ABCMdb

ABCC7 p.Leu1156Phe

ClinVar:	c.3468G>A , p.Leu1156= ? , Conflicting interpretations of pathogenicity, not provided
CF databases:	c.3468G>T , p.Leu1156Phe (CFTR1) ? , A new substitution was detected in the CFTR by DGGE and identified by direct sequencing. The defect is G to T change at nucleotide 3599 in exon 18 which would lead to an leucine to phenylalanine replacement in the protien sequence at residue 1156(L1156F). This mutation has been found in an adult, Japanese healthy female who was studied for couple's infertility.
Predicted by SNAP2:	A: D (59%), C: N (57%), D: D (80%), E: D (75%), F: D (53%), G: D (80%), H: D (75%), I: D (59%), K: D (85%), M: N (78%), N: D (80%), P: D (91%), Q: D (59%), R: D (85%), S: D (75%), T: D (71%), V: D (53%), W: D (80%), Y: D (75%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N,

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[hide] Targeted next-generation sequencing effectively an... Dig Dis Sci. 2015 May;60(5):1297-307. doi: 10.1007/s10620-014-3476-9. Epub 2014 Dec 10. Nakano E, Masamune A, Niihori T, Kume K, Hamada S, Aoki Y, Matsubara Y, Shimosegawa T
Targeted next-generation sequencing effectively analyzed the cystic fibrosis transmembrane conductance regulator gene in pancreatitis.
Dig Dis Sci. 2015 May;60(5):1297-307. doi: 10.1007/s10620-014-3476-9. Epub 2014 Dec 10., [PMID:25492507]

Abstract [show]
BACKGROUND: The cystic fibrosis transmembrane conductance regulator (CFTR) gene, responsible for the development of cystic fibrosis, is known as a pancreatitis susceptibility gene. Direct DNA sequencing of PCR-amplified CFTR gene segments is a first-line method to detect unknown mutations, but it is a tedious and labor-intensive endeavor given the large size of the gene (27 exons, 1,480 amino acids). Next-generation sequencing (NGS) is becoming standardized, reducing the cost of DNA sequencing, and enabling the generation of millions of reads per run. We here report a comprehensive analysis of CFTR variants in Japanese patients with chronic pancreatitis using NGS coupling with target capture. METHODS: Exon sequences of the CFTR gene from 193 patients with chronic pancreatitis (121 idiopathic, 46 alcoholic, 17 hereditary, and nine familial) were captured by HaloPlex target enrichment technology, followed by NGS. RESULTS: The sequencing data covered 91.6 % of the coding regions of the CFTR gene by >/= 20 reads with a mean read depth of 449. We could identify 12 non-synonymous variants including three novel ones [c.A1231G (p.K411E), c.1753G>T (p.E585X) and c.2869delC (p.L957fs)] and seven synonymous variants including three novel ones in the exonic regions. The frequencies of the c.4056G>C (p.Q1352H) and the c.3468G>T (p.L1156F) variants were higher in patients with chronic pancreatitis than those in controls. CONCLUSIONS: Target sequence capture combined with NGS is an effective method for the analysis of pancreatitis susceptibility genes.

