CYSTIC FIBROSIS

Powerpoint Presention By:

Deirdre Murphy

Kimberley Madigan

Lorraine McCarthy

Aleksandra Sikora

Background Cystic fibrosis is an

inherited disease that primarily affects the lungs and the digestive system

Autosomal recessive disorder

A CF sufferer produces thick, sticky mucus that clogs the lungs and digestive tract

1/19 people are carriers of the gene

50 new cases are diagnosed each year

Fig 1.1 Two copies of the abnormal gene must be present for the disease to develop

Causes

Cystic Fibrosis is caused by the mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene

Located on the long arm of chromosome 7, position 7q31

The most common mutation is ΔF508, codes for the deletion of the amino acid – Phenylalanine in position 508 of the protein

Disruption of protein folding and trafficking to the apical membrane

Remains in the Endoplasmic Reticulum (ER)

Symptoms

Respiratory Symptoms

Persistent coughing producing thick mucus

Wheezing and breathlessness

Digestive Symptoms

Intestinal blockage

Severe constipation

Poor weight gain and growth

Protein Structure

Transmembrane glycoprotein

1480 amino acids

Five domains cyclic-AMP

dependent activation channelFig 1.2: Homology model

structure of the CFTR protein (Patrick and Thomas 2012)

Protein Function

Under normal conditions, CFTR protein functions as a chloride channel regulating the transport of Cl- ions into and out of the apical membrane of epithelial cells

Figure 1.3: Normal CFTR function

Protein Function

Under abnormal conditions, non functioning or no production of the CFTR protein alters the chloride balance within the cells

Figure 1.4: Abnormal CFTR function

CYSTIC FIBROSIS TREATMENTS

•Kalydeco•Respiratory Treatments•Implanted Devices•Nutrition•Research Therapies

Kalydeco

Treats patients with one of 9 mutations : G551D, G1244E, G1349D, G178R, G551S, S1251N, S1255P, S549N and S549R

Increases activity of the defective channels

Proved to help restore function of CFTR protein.

Lowers chloride levels in sweat. Thins mucous in the lungs. Only drug to target the cause of

CF, not just focus on it’s symptoms.

Respiratory Treatments

Airway clearance techniques (ATCs): helps patients breathe easier by clearing mucous from lungs. Postural drainage and percussion

TOBI® (tobramycin inhalation solution) and Cayston® (active substance aztreonam) inhaled antibiotics which targets Pseudomonas aeruginosa – cause of lung infection for CF sufferers

Pulmozyme: Bacteria can build up in thick mucous. White blood cells fight bacteria and leave behind extracellular DNA-

which makes mucous more thick and sticky. Pulmozyme acts by cutting up this extracellular DNA.

Hypertonic Saline: Involves inhaling an extra salty mist as CF sufferers airways lack salt and water

Implanted devices

Peripheral Inserted Central Catheter: long, thin, flexible tube placed into one of the large veins in the arm. This tube is threaded into a large vein above the right side of the heart

Implanted Ports: 2 parts, a catheter and a ‘port’. Used for fluids or IV

medication

Nutrition

Young CF sufferers need extra calories to grow and develop

Dairy products and high fat diets: for extra calories

Tube feeding Viatmins A, D E and K: taken daily. Minerals, like calcium,iron, sodium, chloride

and zinc, are essential to maintaining good health

Enzyme capsules-Pancrelipase (Creon) aids digestion when pancreas is malfunctioning Replaces pancreatic enzymes made by a

healthy person

Research

CF patients have less natural antioxidants than non CF sufferers.

Research and clinical trails being carried out on drugs such as glutathione, which are building blocks for antioxidants.

Gene therapy: targets the cause rather than the symptoms. Germ line therapy Somatic gene therapy

CYSTIC FIBROSIS DIAGNOSTICS

•Newborn Screening•Sweat Chloride Test•Oligonucleotide Ligation Assay•DHPLC

Screening for CF

Newborn Screening High levels of

immunoreactive trypsinogen (IRT)

Heel prick test NBS is a screen for CF,

NOT a diagnostic Positive screen leads to

diagnostic Sweat Chloride test

Sweat Chloride Test

Two electrodes containing the sweat-inducing drug Pilocarpine placed on the skin of the forearm.

Electrodes produce a current for five minutes Sweat collected for thirty minutes Sample taken to lab for quantification and

analysis of chloride concentration Chloride (mmol/L)

Normal ≤39

Intermediate 40-59

Abnormal ≥60

Oligonucleotide Ligation Assay (OLA)

First level analysis In vitro diagnostic device Two Phases:

Multiplex PCR OLA

Produces allele-specific, fluorescent-labelled fragments which are then separated by electrophoresis

PCR OLA Process

1. Multiplex PCR2. Ligation reaction

Normal = standard fragment length Abnormal = different fragment length

3. Electrophoresis of ligation fragments

Denaturing High Performance Liquid Chromatography (DHPLC)

Second level analysis Considered the most reliable

technique for detection of mutations Relies on the different

elution properties of DS DNA fragment with/without mutation

High degree of sensitivity

DHPLC Process

1. Wild and mutant type DNA strands amplified separately, mixed, heated and then cooled to form homoduplexes and heteroduplexes

2. PCR products loaded onto polystyrene column and eluted with an acetonitrile (ACN) gradient buffer

3. As ACN concentration increases, DNA fragments are released from the cartridge and pass through UV detector which record absorbance over time

No mutation – all fragments released at the same time. Single Peak

Mutation – fragments released at different time. 2-4 peaks

THE CHLORO-PATCH

•Medical patch: easy and rapid diagnostic technique•Self-diagnosing•Safe to use and non-invasive•All ages

Chloro-Patch Design• A layer to preserve adhesive• Adhesive semipermeable layer allows only

molecules of >40DA to pass into the patch • Carrier layer chamber containing the reaction

substance and is made up from Ethylene-vinyl acetate

• Reacting substance consists of Sodium Bromide solution.

• Observation point visualises the reaction effects

Chloro-Patch mechanism

Colour change caused by fallowing equilibrium [Co(H2O)6]2+(aq)(pink) + 4Cl-(aq) ⇌ [CoCl4]2-(aq)(blue) +

6H2O(l)

Underarm administration Time frame is dependant on location and

activity Children require observation and …..

Helicase Dependant Amplification

Mimics in vivo mechanism The method was used due

to its simplicity and time requirements

Only a short DNA sequence is required for diagnostic purpose

Extraction kit used of purification

Southern Blotting

Visualization on agarose gel after gel electrophoresis

The DNA denaturation (DS -> SS) Transfer of ssDNA to +vely charged nylon

membrane by capillary action transfer Addition of probes and visualization using x ray. The appearance of a band would diagnose the

mutation resulting in a positive test for cystic fibrosis.