ABCMdb

PMID: 21708286

Fresquet F, Clement R, Norez C, Sterlin A, Melin P, Becq F, Kitzis A, Thoreau V, Bilan F
Orphan missense mutations in the cystic fibrosis transmembrane conductance regulator a three-step biological approach to establishing a correlation between genotype and phenotype.
J Mol Diagn. 2011 Sep;13(5):520-7. Epub 2011 Jun 25., [PubMed]

Sentences

No. Mutations Sentence Comment

34 ABCC7 p.Gly551Asp We previously used the expression of green fluorescent protein (GFP)-tagged CFTR in COS-7 cells to study interactions between CFTR and syntaxin 8.16 Moreover, we observed that wild-type (WT)-CFTR and well characterized mutants, p.Phe508del and p.Gly551Asp (where p. indicates protein sequence), exhibit expected intracellular trafficking and pharmacological profile in these cells.17 In the presence of each of the six rare missense mutations, CFTR cellular localization was determined by confocal microscopy using several markers: calreticulin, which is a marker of the endoplasmic reticulum (ER) and cortical actin, which delimits the plasma membrane. Login to comment 40 ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Val562Ile The exception was p.[Val562Ile] (V562I), which we encountered in two unrelated families with distinct genotypes. Login to comment 43 ABCC7 p.Leu102Pro Patient 1 was a 3-year-old girl when the CF diagnosis was established, with a genotype as follows: p.[Leu102Pro] ϩ [Arg553X]. Login to comment 47 ABCC7 p.Leu167Arg The CF diagnosis was confirmed by CFTR molecular analysis, with a genotype as follows: p.[Phe508del] ϩ [Leu167Arg]. Login to comment 50 ABCC7 p.Asn1303Lys ABCC7 p.Pro574Ser The genotype was as follows: p.[Asn1303Lys] ϩ [Pro574Ser]. Login to comment 53 ABCC7 p.Pro574Ser The two boys had the same genotype: p.[Phe508del] ϩ [Pro574Ser]); they presented with congenital bilateral aplasia of the vas deferens (CBAVD). Login to comment 56 ABCC7 p.Arg74Trp ABCC7 p.Pro841Arg CFTR mutational analysis revealed a complex genotype: p.[Arg74Trp;Val201Met;Asp1270Asn] ϩ [Pro841Arg]. Login to comment 60 ABCC7 p.Val562Ile ABCC7 p.Pro574Ser ABCC7 p.Pro841Arg ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg ABCC7 p.Lys696Arg Sequences of Site-Directed Mutagenesis Primers Mutation Sense oligonucleotide sequences L102P 5=-GTACAGCCTCTCTTACCGGGAAGAATCATAGCTTCC-3= L167R 5=-AAGAAGACTTTAAAGCGGTCAAGCCGTGTTCTAG-3= P574S 5=-GCTGATTTGTATTTATTAGACTCTTCTTTTGGATACCTAGATG-3= V562I 5=-AGAATTTCTTTAGCAAGAGCAATATACAAAGATGCTGATTTG-3= K696R 5=-CAGACTGGAGAGTTTGGGGAAAGAAGGAAGAATTCTATTCTC-3= P841R 5=-GATATGGAGAGCATACGAGCAGTGACTACATGG-3= was presented. Login to comment 61 ABCC7 p.Lys696Arg ABCC7 p.Lys696Arg The genotype was as follows: p.[Lys696Arg] (K696R). Login to comment 62 ABCC7 p.Lys696Arg ABCC7 p.Lys696Arg The genotype was as follows: p.Lys696Arg (K696R). Login to comment 66 ABCC7 p.Val562Ile [274-6TϾC(ϩ)1210-12T(5)(ϩ)1684GϾ A] (equaling V562I). Login to comment 67 ABCC7 p.Val562Ile ABCC7 p.Val562Ile [274-6TϾC(ϩ)1210-12T(5)(ϩ)1684GϾ A] [c.1684GϾA equaling p.Val562Ile (V562I)]. Login to comment 72 ABCC7 p.Leu102Pro ABCC7 p.Leu102Pro ABCC7 p.Leu102Pro ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg ABCC7 p.Leu167Arg ABCC7 p.Leu167Arg ABCC7 p.Leu167Arg ABCC7 p.Leu167Arg A calreticulin egremP201L calreticulin