ABCMdb

PMID: 20977904

Schneider A, Larusch J, Sun X, Aloe A, Lamb J, Hawes R, Cotton P, Brand RE, Anderson MA, Money ME, Banks PA, Lewis MD, Baillie J, Sherman S, Disario J, Burton FR, Gardner TB, Amann ST, Gelrud A, George R, Rockacy MJ, Kassabian S, Martinson J, Slivka A, Yadav D, Oruc N, Barmada MM, Frizzell R, Whitcomb DC
Combined bicarbonate conductance-impairing variants in CFTR and SPINK1 variants are associated with chronic pancreatitis in patients without cystic fibrosis.
Gastroenterology. 2011 Jan;140(1):162-71. Epub 2010 Oct 25., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Arg75Gln CFTR wild-type and p.R75Q were cloned and expressed in HEK293 cells, and relative conductances of HCO3 - and Cl- were measured. Login to comment 7 ABCC7 p.Arg75Gln One variant of CFTR not associated with CF, p.R75Q, was found in 16% of subjects and 5.3% of controls (OR, 3.4). Login to comment 8 ABCC7 p.Arg75Gln Coinheritance of CFTR p.R75Q and SPINK1 variants occurred in 8.75% of patients and 0.38% of controls (OR, 25.1). Login to comment 9 ABCC7 p.Arg75Gln Patch-clamp recordings of cells that expressed CFTR p.R75Q showed normal chloride currents but significantly reduced bicarbonate currents (P ϭ .0001). Login to comment 10 ABCC7 p.Arg75Gln CONCLUSIONS: The CFTR variant p.R75Q causes a selective defect in bicarbonate conductance and increases risk of pancreatitis. Login to comment 11 ABCC7 p.Arg75Gln Coinheritance of p.R75Q or CF causing CFTR variants with SPINK1 variants significantly increases the risk of ICP. Login to comment 29 ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln Finally, to identify a physiological reason for the repeated association of the CFTR variant p.R75Q with pancreatitis, we have tested the chloride and bicarbonate conductance of CFTR p.R75Q in a polarized epithelial cell line. Login to comment 38 ABCC7 p.Arg75Gln To confirm sequencing data and expand the control population, 375 additional unrelated NAPS2 controls were tested for the specific CFTR mutations p.F508del and p.R75Q via custom iPLEX Sequenom (San Diego, CA) assay (primer sequences available on request). Login to comment 39 ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln CFTR p.R75Q Patch-Clamp Studies The CFTR p.R75Q mutant was prepared using a QuikChange XL Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA) from full-length human CFTR complementary DNA in pCDNA3.1. Login to comment 40 ABCC7 p.Arg75Gln The CFTR p.R75Q mutation was introduced using polymerase chain reaction mutagenesis and verified via sequencing (primer sequences available on request). Login to comment 41 ABCC7 p.Arg75Gln HEK 293 cells were transfected with CFTR wild-type (WT) or p.R75Q vectors using Lipofectamine 2000 (Invitrogen), and stable cell lines were selected in gentamicin (750 ␮g/mL). Login to comment 43 ABCC7 p.Arg75Gln The expression of CFTR WT and p.R75Q were confirmed by immunoblot, confirming similar expression levels. Login to comment 46 ABCC7 p.Arg75Gln Whole-Cell Recording Whole-cell voltage and current recordings were obtained from HEK 293 cells stably expressing CFTR WT or p.R75Q using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) and standard methods.26 Cells were cultured on glass cover slips, and the recordings were made 24 to 48 hours after plating. Login to comment 62 ABCC7 p.Arg75Gln Genotyping success rate was 100% in all sequenced samples and 99.4% and 98.6% for TaqMan genotyping of p.F508del and p.R75Q. Login to comment 74 ABCC7 p.Arg75Gln The remaining 375 control subjects were screened for SPINK1 exon 3 variants and CFTR variants p.R75Q and p.F508del. Login to comment 89 ABCC7 p.Arg560Thr Two mutations, p.F508del and p.R560T, well characterized as class II CFTR mutations that are often associated with pancreatic-insufficient CF, were categorized as CF severe for statistical calculation. Login to comment 90 ABCC7 