ABCMdb

PMID: 11823443

Dickinson P, Smith SN, Webb S, Kilanowski FM, Campbell IJ, Taylor MS, Porteous DJ, Willemsen R, de Jonge HR, Farley R, Alton EW, Dorin JR
The severe G480C cystic fibrosis mutation, when replicated in the mouse, demonstrates mistrafficking, normal survival and organ-specific bioelectrics.
Hum Mol Genet. 2002 Feb 1;11(3):243-51., 2002-02-01 [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Gly480Cys 3 243-251 The severe G480C cystic fibrosis mutation, when replicated in the mouse, demonstrates mistrafficking, normal survival and organ-specific bioelectrics Paul Dickinson, Stephen N. Smith1, Sheila Webb, Fiona M. Kilanowski, Isla J. Campbell, Martin S. Taylor, David J. Porteous , Rob Willemsen2, Hugo R. de Jonge3, Ray Farley1, Eric W. F. W. Alton1 and Julia R. Dorin* MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK, 1Department of Gene Therapy, National Heart and Lung Institute at Imperial College, London, UK, 2CBG-Department of Clinical Genetics and 3Department of Biochemistry, Faculty of Medicine and Health Sciences, Erasmus University, PO Box 1738, 3000 DR Rotterdam, The Netherlands Received September 13, 2001; Revised and Accepted November 23, 2001 The majority of cystic fibrosis patients produce a mutant form of CFTR (∆F508) which has been shown to be mislocalized in both humans and mice. Login to comment 3 ABCC7 p.Gly480Cys G480C, another clinically 'severe` mutation, has also been demonstrated to be defective in its intracellular processing, but when allowed to traffic in Xenopus oocytes showed similar channel characteristics to that of wild-type CFTR. Login to comment 4 ABCC7 p.Gly480Cys We have replicated the G480C mutation in the murine Cftr gene using the 'hit and run` double recombination procedure. Login to comment 5 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys As expected, the G480C cystic fibrosis mouse model expresses the G480C mutant transcript at a level comparable to that of wild-type Cftr. Login to comment 7 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys Analysis of the mutant protein revealed that the majority of G480C CFTR was abnormally processed and no G480C CFTR-specific immunostaining in the apical membranes of intestinal cells was detected. Login to comment 9 ABCC7 p.Gly480Cys In contrast to ∆F508 'hit and run` homozygotes, the classic defect of forskolin-induced chloride ion transport is not replicated in the caecum, but the response to low chloride in the nose is clearly defective in the G480C mutant animals. Login to comment 10 ABCC7 p.Gly480Cys The mild phenotype of these G480C mutant animals combined with the defective chloride transport in the nose uniquely provides a valuable resource to test novel pharmacological agents aimed at improving trafficking and correcting the electrophysiological defect in the respiratory tract. Login to comment 16 ABCC7 p.Gly480Cys Other 'severe` mutations have been described and G480C is one where an amino acid substitution occurs in exon 10 of CFTR (3). Login to comment 17 ABCC7 p.Gly480Cys Both G480C and ∆F508 mutations show a primary defect in protein processing and trafficking, such that mutant protein is retained and degraded in the endoplasmic reticulum, resulting in a severe reduction at the plasma membrane (4-6). Login to comment 18 ABCC7 p.Gly480Cys When expressed in Xenopus oocytes (where the transport block can be overcome), the G480C protein has an apical plasma membrane Cl-channel activity identical to that of wild-type CFTR (6). Login to comment 20 ABCC7 p.Gly480Cys We created mutant mice that carry the G480C mutation by gene targeting using the 'hit and run` technique (10,11). Login to comment 26 ABCC7 p.Gly551Asp In such mutant mice (e.g. the ∆F508 Cftrtm2Cam and the G551D Cftrtm1G551D mice), it is therefore difficult to assess whether the phenotype is due to the precise mutation or principally related to a reduction in the level of the transcript. Login to comment 30 ABCC7 p.Gly480Cys In this study, an accurate mouse model of the G480C mutation was used to assess the phenotype of another 'severe` CF mutation in vivo and to clarify the organ-specific consequences of a mistrafficking mutant. Login to comment 31 