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Genomics week 10

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winniesmith2's version from 2017-12-31 17:08

Section 1

Question Answer
Key pathophysiology of CF (cystic fibrosis) Very salty-tasting skin. Appetite but poor growth and weight gain. Coughing, wheezing and shortness of breath. Lung infections e.g. pneumonia/bronchitis. All these characteristics have mucus in common. First described in 1938.
What is the sweat test reliable test to diagnose CF. Measure the concentration of chloride (excess of 60 mmol/l indicated CF) and sodium that is excreted in sweat. Blaster like substance put on skin, test repeated on different days. Clinical presentation, family history and patient age (normally between 1-3) must be considered to interpret the results.
Cystic fibrosis inheritance Autosomal recessive (pedigree analysis). Early age of onset, average life expectancy 30-40 years. Approx 1/2000 new births affected 1/20 to 1/30 caucasians carriers. If both parents carry gene, 25% chance child will get CF.
Which population is CF more common inCaucasians, incidence of 1/3300.
CFTR candidate region; how was this found A loosely linked genetic marker was found. By in situ hybridization, this marker mapped to chromosome 7. RFLP markers located on chromosome 7 were tested for linkage to CF. Two markers flanked CF, but they were 1490 kb apart (Met and D7S8). But then they had to identify what was between these two markers.
What is cystic fibrosis gene mapping Finding out what was between the two marks of MET and D7S8.
How did they identify what was between the 2 markers (CF gene identification).BY jumping and walking. 7 jumps of 50 to 75kb were made with each arrival point then serving as a new origin for chromosome walking. They took little fragments and worked out how these all fitted together by how they over lapped. Managed to narrow the region of exploration and cloning of the CF gene. 4 possible candidate genes. 3 downstream on chromo 7 and 1 up. Had to link anything in these gene sequences to the phenotypes of what we know about CF.
Did they find the gene? and in 1989 Francis Collins and Lap-chee tsui, identified the CFTR gene. They found it was long arm of chromosome 7. They also found that it was huge. 3 of the possibles were too small.
what does CFTRcystic fribrosis transmembrane conductance regulator. Chloride channel.
CF pathophysiology summary ; numbersMost common life shortening recessive disease. Complex multisystem disease. approx 10000 patients in UK.
CF pathophysiology summary; what causes it Cystic fibrosis (CF), a systemic, multiorgan disease, is caused by loss of CFTR protein-mediated ion transport (activity). Defective ion transport leads to an imbalance of fluid and electrolytes causing thick, sticky mucus and viscous secretions to accumulate in different organs. This interferes with the proper function of the lungs, pancreas, gastrointestinal system, sinuses, and reproductive system. In the sweat glands, loss of CFTR activity restricts reabsorption of chloride in the duct, limiting the amount of salt that can be reabsorbed. (Chlorine, possibility sodium, and water affected)
From mutation (genotype) to disease (Phenotype)The mutant form of CFTR prevents chloride transport, causing mucus build-up (ASL). This mucus clogs the airways and disrupts the function of the pancreas and intestines. CF patients need physio therapy and have to complete exercises everyday.
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Section 2

Question Answer
locus of CF gene 7q31.2 - The CFTR gene is found in region q31.2 on the long (q) arm of human chromosome 7
gene structure of CF gene is about 250,000 bp long and contains 27 exons. - BIG
mRNA of CF The intron-free mRNA transcript for the CFTR gene is 6129 bp long. -BIG
Coding sequence (CDS) of CF 4443 bp within the mRNA code for the amino acid sequence of the gene's protein product.
Protein size The CFTR protein is 1480 amino acids long and has a molecular weight of 168,173 Da.
Protein function The normal CFTR protein product is a chloride channel protein found in membranes of cells that line passageways of the lungs, liver, pancreas, intestines, reproductive tract, and skin. CFTR is also involved in the regulation of other transport pathways.
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Section 3

