The Suggestion of Cystic Fibrosis - Case Study Example

CFTR gene- Cystic fibrosis transmembrane conductance regulator CFTR gene s for the CFTR protein which is a chloride channel protein seen in membranes of cells lining passageways of lungs, intestine, liver, pancreas, skin and the reproductive tract and is also involved in other transport pathways regulation. Cystic Fibrosis and congenital bilateral aplasia of the vas deferens are the disorders associated with the CFTR gene due to gene mutations. Existence of cystic fibrosis has been known for a very long time from stories of old cleaning ceremonies where foreheads of children were licked to see if they were salty and if they were those children were feared to die soon due to the disease which we today call cystic fibrosis (Busch, 1990, pp. 13-15 ). The illness was first described in the 1930s with the earlier paper was believed to be published by the Swiss paediatrician Dr. Fanconi who called pancreatic changes in children to be ‘celiac syndrome’ (Fanconi et al, 1936, pp.753-756). During this time clinical manifestation of the disease in the form of pancreatic malabsorption considered to be due to pancreatic abnormalities with evidence from histological changes at autopsy was reported (Parmalee, 1935, pp. 1418-1428; Hess and Sapphire, 1935, pp. 1-13). Another important clinical feature included severe respiratory problems in affected children. Other significant report of pancreatic changes with features compatible to that of cystic fibrosis came from Margaret Harper of Sydney who reported congenital steatorrhea due to pancreatic defect (Harper, 1938, pp. 45-56). However the recognition for describing cystic fibrosis as a separate clinical entity goes to Dr Dorothy Andersen who described neonatal intestinal obstruction, respiratory complications and characteristic pancreatic histology in her 1938 report (Andersen, 1938, pp. 344-399). She called it the ‘fibrocystic disease of the pancreases. In the forties it was recognized as a generalized disorder affecting organs other than the pancreas and Dr Sydney Farber who coined the term ‘mucoviscidosis’ for the condition accurately summarized the secondary consequences of Cystic fibrosis to cause clogging of respiratory tract by thick mucus, secondary Staphylococcal infection and failure of proper lubrication of ciliated epithelium (Farber, 1943, pp. 827-833). Cystic fibrosis is an autosomal recessive disorder. In autosomal recessive disorders two copies of an abnormal gene must be present in one of the first 22 nonsex or autosomal chromosomes for the development of the disease. The first suggestion of cystic fibrosis being an inherited disorder came from Philip Howard who reviewed familial occurrence of the fibrocystic disease of the pancreas (Howard, 1944, pp. 330-332). However the first clear report that identified cystic fibrosis to be inherited as a recessive Mendelian trait came from Andersen and Hodges in 1946. They investigated 56 families from literature and 47 of their own families to come to the conclusion that the incidence of the disorder follows the Mendelian recessive inheritance which approximated the incidence in siblings as 25% as expected of a Mendelian recessive condition which required more than one factor for expression (Andersen and Hodges, 194, pp. 62-80). It took almost 8 years to identify the genetic inheritance of the disorder with clinical evidence following its identification as a specific clinical entity. Various research groups including that of Professor Bob Williamson’s in London’s St. Mary’s Hospital and Professor Lap-Chee Tsui’s in Toronto in conjunction with Collaborative Research Inc in Boston attempted to identify the Cystic Fibrosis gene starting from the early eighties. They extracted DNA from the blood samples of patients with cystic fibrosis and their families to look for markers physically close to the gene on the same chromosome. Researchers spent almost five years finding and testing the wrong markers until Hans Eiberg and his colleagues in Copenhagen demonstrated the gene for a protein called paraoxonase (PON) was linked to CF gene (Eiberg et al, 1985, pp. 265-271). Shortly afterwards Tsui and Collaborative Research demonstrated a marker on chromosome 7 linked to both PON and cystic fibrosis (Tsui et al, 1985, pp. 1054-1057). Dr Ray White in Utah found two other markers Met H and Met D (White et al, 382-384) and Dr Williamson’s Lab in London reported DNA probe pJ3.11 much closer to the gene (Wainwright et al, 1985, pp. 384-385). All the three finding were published together in Nature on 29th November 1985. Eventually in 1989, the cystic fibrosis gene was identified