ABCMdb

PMID: 25033378

LaRusch J, Jung J, General IJ, Lewis MD, Park HW, Brand RE, Gelrud A, Anderson MA, Banks PA, Conwell D, Lawrence C, Romagnuolo J, Baillie J, Alkaade S, Cote G, Gardner TB, Amann ST, Slivka A, Sandhu B, Aloe A, Kienholz ML, Yadav D, Barmada MM, Bahar I, Lee MG, Whitcomb DC
Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.
PLoS Genet. 2014 Jul 17;10(7):e1004376. doi: 10.1371/journal.pgen.1004376. eCollection 2014 Jul., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Arg117His ABCC7 p.Asp1270Asn ABCC7 p.Arg75Gln ABCC7 p.Ser1235Arg ABCC7 p.Asp1152His ABCC7 p.Leu997Phe ABCC7 p.Arg170His ABCC7 p.Leu967Ser ABCC7 p.Arg74Gln Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Login to comment 39 ABCC7 p.Arg75Gln Recently, we reported that the variant CFTR R75Q, which was previously classified as benign, is associated with familial and sporadic chronic pancreatitis, either with another CFTR variant (recessive) or with the serine protease inhibitor, Kazal Type 1 (SPINK1) N34S high-risk haplotype (complex genotype)[18]. Login to comment 40 ABCC7 p.Arg75Gln Patch-clamp studies of CFTR R75Q clones under standard conditions demonstrated normal chloride conductance but a selective disruption in bicarbonate conductance[18]. Login to comment 41 ABCC7 p.Arg75Gln Thus, CFTR R75Q causes selective bicarbonate defective (CFTRBD ) conductance and is associated with chronic pancreatitis but not CF[18]. Login to comment 62 ABCC7 p.Arg117His ABCC7 p.Asp1270Asn ABCC7 p.Arg75Gln ABCC7 p.Ser1235Arg ABCC7 p.Asp1152His ABCC7 p.Leu997Phe ABCC7 p.Arg170His ABCC7 p.Leu967Ser ABCC7 p.Arg74Gln Of 43 CFTR variants identified in the NAPS2 cohort (Table 1), nine not associated with typical CF but reported in patients with pancreatitis[25-29] were of particular interest: R74Q, R75Q, R117H (CFTRm-v only when in cis with IVS8-T5[30]; R117H*T5), R170H, L967S, L997F, D1152H, S1235R, and D1270N. Login to comment 66 ABCC7 p.Ile148Thr Other candidate CFTR variants, including I148T, M470V, T854T, Q1463Q and the ''5T`` allele, were either rare or were not associated with pancreatitis in our cohort (Table 1). Login to comment 71 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr I148T was seen in three cases and one control, so an effect could not be detected or excluded; the in cis deletion mutation 3199del6 was not detected in any I148T carriers. Login to comment 72 ABCC7 p.Arg1162* ABCC7 p.Gly1069Arg The IVS8T5 variant was identified in 9.9% of cases and 8.2% of controls, which is not individually significant. There were six N34S/T5 trans-heterozygote controls and no cases, but the combined effect of the SPINK1 N34S variant with IVS8T5 was not significantly higher than N34S alone. Four variants were identified in only one patient and no controls: CF mutations 2184delA, 3120+1G.A, R1162X, and mutation of varying clinical consequence, G1069R. Login to comment 73 ABCC7 p.Arg117His Functional assays on CFTR variants For our functional studies, we cloned the nine CFTR variants and confirmed that they had normal folding, glycosylation (Figure 1a) and chloride channel activities, except for R117H (Figure 1b). Login to comment 78 ABCC7 p.Arg170His In contrast, CFTR PHCO3/PCl failed to increase in CFTR R170H (Figure 1d) and all of the candidate CFTRBD variants (Figures 1e and S2). Login to comment 79 ABCC7 p.Asp1270Asn Furthermore, all CFTRBD candidate variants lowered bicarbonate conductance (GHCO3/ GCl), which