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7 The frequencies of the c.4056G[C (p.Q1352H) and the c.3468G[T (p.L1156F) variants were higher in patients with chronic pancreatitis than those in controls. Login to comment
90 On average, 90.3 % of the coding region was successfully covered by C20 reads Table 2 Non-synonymous CFTR variants detected in this study Exon Non-synonymous variant Amino acid change dbSNP135 Genotype SIFT (score) PolyPhen-2 (score) Alcoholic CP (%) Idiopathic CP (%) Hereditary/ familial CP (%) 2 c.91C[T p.R31C rs1800073 CT D (0.012) PD (0.989) 0/46 (0) 3/121 (2.5) 0/26 (0) 2 c.92G[A p.R31H rs149353983 GA T (0.183) B (0.003) 0/46 (0) 1/121 (0.8) 0/26 (0) 4 c.374T[C p.I125T rs141723617 TC D (0.005) B (0.17) 0/46 (0) 2/121 (1.6) 1/26 (3.8) 10 c.1231A[G p.K411E - AG D (0.015) B (0.233) 0/46 (0) 1/121 (0.8) 0/26 (0) 11 c.1408G[A p.V470M rs213950 GA T (1) B (0) 21/46 (45.7) 65/121 (53.7) 11/26 (42.3) AA 5/46 (10.9) 19/121 (15.7) 1/26 (3.8) 12 c.1666A[G p.I556V rs75789129 AG T (0.536) B (0.334) 2/46 (4.3) 8/121 (6.6) 0/26 (0) GG 0/46 (0) 0/121 (0) 0/26 (0) 13 c.1753G[T p.E585X - GT - - 1/46 (2.2) 0/121 (0) 0/26 (0) 17 c.2869delC p.L957fs - - - 0/46 (0) 1/121 (0.8) 0/26 (0) 21 c.3468G[T p.L1156F rs139729994 GT T (0.163) PD (0.994) 2/46 (4.3) 10/121 (8.3) 2/26 (7.7) TT 1/46 (2.2) 0/121 (0) 0/26 (0) 25 c.4045G[A p.G1349S rs201686600 GA D (0) PD (1) 1/46 (2.2) 0/121 (0) 0/26 (0) 25 c.4056G[C p.Q1352H rs113857788 GC D (0) PD (1) 5/46 (10.9) 11/121 (9.1) 4/26 (15.4) CC 0/46 (0) 0/121 (0) 0/26 (0) 27 c.4357C[T p.R1453W rs4148725 CT D (0) PD (0.999) 3/46 (6.5) 6/121 (5.0) 1/26 (3.8) B benign, CP chronic pancreatitis, D damaging, PD probably damaging, T tolerated, SIFT Sorting Intolerant From Tolerant heterozygous form (Table 6). Login to comment
94 The patient also had the c.3468G[T (p.L1156F) variant in a heterozygous form. Login to comment
99 The frequency of the c.3468G[T (p.L1156F) variant was also higher in patients with CP than that in controls (P = 0.04). Login to comment
100 There were no significant difference for any other non-synonymous or synonymous variants detected in the exons Table 3 Comparison of the non-synonymous variant frequencies between the patients with CP and controls Amino acid change Genotype All CP (%) HGVD (%) P value (vs. HGVD) All CP Alcoholic CP Nonalcoholic CP Idiopathic CP Hereditary/ familial CP p.R31C CT 3/193 (1.6) 12/1102 (1.1) 0.48 [0.99 0.41 0.18 [0.99 p.R31H GA 1/193 (0.5) 0 - - - - - p.I125T TC 3/193 (1.6) 5/1102 (0.5) 0.11 [0.99 0.057 0.15 0.13 p.K411E AG 1/193 (0.5) 0 - - - - - p.V470M GA 97/193 (50.3) 573/1199 (47.8) 0.66 0.57 0.68 0.38 0.12 AA 25/193 (13.0) 185/1199 (15.4) p.I556V AG 10/193 (5.2) 78/1150 (6.8) 0.70 0.79 0.81 [0.99 0.45 GG 0/193 (0) 3/1150 (0.3) p.E585X GT 1/193 (0.5) 0 - - - - - p.L957fs 1/193 (0.5) 0 - - - - - p.L1156F GT 14/193 (7.3) 45/1136 (4.0) 0.04 0.06 0.07 0.11 0.30 TT 1/193 (0.5) 1/1136 (0.1) p.G1349S GA 1/193 (0.5) 4/1094 (0.4) 0.56 0.19 [0.99 [0.99 [0.99 p.Q1352H GC 20/193 (10.4) 57/1153 (4.9) 0.009 0.12 0.037 0.17 0.062 CC 0/193 (0) 1/1153 (0.1) p.R1453W CT 10/193 (5.2) 42/1144 (3.7) 0.32 0.25 0.49 0.45 [0.99 CP chronic pancreatitis, HGVB Human Genetic Variation Database P values were determined versus HGVD by the Fisher`s exact test Table 4 Synonymous variants in the exons of the CFTR gene detected in this study Exon Synonymous variant Amino acid change dbSNP135 Genotype Alcoholic CP (%) Idiopathic CP (%) Hereditary/ familial CP (%) 4 