ABCMdb

PMID: 26089335

Kondo S, Fujiki K, Ko SB, Yamamoto A, Nakakuki M, Ito Y, Shcheynikov N, Kitagawa M, Naruse S, Ishiguro H
Functional characteristics of L1156F-CFTR associated with alcoholic chronic pancreatitis in Japanese.
Am J Physiol Gastrointest Liver Physiol. 2015 Aug 15;309(4):G260-9. doi: 10.1152/ajpgi.00015.2014. Epub 2015 Jun 18., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Leu1156Phe In the present study, we have investigated the association of CFTR variants and chronic pancreatitis in Japanese and the functional characteristics of a Japanese- and pancreatitis-specific CFTR variant, L1156F. Login to comment 8 ABCC7 p.Leu1156Phe Human embryonic kidney-293 cells were transfected with three CFTR variants (M470V, L1156F, and M470Vaf9;L1156F), and the protein expression was examined. Login to comment 11 ABCC7 p.Glu217Gly ABCC7 p.Arg1453Trp ABCC7 p.Ile556Val ABCC7 p.Gln1352His ABCC7 p.Leu1156Phe Six variants (E217G, I556V, M470V, L1156F, Q1352H, and R1453W) were identified in the coding region of the CFTR gene. Login to comment 13 ABCC7 p.Gln1352His ABCC7 p.Leu1156Phe The allele frequencies of L1156F and Q1352H in alcoholic chronic pancreatitis (5.0 and 7.9%) were significantly (P b0d; 0.01) higher than those in normal subjects (0.6 and 1.9%). Login to comment 14 ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe L1156F was linked with a worldwide CFTR variant, M470V. Combination of M470V and L1156F significantly reduced CFTR expression to b03;60%, impaired CFTR-mediated HCO3 afa; /Clafa; transport activity to 50-60%, and impaired CFTR-coupled Clafa; /HCO3 afa; exchange activity to 20-30%. Login to comment 15 ABCC7 p.Leu1156Phe The data suggest that the Japanese-specific CFTR variant L1156F causes mild dysfunction of CFTR and increases the risk of alcoholic chronic pancreatitis in Japanese. Login to comment 16 ABCC7 p.Leu1156Phe CFTR gene; L1156F; alcoholic chronic pancreatitis; Japanese CYSTIC FIBROSIS TRANSMEMBRANE conductance regulator (CFTR) is a cAMP-regulated anion channel that is expressed in the apical membrane of epithelial cells (18). Login to comment 38 ABCC7 p.Arg1453Trp ABCC7 p.Arg1453Trp ABCC7 p.Gln1352His Our laboratory`s previous study (13) indicated the association of two types of CFTR variants, c.4056Gb0e;C, p.Arg1352His (Q1352H) and c.4357Cb0e;T, p.Arg1453Trp (R1453W), and chronic pancreatitis in Japanese. Login to comment 39 ABCC7 p.Arg1453Trp ABCC7 p.Gln1352His Defects of protein expression and ion transport of Q1352H-CFTR were confirmed by heterologous expression system, while R1453W-CFTR showed mild reduction of open probability (27). Login to comment 40 ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe In the present study, we have shown that a Japanese-specific CFTR variant, c.3468Gb0e;T, p.Leu1156Phe (L1156F), is related to alcoholic chronic pancreatitis in Japanese. Login to comment 41 ABCC7 p.Leu1156Phe The risk of the L1156F carrier for developing chronic pancreatitis was 9.0 times higher compared with that of the noncarrier. Login to comment 42 ABCC7 p.Leu1156Phe Since L1156F is linked to c.1408Ab0e;G, p.Met470Val (M470V), a world-wide CFTR variant, we have examined the protein expression and functional characteristics of M470Vaf9;L1156F-CFTR using heterologous expression systems. Login to comment 43 ABCC7 p.Leu1156Phe We have found that combination of M470V and L1156F partially impaired expression and channel function of CFTR and substantially impaired the CFTR-coupled Clafa; /HCO3 afa; exchange activity. Login to comment 57 ABCC7 p.Glu217Gly ABCC7 