is an important metric determining apical bicarbonate efflux in CFTR-expressing epithelia (Figure 1f); the decrease was statistically significant for all variants except D1270N. Login to comment 95 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Trp1282* ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Arg1162* ABCC7 p.Asp1270Asn ABCC7 p.Arg75Gln ABCC7 p.Ser1235Arg ABCC7 p.Asp1152His ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Leu997Phe ABCC7 p.Arg170His ABCC7 p.Leu967Ser ABCC7 p.Arg74Gln CFTR variant %Cases %Uctrls OR p-value %Cases w/N34S OR w/N34S p-value w/N34S CF/BD or BD/BD 2.5 0.1 31.9 ,0.0001 5.5 7.46 0.12 All CF 8.7 3.3 2.76 ,0.0001 16.4 5.65 ,0.0001 F508del CF 6.9 3.1 2.32 ,0.0001 14.5 5.13 ,0.0001 IVS8T5** CF 9.9 8.2 1.24 0.079 10.9 1.37 0.47 2789+5G.A CF 0.3 0.0 0.028 0.0 3849+10kbC.T CF 0.3 0.0 0.028 0.0 N1303K CF 0.3 0.0 0.027 0.0 621+1G.T CF 0.1 0.0 0.13 1.8 ,0.0001 2184delA CF 0.1 0.0 0.13 0.0 3120+1G.A CF 0.1 0.0 0.13 0.0 G551D CF 0.2 0.1 2.50 0.20 0.0 0.00 0.83 W1282X CF 0.2 0.1 2.50 0.20 0.0 0.00 0.83 G542X CF 0.2 0.0 0.059 0.0 R1162X CF 0.1 0.0 0.13 0.0 2183AA.G CF 0.0 0.1 0.17 0.0 0.00 0.83 All BD 14.2 9.8 1.50 0.002 25.5 4.63 ,0.0001 R75Q BD 6.3 6.2 1.02 0.30 16.4 2.97 0.003 S1235R BD 2.4 1.4 1.69 0.052 1.8 1.30 0.80 R117H CF/BD 2.3 0.7 3.49 0.0007 5.5 8.74 0.0002 L967S BD 1.1 0.2 6.87 0.002 1.8 11.17 0.014 L997F BD 0.8 1.0 0.82 0.26 1.8 1.84 0.55 D1152H BD 0.4 0.0 0.014 0.0 D1270N BD 0.3 0.2 1.25 0.29 0.0 0.00 0.71 R170H BD 0.3 0.0 0.028 0.0 R74Q BD 0.3 0.1 3.02 0.17 1.8 21.15 0.002 Other M470V 76.1 74.2 1.11 0.14 70.9 0.85 0.59 T854T 57.3 57.8 0.98 0.29 45.5 0.61 0.071 Q1463Q 39.6 39.5 1.01 0.30 40.0 1.02 0.94 1001+11C.T* 13.4 10.9 1.27 0.016 14.5 1.40 0.42 125G.C 10.3 9.7 1.07 0.26 12.7 1.36 0.45 P1290P 7.6 7.9 0.95 0.28 7.3 0.91 0.86 1716G.A 4.5 4.1 1.10 0.26 1.8 0.43 0.39 R668C 1.0 1.4 0.72 0.19 0.0 0.00 0.38 G576A 0.7 1.2 0.58 0.11 0.0 0.00 0.41 computationally modeled the molecular structure, and studied the dynamics, of wild type (WT) and mutated CFTR channels. Login to comment 102 ABCC7 p.Asp1152His ABCC7 p.Asp1152His ABCC7 p.Leu997Phe ABCC7 p.Leu997Phe MD simulations comparing the channel diameters of the WT and mutants L997F and D1152H (Figure 2c-f) demonstrate that the channel diameter is observed to narrow down from an average value of 10.3 A da; to 7.5 A da; (standard deviation, s = 0.5 A da; ) at the pore region, near the L997F amino acid substitution (Figure 2e), and from an average of 9.9 A da; to 4.3 A da; (s = 1.1 A da; ) for the CFTRBD mutant D1152H (Figure 2f). Login to comment 103 ABCC7 p.Asp1152His ABCC7 p.Leu997Phe Note that in contrast to the WT CFTR and L997F mutant where the structure maintains its stability, the D1152H mutation induces significant fluctuations in local conformation, which are reflected on the changes in the pore diameter at this location within the channel. Login to comment 116 ABCC7 p.Arg74Trp ABCC7 p.Ile148Thr ABCC7 p.Arg258Gly ABCC7 p.Phe1052Val ABCC7 p.Phe508Cys ABCC7 p.Arg31Cys ABCC7 p.Arg297Gln ABCC7 p.Ile1027Thr ABCC7 p.Arg1162Leu ABCC7 p.Val201Met ABCC7 p.Gly1069Arg ABCC7 p.Ile807Met CFTR variant %Cases %Uctrls OR p-value %Cases w/N34S OR w/N34S p-value w/N34S F508C 0.5 0.3 1.58 0.21 0.0 0.00 0.67 R1162L 0.5 0.5 1.13 0.29 1.8 4.03 0.17 I1027T 0.5 0.3 1.99 0.17 0.0 0.00 0.70 R31C 0.3 0.7 0.42 0.088 0.0 0.00 0.52 I148T 0.3 0.4 0.75 0.27 0.0 0.00 0.63 R297Q 0.3 0.2 1.89 0.21 0.0 0.00 0.76 R74W 0.2 