ABCMdb

PMID: 24556927

Molinski SV, Gonska T, Huan LJ, Baskin B, Janahi IA, Ray PN, Bear CE
Genetic, cell biological, and clinical interrogation of the CFTR mutation c.3700 A>G (p.Ile1234Val) informs strategies for future medical intervention.
Genet Med. 2014 Aug;16(8):625-32. doi: 10.1038/gim.2014.4. Epub 2014 Feb 20., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp VX-770 (or Ivacaftor) has been approved in North America and Europe as a drug for patients bearing the relatively rare mutation: p.Gly551Asp.7,8 The p.Gly551Asp mutation causes defective channel gating,9-11 and VX-770 is thought to be effective in partially restoring lung function in patients with p.Gly551Asp because it enhances the channel activity of this mutant.12-15 The most common mutation in Europe and North America, p.Phe508del, has been studied extensively and has been found to impair CFTR protein folding during synthesis.16-19 Knowledge regarding the molecular defects caused by p.Phe508del has driven the development of targeted, interventional compounds, such as VX-809 (or Lumacaftor). Login to comment 5 ABCC7 p.Ile1234Val Submitted 18 October 2013; accepted 6 January 2014; advance online publication 20 February 2014. doi:10.1038/gim.2014.4 Purpose: The purpose of this study was to determine the molecular consequences of the variant c.3700 A>G in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, a variant that has been predicted to cause a missense mutation in the CFTR protein (p.Ile1234Val). Login to comment 8 ABCC7 p.Ile1234Val CFTR complementary DNA clones containing the missense mutation p.Ile1234Val or a truncated exon 19 (p.Ile1234_ Arg1239del) were constructed and heterologously expressed to test CFTR protein synthesis and processing. Login to comment 15 ABCC7 p.Ile1234Val ABCC7 p.Ile1234Val Correspondence: Christine E. Bear (bear@sickkids.ca) Genetic, cell biological, and clinical interrogation of the CFTR mutation c.3700 A>G (p.Ile1234Val) informs strategies for future medical intervention Steven V. Molinski, MSc1,2 , Tanja Gonska, MD3,4 , Ling Jun Huan, BSc1 , Berivan Baskin, PhD5 , Ibrahim A. Janahi, MD6 , Peter N. Ray, PhD7,8 and Christine E. Bear, PhD1,2,9 A recent, large-scale study showed that individuals in North America and Europe bearing the rare variant c.3700 A>G, predicted to cause the missense mutation p.Ile1234Val or alternativesplicing,exhibitedvariableCFdiseaseseverity.6 Thisvariant, although rare in North America (present in only 15 patients, http://cftr2.org), is relatively common in the Middle East. Login to comment 17 ABCC7 p.Ile1234Val who.int/genomics/publications) report p.Ile1234Val as the second most common CF-causing mutation in the Middle East (12.3% occurrence in patients from Jordan, Kuwait, Lebanon, Oman, Qatar, Saudi Arabia, and United Arab Emirates), with the exception of two countries, Bahrain and Israel, in which the occurrence is less than 3.8 and 0.06%, respectively. Login to comment 18 ABCC7 p.Trp1282* The mutation 1548delG is most common in these seven countries (17.2%),whereas2043delGismostcommoninBahrain(30.8%) and W1282X is most common in Israel (36.1%). Login to comment 19 ABCC7 p.Ile1234Val ABCC7 p.Ile1234Val Furthermore, the c.3700 A>G (p.Ile1234Val) mutation is specific to Middle Eastern individuals originating from Bedouin tribes, and although diagnostic tests are sensitive enough to identify this mutation at an early age, currently there is no effective treatment for patients with this CF-causing genotype.23 Interestingly, Sosnay et al.6 recently showed that there were no functional consequences of introducing the missense mutation (p.Ile1234Val) in CFTR complementary DNA (cDNA) with respect to CFTR protein synthesis, processing, and/or function. Login to comment 37 ABCC7 p.Ile1234Val Generation of mutant CFTR constructs p.Ile1234Val-CFTR was generated in human wild-type (WT)- CFTR cDNA (pcDNA3.1) by Norclone Biotech Laboratories (London, ON, Canada). Login to comment 41 ABCC7 p.Ile1234Val Studies of CFTR protein processing and function Human embryonic kidney cells were transiently transfected with WT-CFTR or p.Ile1234Val-CFTR using PolyFect Transfection Reagent, according to the manufacturer`s protocol (Qiagen, Venlo, the Netherlands). Login to comment 45 ABCC7 p.Ile1234Val Human embryonic kidney cells expressing WT-CFTR or p.Ile1234Val-CFTR and BHK cells expressing WT-CFTR, p.Phe508del-CFTR, or p.Ile1234_Arg1239del-CFTR were grown at 37 &#b0;C for 24ߙ h and subsequently lysed in modified radioimmunoprecipitation assay buffer (50 mmol/l Tris-HCl, 150 mmol/l NaCl, 1 mmol/l ethylenediaminetetraacetic acid (pH 7.4), 0.2% (v/v) sodium dodecyl sulfate, and 0.1% (v/v) Triton X-100) containing a protease inhibitor cocktail (Roche, Indianapolis, IN) for 