A Study of Cystic Fibrosis Using Web-Based Tools

1. A Study of Cystic Fibrosis Using Web-Based Tools Anuradha Datta Murphy Graduate Student, Dept. of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign

2. Introduction: • We will use several medical and computational biology web sites to obtain information on cystic fibrosis, an inherited respiratory disorder. • Outline of presentation: • What is cystic fibrosis (CF)? • Gather information on cause and symptoms of CF from medical web sites • Search for cystic fibrosis transmembrane receptor (CFTR) protein, locate CF-causing mutation, view CFTR structure using web-based tools • Obtain information on other inherited disorders of interest

3. What is cystic fibrosis? • Cystic fibrosis (CF) is an inherited genetic disease that affect cells producing sweat, saliva, mucus, and digestive fluids. Normally thin and slippery, in CF patients these secretions are sticky and block tubes and passageways. Most CF patients die of bacterial infections resulting from blocked airways in the lungs. • Most children with CF have frequent coughing, wheezing, and respiratory infections.

4. Symptoms of CF, contd. • CF also affects the pancreas, liver, and intestines, leading to poor absorption of nutrients and poor weight gain. CF patients have salt crystal formation on their skin when they sweat excessively. • CF slide show available at: http://www.healthcentral.com/library/April_03/flash_content/hcplay.asp?id=110

5. What causes CF? • A mutation in the gene coding for the CFTR (cystic fibrosis transmembrane receptor) protein causes CF. • CF is caused when a child inherits two copies of this defective (mutated) gene, one from each parent. • The CFTR protein is a Cl- channel located on the plasma membrane of epithelial cells of the lungs, pancreas, sweat glands, and other tissues. • In non-CF individuals, Cl- transport from the cell is increased in the presence of ATP. In CF patients this transport malfunctions, causing salt and water imbalance in and around the cell.

6. The CFTR protein: • A single mutation in the CFTR gene results in cystic fibrosis. • This mutation involves deletion of three basepairs of DNA, resulting in deletion of a phenylalanine residue (delta-F508 mutation) from a normal (wild type) CFTR sequence. • Theory: deletion of Phe508 prevents the CFTR protein from being properly delivered to the plasma membrane, thus causing Cl- transport malfunction.

7. Properties of the CFTR protein: • The CFTR gene is located on chromosome 7 in the human genome • This protein is made up of 1480 amino acids and contains two domains: (1) membrane association, and (2) ATP-binding • In CF patients, deletion of Phe 508 causes defective Cl- transport.

8. Locating and viewing the CFTR protein: • In the Biology Student Workbench, search for the CFTR protein using the following: • search words: “cystic fibrosis homo sapiens” • search database: Non-Redundant protein sequence database • tool: Ndjinn • Select SDSCNR:315424, Cystic Fibrosis Transmembrane Conductance Regulator …, import sequence. • View sequence, note accession number, P13569. Use other tools on BSW to learn about CF.

9. Using the NCBI web site: • The National Center for Biotechnology Center is available at the following url: http://www.ncbi.nlm.nih.gov • Many computational biology tools are available here, including PubMed (a database of biological literature), Entrez (a search engine for information on protein and nucleotide sequences across databases), BLAST (alignment search tool for protein and nucleotide sequences), OMIM (Online Mendelian Inheritance in Man, a directory of human genes and genetic disorders), and Cn3D (structure viewer).

10. Study of CFTR using tools on NCBI: • In the NCBI home page search Entrez for cystic fibrosis: select Entrez, type “human cystic fibrosis” in the Search window, click on “Go”. • In the results page; note that our keyword search has yielded hits on multiple databases contained within NCBI. • Since CF is an inherited genetic disorder, click on OMIM to view results within the Online Mendelian Inheritance in Man database. • In the OMIM hits list page, click on the top hit, *602421.

11. Using OMIM: • Note the exhaustive information on CF available in OMIM, including cause and symptoms of the disease, molecular basis, summary of literature on CFTR mutation research, study of other related diseases generated by mutations on CFTR, etc. • Click on “Gene map locus 7q31.2” near the top of the page. This gives the CFTR gene location on the human genome. Click on “Location” in the following page to get more detailed information, including a figure of human chromosome 7 and other genes located in the vicinity.

12. Using OMIM: • Return to the OMIM results page. Scroll down the page to locate literature on F508 deletion (you can click on “Edit” in the toolbar header, click on “Find on This Page” and type delF508). Research indicates that this deletion causes CF. Click on (602421.0001). This is the mutated CFTR gene causing CF. In the displayed page, there is more literature on CFTR mutation research.

13. Using NCBI tools: • Return to the Entrez results page. Click on “Protein” to examine the CFTR protein sequence. In the hits page, click on P13569. • Note presence of a phenylalanine residue (F) at position 508 in the CFTR sequence. There is also detailed information on various CFTR sequence mutations and their results.

14. Using Cn3D structure viewer: • Return to the Entrez results page. Click on Structure. • Note presence of multiple CFTR structures based on Phe508 mutation. • Click on 1CKZ, view structure of CFTR segment affected by Phe508 mutation using Cn3D. • In the MMDB Structure Summary page, click on “View 3D Structure” button. • Click on amino acid residues below to observe location on structure. Note absence of “F” residue.

15. Using NCBI tools: • Return to the OMIM page. • On the left column, click on “Search Morbid Map” • In the “Search for” window, type in keywords for any inherited disease of interest (ex., sickle cell anemia, muscular dystrophy, diabetes, etc.) • Locate the affected gene on the human genome, view location on the chromosome, view structure of protein (if available) using Cn3D, and read up on literature of research (both clinical trials and gene mutations), all using tools on NCBI!

16. Closing Notes: • For more information on CF symptoms and treatment, see: • http://www.merck.com/mrkshared/CVMHighLight?file=/mrkshared/mmanual_home2/sec04/ch053/ch053a.jsp%3Fregion%3Dmerckcom&word=cystic&word=fibrosis&domain=www.merck.com#hl_anchor • http://www.healthcentral.com/library/librarycontent.cfm?id=222 • http://www.healthcentral.com/library/librarycontent.cfm?id=222