p.Glu217Gly ABCC7 p.Arg1453Trp ABCC7 p.Ile556Val ABCC7 p.Ile556Val ABCC7 p.Gln1352His ABCC7 p.Leu1156Phe Six CFTR variants, c.650Ab0e;G, p.Glu217Gly (E217G); c.1666Ab0e;G, p.Ile556Val (I556V); M470V; L1156F; Q1352H; and R1453W, were detected. Login to comment 75 ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe CFTR mutations (M470V, L1156F, and both of M470V and L1156F) were introduced by site-directed mutagenesis (QuickChange mutagenesis kit, Stratagene, La Jolla, CA). Login to comment 77 ABCC7 p.Leu1156Phe The primers used for L1156F are as follows: sense 5=-ATAGATGTGGATAGCTTTAT- GCGATCTGTGAGCCGAGTCT-3=, antisense 5=-AGACTCGGCT- CACAGATCGCATAAAGCTATCCACATCTAT-3=. Login to comment 132 ABCC7 p.Glu217Gly ABCC7 p.Arg1453Trp ABCC7 p.Ile556Val ABCC7 p.Gln1352His ABCC7 p.Leu1156Phe Six variants (E217G, I556V, M470V, L1156F, Q1352H, and R1453W) were identified in coding regions of the CFTR gene (Table 2). Login to comment 133 ABCC7 p.Gln1352His ABCC7 p.Leu1156Phe The allele frequencies of L1156F and Q1352H in alcoholic chronic pancreatitis (5.0 and 7.9%) were significantly (P b0d; 0.01) higher than those in normal subjects (0.6 and 1.9%). Login to comment 134 ABCC7 p.Arg1453Trp The allele frequency of R1453W in idiopathic pancreatitis (11.1%) was significantly (P b0d; 0.01) higher than that in normal subjects (1.9%). Login to comment 135 ABCC7 p.Glu217Gly ABCC7 p.Ile556Val The allele frequencies of E217G, I556V, and M470V were not different among groups. Login to comment 136 ABCC7 p.Leu1156Phe Genotypes, pancreatic exocrine function, and sweat Clafa; of chronic pancreatitis patients carrying the L1156F. Login to comment 137 ABCC7 p.Leu1156Phe Table 3 shows the characteristics of eight patients with alcoholic (7 men) and idiopathic (1 woman) chronic pancreatitis who carry the L1156F variant. Login to comment 138 ABCC7 p.Gln1352His ABCC7 p.Leu1156Phe Genotypes of CFTR (presence of L1156F and Q1352H, M/V470, poly T, and TG repeats), sex, age, etiology of pancreatitis, presence or absence of pancreatic stone, pancreatic exocrine function (secretin test), sweat Clafa; concentration, and mutations in SPINK1 and PRSS1 are shown. Login to comment 141 ABCC7 p.Glu217Gly ABCC7 p.Arg1453Trp ABCC7 p.Ile556Val ABCC7 p.Gln1352His ABCC7 p.Leu1156Phe The allele frequencies of polymorphisms in the coding regions of CFTR gene ACP ICP NS n 140 36 360 E217G (exon 6a) Glu 137 (97.9) 36 (100) 354 (98.3) Gly 3 (2.1) 0 (0) 6 (1.7) M470V (exon 10) Met 60 (42.9) 14 (38.9) 143 (39.7) Val 80 (57.1) 22 (61.1) 217 (60.3) I556V (exon 11) Ile 138 (98.6) 36 (100) 348 (96.7) Val 2 (1.4) 0 (0) 12 (3.3) L1156F (exon 18) Leu 133 (95.0) 35 (97.2) 358 (99.4) Phe 7 (5.0)* 1 (2.8) 2 (0.6) Q1352H (exon 22) Gln 129 (92.1) 35 (97.2) 353 (98.1) His 11 (7.9)* 1 (2.8) 7 (1.9) R1453W (exon 24) Arg 138 (98.6) 32 (88.9) 353 (98.1) Trp 2 (1.4) 4 (11.1)* 7 (1.9) Values are no. Login to comment 147 ABCC7 p.Leu1156Phe Characteristics of 8 patients who carry L1156F Patient No. Login to comment 148 ABCC7 p.Gln1352His ABCC7 p.Gln1352His ABCC7 p.Gln1352His ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe CFTR Genotypes Sex Age, yr Etiology Pancreatic Stone Secretin Test* Sweat [Clafa; ] SPINK1 Mutation PRSS1 Mutation Volume MBC Amylase 1 L1156F af9; Q1352H af9; V/V470 af9; 7/7T af9;11/11(TG) M 59 Alcoholic af9; - - 2 L1156F af9; V/V470 af9; 7/7T af9;11/11(TG) M 65 Alcoholic af9; 174.5 96.9 21,524 42.0 - - 3 L1156F af9; V/V470 af9; 7/7T af9;11/12(TG) M 49 Alcoholic - - - 4 L1156F af9; Q1352H af9; M/V470 af9; 7/7T af9;11/12(TG) M 57 Alcoholic af9; 88.9 - 5 L1156F