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys RESULTS Generation of Cftrtm2Hgu mice which carry the G480C mutation ES cells modified at the Cftr locus to possess the G480C mutation in exon 10 by 'hit and run` gene targeting have previously been described by Dickinson et al. (11). Login to comment 32 ABCC7 p.Gly480Cys Four ES cell clones modified to possess the G480C mutation which had normal karyotypes were used for blastocyst injections. Login to comment 34 ABCC7 p.Gly480Cys Homozygous G480C mutant mice are designated Cftrtm2Hgu following the Mouse Nomenclature Committee guidelines. Login to comment 35 ABCC7 p.Gly480Cys A novel NsiI restriction enzyme site was created at the site of the G480C mutation (Fig. 1A) and restriction enzyme digestion verified the faithful replacement of the wild-type exon 10 with the mutant exon 10 (Fig. 1B-D). Login to comment 37 ABCC7 p.Gly480Cys Generation of G480C Cftr mutant mice by 'hit and run` gene targeting. Login to comment 46 ABCC7 p.Gly480Cys Wild-type Cftr genomic structures are shown for strains 129, in which gene targeting was performed, C57 Bl/6, which was used for subsequent breeding, and 129/G480C 'hit and run` gene targeted locus. Login to comment 48 ABCC7 p.Gly480Cys (C) Germline transmission of the Cftr G480C allele. Login to comment 50 ABCC7 p.Gly480Cys B, C57 Bl/6 offspring; C, CGR8 parental ES cells; E, G480C targeted ES cells used for chimera injection; H, heterozgous Cftrtm2Hgu/+ offspring; 1/B, 129/C57 Bl/6 offspring; M, λHindIII molecular weight marker. Login to comment 53 ABCC7 p.Gly480Cys (E) PCR genotyping of G480C mice. Heterozygote Cftrtm2Hgu/+ mice were intercrossed and litters genotyped by PCR and subsequent NsiI digestion. Login to comment 55 ABCC7 p.Gly480Cys wild-type band from the G480C-containing band, which can be digested with the enzyme (Fig. 1E). Login to comment 56 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys G480C mutant mice express the mutant allele at wild-type levels Both male and female mice heterozygous for the G480C allele were included in the study and a range of tissues investigated. Login to comment 60 ABCC7 p.Gly480Cys G480C products were distinguished from wild-type by the presence of the novel NsiI restriction site polymorphism engineered adjacent to the missense change. Login to comment 62 ABCC7 p.Gly480Cys These results indicate that the modified allele was expressed at the same level as the wild-type allele and therefore the presence of the G480C mutation has no effect on the level of expression. Login to comment 63 ABCC7 p.Gly480Cys G480C CFTR is incompletely processed Analysis of CFTR processing in isolated jejunal enterocytes of wild-type mice by western blotting demonstrated a normal pattern of CFTR isoforms with the core-glycosylated isoform of CFTR (Fig. 3, band B) in the ER, and the mature, fully-glycosylated isoform (Fig. 3, band C) in the plasma membrane. Login to comment 65 ABCC7 p.Gly480Cys In contrast, intestinal epithelium from homozygous G480C mice showed a normal intensity of the B band of CFTR in the crude jejunal membrane fraction (Fig. 3, lane 2), but a strongly reduced intensity of the C band [measured as 8% (±2), n = 4, residual CFTR compared to wild-type as determined by dilution-calibrated scanning of the bioluminescence] in the BBMV (Fig. 3, lanes 2 and 4). Login to comment 66 ABCC7 p.Gly480Cys This outcome clearly demonstrates that the G480C CFTR mutant protein is retained in the ER of the enterocytes in vivo and that only a very small fraction is able to escape the quality control mechanism in the ER and reach the cell surface. Login to comment 69 ABCC7 p.Gly480Cys Expression analysis of the Cftr G480C allele. Login to comment 70 ABCC7 p.Gly480Cys Real-time quantitative RT-PCR expression analysis of the G480C allele was performed. Login to comment 75 ABCC7 p.Gly480Cys Quantification of expression of G480C allele Results from ileum, jejunum and testis show no evidence of an allele bias, only small and inconsistent fluctuations around a 1:1 ratio. Login to comment 77 ABCC7 p.Gly480Cys Tissue Mouse Wild-type (%) G480C (%) Ileum 1 (rp1) 47.13 52.68 1 (rp2) 44.55 55.45 Jejunum 1 47.56 52.44 2 52.13 47.17 Lung 1 