egremP201L L167R calreticulin mergeL167R calreticulin merge CB F508del L102P L167R WT F508del L102P L167R *** *** ***WT + inh172 L102P L167R *** *** ***WT + inh172 L102P L167R B C CFTR B C CFTR % of maximal activation 0 50 100 WT 0 50 100 WTNaKATPaseNaKATPase Figure 1. Login to comment 73 ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg L102P and L167R amino acid substitutions impair CFTR protein maturation. Login to comment 81 ABCC7 p.Asn1303Lys ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp ABCC7 p.Pro574Ser ABCC7 p.Pro841Arg ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg Summary of the Patients` Data, Concerning Genotype, Phenotype, and Protein Dysfunction Patient no./sex/age at molecular diagnostics (years) Genotype Phenotype Protein dysfunctions Channel activity* Maturation† Intracellular localization‡ 1/F/3 p.[Leu102Pro] ϩ [Arg553X] Positive sweat test result, bacterial lung colonization, no pancreatitis ϩϩ ϩϩ ϩϩ 2/F/newborn p.[Phe508del] ϩ [Leu167Arg] Positive sweat test result, recurrent pancreatitis, no lung infection ϩϩ ϩϩ ϩϩ 3/F/3 p.[Asn1303Lys] ϩ [Pro574Ser] Normal sweat test result, asymptomatic ϩϩ ϩ ϩ 4/M/31 p.[Arg74Trp;Val201Met; Asp1270Asn] ϩ [Pro841Arg]; c. Login to comment 82 ABCC7 p.Lys696Arg [1210-12T(7)] ϩ [1210-12T(7)] CBAVD ϩ Ø Ø 5/F/37 p.[Lys696Arg] Asymptomatic (prenatal CF diagnostics) ϩ Ø Ø 6/M/33 c. Login to comment 83 ABCC7 p.Val562Ile [274-6TϾC(ϩ)1210-12T(5) (ϩ)1684GϾA (ϭp.[Val562Ile])] Infertility, no CBAVD, previously treated bilateral cryptorchidism Ø Ø Ø In the genotype column, the amino acid substitutions that we investigated are boldfaced. Login to comment 95 ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg Results Defective Activation and Maturation of L102P and L167R CFTR Mutants Details of the six patients, their genotypes and phenotypes, and characteristics of their CFTR mutations are provided (Table 2). Login to comment 97 ABCC7 p.Val562Ile ABCC7 p.Pro574Ser ABCC7 p.Pro841Arg ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg ABCC7 p.Lys696Arg Sequences of Site-Directed Mutagenesis Primers Mutation Sense oligonucleotide sequences L102P 5=-GTACAGCCTCTCTTACCGGGAAGAATCATAGCTTCC-3= L167R 5=-AAGAAGACTTTAAAGCGGTCAAGCCGTGTTCTAG-3= P574S 5=-GCTGATTTGTATTTATTAGACTCTTCTTTTGGATACCTAGATG-3= V562I 5=-AGAATTTCTTTAGCAAGAGCAATATACAAAGATGCTGATTTG-3= K696R 5=-CAGACTGGAGAGTTTGGGGAAAGAAGGAAGAATTCTATTCTC-3= P841R 5=-GATATGGAGAGCATACGAGCAGTGACTACATGG-3= CFTR Missense Mutation Biological Assay 3 JMD Month 2011, Vol. xx, No. Login to comment 98 ABCC7 p.Leu102Pro ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg ABCC7 p.Leu167Arg x 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 AQ: 15 AQ: 16 AQ:17-18 AQ: 19 AQ: 20 T1 AQ: 21 AQ: 22 AQ: 23 AQ: 24 AQ: 25 AQ: 26 AQ: 27 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 AQ: 28 AQ: 29 AQ: 30 T2 variants that we investigate herein, two affect membrane-spanning domain 1: p.[Leu102Pro] (L102P) and p.