Cystic Fibrosis Summary PDF

Cystic fibrosis intro - progressive, autosomal-recessive disorder - primarily affects the lungs (Obstructive airways disease) and pancreas - mutations in the CFTR gene leading to dysfunctional CFTR protein. - F508del mutation is the most common cause of cystic fibrosis Common sympto...

Cystic fibrosis intro - progressive, autosomal-recessive disorder - primarily affects the lungs (Obstructive airways disease) and pancreas - mutations in the CFTR gene leading to dysfunctional CFTR protein. - F508del mutation is the most common cause of cystic fibrosis Common symptoms 1. respiratory issues - chronic inflammation/bronchial wall thickening - air trapping/bronchiectasis - Chronic cough, increased sputum production (thick, purulent, or bloody), shortness of breath, wheezing, and chest pain. - chronic rhinosinusitis(CRS) - majority of deaths in CF are due to respiratory failure. 2. gastrointestinal problems a. Pancreatic Insufficiency - Difficulty in maintaining weight, bowel issues, fatty infiltration of the pancreas. b. Large bowel - - c. Small bowel - d. Upper GI - - 3. increased susceptibility to lung infections. - Pseudomonas aeruginosa, - Staphylococcus aureus, - Stenotrophomonas maltophilia, - Burkholderia cepacia complex, - atypical mycobacteria, Aspergillus, - Candida. **Clinical Manifestations:** 1. **Presentation in Childhood**: - Failure to thrive, recurrent chest infections, meconium ileus, and failure to reach growth milestones. 2. **Adult Symptoms**: - **Respiratory**: Pan-bronchiectasis, decreased mucociliary clearance, obstructive airway disease, respiratory failure. - **Gastrointestinal**: Pancreatic insufficiency, malabsorption, GORD, rectal prolapse, DIOS. - **Other Manifestations**: CF-related diabetes, liver disease, osteoporosis, nasal polyps, infertility in males (CBAVD). **CF Exacerbation Symptoms:** Respiratory symptoms - ↑ Productive cough - Sputum - ↑ volume, ↑ purulence, ↑ viscidity - ↑ SOB - ± Wheeze - ± Haemoptysis - Nasal/ sinus symptoms - Duration of onset -- few days- couple of weeks (Absence of febrile symptoms -- common) CF: Non-infectious respiratory complications - Haemoptysis -- can be intermittent, mild/moderate/severe - Pneumothorax (pleuradiesis contraindicated) - Allergic bronchopulmonary aspergillosis -- wheeze, unresponsive to antibiotics, thick mucus plugs, raised IgE - Atelectasis -- due to mucus plugging. Can cause acute SOB and hypoxaemia (desaturation) - Advanced disease -- Type II Respiratory failure, Pulmonary hypertension with Cor Pulmonale Other manifestations: - **Liver disease** -- focal biliary cirrhosis, fatty infiltration, can lead to cirrhosis with splenomegaly, varices - Any jaundice, itch, abdominal pain )gallstones), hematemesis - **CF related diabetes** - Polyuria, polydipsia, lack of energy, failure to gain weight - **Low bone mineral density/ osteoporosis --** - usually assymptomatic, significance unknown, no increase in incidence of fractures - **Growth retardation/ Delayed puberty** - **Obstructive azospermia --** functional sterility in males (due to CBAVD) - Do you have any children? - **CF Renal disease --** distinct entity, compounded by aminoglycoside usage, and diabetes **Physical Examination finding** General Inspection - ↓BMI, Delayed puberty - Clubbed, ±Cyanosis +/- oxygen - Tremor - Tachypnea, using accessory muscles of respiration Chest - Hyperinflation, ±Portacath - Crepitations, Wheeze Abdomen - ± Hepatosplenomegaly - ± PEG tube CFTR gene mutations \- (Cystic Fibrosis Transmembrane Conductance Regulator) \- results in production of dysfunctional CFTR protein CFTR Protein - Member of the ATP-binding cassette (ABC) transporter superfamily - Located at the apical membrane of polarised epithelial cells - Cyclic AMP-regulated (phosphorylated by Protein Kinase A) - Acts as a chloride ion channel - ion channel moves Cl- from inside the cell to outside the cell, attracting a layer of water that allows cilia on lung cells to move and sweep mucus out of the airways. - The water layer allows tiny cilia on the surface of lung cells to move back and forth. CFTR Protein mutations - reduced chloride movement to the cell surface, causing reduced volume and increased hyper-viscosity of mucosal secretions Diagnosing