c.372C[T p.G124= - CT 0/46 (0) 1/121 (0.8) 0/26 (0) 13 c.1731C[T p.Y577= rs55928397 CT 0/46 (0) 1/121 (0.8) 0/26 (0) 15 c.2562T[G p.T854= rs1042077 TG 20/46 (43.5) 69/121 (57.0) 12/26 (46.2) GG 6/46 (13.0) 18/121 (14.9) 0/26 (0) 23 c.3723C[A p.G1241= rs185065886 CA 1/46 (2.2) 0/121 (0) 0/26 (0) 25 c.3975A[G p.R1325= - AG 0/46 (0) 1/121 (0.8) 0/26 (0) 27 c.4254G[A p.E1418= - GA 0/46 (0) 1/121 (0.8) 0/26 (0) 27 c.4389G[A p.Q1463= rs1800136 GA 1/46 (2.2) 3/121 (2.5) 0/26 (0) CP chronic pancreatitis between all patients with CP and controls (Tables 3, 5). Login to comment
114 Comprehensive analysis by targeted NGS enabled us to identify novel and Table 5 Comparison of the synonymous variant frequencies between the patients with CP and controls Synonymous variant Genotype All CP (%) HGVD (%) P value (vs. HGVD) All CP Alcoholic CP Nonalcoholic CP Idiopathic CP Hereditary/ familial CP c.C372T CT 1/193 (0.5) 0 - - - - - c.1731C[T CT 1/193 (0.5) 0 - - - - - c.2562T[G TG 101/193 (52.3) 528/1154 (45.8) 0.22 0.81 0.11 0.045 0.033 GG 24/193 (12.4) 181/1154 (15.7) c.3723C[A CA 1/193 (0.5) 3/671 (4.5) [0.99 0.23 [0.99 [0.99 [0.99 c.3975A[G AG 1/193 (0.5) 0 - - - - - c.4254G[A GA 1/193 (0.5) 0 - - - - - c.4389G[A GA 4/193 (2.1) 40/1112 (3.6) 0.48 [0.99 0.53 0.81 [0.99 AA 0/193 (0) 1/1112 (0.1) CP chronic pancreatitis, HGVD Human Genetic Variation Database P values were determined against HGVD by the Fisher`s exact test Table 6 Total CFTR sequencing results of patients carrying rare non-synonymous CFTR variants a Pancreatitis-associated mutations in the PRSS1, SPINK1, CTRC, and CPA1 genes Case# Etiology Age at onset Rare variant Additional non-synonymous variants c.1210-34TG(9_13) c.1210-12T(5_9) Mutation in other pancreatitis susceptibility genesa A1 Idiopathic 34 p.R31C/- p.R1453W/- TG11/TG11, 7T/7T - A2 Idiopathic 8 p.R31C/- - TG11/TG12, 7T/7T - A3 Idiopathic 16 p.R31C/- - TG11/TG12, 7T/7T - A4 Idiopathic 10 p.R31H/- - TG11/TG12, 7T/7T - A5 Idiopathic 16 p.I125T/- p.L1156F/- TG11/TG12, 7T/7T CTRC p.R29Q/- A6 Idiopathic 2 p.I125T/- - TG11/TG12, 7T/7T - A7 Hereditary 28 p.I125T/- p.R1453W/- TG11/TG12, 7T/7T - A8 Idiopathic 19 p.K411E/- p/L1156F/- TG11/TG12, 7T/7T - A9 Alcoholic 28 p.E585X/- p.I556V/- TG11/TG11, 7T/7T - A10 Idiopathic 21 p.L957fs/- p.Q1352H/- TG11/TG12, 7T/7T - A11 Alcoholic 40 p.G1349S/- - TG11/TG11, 7T/7T - rare variants in the CFTR gene. Login to comment
120 Importantly, the clinical phenotype of this patient might be complicated by the presence of another variant, p.L1156F. Login to comment
139 Similarly, we found that the p.L1156F variant was overexpressed in patients with CP. Login to comment
140 A functional study reported reduced Cl- / HCO3 - permeability in the presence of the p.L1156F variant [41]. Login to comment
142 Indeed, seven out of 25 patients carrying the SPINK1 variant(s) had the CFTR p.Q1352H and/or p.L1156F variants. Login to comment
143 One patient was trans-heterozygous for the CTRC p.R29Q and CFTR p.I125T/p.L1156F variants. Login to comment