[Leu167Arg] (L167R). Login to comment 100 ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg Thus, L102P and L167R mutants appear unable to escape the ER. Login to comment 102 ABCC7 p.Pro574Ser ABCC7 p.Pro574Ser ABCC7 p.Pro574Ser ABCC7 p.Pro574Ser ABCC7 p.Pro574Ser ABCC7 p.Pro574Ser Fluorescence was examined with a spectral confocal station FV-1000 in- B A calreticulin egremS475P calreticulin egremS475P WT F508del P574S CFTR C WT F508del P574S CFTR C B CFTR NaKATPase B CFTR NaKATPase C WT P574S *** WT P574S ***** 00 WT P574S 0 00 00 00 00 **** 00 WT P574S 0 00 00 00 00 0 50 100 WT % of maximal activation 0 50 100 WT % of maximal activationB/(B+C) (%) 100 0 200 300 400 500 B/(B+C) (%) 100 0 200 300 400 500 Figure 2. Login to comment 103 ABCC7 p.Pro574Ser P574S amino acid substitution decreases CFTR protein maturation. Login to comment 104 ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg Although 100% activation is attributed to the maximal activation peak of WT-CFTR, activation is reduced to 7.45% Ϯ 3.76% (n ϭ 4) for L102P and to 27.84% Ϯ 1.96% (n ϭ 4) for L167R. Login to comment 109 ABCC7 p.Pro574Ser ABCC7 p.Pro574Ser ABCC7 p.Pro574Ser ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg ABCC7 p.Lys696Arg ABCC7 p.Lys696Arg ABCC7 p.Lys696Arg ABCC7 p.Lys696Arg ABCC7 p.Lys696Arg ABCC7 p.Lys696Arg ABCC7 p.Lys696Arg Mixed Phenotype with P574S CFTR Variant The missense substitution p.[Pro574Ser] (P574S) lies within nucleotide-binding domain 1. Login to comment 110 ABCC7 p.Pro841Arg ABCC7 p.Lys696Arg K696R and P841R amino acid substitutions affect CFTR channel activity. Login to comment 112 ABCC7 p.Pro574Ser Moreover, the P574S mutant does not fully colocalize with the ER marker and seems to escape this cellular compartment because a strong green stain can be observed, which clearly differs from the calreticulin red signal. Login to comment 113 ABCC7 p.Pro574Ser After immunoblotting (Figure 2B), we quantify the ratio of immature core-glycosylated CFTR/total CFTR [band B/(band B ϩ band C)]: attributing 100% to WT-CFTR, this ratio increases to 349.5% Ϯ 88% (n ϭ 3) for P574S. Login to comment 115 ABCC7 p.Pro574Ser Surprisingly, the iodide efflux assay exhibits a dramatic inhibition of CFTR channel activity: when 100% activation is attributed to the maximal activation peak of WT-CFTR, activation is reduced to 13.07% Ϯ 5.23% (n ϭ 4) for P574S (Figure 2C). Login to comment 116 ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Pro574Ser The lowered maturation of P574S suggests an attenuated class II mutation. Login to comment 117 ABCC7 p.Val562Ile The V562I amino acid substitution does not alter CFTR phenotype. Login to comment 118 ABCC7 p.Pro574Ser These data should be confronted to the patient genotype to evaluate the pathogenicity of the P574S mutation. Login to comment 119 ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg ABCC7 p.Lys696Arg ABCC7 p.Lys696Arg Decreased Activity of K696R and P841R CFTR Mutants Two missense mutations, K696R and p.[Pro841Arg] (P841R), are situated within the cytoplasmic regulator domain of CFTR. Login to comment 122 ABCC7 p.Asn1303Lys ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp ABCC7 p.Pro574Ser ABCC7 p.Pro841Arg ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg Summary of the Patients` Data, Concerning Genotype, Phenotype, and Protein Dysfunction Patient no./sex/age at molecular diagnostics (years) Genotype Phenotype Protein dysfunctions Channel activity* Maturation† Intracellular localization‡ 1/F/3 p.[Leu102Pro] ϩ [Arg553X] Positive sweat test result, bacterial lung colonization, no pancreatitis ϩϩ ϩϩ ϩϩ 2/F/newborn p.