p.Met952Thr ABCC7 p.Phe508Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr ABCC7 p.Ile807Met Also identified were 6 mutations (IVS8 T5, p.D443Y, p.G576A, p.F508C, p.I807M, p.M952T) reported to cause a milder form of CF or other CF-related diseases (such as congenital absence of the vas deferens), which we have categorized as CF mild. Login to comment 92 ABCC7 p.Ile148Thr ABCC7 p.Arg75Gln ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Asp443Tyr Two peculiar mutations that occurred in both populations, c.1584GtoA (1716GtoA legacy name) and p.R75Q, have been generally regarded as benign sequence variations28 (www.genet.sickkids. on.ca) but repeatedly show association to CF-related diseases, pancreatitis,29-31 and some patients with atypical CF.32 Two individual nonsynonymous sequence changes, p.R668C and p.I148T, were identified with CFTR full sequencing in one control each but without additional mutations found in cis (p.D443Y ϩ p.G576A and c.3067del6 [ie, 3199del6], respectively). Login to comment 99 ABCC7 p.Arg560Thr ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Met952Thr ABCC7 p.Phe508Cys ABCC7 p.Ile807Met Total CFTR Sequencing Results of Patients With SPINK1 Mutations Diagnosis Age at diagnosis (y) CFTR mutations SPINK1 mutations 1 SP 12 -/- N34S/P55S 2 SP 46 -/- N34S/P55S 3 SP 13 -/- N34S/N34S 4 FP Infant -/- N34S/N34S 5 FP 8 R560T/- N34S/P55S 6 FP 15 M952T/- N34S/N34S 7 SP 19 R75Q/-a N34S/N34S 8 SP 3 F508del/-a P55S/- 9 SP 3 F508del/1584GtoAa N34S/- 10 SP 19 F508del/-a N34S/- 11 FP 12 F508del/I807M, 3139ϩ42AtoTa N34S/- 12 SP 14 D443YϩG576AϩR668Cb N34S/- 13 SP 1 F508C/-a N34S/- 14 SP 20 IVS8-T5-TG12/-a N34S/- 15 SP 16 R75Q/-a P55S/- 16 SP 9 R75Q/-a N34S/- 17 SP 9 R75Q/-a N34S/- 18 SP 16 R75Q/ϩ1584GtoAa N34S/- 19 FP 7 R75Q/-a N34S/- 20 FP 35 R75Q/-a N34S/- 21 FP 2 1584GtoA/-a N34S/- 22 FP Child 1584GtoA/-a N34S/- 23 SP 14 1584GtoA/-a N34S/- 24 FP 14 3139ϩ42AtoT/- N34S/- 25 FP 28 -/- N34S/- 26 FP 36 -/- N34S/- 27 SP 8 -/- N34S/- 28 SP 9 -/- N34S/- 29 SP 3 -/- N34S/- FP, familial pancreatitis; SP, sporadic pancreatitis. Login to comment 104 ABCC7 p.Arg75Gln ABCC7 p.Phe508Cys In patients with SPINK1 mutations, CFTR variants were most commonly observed in exons 3 (eg, p.R75Q) and 10 (eg, p.F508C, c.1584GtoA, p.F508del) and IVS8/exon 9 (T5/TG12 or TG13) (Tables 1 and 3). Login to comment 108 ABCC7 p.Arg75Gln Risk Analysis of Individual CFTR Mutations Recognizing that many single nucleotide polymorphisms in CFTR may be physiologically harmless and not cause disease, we calculated the individual risks of our most commonly identified CFTR variants p.F508del, p.R75Q, c.1584GtoA, and IVS8 T5 for their effects on ICP (see Table 3) regardless of SPINK1 status. Login to comment 111 ABCC7 p.Arg75Gln The common CFTR variant p.R75Q, however, was significantly overrepresented in patients vs controls (OR, 3.4; P ϭ .001). Login to comment 112 ABCC7 p.Arg75Gln Combined Effect of CFTR and SPINK1 Variants We observed a striking increase in risk of pancreatitis by comparing the expected with the observed frequency of combined SPINK1 mutations with either CFTR p.F508del or p.R75Q (OR, 84.4; 95% CI, 35.8-199; P ϽϽ .0001; Table 3). Login to comment 115 ABCC7 p.Arg75Gln When analyzed individually, the c.1584GtoA mutation did not confer significant risk either with or without a corresponding SPINK1 mutation, while the CFTR p.R75Q mutant conferred a significant risk of pancreatitis both when considered individually and with a concurrent SPINK1 mutation (OR, 3.4 and 25.1; Table 3). Login to comment 116 ABCC7 p.Arg75Gln Given the carrier frequency of the aforementioned mutations p.F508del and p.R75Q, the expected frequency of a compound CFTR/SPINK1 genotype is approximately 2 in 1000. Login to comment 117 ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln Of all 525 controls, two subjects carried mutations in both CFTR and