0.2 0.85 0.29 0.0 0.00 0.71 F1052V 0.1 0.2 0.63 0.27 0.0 0.00 0.76 I807M 0.1 0.1 1.26 0.30 0.0 0.00 0.83 R258G 0.1 0.1 1.26 0.30 0.0 0.00 0.83 G1069R 0.1 0.0 0.13 0.0 V201M 0.0 0.1 0.17 0.0 0.00 0.83 Of the 81 CFTR mutations tested in the cohort, 43 were observed at least once in cases or controls. Login to comment 119 ABCC7 p.Arg117His Blank cells indicate undefined (e.g. x40) **IVS8 T5 is reported but causes CF only when in cis with either R117H or IVS8 TG12or13. Intronic mutations are reported in standard nomenclature ''####+/2##N.N`` except IVS8-T5 (1210-12T[5]). Login to comment 124 ABCC7 p.Arg117His ABCC7 p.Gly542* ABCC7 p.Arg75Gln ABCC7 p.Ser1235Arg ABCC7 p.Asp1152His ABCC7 p.Leu997Phe ABCC7 p.Leu967Ser We identified the R75Q, R117H, L967S, L997F, D1152H, and S1235R CFTRBD variants as well as CFTRCF -associated variants (e.g., F508del, G542X) in cases with rhinosinusitis. Login to comment 129 ABCC7 p.Arg117His ABCC7 p.Gly542* ABCC7 p.Arg75Gln ABCC7 p.Ser1235Arg We identified R75Q, R117H, and S1235R as well as the CFTRCF variants F508del, G542X and 2789+5G,A in male cases with infertility. Login to comment 158 ABCC7 p.Arg170His Whole-cell currents of R170H-CFTR were measured in HEK 293T cells using the same protocol shown in panel c. Login to comment 170 ABCC7 p.Arg75Gln ABCC7 p.Arg1162Leu ABCC7 p.Arg170His ABCC7 p.Leu967Ser ABCC7 p.Arg74Gln Five variants (R74Q, R75Q, R170H, L967S, and R1162L) were located in the hinge region that modulates the collective movements of the NBDs with respect to the MSDs (Figure 3). Login to comment 171 ABCC7 p.Arg74Gln ABCC7 p.Arg74Gln R74Q was previously reported in a single chronic pancreatitis patient [53] but not in the CFTR2 database. CFTR R74Q was identified by us in two cases and no controls (p = ns) and in one case who was a SPINK1 N34S carrier (p = 0.006). Login to comment 172 ABCC7 p.Arg75Gln R75Q is considered to be a non-CF causing mutation according to the CFTR2 mutation database [54]. Login to comment 173 ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln CFTR R75Q was identified in 61/906 cases and 75/1214 controls (6.3 vs. 6.2%, p = ns) but was also detected in nine SPINK1 N34S/- mutation carriers (9/55, 16.4%), with strong combined effect (SPINK1 OR 3.7, SPINK1+ R75Q compound OR 12.2, p 0.002). Login to comment 175 ABCC7 p.Arg75Gln R75Q was also identified in four cases with a concurrent severe CF-causing mutation and in no compound controls. Login to comment 176 ABCC7 p.Arg170His R170H was first reported Figure 3. Login to comment 188 ABCC7 p.Asp1152His Panel c shows the charge distribution around D1152H: this negatively charged residue (left; shown in red space-filling representation) is surrounded by several positively charged residues (green), especially on its side of the cavity, creating an attractive force that keeps the residue from extending into the cavity. Login to comment 190 ABCC7 p.Asp1152His Panel d shows the corresponding scene for the variant residue, D1152H (cyan), which can move toward the center of the cavity, thus leading to a constriction in the channel diameter. Login to comment 192 ABCC7 p.Asp1152His On panel f, the same information for the WT and variant D1152H is shown. Login to comment 195 ABCC7 p.Arg170His doi:10.1371/journal.pgen.1004376.g002 in two cases of congenital bilateral aplasia of vas deferens in England [53] but is not currently in the CFTR2 mutation database. CFTR R170H was identified in