20.20 79.80 2 55.20 44.80 Testis 3 (rp1) 56.77 43.23 3 (rp2) 56.65 43.35 Figure 3. Login to comment 78 ABCC7 p.Gly480Cys Western blot analysis of CFTR processing in wild-type and G480C Cftr mice. Login to comment 79 ABCC7 p.Gly480Cys Abnormal processing of G480C CFTR in mouse jejunum. Login to comment 80 ABCC7 p.Gly480Cys Crude epithelial membranes (lanes 1 and 2) and BBMV (lanes 3 and 4) isolated from wild-type (CFTR+/+; lanes 1 and 3) or homozygous G480C mutant mice (lanes 2 and 4) were subjected to western blot analysis as described in Materials and Methods. Login to comment 86 ABCC7 p.Gly480Cys In contrast, CFTR expression in crypts and villi from homozygous G480C mutant mice remained below the detection level of the immunocytochemical technique (Fig. 4B). Login to comment 87 ABCC7 p.Gly480Cys This finding confirms the results of the western blotting shown in Figure 3 and is in line with the concept of a processing defect affecting the maturation of G480C CFTR in both the crypt and villus compartments. Login to comment 88 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys Phenotype of the CF mutant mice homozygous for the G480C mutation Figure 5 demonstrates that genotypes of the litters produced from matings between G480C heterozygous mice did not deviate from the expected Mendelian ratio of wild-type:heterozygotes:homozygotes of 1:2:1, and no reduction in the number of homozygotes was observed. Login to comment 90 ABCC7 p.Gly480Cys Furthermore, homozygous G480C mice did not show any increased mortality over wild-type animals (pre-or post-weaning) over an 18 month period. Login to comment 92 ABCC7 p.Gly480Cys Histological analysis of intestinal sections demonstrated focal hypertrophy of goblet cells in the G480C homozygous mutant mice. Login to comment 93 ABCC7 p.Gly480Cys Sections from the G480C mutants could easily be distinguished from wild-type by this criterion alone (Fig. 4). Login to comment 98 ABCC7 p.Gly480Cys However, the incisor teeth of the G480C mutant mice were not abnormally white (data not shown). Login to comment 99 ABCC7 p.Gly480Cys Electrophysiological characteristics of G480C CFTR mice The reduced chloride permeability of the epithelium due to CFTR dysfunction, causes typical abnormalities in the ion transport of different epithelia in both CF individuals and Cftr Figure 4. Login to comment 100 ABCC7 p.Gly480Cys Immunohistological analysis of G480C Cftr expression. Login to comment 101 ABCC7 p.Gly480Cys Immunocytochemical staining of CFTR in the jejunum from wild-type mice (A) and homozygous G480C mutant mice (B). Login to comment 103 ABCC7 p.Gly480Cys Crypts and the lower and mid-portion of the villi show intense staining of the apical border of the epithelial cells in wild-type, but not in G480C mouse intestine. Login to comment 105 ABCC7 p.Gly480Cys Similar differences in the CFTR staining pattern were found in four couples of wild-type and G480C mutant mice. Login to comment 108 ABCC7 p.Gly480Cys The G480C CF mice do not suffer from the intestinal blockage (the first signs of which are located in the caecum) that is seen in mice with a complete disruption of Cftr expression, so we examined the electrophysiological profile of these animals in the intestine. Login to comment 109 ABCC7 p.Gly480Cys In the caecum, Ussing chamber measurements revealed that the initial baseline Isc (short circuit current) was significantly (P = 0.0001) reduced in the G480C homozygous mutants compared to controls (Fig. 6A). Login to comment 122 ABCC7 p.Gly480Cys The response to a low chloride gradient was significantly (P = 0.0001) reduced in the G480C mutant animals, in contrast to the normal response reported in the Cftrtm1Eur ∆F508 mouse. Login to comment 123 ABCC7 p.Gly480Cys DISCUSSION The mutant mice we present here mimic human CF individuals with the 'severe` G480C mutation. Login to comment 124 ABCC7 p.Gly480Cys The G480C mutant protein was detected in a pancreatic insufficient African-American CF patient (6). Login to comment 126 ABCC7 p.Gly480Cys This suggested that the G480C protein was similar to the ∆F508 protein and subject to defective intracellular Figure 5. Login to comment 127 ABCC7 p.Gly480Cys G480C mice show good