SPINK1 concurrently (p.N34S/ p.R75Q and p.P55S/p.R75Q). Login to comment 118 ABCC7 p.Arg75Gln The p.P55S/p.R75Q healthy carrier was a 32 year old female non-smoker and non-drinker. Login to comment 119 ABCC7 p.Arg75Gln A further review of the medical history of the control carrying both the SPINK1 p.N34S and CFTR p.R75Q mutations revealed recurrent abdominal pain requiring hospitalization and abdominal surgery (cholecystectomy). Login to comment 121 ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln Anion Transport By p.R75Q CFTR in HEK 293 Cells To assess the physiological properties of the p.R75Q variant, we stably expressed mutant and WT CFTR in HEK293 cells and tested the relative conductances of each CFTR protein to HCO3 - and Cl-. Login to comment 123 ABCC7 p.Ile148Thr ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg668Cys ABCC7 p.Thr1086Ala ABCC7 p.Cys76Trp ABCC7 p.Asn1432Lys Total CFTR Sequencing Results of Healthy Controls CFTR mutations SPINK1 mutations 1 -/- N34S/- 2 1584GtoA/-a N34S/- 3 F508del/-a -/- 4 F508del/-a -/- 5 G576AϩR668C/- -/- 6 IVS8 T5-TG12/-a -/- 7 IVS8 T5/TG12/- -/- 8 R75Q/-a -/- 9 R75Q/-a -/- 10 R75Q/-a -/- 11 R75Q/-a -/- 12 R75Q/-a -/- 13 R75Q/-a -/- 14 R75Q/-a -/- 15 R75Q/-a -/- 16 R75Q/-a -/- 17 9CtoT/- -/- 18 C76W/-a -/- 19 T1086A/- -/- 20 R668C/- -/- 21 N1432K/- -/- 22 I148T/- -/- 23 2657ϩ22GtoA/- -/- 24-95 -/- -/- NOTE. Login to comment 126 ABCC7 p.Arg75Gln p.R75Q and studied under conditions where chloride or HCO3 are the major permeant anions. Login to comment 127 ABCC7 p.Arg75Gln Currents in Cl- media at -60 mV for CFTR WT and p.R75Q were not significantly different (mean, -37.6 vs -28.6; P ϭ .3) (Figure 2C). Login to comment 128 ABCC7 p.Arg75Gln When recorded using HCO3 - solutions in the pipette and bath, the current for CFTR WT was significantly less than in Cl- media but significantly greater than that of the p.R75Q variant (mean, -8.23 vs -1.53; P ϭ .0001). Login to comment 130 ABCC7 p.Arg75Gln Viewed in another way (Figure 2C), the current ratio in Cl- media for p.R75Q/WT was Table 3. Login to comment 131 ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln Statistical Analysis of SPINK1 and CFTR Genotyping Data CFTRa SPINK1b Patient carriers Patient noncarriers Control carriersc Control noncarriersc OR 95% confidence interval P value SPINK1 only Mut/any 29 51 6 145 16.5 6.1-44.9 ϽϽϽ.0001 Mut/Mut 7 73 9d 9991d 106.5 38.6-293.5 ϽϽ.0001 CFTR only, full sequencing results All variants 20 9 22 73 7.4 2.3-18.5 Ͻ.0001 CF severe 5 24 2 93 9.7 1.8-53.0 .002 CF mild 5 24 3 92 6.4 1.4-28.6 .007 Other 2 27 7 88 0.9 0.2-4.8 .93 CFTR analysis of individual mutations F508del/any 7 73 12 513 4.10 1.6-10.7 .007 R75Q/any 13 67 28 523 3.44 1.7-7.0 .0001 1584GtoA/any 7 73 5 145 2.8 0.9-9.1 .12 IVS8 T5-TG12/any 4 76 4 146 1.9 0.5-7.9 .45 Combined effects of SPINK1 and CFTR mutations F508del/any Mut/any 4 76 7d 9993d 75.1 21.5-176.9 .0003 R75Q/any Mut/any 7 73 2 523 25.1 5.1-123.0 Ͻ.0001 1584GtoA/any Mut/any 5 75 3 147 3.27 0.76-14.0 .09 IVS8 T5-TG12/any Mut/any 1 79 9d 9991d 14.0 1.7-112.2 .348 aCFTR genotypes categorized according to severity of CFTR phenotype.42,43 bSPINK1 N34S and/or P55S heterozygous/homozygous/compound carriers all considered (Mut/any). Login to comment 136 ABCC7 p.Ile148Thr ABCC7 p.Arg560Thr ABCC7 p.Arg75Gln ABCC7 p.Met952Thr ABCC7 p.Phe508Cys ABCC7 p.Arg668Cys ABCC7 p.Ile807Met ABCC7 p.Thr1086Ala ABCC7 p.Cys76Trp ABCC7 p.Asn1432Lys CFTR Mutation Class Types and Corresponding Disease Severity CFTR mutation Exon CF mutation class Disease association % Carriers, case (n) % Carriers, controls (n) p.R75Q 3 "CP" 16.2 (80) 5.3 (525) c.1584GtoA (p.E528E) 10 "CP" 8.7 (80) 3.3 (150) p.F508del 10 II CF severe 8.7 (80) 2.3 (525) p.R560T 11 II CF severe 3.4 (29) 0 (95) IVS8 T5/TG12or13 i8 V CF mild 5.0 (80) 2.7 (150) p.F508C 10 CF mild 1.2 (80) 0 (150) p.I807M 13 CF mild 3.4 (29) 0 (95) p.D443YϩG576AϩR668Ca 9;12;13 CF mild 3.4 (29) 0 (95) p.G576AϩR668Ca 12;13 CF mild 0 (29) 1 (95) p.M952T 15 CF mild 3.4 (29) 0 (95) p.R668C 13 Other 0 (29) 1 (95) c.3139ϩ42AtoT i17a Other 3.4 (29) 0 (95) p.N1432K 24 Other 0 (29) 1 (95) c.