cystic fibrosis Newborn screening (NBS) - heel prick/ Guthrie test - IRT test measures immunoreactive trypsinogen levels in the blood; elevated levels indicate potential cystic fibrosis due to blocked pancreatic ducts. - If IRT is raised, to confirm diagnosis- - testing for common CF gene mutations : A diagnosis of CF is confirmed if two mutations are identified. - a sweat test - \>60mmol/L indicating CF - 30-59mmol/L being inconclusive - \ 40 and \< 60 require a repeat test - \> 60 mEq/L are considered abnormal. - If Genetic testing and sweat test are inconclusive- - nasal potential difference (NPD) : voltage difference across the nasal epithelium to assess CFTR protein function by evaluating ion movement (Cl-) - intestinal current measurements (ICM): assesses chloride ion transport across rectal or colonic biopsies in vitro Management 1. Hydrators and mucolytics - hypertonic saline: a. hydrate and thin the mucus in the airways b. (3%-7%) 2-3x/d - Pulmozyme : c. contains recombinant human deoxyribonuclease I, which breaks down the DNA in the mucus, reducing its viscosity d. Nebulized DNAase -- Dornase Alpha ('Pulmozyme') 1x/d - Mannitol (Bronchitol): e. an osmotic agent that draws water into the airways, which helps to thin and clear mucus, thereby improving airway clearance f. (Bronchitol^TM^) 2x/d - Inhaled/ Nebulized bronchodilators -- Salbutamol, Ipratropium 2. Anti Inflammatories: - - 3. Antibiotics for chronic bacterial infections: i.v. antibiotics -- 2 weeks/ 2 agents - ***H. influenza, S. aureus*** - ***P. aeruginosa (CAT)*** - Tobramcyin + anti-pseudomonal semi-synthetic penicillin or 3^rd^ generation cephalosporin or carbapenem - Why is pseudomonas so bad? - - - - ***Aspergillus*** - Allergic Bronchopulmonary Aspergillosis - Daily oral prednisolone or Monthly iv Methylprednisolone ± Oral Itraconozole (12-18 months) - Stenotrophomonas: sulphamethoxazole/trimethoprim-Septrin  - MRSA use - vancomycin or linezolid. - If MSRA use flucloxacillin 4. **CFTR Modulators (Precision Medicine):** - Improve function at the cell membrane - **Ivacaftor/Kalydeco** - patients aged 6 and up with **one copy of the G551D mutation, a class III (gating) mutation** - - Help with protein folding and trafficking within the cell (e.g., **Lumacaftor** and **Tezacaftor**). - **Orkambi** : Lumacaftor (Corrector) + Ivacaftor (Potentiator) - **Symkevi** : Tezacaftor (Corrector) + Ivacaftor (Potentiator) - Homozygous F508del - A combination of two correctors (Elexacaftor, Tezacaftor) and a potentiator (Ivacaftor) for F508del mutation. Vanza: CFTR modulator therapy (vanzacaftor, tezacaftor and deutivacaftor) 5. **Physical therapies:** Airway clearance techniques, physiotherapy. 6. **Dietary supplements** pancreatic enzymes, fat-soluble vitamins, high fat diet a. Pancreatic enzyme tablets -- 'Creon' b. Vitamin ADEK -- 'Aquadek' c. Proton pump inhibitors d. Oral nutritional supplements e. PEG feeds -- 'Perative', 'Nutrison' 7. [Liver disease-]Ursofalk **Monitoring and Outpatient Care:** - Regular spirometry (FEV1), sputum culture, liver ultrasound, bone densitometry, chest X-ray, and annual measurements (OGTT, vitamin levels, liver function tests). **Outcomes:** - CF-related outcomes predicted by lung function (FEV1), BMI, exacerbation frequency, and infection control. Transplantation may be considered in severe cases. Common mutations **Mutations in CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) Gene:** - Over 2000 mutations identified. - CFTR mutations categorized into **seven classes** based on structural and functional consequences: - **Class I:** No CFTR protein produced (e.g., G542X mutation). - **Class II:** Defective protein processing (e.g., F508del mutation). - **Class III:** Defective gating (e.g., G551D mutation). - **Class IV:** Reduced ion conductance. - **Class V:** Reduced protein synthesis. - **Class VI:** Decreased CFTR stability at the membrane. - **Class VII:** No mRNA production. Novel Genomics 1. **Sanger Sequencing (First Generation)**: - Uses chain-termination PCR with dideoxynucleotides (ddNTPs) to sequence DNA by synthesizing fragments that terminate randomly. - Uses DNA polymerase, primers and fluorescently-labelled nucleotides - DNA is made of 4 nucleotides calld dNTPs - dNTPs have 1 less O than ribose - ddNTPs have 2 less O than ribose - incoming dNTPs react with bound dNTPs and goes on - But if ddNTPS gets joined, it terminates because there are no O to continue. 