10ߙ min, and the soluble fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 6% gels. Login to comment 53 ABCC7 p.Ile1234Val To test function, human embryonic kidney cells overexpressing WT-CFTR or p.Ile1234Val-CFTR were grown in 12-well plates, and on formation of a monolayer, the cells were incubated overnight with 10 mmol/l of the halide-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ; Invitrogen Molecular Probes, Carlsbad, CA), at 37 &#b0;C and 5% CO2 . Login to comment 68 ABCC7 p.Ile1234Val RESULTS Siblings in a Qatari family homozygous for the CFTR variant c.3700 A>G exhibit clinical features of CF and lack of CFTR function in in vivo measurements A 27-year-old man and his 15-year-old sister, both from Qatar and homozygous for the mutation c.3700 A>G (p.Ile1234Val), presentedforfurtherCFdiagnosticevaluation.Theman,patient 1, was diagnosed at 4 months of age and had experienced recurrent lung infection over the years. Login to comment 86 ABCC7 p.Ile1234Val ABCC7 p.Ile1234Val Determining the consequences of p.Ile1234Val on CFTR folding, processing, and function The CFTR variant c.3700 A>G has been predicted to create a missense mutation (p.Ile1234Val). Login to comment 95 ABCC7 p.Ile1234Val Figure 1ߒ Qatari siblings with cystic fibrosis transmembrane conductance regulator (CFTR) variant c.3700 A>G (predicted to cause the CFTR missense mutation: p.Ile1234Val) exhibit loss of CFTR function in nasal potential difference (NPD) and sweat secretion assays. Login to comment 114 ABCC7 p.Ile1234Val This clinical phenotype is incompatible with the prediction that this variant caused the missense mutation, p.Ile1234Val. Login to comment 115 ABCC7 p.Ile1234Val Cell-based studies of the predicted missense mutation introduced into CFTR cDNA showed that the predicted missense mutation, p.Ile1234Val, caused no apparent defects in protein folding, processing, or function.Thesefindingspromptedadetailedanalysisoftheentire CFTRgeneandCFTRmRNAobtainedfromthenasalepithelium of a homozygous patient with the detection of aberrant splicing. Login to comment 120 ABCC7 p.Ile1234Val The effect of p.Ile1234_Arg1239del on protein folding and processing is consistent with bioinformatics predictions Examination of homology models and bioinformatics analyses predict that p.Ile1234_Arg1239del will cause significant Figure 2ߒ In vitro characterization of p.Ile1234Val missense mutation introduced into cystic fibrosis transmembrane conductance regulator (CFTR) complementary DNA and expressed in heterologous expression system. Login to comment 121 ABCC7 p.Ile1234Val ABCC7 p.Ile1234Val (a) Immunoblots show the processing of wild-type (WT)-CFTR and p.Ile1234Val-CFTR in human embryonic kidney (HEK) cells after 24ߙh at 37 &#b0;C. WT-CFTR and p.Ile1234Val-CFTR were expressed as both the Golgi-modified, complex glycosylated (mature) band C form (broad 170-kDa band) as well as the endoplasmic reticulum-modified, core glycosylated (immature), band B form (sharp 150-kDa band). Login to comment 122 ABCC7 p.Ile1234Val Maturation (expressed as percentage of band C/ (band B + band C)) of WT-CFTR and p.Ile1234Val-CFTR was quantified for three independent trials, and there was no significant difference between the two (P > 0.05). Login to comment 123 ABCC7 p.Ile1234Val (b) Fluorescence-based anion flux assay in HEK cells show WT-CFTR and p.Ile1234Val-CFTR function after stimulation using a cAMP agonist (gray bar, forskolin, 10 bc;mol/l) or vehicle (dimethyl sulfoxide) alone (empty bar). Login to comment 124 ABCC7 p.Ile1234Val Flux responses in the first 4ߙmin after cAMP stimulation (reported as an initial rate of change in relative fluorescence units (RFU)) for WT-CFTR and p.Ile1234Val-CFTR were quantified for three independent trials (four technical replicates each trial), and there was no significant difference between the two (P > 0.05). Login to comment 131 ABCC7 p.Ser1235Arg ABCC7 p.Gly1237Ser ABCC7 p.Gln1238Arg ABCC7 p.Arg1239Ser Interestingly, the CF Mutation Database5 contains several disease-associated mutations within the p.Ile1234_Arg1239 sequence and includes c.3705T>G (p.Ser1235Arg), c.3709G>A (p.Gly1237Ser), c.3713A>G (p.Gln1238Arg), c.3712C>T (p.Gln1238X), and c.3717G>C (p.Arg1239Ser). Login to comment 132 ABCC7 p.Gly1237Ser These mutations were each found in one or two individuals of European descent (i.e., Belgian, Spanish, French, and English), and the clinical presentation varied from a mild phenotype, in which the mutation was detected at 40 years of age (p.Gly1237Ser), with pancreatic sufficiency and forced expiratory volume in 1 s >70%, to a more severe CF phenotype (p.Gln1238X, in trans with p.Phe508del) that was diagnosed at birth and manifested with pancreatic insufficiency. Login to comment