survival and no weight reduction. Login to comment 133 ABCC7 p.Gly480Cys Bioelectric characteristics of (A) caecum, (B) jejunum and (C) nose of wild-type (black bars) and G480C Cftrtm2Hgu homozygous mutant mice (white bars). Login to comment 136 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys Numbers of animals used, jejunum and caecum baselines and forskolin responses: G480C 12, littermate controls 11, carbachol responses; G480C 6, littermate controls 7; nose baseline: G480C 13, controls 45, nose low chloride: G480C 16, control 38. processing. Login to comment 137 ABCC7 p.Gly480Cys We demonstrate that when replicated in the mouse, the G480C mutant CFTR is mislocalized and the defect in chloride ion transport characteristic of CF varies between tissues and is present in the nose and jejunum but absent from the caecum with no evidence of fatal gut blockage. Login to comment 138 ABCC7 p.Gly480Cys G480C mutant mice express normal levels of the mutant allele The 'hit and run` procedure used to generate these mice results in the only genomic alteration being at the site of the mutation. Login to comment 147 ABCC7 p.Gly480Cys In contrast to the ∆F508 mice generated by replacement gene targeting, the Cftrtm1Eu ∆F508 'hit and run` mice and the Cftrtm2Hgu G480C 'hit and run` mice generated here both express normal levels of the mutant allele. Login to comment 148 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys G480C mutant protein is mislocalized The majority of G480C CFTR when subjected to western blot analysis is clearly mislocalized in vivo in the mouse intestine. Login to comment 150 ABCC7 p.Gly480Cys This outcome clearly suggests that the majority of G480C CFTR mutant protein is retained in the ER of the enterocytes in vivo but that a significant fraction is able to escape the quality control mechanism in the ER and travel to the cell surface. Login to comment 152 ABCC7 p.Gly480Cys This suggests that the G480C processing defect in the intestine is slightly less severe than that of the ∆F508 mutant in vivo. Login to comment 153 ABCC7 p.Gly480Cys It should be noted that although 8% of normal levels of mature G480C was detectable in western blot analysis of BBMV, only cytoplasmically localized protein could be detected by immunohistochemistry. Login to comment 154 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys The phenotype of the Cftrtm2Hgu G480C mutant mice is mild The Cftrtm2Hgu G480C mutant mice do not demonstrate a phenotype of death from gut blockage and unlike the ∆F508 Cftrtm1Eur mice do not even display any evidence of growth retardation at weaning. Login to comment 155 ABCC7 p.Gly480Cys The histology of the G480C intestine is not severely abnormal unlike the Cftrtm1Unc 'null` mice, which display extensive goblet cell hyperproliferation, increased mucus accumulation and luminal obstruction. Login to comment 157 ABCC7 p.Gly480Cys The G480C mice (Fig. 4), also do not have any gross abnormalities but do display a mild focal hypertrophy of goblet cells comparable to the data reported for the ∆F508 Cftrtm1Eur homozygotes. Login to comment 158 ABCC7 p.Gly480Cys The classic CF chloride transport defect is not present in the caecum and may account for the lack of intestinal blockage The G480C mutant mice do not show a defect in their forskolin response in the caecum, although baseline and carbachol response are altered compared to wild-type. Login to comment 159 ABCC7 p.Gly480Cys The ∆F508 'hit and run` mutant mice, in contrast to the G480C mice, have a significant but markedly reduced (by 85%) forskolin-activated chloride ion conductance in the caecum compared to wild-type (13). Login to comment 162 ABCC7 p.Gly480Cys Variation between the bioelectric phenotypes of ∆F508 and G480C 'hit and run` mutant mice could be explained by the effect of modifier genes of residual chloride secretion present in the genetic backgrounds on which the mutations have been bred. Login to comment 165 ABCC7 p.Gly480Cys Finally, the G480C mice show 100% survival on a mixed 129/C57Bl/6J background (the same as that reported for the Cftrtm1Eur mice with 100% survival) and this does not alter after four backcrosses onto the C57Bl/6J background. Login