2. **Next Generation Sequencing (NGS)**/ Massively parallel sequencing/ Deep sequencing - Isolate DNA and divide into smaller fragments - Fragments get ligated by oligonucleotides (sequence binding site + complementary sequencing: hybridizes with flow cell) - Denatures double stranded fragments to single strands - Binds to flow cell - DNA polymerase synthesizes complementary strands by PCR - Denatures: single strands not attached to the flow cell washed away - Bridge building: 2 ends are stuck to the flow cell - DNA polymerase - Denatures - Repeat - More efficient than Sanger for large-scale genomic sequencing. - Short-read sequencing with high accuracy but difficulty in covering repetitive genomic regions -  **Illumina (75-300bp)** Illumina\'s NGS platforms use a **reversible termination sequencing** method. This involves: - **Sequencing by synthesis:** Short DNA fragments (reads) are sequenced one nucleotide at a time. - **Reversible termination:** Each nucleotide has a removable blocking group that prevents further extension until the correct base is incorporated. - **Optical detection:** As each base is added, a fluorescent signal is emitted and detected, allowing for identification of the specific nucleotide.  3. **Third Generation Sequencing**: - a subset of NGS technologies that directly sequence single molecules without amplification - focusing on long-read sequencing for complex genomic regions like repetitive sequences - **Nanopore** and **PacBio** sequencing, **Nanopore Sequencing** - Uses a protein pore and electrical disturbances to sequence DNA (or RNA) - g. **5mC (5-Methylcytosine)** h. **5hmC (5-Hydroxymethylcytosine)** i. **5mA (5-Methylcytosine with N6-methyladenine)** **Procedure:** 1. Extract DNA from a tissue sample 2. End-prep and nick repair (*DNA polymerases repair overhangs, Stabilise DNA molecules )* 3. Add sequencing adapters and motor proteins *(Regulate speed of sequencing )* 4. Load the library onto a flow cell **Principle:** - Protein pore embedded in a membrane.  - You add your DNA, which is negatively charged.  - Apply a current to the space beneath the pore \--\> DNA is pulled through the pore - As the DNA passes through the pore, it creates an electrical disturbance that is picked up by a computer chip in the flow cell. Each nucleotide has a characteristic signal.  - If the DNA passes through the pore too quickly, the computer can\'t tell what nucleotide just passed through.  - So we add a motor protein to the DNA.  - **DNA methylation detection using Single-Molecule Real-Time (SMRT) sequencing**. - direct detection of DNA methylation modifications without the need for chemical treatments -     *PacBio long reads: 10-25kb* 1. **Start with high-quality double-stranded DNA** 2. **Prepare SMRTbell libraries:** 3. **Anneal primers and bind DNA polymerase** 4. **Circularized DNA is sequenced in repeated passes:** 5. **The polymerase reads are trimmed of adapters to yield subreads:** 6. **Consensus and methylation status are called from subreads:** -- -- -- -- -- -- 4. 4th generation sequencing A term sometimes used to describe the latest advancements in sequencing technology T2T: Telomere-to-Telomere  **Understand genome evolution** - *Complete image of long, conserved blocks of DNA sequence flanking genes* - *Study how genes evolved between species* **Construct more accurate gene regulatory networks** - *Regulatory regions are typically repetitive DNA sequences, e.g. high GC content* **Better capture complex structural variation,  chromosomal instability** *e.g. higher resolution of chromosomal shattering (chromothripsis) in cancer* Chromothripsis is a process in which hundreds to thousands of clustered chromosomal rearrangements occur in a single event in a confined genomic regions in one or a several chromosomes, and is known to be involved in both cancer and congenital diseases \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-- **RNA-seq** - sequencing of total RNA or mRNAs from a sample. - Used to quantify gene expression. - Detection of fusion transcripts. (**Fusion