[Phe508del] ϩ [Leu167Arg] Positive sweat test result, recurrent pancreatitis, no lung infection ϩϩ ϩϩ ϩϩ 3/F/3 p.[Asn1303Lys] ϩ [Pro574Ser] Normal sweat test result, asymptomatic ϩϩ ϩ ϩ 4/M/31 p.[Arg74Trp;Val201Met; Asp1270Asn] ϩ [Pro841Arg]; c. Login to comment 123 ABCC7 p.Lys696Arg [1210-12T(7)] ϩ [1210-12T(7)] CBAVD ϩ Ø Ø 5/F/37 p.[Lys696Arg] Asymptomatic (prenatal CF diagnostics) ϩ Ø Ø 6/M/33 c. Login to comment 124 ABCC7 p.Val562Ile [274-6TϾC(ϩ)1210-12T(5) (ϩ)1684GϾA (ϭp.[Val562Ile])] Infertility, no CBAVD, previously treated bilateral cryptorchidism Ø Ø Ø In the genotype column, the amino acid substitutions that we investigated are boldfaced. Login to comment 133 ABCC7 p.Pro841Arg ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg ABCC7 p.Lys696Arg After using Western blot analysis (Figure 3B), the proportions of core-glycosylated protein for K696R and P841R appear insignificantly different from that of WT-CFTR (n ϭ 3, data not shown). Login to comment 134 ABCC7 p.Pro841Arg ABCC7 p.Leu102Pro ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg ABCC7 p.Leu167Arg ABCC7 p.Lys696Arg Moreover, we observe by iodide efflux experiments that both mutations have similar functional consequence on CFTR activity: the maximum activation is reduced to 49.67% Ϯ 2.61% (n ϭ 4) for K696R and to 45.10% Ϯ 4.94% (n ϭ 4) for P841R (Figure 3C). Login to comment 136 ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg Thus, L102P and L167R mutants appear unable to escape the ER. Login to comment 137 ABCC7 p.Val562Ile ABCC7 p.Val562Ile Normal Phenotype for the V562I CFTR Variant The controversial V562I substitution lies inside nucleotide-binding domain 1. Login to comment 139 ABCC7 p.Val562Ile By using Western blot analysis, the maturation process of V562I-CFTR appears similar to the one visualized for WT-CFTR (Figure 4B). Login to comment 140 ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg When 100% activation is attributed to the maximal activation peak of WT-CFTR, activation is reduced to 7.45% Ϯ 3.76% (n ϭ 4) for L102P and to 27.84% Ϯ 1.96% (n ϭ 4) for L167R. Login to comment 142 ABCC7 p.Val562Ile In our cell model, according to the three techniques that we used, V562I substitution behaves like a polymorphism with no evidence for cellular or functional impact. Login to comment 145 ABCC7 p.Val562Ile ABCC7 p.Pro574Ser ABCC7 p.Pro574Ser ABCC7 p.Pro574Ser This confirms that V562I substitution should be a polymorphism with no structural or functional impact on the CFTR protein. Login to comment 151 ABCC7 p.Pro574Ser ABCC7 p.Leu102Pro ABCC7 p.Leu102Pro ABCC7 p.Leu102Pro ABCC7 p.Leu102Pro ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg ABCC7 p.Leu167Arg ABCC7 p.Leu167Arg ABCC7 p.Leu167Arg ABCC7 p.Leu167Arg In our model, two missense mutations, L102P and A calreticulin egremP201L calreticulin egremP201L L167R calreticulin mergeL167R calreticulin merge CB F508del L102P L167R WT F508del L102P L167R *** *** ***WT + inh172 L102P L167R *** *** ***WT + inh172 L102P L167R B C CFTR B C CFTR % of maximal activation C. Login to comment 152 ABCC7 p.Pro574Ser After immunoblotting (Figure 2B), we quantify the ratio of immature core-glycosylated CFTR/total CFTR [band B/(band B ϩ band C)]: attributing 100% to