 1: 19,000 [1972] - Maple Syrup Urine Disease 1: 125,000 [1972] - Congenital Hypothyroidism 1: 3,500 [1979] - Glutaric acidiuria type 1 (GA1) 1: 54,000 [2018] - MCADD (medium-chain acyl-CoA dehydrogenase deficiency) 1: 66,000 [2018] New-born screening for CF - IRT screening has low specificity (increased IRT in CF) - Relatively high false positive rate - Combined with mutational analysis vastly improves specificity - ~2% of carriers of CF born each year will also be detected Why no CF population carrier screening? - The condition – serious, well understood and relatively common (cost/benefit) (YES) - The test – acceptable, easy and cheap, valid and reliable (NO) - The intervention – effective treatment/counselling, prenatal diagnosis and ART available (YES) - The screening program – effective (clinical data), ethical, benefit outweigh harms (NO) - Implementation criteria – accessibility, resources for diagnosis and treatment, communication of results, data privacy, quality assurance (NO) Bottom line – complementary approach works best – miss fewer infants and less ethical concerns Screening Adults for Carriage - 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 - Other common mutations – 38 mutation panel detects ~93.5% of the CF mutations found in the Irish population - But what about the other >1900? - What can you do if none are detected? Sequencing NGS & Cystic Fibrosis - Despite extensive screening, 1–5% of CF patients lack a definite molecular diagnosis. - 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 Screening for Carriage - Which mutations to screen for depends on who you are looking at (ethnic background) - In non-Hispanic US Caucasians - ∆F508 = ~70% carriage rate - In US Hispanics = ~46% - The next 5 most common are G542X, G551D, 621+1G>T, W1282X and N1303K - In US Ashkenazi Jews (carriage rate of 1/25) o W1282X (45.92%) o ΔF508 (31.41%), o G542X (7.55%) o 3842+10kbC>T (4.77%) o N1303K (2.78%) - Why is DF508 not the predominant mutation? Genetic drift and founder effect Haemochromatosis - Clinical condition characterised by accumulation of Iron (liver, skin and other tissues) - Clinical manifestations develop in adult life (age of onset very variable) - Hepatic failure, Cardiac failure - Skin pigmentation - Joint Disease - It is relatively common - Diagnosis: o Elevated transferrin saturation o Elevated serum ferritin levels - Treatment – phlebotomy (More common in males – females menstruate) - - Classical Haemochromatosis is associated with variant alleles of the HFE gene 6p21.3 Autosomal recessive pattern HFE regulates iron absorption from the diet and iron storage Deficiency = iron overload 2 key mutations: 1. G to A transition at nucleotide 845 (c.845G>A) - Cysteine to Tyrosine (p.C282Y) 2. C to G at nucleotide 187 (c.187C>G) - Histidine to Aspartic acid 63 (p.H63D) C282Y homozygous in 80-85% hemochromatosis cases (3x increase iron absorption) Also C282Y and H63D Compound Heterozygotes Homozygous H63D does not result in clinically manifest haemochromatosis Penetrance may be as low as 1%, even in homozygotes Is haemochromatosis a hereditary disease? Outcome critically dependent on lifestyle factors “exposome” (e.g. red meat diet vs vegan diet) Should there be a screening program? No – very low penetrance – no actionable knowledge Population Screening - Something that is a useful diagnostic test is not automatically a good population screening test - Likelihood of discovering undiagnosed patient with HH is <1 in 1000 - No evidence of clinical benefit for treatment of asymptomatic carriers - Contraindications for screening: o Low positive predictive value – e.g. Haemochromatosis H63D o 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 o 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 o No actionable knowledge – no way to improve prognosis Principles of Screening CDC's ACCE framework: - 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? Screening is not a diagnosis – need a specific diagnosis for treatment Screening is only good if can do something with the information Things to remember: 1. Screening is not diagnosis and you need to confirm the diagnosis. 2. Genetic drift is 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. 3. Founder effect can cause some rare heritable disorders to occur with greater frequency, but can also lead to a lower incidence of common heritable disorders in the founder population 4. Haemochromatosis is a good example of a genome based predisposition to disease with outcome critically dependent on “exposome” 5. Contraindications for screening include a low positive predictive value, low population attributable risk and lack of clinical utility