transcripts** are abnormal RNA molecules that arise when parts of two or more genes are joined together. Caused by chromosomal rearrangements, translocations, or gene fusions etc) **Types of Genomic Changes in Disease** **[These alterations affect disease genes]** a. **Single nucleotide variants:** A single nucleotide change in the DNA sequence. - **Silent mutations:** No change in A.A sequence due to redundancy in the genetic code. - **Missense mutations:** single nucleotide substitution results in diff A.A - **Nonsense mutations:** Termination, leading to a truncated protein( shortened protein due to translation process terminating prematurely) - **Splice site mutations:** Affect the splicing process, leading to abnormal mRNA transcripts. b. **Small insertions / deletions** - Frameshift mutations : number of nucleotides in a DNA sequence is not a multiple of 3 - Addition/removal of nucleotides alters how DNA sequence is read into codons, leading to a shift in the reading frame c. **Chromosomal gains or losses** d. **Structural variants** Large-scale rearrangements of DNA, such as deletions, duplications, inversions, or translocations (**Inversions:** A segment of DNA is flipped, reversing the order of genes. **Translocations:** A segment of DNA is moved from one chromosome to another.) **Epigenomics** study of all epigenetic changes in the cell that alter gene expression without changing the DNA sequence **Examples of epigenomic markers** - **DNA Methylation:** Methylation of 5^th^ C on cytosine base/ represses gene expression - **Histone modifications** - Multiple different markers across the tails of histones - Can repress or activate gene expression depending on location/mark - Generally contribute to chromatin remodelling **Epigenomic sequencing techniques** a. Histone Modification Sequencing - Identify gene activation/repression sites - Reveal location of regulatory elements - Promoters/Enhancer regions **Eg: ChIP-seq (Chromatin Immunoprecipitation Sequencing):** Identifies the binding sites of proteins, such as transcription factors and histone modifiers, on DNA. - Fragment chromatin - Target with antibody specific to modification of interest - Purify DNA - PCR amplification - Sequence regions associated with modification of interest - Requires a large number of cells - Lots of steps with lots of optimisation (very difficult) - Poor reliability b. **Bisulfite sequencing:** Detects the methylation status of cytosine bases in DNA**.** **Mechanism** - Fully denature DNA to single strand - Treat with sodium bisulphite - PCR amplification - Sequencing - Identify which residues are still cytosine and were therefore methylated - Unmethylated cytosines turns into uracil and then thymine **Issues with bisulphite sequencing** - As unmethylated cytosines are converted to thymine, there is reduced sequence complexity and alignment is harder - SNPs where a cytosine is converted to a thymine will be missed - Not able to distinguish between 5mC and 5hmC c. **Chromatin accessibility sequencing** - Identify the DNA regions where chromatin is accessible (open) - Identify novel gene regulatory regions- Promoters/enhancers **Eg: ATAC-seq (Assay for transposase accessible chromatin with sequencing)** Maps the accessibility of chromatin, which can influence gene expression. **Mechanism** - Expose cells to Tn5 transposase which TAGs DNA fragments - Purify DNA - PCR amplification - Sequencing of open DNA fragments - Chromatin might open during processing - Only 50% of molecules are in the correct orientation for amplification  d. **Single-Cell Sequencing in CF:** - **Single-Cell RNA Sequencing (scRNA-seq)**: Analyzes gene expression at the single-cell level, offering insights into CF airway epithelial cells and altered cell states. - **Single-Cell ATAC Sequencing (scATAC-seq)**: Used alongside scRNA-seq to map chromatin accessibility and regulatory elements in CF tissues. **Multi-omic analysis using multiple different epigenomic sequencing methods can be used to create the full picture** **Summary:** - Epigenomic techniques like DNA methylation sequencing and ChIP-seq reveal regulatory mechanisms affecting CF. - Single-cell sequencing offers a high-resolution view of gene expression and chromatin accessibility in CF cells.