WT-CFTR, this ratio increases to 349.5% Ϯ 88% (n ϭ 3) for P574S. Login to comment 154 ABCC7 p.Pro574Ser ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg L102P and L167R amino acid substitutions impair CFTR protein maturation. Login to comment 155 ABCC7 p.Pro574Ser The lowered maturation of P574S suggests an attenuated class II mutation. Login to comment 157 ABCC7 p.Pro574Ser These data should be compared to the patient genotype to evaluate the pathogenicity of the P574S mutation. Login to comment 158 ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg ABCC7 p.Lys696Arg ABCC7 p.Lys696Arg ABCC7 p.Lys696Arg Decreased Activity of K696R and P841R CFTR Mutants Two missense mutations, p.Lys696Arg (K696R) and p.Pro841Arg (P841R), are situated within the cytoplasmic regulator domain of CFTR. Login to comment 161 ABCC7 p.Pro574Ser ABCC7 p.Pro574Ser ABCC7 p.Pro574Ser ABCC7 p.Pro574Ser ABCC7 p.Pro574Ser ABCC7 p.Pro574Ser ABCC7 p.Pro841Arg ABCC7 p.Lys696Arg B A calreticulin egremS475P calreticulin egremS475P WT F508del P574S CFTR C WT F508del P574S CFTR C B CFTR NaKATPase B CFTR NaKATPase C WT P574S *** WT P574S ***** 00 WT P574S 0 00 00 00 00 **** 00 WT P574S 0 00 00 00 00 0 50 100 WT % of maximal activation 0 50 100 WT % of maximal activationB/(B+C) (%) 100 0 200 300 400 500 B/(B+C) (%) 100 0 200 300 400 500 Figure 2. Login to comment 162 ABCC7 p.Pro574Ser ABCC7 p.Pro841Arg ABCC7 p.Lys696Arg P574S amino acid substitution decreases CFTR protein maturation. Login to comment 165 ABCC7 p.Val562Ile ABCC7 p.Val562Ile Normal Phenotype for the V562I CFTR Variant The controversial V562I substitution lies inside nucleotide-binding domain 1. Login to comment 167 ABCC7 p.Val562Ile By using Western blot analysis, the maturation process of V562I-CFTR appears similar to the one visualized for WT-CFTR (Figure 4B). Login to comment 169 ABCC7 p.Leu167Arg x 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 F4 C O L O R 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 AQ: 33 F5 C O L O R L167R, exhibit an F508del-like phenotype: the proteins are blocked at the ER level, and their absence at the plasma membrane elicits reduced channel activity. Login to comment 170 ABCC7 p.Val562Ile In our cell model, according to the three techniques that we used, V562I substitution behaves like a polymorphism with no evidence for cellular or functional impact. Login to comment 172 ABCC7 p.Leu102Pro One of these patients, with severe respiratory tract symptoms and long-term lung colonization, was diagnosed as having CF; his genotype is p.[Leu102Pro] ϩ [Arg553X]. Login to comment 173 ABCC7 p.Val562Ile ABCC7 p.Leu102Pro This finding is in accordance with the dramatically reduced activity that we found for L102P-CFTR protein in cell culture. Login to comment 174 ABCC7 p.Leu167Arg Another patient, with chronic sinusitis and pancreatic failure, has the genotype p.