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The ?F508 mutation causes CFTR misprocessing and cystic fibrosis-like disease in pigs.

ABSTRACT: Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. The most common CF-associated mutation is ?F508, which deletes a phenylalanine in position 508. In vitro studies indicate that the resultant protein, CFTR-?F508, is misprocessed, although the in vivo consequences of this mutation remain uncertain. To better understand the effects of the ?F508 mutation in vivo, we produced CFTR(?F508/?F508) pigs. Our biochemical, immunocytochemical, and electrophysiological data on CFTR-?F508 in newborn pigs paralleled in vitro predictions. They also indicated that CFTR(?F508/?F508) airway epithelia retain a small residual CFTR conductance, with maximal stimulation producing ~6% of wild-type function. Cyclic adenosine 3',5'-monophosphate (cAMP) agonists were less potent at stimulating current in CFTR(?)(F508/)(?)(F508) epithelia, suggesting that quantitative tests of maximal anion current may overestimate transport under physiological conditions. Despite residual CFTR function, four older CFTR(?F508/?F508) pigs developed lung disease similar to human CF. These results suggest that this limited CFTR activity is insufficient to prevent lung or gastrointestinal disease in CF pigs. These data also suggest that studies of recombinant CFTR-?F508 misprocessing predict in vivo behavior, which validates its use in biochemical and drug discovery experiments. These findings help elucidate the molecular pathogenesis of the common CF mutation and will guide strategies for developing new therapeutics.

SUBMITTER: Ostedgaard LS

PROVIDER: S-EPMC3119077 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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The ΔF508 mutation causes CFTR misprocessing and cystic fibrosis-like disease in pigs.

Ostedgaard Lynda S LS Meyerholz David K DK Chen Jeng-Haur JH Pezzulo Alejandro A AA Karp Philip H PH Rokhlina Tatiana T Ernst Sarah E SE Hanfland Robert A RA Reznikov Leah R LR Ludwig Paula S PS Rogan Mark P MP Davis Greg J GJ Dohrn Cassie L CL Wohlford-Lenane Christine C Taft Peter J PJ Rector Michael V MV Hornick Emma E Nassar Boulos S BS Samuel Melissa M Zhang Yuping Y Richter Sandra S SS Uc Aliye A Shilyansky Joel J Prather Randall S RS McCray Paul B PB Zabner Joseph J Welsh Michael J MJ Stoltz David A DA

Science translational medicine 20110301 74

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. The most common CF-associated mutation is ΔF508, which deletes a phenylalanine in position 508. In vitro studies indicate that the resultant protein, CFTR-ΔF508, is misprocessed, although the in vivo consequences of this mutation remain uncertain. To better understand the effects of the ΔF508 mutation in vivo, we produced CF ...[more]

PMID: 21411740

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Project description:The distribution and frequency of the CFTR gene mutations vary considerably between countries and ethnic groups. Russians are an East Slavic ethnic groups are native to Eastern Europe. Russians, the most numerous people of the Russian Federation (RF), make about 80% of the population. The aim is to reveal the molecular causes of CF in ethnic Russian patients as comprehensively as possible. The analysis of most common CFTR mutations utilized for CF diagnosis in multiethnic RF population accounts for about 83% of all CF-causing mutations in 1384 ethnic Russian patients. Variants c.1521_1523delCTT (F508del), c.54-5940_273+10250del21kb (CFTRdele2,3), c.2012delT (2143delT), c.2052_2053insA (2184insA), and c.3691delT (3821delT) are most typical for CF patients of Russian origin. DNA of 154 CF patients, Russian by origin, in whom at least one mutant allele was not previously identified (164 CF alleles), was analyzed by Sanger sequencing followed by the multiplex ligase-dependent probe amplification (MLPA) method. In addition to the 29 variants identified during the previous test for common mutations, 91 pathogenic CFTR variants were also revealed: 29 missense, 19 nonsense, 14 frame shift in/del, 17 splicing, 1 in frame ins, and 11 copy number variations (CNV). Each of the 61 variants was revealed once, and 17 twice. Each of the variants c.1209G>C (E403D), c.2128A>T (K710X), c.3883delA (4015delA), and c.3884_3885insT (4016insT) were detected for three, c.1766+1G>A (1898+1G>A) and c.2834C>T (S945L) for four, c.1766+1G>C (1898+1G>C) and c.(743+1_744-1)_(1584+1_1585-1)dup (CFTRdup6b-10) for five, c.2353C>T (R785X) and c.4004T>C (L1335P) for six, c.3929G>A (W1310X) for seven, c.580-1G>T (712-1G>T for eight, and c.1240_1244delCAAAA (1365del5) for 11 unrelated patients. A comprehensive analysis of CFTR mutant alleles with sequencing followed by MLPA, allowed not only the identification of 163 of 164 unknown alleles in our patient sample, but also expansion of the mutation spectrum with novel and additional frequent variants for ethnic Russians.

Project description:Cystic fibrosis (CF) is the most common genetic disease among Caucasians, and accordingly the cystic fibrosis transmembrane conductance regulator (CFTR) protein has perhaps the best characterized disease mutation spectrum with more than 1,500 causative mutations having been identified. In this study, we took advantage of that wealth of mutational information in an effort to relate site-specific evolutionary parameters with the propensity and severity of CFTR disease-causing mutations. To do this, we devised a scoring scheme for known CFTR disease-causing mutations based on the Grantham amino acid chemical difference matrix. CFTR site-specific evolutionary constraint values were then computed for seven different evolutionary metrics across a range of increasing evolutionary depths. The CFTR mutational scores and the various site-specific evolutionary constraint values were compared in order to evaluate which evolutionary measures best reflect the disease-causing mutation spectrum. Site-specific evolutionary constraint values from the widely used comparative method PolyPhen2 show the best correlation with the CFTR mutation score spectrum, whereas more straightforward conservation based measures (ConSurf and ScoreCons) show the greatest ability to predict individual CFTR disease-causing mutations. While far greater than could be expected by chance alone, the fraction of the variability in mutation scores explained by the PolyPhen2 metric (3.6%), along with the best set of paired sensitivity (58%) and specificity (60%) values for the prediction of disease-causing residues, were marginal. These data indicate that evolutionary constraint levels are informative but far from determinant with respect to disease-causing mutations in CFTR. Nevertheless, this work shows that, when combined with additional lines of evidence, information on site-specific evolutionary conservation can and should be used to guide site-directed mutagenesis experiments by more narrowly defining the set of target residues, resulting in a potential savings of both time and money.

Dataset Information

The ?F508 mutation causes CFTR misprocessing and cystic fibrosis-like disease in pigs.

Publications

The ΔF508 mutation causes CFTR misprocessing and cystic fibrosis-like disease in pigs.

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