Question Answer
Common CFTR mutation 3 base pair deletion at codon 508. Deleted a Phe residue but maintains reading frame. - Most common is the deletion of phenyalanine (Delta (triangle) F508) ; Causes 70% of CF cases.
Why is the DeltaF508 most common The mutation is most likely to occur in northern Europe. most likely over 50,000 years ago. Individuals with 2 copies of the gene get CF and often cannot reproduce. But having one copy of the gene reduces water loss during cholera, greatly increasing the chance of survival- selection pressure. Still most common in world. 50% of all cases in Europe have one of these mutations.
How many CFTR mutations are thereover 1500 mutations have been found. Massive gene. A DeltaF508 accounts for 70% of CF cases. Next highest is unknown. TOO VARIABLE. Unknown in part due to magnitude of the gene, high probability you can pick up rouge mutation. Have to hit 1% of population to become polymorphism- probably not being able to put right. 3rd one G542X - X = early stop codon. ANY 2 mutations lead to defective protein.
What are the effects of the F508del mutationHas an absolute DNA change but results in multiple CFTR protein defects.
Class ll mutations (2) of F508F508del has a severe defect in CFTR processing and trafficking with degradation of immature CFTR proteins in the ER, typical of a class ll mutation. Few to no CFTR channels are present at the apical cell surface.
Class lll mutation (3 of F508F508del has also been shown to be a class lll mutation, resulting in CFTR proteins with reduced channel-open probability (or gating).
Class Vl mutation (6) of F508F508del also manifests characteristics of a class Vl mutation so that the few CFTR proteins that reach the cell surface have decreased surface stability.
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Section 4

Question Answer
class 1 mutation CFTR is either not synthesized. Try to read DNA but cannot generate mRNA
class 2 mutation inadequately processed (as we pass from nucleus to ER, to be processed, cant be proessed properly).
class 3 mutation not regulated (doesn't show normal regulation of opening/closing)
class 4 mutation Shows abnormal conductance (misregulated amount of ions on one side of the surface).
class 5 mutation Has partially defective production.
class 6 mutation displays accelerated degradation.
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Section 5

Question Answer
G542X second most common KNOWN mutation for CFTR.
how common is G542X4% of patients have atleast one copy of this mutation. Doesn't look like its been selected for at any point.
What is the G542X mutation Class 1 mutation. Rarely makes it out of the nucleus. A nonsense mutation which produces a premature stop codon. The cell cannot synthesize a full length CFTR protein (class 1). As a result, few to no CFTR proteins are present at the apical cell surface. More severe mutation than phenyalanine but still common
Clinical phenotypes vary widely across mutations - how 1500 combinations crossed with another 1500 (need the 2 genes- recessive), with a 20% prevalence of unknowns that arent reaching the 1%.
Describe the spectrum of phenotypes associated with total CFTR activity could have no CF disease, some CFTR-related disorders or full on CF. Some CFTR mutations result in residual or partial CFTR activity. Some result in little to no CFTR activity. Both CFTR alleles play a role in determining phenotype or disease severity.
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Section 6

Question Answer
Therapies of CF - genetic link Linked to genotypes and phenotypes. Personalised medicine depending on genome.
Current therapies for CF manifestations Medication are often aerosol based and can be inhaled: -Bronchodilators -Mucolytics -Decongestants -Antibiotics to fight lung infections -Enzyme supplements. Airway clearance using chest physiotherapy (CPT) and a variety of airway clearance techniques (ACTs)
Possible future therapies Gene therapy; target to lung - bioavailabitiy and vector. Stem cell therapy; iPSCs and CRISPr gene editing. Personalised protein therapy by mutation.
Personalised medicine and CF pg 28
Future therapies for CF - gene therapyto induce the wt CTFR via non viral liposomal vector. Phase llb, pg 29.
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Section 7

Question Answer
CF follows what type of inheritance autosomal recessive
CF is characterised in patients by which pathophysiology excessive mucus around multiple organs
The gene causing CF was identified by using what techniques Cytogenetics, linkage analysis and molecular cloning.
In European populations the most common CF causing allele is deltaF508
CFTR stands forcystic fibrosis transmembrane conductance regulator
3 key wordslocation. Linkage (analysis). Variation.
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