af9; M/V470 af9; 7/7T af9;11/12(TG) M 60 Alcoholic af9; 53.0 50.9 1,627 67.0 - - 6 L1156F af9; M/V470 af9; 7/7T af9;11/12(TG) M 56 Alcoholic af9; 149.0 71.3 6,891 98.9 - - 7 L1156F af9; M/V470 af9; 7/7T af9;11/12(TG) M 51 Alcoholic af9; N34S 8 L1156F af9; M/V470 af9; 7/7T af9;11/12(TG) F 73 Idiopathic af9; 58.9 - - Genotypes of CFTR indicate presence of L1156F and Q1352H, M/V470, poly T, and TG repeats. Login to comment 155 ABCC7 p.Leu1156Phe There was no homozygote of L1156F. Login to comment 156 ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe All patients carrying the L1156F had M470V on at least one allele, suggesting that L1156F is linked to M470V. Login to comment 157 ABCC7 p.Gln1352His Two patients had the Q1352H variant, which is also associated with alcoholic chronic pancreatitis. Login to comment 163 ABCC7 p.Leu1156Phe The protein expression of wild-type CFTR and three CFTR variants (M470V-CFTR, L1156F-CFTR, M470Vaf9;L1156F-CFTR) transfected in HEK-293 cells was examined by Western blot analysis (Fig. 1). Login to comment 166 ABCC7 p.Leu1156Phe The expression of M470V-CFTR and L1156F-CFTR was not significantly different from that of wild-type CFTR. Login to comment 175 ABCC7 p.Leu1156Phe Clafa; channel current of wild-type CFTR and three CFTR variants (M470V-CFTR, L1156F-CFTR, M470Vaf9;L1156F-CFTR) were measured by the whole cell configuration (Fig. 4). Login to comment 178 ABCC7 p.Leu1156Phe A: HEK-293 cells transfected with wild-type CFTR and 3 CFTR variants (M470V-CFTR, L1156F-CFTR, M470Vaf9;L1156F-CFTR) were lysed, and the separated proteins were blotted with antibodies against the nucleotide-binding domain (NBD) 2 domain of CFTR (M3A7, Millipore, Billerica, MA) and beta-actin. Login to comment 190 ABCC7 p.Leu1156Phe The magnitude of Clafa; current by M470V-CFTR and L1156F-CFTR was similar to that by wild-type CFTR. Login to comment 193 ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe HCO3 afa; and Clafa; fluxes were examined in Xenopus laevis oocytes expressing wild-type CFTR or three CFTR variants (M470V-CFTR, L1156F-CFTR, M470Vaf9; L1156F-CFTR) (Fig. 5). Login to comment 199 ABCC7 p.Leu1156Phe M470V and L1156F by themselves did not affect CFTR-mediated HCO3 afa; /Clafa; transport. Login to comment 215 ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe B A Current density (pA/pF) 0 20 40 60 80 100 100 sec 500 pA Forskolin Glibenclamide Wild-type CFTR L1156F M470V M470V + L1156F Mock Fig. 4. Login to comment 217 ABCC7 p.Leu1156Phe Clafa; channel activity of wild-type CFTR (black trace) and 3 CFTR variants (M470V-CFTR, blue; L1156F-CFTR, green; M470Vaf9;L1156F-CFTR, red) were measured in the whole cell configuration. Login to comment 223 ABCC7 p.Leu1156Phe The AE activity in cells transfected with three CFTR variants, M470V, L1156F, and M470Vaf9;L1156F-CFTR, was all much smaller (P b0d; 0.01) compared with cells transfected with wild-type CFTR. Login to comment 224 ABCC7 p.Leu1156Phe The AE activity coupled with M470V, L1156F, and M470Vaf9;L1156F-CFTR was 33, 35, and 26%, respectively, of that coupled with wild-type CFTR. Login to comment 225 ABCC7 p.Leu1156Phe The AE activity coupled with M470Vaf9;L1156F-CFTR was significantly (P b0d; 0.05) smaller compared with that with L1156F-CFTR. Login to comment 227 ABCC7 p.Leu1156Phe To examine the possible effects of M470V and L1156F on CFTR function at the molecular level, we constructed a homology model of CFTR in the inward-facing conformation (closed state). Login to comment 229 ABCC7 p.Leu1156Phe Thus M470V may affect ATP binding to