three cases and no controls (p = ns). Login to comment 196 ABCC7 p.Leu967Ser L967S has been reported in a single case of azoospermia from the CF mutation database [53] but is not in the CFTR2 mutation database. Login to comment 197 ABCC7 p.Leu967Ser L967S was identified in ten cases (one trans-heterozygote), two controls (OR 6.9 p = 0.004), and one N34S case carrier. Login to comment 198 ABCC7 p.Arg1162Leu R1162L is predicted to be a highly deleterious variant by SIFT and damaging by PolyPhen modeling [55] and is included in the CFTR2 mutation database and classified as a variant not causing CF. Login to comment 199 ABCC7 p.Asp1152His ABCC7 p.Leu997Phe Although located in a critical portion of the CFTR molecule, the association and functional threshold for inclusion as a CFTRBD variant were not fully met. Two variants (L997F and D1152H) appeared to reduce channel diameter. Login to comment 200 ABCC7 p.Leu997Phe L997F is considered a mutation of varying clinical consequences for CF, with low rates of pancreatic insufficiency and retention of chloride conductance [54]. Login to comment 201 ABCC7 p.Leu997Phe ABCC7 p.Leu997Phe In this study L997F was identified both in the cases (0.7%) and controls (1.0%), additionally, L997F was identified in one N34S case carrier and three compound heterozygous mutation case carriers, but independent statistical association with pancreatitis was not demonstrated in this study. Login to comment 202 ABCC7 p.Asp1152His D1152H is a mutation of varying clinical consequence for CF and is associated with low rates of pancreatic insufficiency and retention of chloride conductance [53]. Login to comment 203 ABCC7 p.Asp1152His CFTR D1152H was identified in four cases and no controls (p = 0.014). Login to comment 205 ABCC7 p.Asp1270Asn Two variants (S1235 and D1270N) were on the surface of NBD2 (Figure 2). Login to comment 207 ABCC7 p.Ser1235Arg While this did not reach statistical significance in this cohort, multiple previous reports of CFTR S1235R in idiopathic pancreatitis patients[56,57] and complex functional features [27] were noted. Login to comment 208 ABCC7 p.Asp1270Asn D1270N is of varying clinical consequences for CF, with low rates of pancreatic insufficiency and retention of chloride conductance [54]. Login to comment 209 ABCC7 p.Asp1270Asn D1270N was identified both in the cases (0.3%) and controls (0.2%). Login to comment 212 ABCC7 p.Arg117His The final variant (R117H) is located in an extracellular domain and has functional effects beyond the other CFTRBD variants. Login to comment 213 ABCC7 p.Arg117His R117H is a complex variant that is associated with CF only when found in cis with a T5 tract in intron 8. Login to comment 214 ABCC7 p.Arg117His ABCC7 p.Arg117His The CFTR R117H variant was identified in 22 cases (2.3%) and 8 controls (0.7%) (p = 0.001), with only 3 cases and 1 control having the CF-associated R117H*T5 haplotype (p = ns), which links the CFTR variant R117H to pancreatitis regardless of the intron 8 T5 haplotype. Login to comment 216 ABCC7 p.Arg117His The R117H variant was the only one with reduced chloride current density (Figure 1b). Login to comment 224 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr I148T was seen in three cases and one control, so an effect could not be detected or excluded; the in cis deletion mutation 3199del6 was not detected in any I148T carriers. Login to comment 225 ABCC7 p.Arg1162* ABCC7 p.Gly1069Arg The