by teams headed by Professor Lap-Chi Tsui, Dr Francis Collin and Professor Jack Riordan (Kerem et al, 1989, pp. 1073-1080; Rommens et al, 1989, pp. 1059-1065; Riordan et al, 1989, pp. 1066-1073). In a poster presented in the 8th International Cystic Fibrosis Congress in Toronto in 1980 suggested the primary defect in cystic fibrosis to be the abnormal epithelial electrolyte transport which went onto say the clinical symptom hyper viscous mucus as being caused due to excessive absorption of Na Cl and water (Hopfer et al, 1980). A direct evidence of the epithelial electrolyte transport dysfunction was demonstrated as an abnormally high potential difference in the nasal mucosa of patients by Michael Knowles and colleagues of the University of North Carolina (Knowles et al, 1981, pp. 1489-1495). Paul Quinton in 1983 confirmed that there was a defect in chloride permeability which results in raised salt content in the sweat of the patients (Quinton, 1983, pp. 421-422). The cystic fibrosis gene was identified by teams headed by Professor Lap-Chi Tsui, Dr Francis Collin and Professor Jack Riordan in 1989. Tsui and his colleagues used a technique called saturation mapping to isolate large number of clones from chromosome 7. He collaborated with Francis Collins who was expert in the chromosome jumping techniques that made cloning of DNA sequences of considerable distance from the starting fragment possible. Identification of a DNA sequence in the middle of their region of mapping was seen to be conserved in other animals suggesting the possibility of an expressed gene in the region. However the evidence that confirmed it to be the cystic fibrosis gene was the identification of the same sequence in libraries of cloned fragments from sweat glands by Riordan. 19 related clones were isolated by the researchers in the following months which contributed to the building of the full sequence of the cystic fibrosis gene. The abnormal gene in cystic fibrosis encodes a 1480 amino acid protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein (Riordan, 1989). More than 1500 mutations have been identified in cystic fibrosis patients since 1989. About 70% of the mutation result from deletion of three base pairs in its nucleotide sequence causing the loss of phenylalanine at position 508 of the protein and hence the mutation is referred to as delta F508. Research groups worked to correct the basic defect laying foundation for future drug and gene replacement therapy (Gregory et al, 1990, pp. 382-386; Drumm et al, 1990, pp. 1227-1233). The defective chloride channels were first corrected by Welsh and his Colleagues (Rich et al, 1990, pp. 358-363).Cheng and colleagues (1990, pp. 827-834) found that DF508 CFTR was not processed completely due to faulty intracellular processing and prevented it from reaching the cell membrane (Kartner et al, 1992, pp. 321-327). Welsh and colleagues also reported that growing cystic fibrosis cells at reduced temperatures resulted in an improved CFTR chloride channels expression (Denning et al, 1992, pp. 761-764). Bear and co-workers in 1992 confirmed CFTR to be a chloride channel and not just a regulator (Bear, 1992, pp. 809-818). Their work resulted in the reconstitution of CFTR protein into artificial membranes that demonstrated wild type CFTR gene function. The Cystic Fibrosis Transmembrane Conductance Regulator gene is located in region q31.2 on the long (q) arm of human chromosome 7(Heng et al, 1993, pp. 108-109). The gene is found to be 250 kb in size and contains 27 exons (Zielenski et al, 1991, pp. 214-228). Intron-free mRNA transcript of the gene is 6129 bp long of which 4443 bp code for the gene’s protein product of 1480 amino acid protein that functions as a chloride channel. There has been significant advancement in the understanding of the disease since the identification of the gene in 1989. Although a complete cure by means of gene replacement therapy is still not available though many attempts have been made a number of practical benefits including detection of carriers, accurate antenatal diagnosis and DNA testing in neonatal screening programmes all have been made possible following the identification of the CFTR gene. CFTR protein CFTR protein is classified as a traffic ATPase or ATP-binding cassette transporter (ABC transporter). Molecules including sugars, chlorides, metal cations and inorganic phosphates are transported across the cellular membranes by these proteins. CFTR transports chloride ions across membranes of cells in lungs, pancreas, intestine, skin, liver, digestive and reproductive tract. Structure of CFTR though not experimentally determined due to difficulty