151 Sequence capture eliminates the necessity of setting up hundreds of PCR, instead allowing for parallel Table 8 Total CFTR sequencing results of patients with SPINK1, PRSS1, CTRC, or CPA1 mutations Case# Etiology CFTR variantsa c.1210-34TG(9_13) c.1210-12T(5_9) SPINK1 PRSS1 CTRC CPA1 B1 Familial p.Q1352H/- TG11/TG12, 7T/7T p.N34S/p.N34S B2 Idiopathic - TG12/TG12, 7T/7T p.N34S/p.N34S B3 Idiopathic - TG11/TG12, 7T/7T p.N34S/p.N34S B4 Idiopathic p.L1156F/-, p.Q1352H/- TG11/TG11, 7T/7T p.N34S/- B5 Idiopathic p.Q1352H/- TG11/TG12, 7T/7T p.N34S/- B6 Idiopathic p.Q1352H/- TG11/TG12, 7T/7T p.N34S/- B7 Idiopathic - TG11/TG12, 7T/7T p.N34S/- B8 Idiopathic - TG11/TG12, 7T/7T p.N34S/- B9 Idiopathic - TG11/TG12, 7T/7T p.N34S/- B10 Idiopathic - TG11/TG12, 7T/7T p.N34S/- B11 Idiopathic - TG11/TG12, 7T/7T p.N34S/- B12 Idiopathic - TG11/TG12, 7T/7T p.N34S/- B13 Idiopathic - TG11/TG12, 7T/7T p.N34S/- B14 Alcoholic - TG12/TG13, 5T/7T p.N34S/- B15 Idiopathic - TG11/TG12, 7T/7T p.N34S/IVS3?2T[C B16 Idiopathic p.R1453W/- TG11/TG11, 7T/7T p.N34S/IVS3?2T[C B17 Idiopathic - TG11/TG12, 7T/7T IVS3?2T[C/IVS3?2T[C B18 Idiopathic - TG11/TG12, 7T/7T IVS3?2T[C/IVS3?2T[C B19 Hereditary p.I125T/-, p.L1156F/- TG11/TG12, 5T/7T IVS3?2T[C/- B20 Familial p.L1156F/- TG11/TG12, 7T/7T IVS3?2T[C/- B21 Idiopathic - TG11/TG12, 7T/7T IVS3?2T[C/- B22 Alcoholic p.Q1352H/- TG11/TG12, 7T/7T IVS3?2T[C/- B23 Alcoholic - TG11/TG12, 7T/7T IVS3?2T[C/- B24 Idiopathic - TG11/TG12, 7T/7T p.P45S/- B25 Idiopathic - TG12/TG12, 7T/7T IVS3?2T[C/- p.R122H/- B26 Hereditary TG11/TG12, 7T/7T p.R122H/- B27 Idiopathic p.I556V/- TG11/TG12, 7T/7T p.N29I/- B28 Idiopathic p.I125T/-, p.L1156F/- TG11/TG12, 7T/7T p.R29Q/- B29 Idiopathic - TG11/TG12, 7T/7T p.T368_Y369ins20/- Nine patients had the non-synonymous CFTR variants, which are probably damaging based on the SIFT or the PolyPhen-2 prediction The p.I556V variant appeared to be benign based on the SIFT or the PolyPhen-2 prediction Case B28 is the same as A5 in Table 6 a We excluded the p.V470M variant from the list because of its similar frequencies in patients and controls enrichment of target regions in a single experiment. Login to comment

[hide] Functional characteristics of L1156F-CFTR associat... Am J Physiol Gastrointest Liver Physiol. 2015 Aug 15;309(4):G260-9. doi: 10.1152/ajpgi.00015.2014. Epub 2015 Jun 18. 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., [PMID:26089335]

Abstract [show]
Although cystic fibrosis is rare in Japanese, measurement of sweat Cl(-) has suggested mild dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR) in some patients with chronic pancreatitis. 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. Seventy patients with alcoholic chronic pancreatitis, 18 patients with idiopathic chronic pancreatitis, and 180 normal subjects participated. All exons and their boundaries and promoter region of the CFTR gene were sequenced. Human embryonic kidney-293 cells were transfected with three CFTR variants (M470V, L1156F, and M470V+L1156F), and the protein expression was examined. Xenopus laevis oocytes were injected with the CFTR variants, and bicarbonate (HCO3 (-)) transport activity was examined. CFPAC-1 cells were transfected with the CFTR variants and Cl(-)/HCO3 (-) exchange activity was examined. Six variants (E217G, I556V, M470V, L1156F, Q1352H, and R1453W) were identified in the coding region of the CFTR gene. Cystic fibrosis-causing mutations were not found. The allele frequencies of L1156F and Q1352H in alcoholic chronic pancreatitis (5.0 and 7.9%) were significantly (P < 0.01) higher than those in normal subjects (0.6 and 1.9%). L1156F was linked with a worldwide CFTR variant, M470V. Combination of M470V and L1156F significantly reduced CFTR expression to approximately 60%, impaired CFTR-mediated HCO3 (-)/Cl(-) transport activity to 50-60%, and impaired CFTR-coupled Cl(-)/HCO3 (-) exchange activity to 20-30%. 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.