IVS8T5 variant was identified in 9.9% of cases and 8.2% of controls, which is not individually significant. There were six N34S/T5 trans-heterozygote controls and no cases, but the combined odds ratio (OR 3.9) of the SPINK1 N34S variant with IVS8T5 was not significantly higher than N34S alone. Four additional variants were identified in only one patient and no controls: CF mutations 2184delA, 3120+1G.A, R1162X and a mutation of varying clinical consequence, G1069R. Login to comment 228 ABCC7 p.Arg117His In addition, other possible mechanisms of CFTR channel dysfunction linked to altered bicarbonate conductance are possible, such as mechanisms linked to CFTR R117H. Login to comment 237 ABCC7 p.Asp1152His ABCC7 p.Leu997Phe In particular, the L997F and D1152H mutants showed channel pore size reductions in their neighborhoods that would directly affect conductance properties. Login to comment 248 ABCC7 p.Leu997Phe A decrease in the CFTR pore diameter, as shown in L997F, can affect the permeability of HCO3 2 in many ways, such as by limiting the accessibility of large, asymmetrically charged HCO3 2 to the channel pore. Login to comment 269 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ala455Glu ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Arg1162* ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp ABCC7 p.Arg352Gln ABCC7 p.Ile148Thr ABCC7 p.Gly85Glu ABCC7 p.Arg560Thr ABCC7 p.Arg75Gln ABCC7 p.Pro67Leu ABCC7 p.Met952Thr ABCC7 p.Arg258Gly ABCC7 p.Thr338Ile ABCC7 p.Leu1065Pro ABCC7 p.Arg1070Gln ABCC7 p.Ser1235Arg ABCC7 p.Ser945Leu ABCC7 p.Asp1152His ABCC7 p.Met952Ile ABCC7 p.Phe1052Val ABCC7 p.Phe508Cys ABCC7 p.Arg668Cys ABCC7 p.Gly576Ala ABCC7 p.Arg31Cys ABCC7 p.Asp443Tyr ABCC7 p.Arg117Cys ABCC7 p.Arg297Gln ABCC7 p.Leu997Phe ABCC7 p.Ile1027Thr ABCC7 p.Arg1162Leu ABCC7 p.Arg170His ABCC7 p.Val201Met ABCC7 p.Phe1074Leu ABCC7 p.Ile336Lys ABCC7 p.Leu967Ser ABCC7 p.Gly1069Arg ABCC7 p.Ser492Phe ABCC7 p.Gly178Arg ABCC7 p.Asp110His ABCC7 p.Ser977Phe ABCC7 p.Asp579Gly ABCC7 p.Ile807Met ABCC7 p.Arg74Gln ABCC7 p.Ile1131Leu ABCC7 p.Lys1180Thr ABCC7 p.Ser485Arg 67 SNPs (125GtoC, 1716G.A, 1717-1G.A, 1898+1G.A, 2183AA.G, 2184delA, 2789+5G.A, 3120+1G.A, 3659delC, 3849+10kbC.T, 621+ 1G.T, 711+5G.A, A455E, D110H, D1152H, D1270N, D443Y, D579G, F1052V, F1074L, F508C, F508del, G1069R, G1244E, G1349D, G178R, G542X, G551D, G551S, I1131L/V, I148T, I336K/T, I507del, I807M, IVS8T5, K1180T, L1065P, L967S, L997F, M1V, M470V, M952I, M952T, N1303K, P67L, Q1463Q, R1070Q, R1162X, R117C, R117H, R170H, R258G, R297Q, R31C, R352Q, R553X, R668C, R74W, R75Q, S1235R, S1255P, S485R, S977F, T338I, T854T, V201M, W1282X) were multiplexed into 6 wells; 14 SNPs (S492F, S945L, R74Q, R560T, R1162L, G85E, I1027T, R334W, R347P, G576A, 711+1G.T, 1001+11C.T, P1290P, 3199del6) were ascertained separately via TaqMan Gene Expression Assays, with repeat confirmation of all positive results. Login to comment 270 ABCC7 p.Ile148Thr 3199del6 was genotyped via TaqMan on all samples that tested positive for I148T. Login to comment 309 ABCC7 p.Asp1152His ABCC7 p.Leu997Phe Using this model for WT CFTR, we generated in silico models for the mutants L997F and D1152H. Login to comment 356 ABCC7 p.Arg117His *IVS8 T5 and R117H are reported but CF disease causing only when in cis with each other or IVS8 T5 with IVS8 TG12or13. Intronic mutations are reported in standard nomenclature ''####+/2##N.N`` except IVS8-T5, (1210-12T[5]). Login to comment