in crystallizing its hydrophobic regions, models have been proposed by comparing the protein sequence of CFTR with other ABC transporters. The proposed model structure is made up of 5 domains the chloride ion channel forming membrane spanning domains MSD1 and MSD2, ATP binding and hydrolyzing domains NBD1 and NBD2 domain and an R domain which is regulatory domain. Cystic Fibrosis causing mutation delta F508 occurs in first nucleotide binding domain NBD1 coding sequence. CFTR is the only ABC transporter protein to possess a fifth regulatory domain, the modification of which through addition or removal of chemical phosphate groups regulates the chloride ion movement across membranes as has been demonstrated (Sheppard and Welsh, 1999. pp. S23-45). Appendix Amino Acid sequenceof human CFTR protein in FASTA format http://www.uniprot.org/uniprot/P13569 Isoform 1 >P13569-1 MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWDRE LASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEERSIA IYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISIGQL VSLLSNNLNKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLALFQAGL GRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQTELKLTRKAA YVRYFNSSAFFFSGFFVVFLSVLPYALIKGIILRKIFTTISFCIVLRMAVTRQFPWAVQT WYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEEGFGELFEKAKQNNNNRK TSNGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTGAGKTSLLMVIMGELEPSEG KIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKACQLEEDISKFAEKDNIV LGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTR ILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDFSSKLMGCDSFDQFSAERRNS ILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKRKNSILNPINSIRKFSIVQKTPLQ MNGIEEDSDEPLERRLSLVPDSEQGEAILPRISVISTGPTLQARRRQSVLNLMTHSVNQG QNIHRKTTASTRKVSLAPQANLTELDIYSRRLSQETGLEISEEINEEDLKECFFDDMESI PAVTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAASLVVLWLLGNTPLQDKGNSTHSRN NSYAVIITSTSSYYVFYIYVGVADTLLAMGFFRGLPLVHTLITVSKILHHKMLHSVLQAP MSTLNTLKAGGILNRFSKDIAILDDLLPLTIFDFIQLLLIVIGAIAVVAVLQPYIFVATV PVIVAFIMLRAYFLQTSQQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHK ALNLHTANWFLYLSTLRWFQMRIEMIFVIFFIAVTFISILTTGEGEGRVGIILTLAMNIM STLQWAVNSSIDVDSLMRSVSRVFKFIDMPTEGKPTKSTKPYKNGQLSKVMIIENSHVKK DDIWPSGGQMTVKDLTAKYTEGGNAILENISFSISPGQRVGLLGRTGSGKSTLLSAFLRL LNTEGEIQIDGVSWDSITLQQWRKAFGVIPQKVFIFSGTFRKNLDPYEQWSDQEIWKVAD EVGLRSVIEQFPGKLDFVLVDGGCVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDPVT YQIIRRTLKQAFADCTVILCEHRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSLFRQA ISPSDRVKLFPHRNSSKCKSKPQIAALKEETEEEVQDTRL Isoform 2 >P13569-2 MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWDRE LASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEERSIA IYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISIGQL VSLLSNNLNKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLALFQAGL GRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQTELKLTRKAA YVRYFNSSAFFFSGFFVVFLSVLPYALIKGIILRKIFTTISFCIVLRMAVTRQFPWAVQT WYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEETSLLMVIMGELEPSEGK IKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKACQLEEDISKFAEKDNIVL GEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRI LVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDFSSKLMGCDSFDQFSAERRNSI LTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKRKNSILNPINSIRKFSIVQKTPLQM NGIEEDSDEPLERRLSLVPDSEQGEAILPRISVISTGPTLQARRRQSVLNLMTHSVNQGQ NIHRKTTASTRKVSLAPQANLTELDIYSRRLSQETGLEISEEINEEDLKECFFDDMESIP AVTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAASLVVLWLLGNTPLQDKGNSTHSRNN SYAVIITSTSSYYVFYIYVGVADTLLAMGFFRGLPLVHTLITVSKILHHKMLHSVLQAPM STLNTLKAGGILNRFSKDIAILDDLLPLTIFDFIQLLLIVIGAIAVVAVLQPYIFVATVP VIVAFIMLRAYFLQTSQQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHKA LNLHTANWFLYLSTLRWFQMRIEMIFVIFFIAVTFISILTTGEGEGRVGIILTLAMNIMS TLQWAVNSSIDVDSLMRSVSRVFKFIDMPTEGKPTKSTKPYKNGQLSKVMIIENSHVKKD DIWPSGGQMTVKDLTAKYTEGGNAILENISFSISPGQRVGLLGRTGSGKSTLLSAFLRLL NTEGEIQIDGVSWDSITLQQWRKAFGVIPQKVFIFSGTFRKNLDPYEQWSDQEIWKVADE VGLRSVIEQFPGKLDFVLVDGGCVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDPVTY QIIRRTLKQAFADCTVILCEHRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSLFRQAI SPSDRVKLFPHRNSSKCKSKPQIAALKEETEEEVQDTRL Isoform 3 >P13569-3 MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWDRE LASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEERSIA IYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISIGQL VSLLSNNLNKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLALFQAGL GRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQTELKLTRKAA YVRYFNSSAFFFSGFFVVFLSVLPYALIKGIILRKIFTTISFCIVLRMAVTRQFPWAVQT WYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEEGFGELFEKAKQNNNNRK TSNGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTGAGKTSLLMVIMGELEPSEG KIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKACQLEEDISKFAEKDNIV LGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLTEKEIFERRRCSCLLDRNK KTIF Bibliography Andersen, D. (1938). Cystic fibrosis of the pancreas and its relation to celiac disease: a clinical and pathological study. American Journal of Diseases of Children, 56, 344-399.  