to comment 167 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys It is probable that the normal forskolin response in the G480C mice compared to the abnormal response in Cftrtm1Eur ∆F508 mice is due to slightly more G480C (8% versus 3%) being correctly processed and reaching the apical membrane. Login to comment 168 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys However, the results from Xenopus oocyte experiments using human CFTR mRNA, strongly suggested that this observed difference between the G480C and ∆F508 response to forskolin was consistent with a trafficking/processing defect in G480C CFTR, and an additional conductance defect in ∆F508 CFTR (6). Login to comment 171 ABCC7 p.Gly480Cys One possible explanation is that the G480C and ∆F508 mouse phenotypes appear to be different to 'null` mice because of differences in human/mouse physiology and gut architecture, and the mouse is more sensitive to small increases in CFTR function. Login to comment 172 ABCC7 p.Gly480Cys Electrophysiological phenotype of the murine G480C mutant protein varies between tissues An unexpected finding was the organ-specific differences in CFTR-related electrophysiology in the mutant mice. Login to comment 181 ABCC7 p.Gly480Cys This must reflect either tissue-specific alterations in the level of mature G480C CFTR with organ-specific subtle translational/post-translational differences, or compensatory pathways altering the bioelectric phenotype. Login to comment 183 ABCC7 p.Gly480Cys ABCC7 p.Gly480Cys The Cftrtm2Hgu G480C mutant mouse is a valuable tool for therapy testing Both the defects in sodium absorption and in chloride secretion are evident in the nose of the G480C mutant mouse and this is widely held to be the mouse tissue that mimics the human respiratory tract phenotype most closely (25). Login to comment 187 ABCC7 p.Gly480Cys The fact that this G480C mutant mouse combines a mistrafficked CFTR mutation (similar to the ∆F508 CFTR) with normal survival means that it is an excellent in vivo model for testing drugs aimed at mutant CFTR relocation strategies. Login to comment 188 ABCC7 p.Gly480Cys In conclusion, the introduction of the G480C mutation into the mouse Cftr gene, using the 'hit and run` technique mimics the human allele with normal levels of Cftr mRNA production. Login to comment 189 ABCC7 p.Gly480Cys This has allowed us to demonstrate that the majority of the G480C mutant protein is mislocalized, but a low level of mature CFTR is detectable by immunoblot. Login to comment 191 ABCC7 p.Gly480Cys The G480C homozygous mutant protein has different ion transport effects in different organs with pronounced effects on the baseline in the nose and the caecum. Login to comment 193 ABCC7 p.Gly480Cys Reduced stimulation of chloride secretion has been found in the nose and the jejunum but a normal response was found in the caecum and this is most likely responsible for the lack of fatal intestinal blockage and the normal weight of the G480C mutant mice. Login to comment 195 ABCC7 p.Gly480Cys Screening of litters for transmission of the G480C allele was performed by PCR using 25 base pair primers used to amplify exon 10 from positions 1530 to 1720 in the Cftr gene, described previously by Dickinson et al. (26), followed by NsiI digestion. Login to comment 196 ABCC7 p.Gly480Cys Real-time RT-PCR analysis Cftrtm2Hgu/+ heterozygous G480C mice were killed by CO2 asphyxiation. Login to comment 218 ABCC7 p.Gly480Cys Western blot analysis Wild-type mice and littermate mice (backcrossed for four generations onto the C57Bl/6 strain background) carrying the G480C mutation were anaesthesized with a hypnorm/diazepam mixture. Login to comment 233 ABCC7 p.Gly480Cys Immunocytochemical analysis Wild-type mice and littermate mice carrying the G480C mutation were killed by cervical dislocation, the intestine was dissected and the jejunum was rinsed with ice-cold saline and fixed in 3% (w/v) paraformaldehyde for 16 h, prior to standard paraffin embedding. Login to comment 241 ABCC7 p.Gly480Cys Electrophysiological analysis G480C homozygous animals were assessed in vivo (nose) and in vitro (jejunum, caecum) and compared with littermate controls. Login to comment