NBD1, and L1156F may affect conformational transition between closed and open states. Login to comment 230 ABCC7 p.Leu1156Phe However, M470 and L1156 are not close to each other, and it is not clear why the double mutation (combination of M470V and L1156F), but not single mutations, affected the transport Clafa; and HCO3 afa; (Fig. 5). Login to comment 238 ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe In a previous study, our laboratory has performed haplotype anal- Current HCO3 - ClC Wild-type CFTR 6.8 7.0 7.2 7.4 pHi HCO3 - 0 Cl- Forskolin A 0 10 20 30 40 Cli 10 min B M470V+L1156F Wild-type CFTR * * 0.0 2.5 5.0 7.5 0.0 2.5 5.0 7.5 Wild-type CFTR L1156F M470V M470V + L1156F Current (bc;A) HCO 3 - /Cl - transport (mM/min) Mock Mock M470V+L1156F Fig. 5. Login to comment 240 ABCC7 p.Leu1156Phe Xenopus oocytes expressing wild-type CFTR or 3 CFTR variants (M470V-CFTR, L1156F-CFTR, M470Vaf9;L1156F-CFTR) were first bathed in HCO3 afa; -free media. Login to comment 246 ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe Cl--free 0.5 pH units B A 3 min Wild-type CFTR L1156F M470V M470V + L1156F Mock 0.0 1.0 2.0 3.0 4.0 ƊpH min -1 # * * * Fig. 6. Login to comment 247 ABCC7 p.Leu1156Phe Clafa; /HCO3 afa; exchange activity in CFPAC-1 cells transfected with CFTR variants. CFPAC-1 cells transfected with wild-type CFTR (black trace), 3 CFTR variants (M470V-CFTR, blue; L1156F-CFTR, green; M470Vaf9;L1156F-CFTR, red), and vector alone (gray) were first bathed in the standard HCO3 afa; - CO2-buffered solution containing 1 òe;M forskolin. Login to comment 253 ABCC7 p.Leu1156Phe #P b0d; 0.05, compared with L1156F-CFTR. Login to comment 258 ABCC7 p.Arg1453Trp ABCC7 p.Gln1352His ABCC7 p.Leu1156Phe The association of chronic pancreatitis and three Japanese/ Asian types of CFTR variants (L1156F, Q1352H, and R1453W) were demonstrated in our present and previous (13) studies. Login to comment 262 ABCC7 p.Leu1156Phe Epidemiology of L1156F-CFTR. Login to comment 263 ABCC7 p.Arg1453Trp ABCC7 p.Gln1352His ABCC7 p.Leu1156Phe While Q1352H and R1453W are also found in Koreans (27) and thus categorized to Asian-type CFTR variants, L1156F is probably a Japanese-specific CFTR variant and has not been reported from other countries. Login to comment 265 ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe L1156F was previously found in an adult, Japanese healthy female (Cystic Fibrosis Mutation Database), and a recent study reported the association of L1156F and chronic pancreatitis in Japanese (32). Login to comment 266 ABCC7 p.Leu1156Phe In the present study, we have found that L1156F is associated with alcoholic chronic pancreatitis in Japanese (the odds ratio afd; 9.0 compared with normal subjects, Table 2). Login to comment 267 ABCC7 p.Leu1156Phe L1156F was found in 10.0% in patients with alcoholic chronic pancreatitis. Login to comment 268 ABCC7 p.Arg1453Trp ABCC7 p.Gln1352His ABCC7 p.Leu1156Phe While Q1352H was also found in patients with congenital bilateral absence of the vas deferens (1) and diffuse panbronchiolitis and R1453W in patients with diffuse panbronchiolitis (Cystic Fibrosis Mutation Database), L1156F has not been found in other CFTR-related diseases. Login to comment 269 ABCC7 p.Leu1156Phe Thus L1156F is probably a pancreatitis-specific CFTR variant. Login to comment 270 ABCC7 p.Leu1156Phe Functional characteristics and molecular modeling of L1156F-CFTR. Login to comment 272 ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe Since L1156F is most likely linked to M470V (Table 2), we analyzed the protein