Andersen, D. and Hodges, R.C. (1946). Celiac syndrome V. Genetics of cystic fibrosis of the pancreas with consideration of the etiology. American Journal of Diseases of Children ,72, 62-80.  Bear, C.E, Li, C, et al. (1992). Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR). Cell, 68, 809-818.  Busch, R. (1990). On the history of cystic fibrosis. Acta Universitatis Carolinae Medica , 36. 1-4, 13-15. Cheng, S.H, Gregory, R.J., et al. (1990). Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell, 63, 827-834. Denning, G.M., Anderson, M.P., et al. (1992). Processing of mutant cystic fibrosis conductance regulator is temperature sensitive. Nature, 358, 761-764. Drumm, M.L., Pope, H.A., et al. (1990). Correction of cystic fibrosis defect by retrovirus-mediated gene transfer. Cell, 62, 1227-1233. Eiberg, H., Mohr, J., et al. (1985). Linkage relationships of paraoxinase (PON) with other markers: evidence of PON-cystic fibrosis synteny? Clinical Genetics, 28, 265-271. Fanconi, G., Uehlinger E., et al. (1936). Das Coeliakie-syndrom bei angeborener zystischer Pankreasfibromatose und Bronchiektasien(Celiac syndrome with congenital cystic fibromatosis of the pancreas and bronchiectasis). Wien Med Wochenschr, 86, 753-756. Farber, S. Shwachman, H., and Maddock, C.L. (1943).Pancreatic function and disease in early life. I. Pancreatic enzyme activity and the celiac syndrome. Journal of Clinical Investigation, 20, 827-833. Gregory, R.J., Cheng, S.H., et al. (1990). Expression and characterisation of the cystic fibrosis transmembrane conductance regulator. Nature, 347, 382-386. Harper, M. (1938). Congenital steatorrhoea due to pancreatic defect. Archives of Disease in Childhood, 13, 45-56 Heng, H.H., Shi, X.M., and Tsui, L.C. (1993). Fluorescence in situ hybridization mapping of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to 7q31.3. Cytogenetics and Cell Genetic, 62, 108-109. Hess J.H., and Saphire O. (1935).Celiac disease: Chronic intestinal indigestion. A report of three cases with autopsy findings. Journal of Pediatrics, 6, 1-13.  Hopfer, U., Will, P.C., et al. (1980). Cystic fibrosis: A secondary endocrine or target tissue sensitivity problem in epithelial electrolyte transport. In Sturgess JM (ed.): Perspectives in Cystic Fibrosis: Proceedings of the Eighth International Congress on CF Toronto. Canadian CF Foundation.  Howard, P. (1944). Familial character of fibrocystic disease of the pancreas. American Journal of Diseases of Children, 68, 330-332. Kartner, N., Augustinas, O., et al. (1992). Mislocalization of DF508 CFTR in cystic fibrosis sweat gland. Nature Genetics (1) , 321-327. Kerem, B., Rommens, J.M., et al. (1989).Identification of the cystic fibrosis gene: genetic analysis. Science, 245, 1073-1080. Knowles, M., Gatzy, J.T., and Boucher, R.C. (1981) increased bioelectric potential difference across respiratory epithelia in cystic fibrosis. New England Journal of Medicine, 305, 1489-1495. Parmalee, A.H. (1935). The pathology of steatorrhoea. American Journal of Diseases of Children, 50, 1418 -1428. Quinton, P. (1983). Chloride impermeability in cystic fibrosis. Nature, 301, 421-422. Rich, D.P., Anderson, M.P., et al. (1990). Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells. Nature, 347, 358-363. Riordan, J., Rommens, J.M., et al. (1989). Identification of the cystic fibrosis gene: cloning and characterization of the complementary DNA. Science, 245, 1066-1073. Rommens, J.M., Iannuzzi, M.C., et al. (1989). Identification of the cystic fibrosis gene: chromosome walking and jumping. Science, 245, 1059-1065.  Sheppard, D.N. and Welsh, M.J. (1999). Structure and Function of the CFTR Chloride Channel. Physiological Review 79 (1 Suppl): S23-45. Tsui, L., Buchwald, M., et al. (1985). Cystic fibrosis locus defined by a genetically linked polymorphic DNA marker. Science, 230, 1054-1057. Wainwright, B., Scambler, P.J., et al. (1985). Localization of cystic fibrosis locus to human chromosome 7cen-q22. Nature, 318, 384-385.  White, R., Woodward, S., et al. (1985). A closely linked genetic marker for cystic fibrosis. Nature, 318 382-384. Zielenski, J., Roznahel, R., Bozon D., et al. (1991). Genomic DNA Sequence of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Genomics 10.1, 214-228. Read More