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5 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 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 Six variants (E217G, I556V, M470V, L1156F, Q1352H, and R1453W) were identified in the coding region of the CFTR gene. Login to comment
13 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 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 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 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
40 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 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 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 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 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 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 The primers used for L1156F are as follows: sense 5=-ATAGATGTGGATAGCTTTAT- GCGATCTGTGAGCCGAGTCT-3=, antisense 5=-AGACTCGGCT- CACAGATCGCATAAAGCTATCCACATCTAT-3=. Login to comment
132 Six variants (E217G, I556V, M470V, L1156F, Q1352H, and R1453W) were identified in coding regions of the CFTR gene (Table 2). Login to comment
133 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
136 Genotypes, pancreatic exocrine function, and sweat Clafa; of chronic pancreatitis patients carrying the L1156F. Login to comment
137 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 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 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 Characteristics of 8 patients who carry L1156F Patient No. Login to comment
148 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 There was no homozygote of L1156F. Login to comment
156 All patients carrying the L1156F had M470V on at least one allele, suggesting that L1156F is linked to M470V. Login to comment
163 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 The expression of M470V-CFTR and L1156F-CFTR was not significantly different from that of wild-type CFTR. Login to comment
175 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 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 The magnitude of Clafa; current by M470V-CFTR and L1156F-CFTR was similar to that by wild-type CFTR. Login to comment
193 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 M470V and L1156F by themselves did not affect CFTR-mediated HCO3 afa; /Clafa; transport. Login to comment
215 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 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 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 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 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 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 Thus M470V may affect ATP binding to NBD1, and L1156F may affect conformational transition between closed and open states. Login to comment
230 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 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 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 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 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 #P b0d; 0.05, compared with L1156F-CFTR. Login to comment
258 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 Epidemiology of L1156F-CFTR. Login to comment
263 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 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 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 L1156F was found in 10.0% in patients with alcoholic chronic pancreatitis. Login to comment
268 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 Thus L1156F is probably a pancreatitis-specific CFTR variant. Login to comment
270 Functional characteristics and molecular modeling of L1156F-CFTR. Login to comment
272 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 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 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 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 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 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 L1156F-CFTR variant and chronic pancreatitis. Login to comment
286 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 The present eight cases of adult-onset chronic pancreatitis carrying L1156F are probably not categorized to atypical CF. Login to comment
296 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 In addition, CFTR-coupled Clafa; /HCO3 afa; exchange activity was substantially affected by M470V, L1156F, and the double mutation (Fig. 6). Login to comment
311 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 In the present study, we have for the first time identified the alcoholic chronic pancreatitis-susceptibility genotype, M470Vaf9; L1156F-CFTR. Login to comment
315 The association of L1156F and EtOH needs to be further investigated. Login to comment
316 In summary, our present data suggest that the Japanese-specific CFTR variant, L1156F, is associated with alcoholic chronic pancreatitis. Login to comment
317 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 The molecular mechanisms underlying the synergistic effects of L1156F and M470V need to be investigated. Login to comment