expression, Clafa; channel activity, HCO3 afa; /Clafa; transport activity, and CFTR-coupled Clafa; /HCO3 afa; exchange activity of three CFTR variants (M470V-CFTR, L1156F-CFTR, M470Vaf9;L1156F-CFTR) using heterologous expression systems. Login to comment 273 ABCC7 p.Leu1156Phe The combination of M470V and L1156F reduced the expression of mature "band C" form of CFTR protein to 60b03;70% (Figs. 1-3). Login to comment 274 ABCC7 p.Leu1156Phe The effects of M470V and L1156F by themselves on the protein expression were not significant due to a considerable variation between experiments. Login to comment 276 ABCC7 p.Leu1156Phe The M470Vaf9;L1156F-CFTR variant expressed in Xenopus laevis oocytes showed a reduction of HCO3 afa; /Clafa; transport activity to 50b03;60% of wild-type CFTR (Fig. 5), while L1156F by itself caused almost no reduction. Login to comment 279 ABCC7 p.Leu1156Phe To the contrary, CFTR-coupled Clafa; /HCO3 afa; exchange activity was substantially impaired by M470V, L1156F, and the double mutation (M470Vaf9;L1156F, Fig. 6). Login to comment 280 ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe M470 and L1156F are not close to each other in the inward-facing conformation (closed state) in a predicted model of CFTR (Fig. 7), which do not explain why the combination of M470V and L1156F impaired Clafa; /HCO3 afa; transport activity. Login to comment 281 ABCC7 p.Leu1156Phe L1156F-CFTR variant and chronic pancreatitis. Login to comment 286 ABCC7 p.Leu1156Phe The CFTR variant, L1156F, identified in the present study is associated with chronic pancreatitis but not CF, which is consistent with the idea. Login to comment 287 ABCC7 p.Leu1156Phe The present eight cases of adult-onset chronic pancreatitis carrying L1156F are probably not categorized to atypical CF. Login to comment 296 ABCC7 p.Leu1156Phe Thus the predicted total CFTR function of the present eight cases with chronic pancreatitis carrying L1156F may vary from b03;30 to b03;90% of the wild type. Login to comment 297 ABCC7 p.Leu1156Phe In addition, CFTR-coupled Clafa; /HCO3 afa; exchange activity was substantially affected by M470V, L1156F, and the double mutation (Fig. 6). Login to comment 300 ABCC7 p.Arg75Gln A study from the United States reported that a risk of chronic pancreatitis was increased by the presence of p.R75Q, a non CF-causing, HCO3 afa; -conductance impairing CFTR variant (40). Login to comment 311 ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe Although 1 of 18 patients (5.6%) with idiopathic chronic pancreatitis had L1156F, L1156F was more prevalent (10.0%) in the patients with alcoholic chronic pancreatitis in the present study (Table 2). Login to comment 313 ABCC7 p.Leu1156Phe In the present study, we have for the first time identified the alcoholic chronic pancreatitis-susceptibility genotype, M470Vaf9; L1156F-CFTR. Login to comment 315 ABCC7 p.Leu1156Phe The association of L1156F and EtOH needs to be further investigated. Login to comment 316 ABCC7 p.Leu1156Phe In summary, our present data suggest that the Japanese-specific CFTR variant, L1156F, is associated with alcoholic chronic pancreatitis. Login to comment 317 ABCC7 p.Leu1156Phe ABCC7 p.Leu1156Phe L1156F is linked with the worldwide CFTR variant M470V. Combination of L1156F and M470V impairs protein expression and HCO3 afa; /Clafa; transport activity of CFTR and CFTR-coupled Clafa; /HCO3 afa; exchange activity. Login to comment 318 ABCC7 p.Leu1156Phe The molecular mechanisms underlying the synergistic effects of L1156F and M470V need to be investigated. Login to comment