-9CtoT 1 Other 0 (29) 1 (95) p.C76W 3 Other 0 (80) 0.7 (150) p.I148T 4 Other 0 (29) 1 (95) c.2657ϩ22GtoA i14b Other 0 (29) 1 (95) p.T1086A 17b Other 0 (29) 1 (95) NOTE. Login to comment 140 ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln 0.76, which was not statistically different from 1.0; however, the current ratio in bicarbonate media for p.R75Q/WT was 0.18 and the HCO3/Cl current ratio for p.R75Q was 0.053, 4 times lower than that for CFTR WT. Login to comment 141 ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln Discussion In the present study of ICP in patients without clinical evidence of CF, we confirmed that the combination of trans-heterozygous CFTR and SPINK1 variants markedly increases the risk of pancreatitis, as first observed by Noone et al.33 However, we show for the first time a very high risk of pancreatitis in patients with specific CFTR mutations (p.R75Q, p.F508del) and show that CFTR bicarbonate conductance is specifically impaired in the relatively common CFTR variant p.R75Q. Login to comment 148 ABCC7 p.Arg75Gln The R75Q mutation results in a selective decrease in CFTR HCO3 conductance. Login to comment 151 ABCC7 p.Arg75Gln (B) Whole-cell I-V relations from HEK cells stably expressing R75Q CFTR. Login to comment 155 ABCC7 p.Arg75Gln Cl media; **additional significant difference between currents in HCO3 media comparing WT and R75Q CFTR expressing cells. Login to comment 157 ABCC7 p.Arg75Gln The CFTR p.R75Q variant was associated with a high risk of pancreatitis in the presence of a SPINK1 mutation in the present study, and it was also observed in the series reported by Noone et al,33 Weiss et al,36 and Tzetis et al34 in patients with CP. Login to comment 158 ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln Although this variant has been investigated previously for causing CF,28 a number of researchers have determined that CFTR p.R75Q is processed and matures much like CFTR WT in cells, and physiological studies show no gating or Cl-channel dysfunction.39 This type of data strengthens the argument that p.R75Q is a sequence variant not contributing to the autosomal recessive disorder CF28 (www.genet. Login to comment 160 ABCC7 p.Arg75Gln However, the fact that CFTR p.R75Q is repeatedly reported as a CFTR variant in a number of patients with atypical CF32 and CF-related disorders such as sarcoidosis,29 chronic obstructive pulmonary disease,30 and CP31 suggests that normal function is disrupted in some way. Login to comment 161 ABCC7 p.Arg75Gln Here we show for the first time that p.R75Q alters bicarbonate but not chloride conductance. Login to comment 162 ABCC7 p.Arg75Gln This is consistent with our CP model that recognizes CFTR as a pancreatic duct cell bicarbonate channel and predicts that CFTR mutations disrupting bicarbonate conductance will markedly increase the risk of pancreatic disease either through total disruption of protein processing (eg, p.F508del) or alteration in channel properties (eg, p.R75Q).16 This finding also suggests that there may be additional CFTR mutations that specifically disrupt bicarbonate conductance and thus are also specific risk factors for CP but not CF. Login to comment 164 ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln Our data suggest that the correlation of the relatively common CFTR variant p.R75Q with a SPINK1 mutation increases the risk of CP in a multiplicative manner (SPINK1 alone OR, 18.1; p.R75Q alone OR, 3.4; combined with SPINK1: OR, 25.1; Table 3), whereas that of heterozygous CFTR p.R75Q or CFTR p.F508del variants becomes insignificant in a SPINK1 WT background. Login to comment