[Leu167Arg] ϩ [Phe508del]. Login to comment 175 ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg The L167R allele exhibits, as described for L102P, a maturation and trafficking defect leading to a sharp decrease of CFTR activity compared with that of WT-CFTR. Login to comment 176 ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg Nevertheless, iodide efflux experiments on L167R exhibit a residual activity slightly more elevated (27.84%) than that for L102P (7.45%). Login to comment 179 ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg In our model, two missense mutations, L102P and L167R, exhibit an F508del-like phenotype: the proteins are blocked at the ER level, and their absence at the plasma membrane elicits reduced channel activity. Login to comment 181 ABCC7 p.Pro574Ser The results obtained for P574S are more complex to interpret. Login to comment 182 ABCC7 p.Leu102Pro One of these patients, with severe respiratory tract symptoms and long-term lung colonization, was diagnosed as having CF; his genotype is p.[Leu102Pro] ϩ [Arg553X]. Login to comment 183 ABCC7 p.Pro574Ser ABCC7 p.Leu102Pro ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg Contrary to L102P and L167R, P574S is not fully retained in the ER compartment because we can see the presence of the fully glycosylated mature form of CFTR by using Western blot analysis. Login to comment 184 ABCC7 p.Pro574Ser ABCC7 p.Leu167Arg This observation is sustained by confocal microscopic imaging clearly showing different staining patterns for P574S- CFTR and the ER resident calreticulin. Login to comment 185 ABCC7 p.Pro574Ser ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg Taken together, these results strengthen the idea that a small part of P574S could reach post-Golgi compartments and, thus, the plasma membrane. Login to comment 186 ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg Nevertheless, iodide efflux experiments on L167R exhibit a residual activity slightly more elevated (27.84%) than that for L102P (7.45%). Login to comment 187 ABCC7 p.Asn1303Lys ABCC7 p.Pro574Ser His genotype was p.[Asn1303Lys] ϩ [Pro574Ser], and the allele in cis is considered a CF-causing mutation.21 Five sweat test results were negative, and this patient is now a healthy 3-year-old girl, asymptomatic of CF disease. Login to comment 189 ABCC7 p.Pro574Ser Their genotype was p.[Phe508del] ϩ [Pro574Ser]. Login to comment 190 ABCC7 p.Pro574Ser These data clearly indicate that P574S induces a CF-related disorder phenotype (CBAVD) for males, consistent with a normal sweat test result and no symptoms of CF disease for the girl. Login to comment 191 ABCC7 p.Pro574Ser ABCC7 p.Pro574Ser However, if the results of our Western blot analysis indicate a mild phenotype, P574S-CFTR-impaired channel activation would suggest a severe CF phenotype. Login to comment 192 ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg ABCC7 p.Lys696Arg ABCC7 p.Lys696Arg ABCC7 p.Lys696Arg ABCC7 p.Lys696Arg ABCC7 p.Lys696Arg ABCC7 p.Lys696Arg ABCC7 p.Lys696Arg One possible explanation could be that iodide effluxes are not sensitive enough to precisely evaluate the CFTR chloride channel activation process; notably, CFTR channel open probability could not be studied directly by this A B C P841R actin merge +TO-PRO K696R actin merge +TO-PRO P841R actin merge +TO-PRO K696R actin merge +TO-PRO % of maximal activation ** ** 0 50 100 WT K696R P841R % of maximal activation ** ** 0 50 100 WT K696R P841R % of maximal activation ** ** 0 50 100 WT K696R P841R WT F508del P841R K696R B CFTR NaKATPase C WT F508del P841R K696R B CFTR NaKATPase C Figure 3. Login to comment 193 ABCC7 p.Pro574Ser ABCC7 p.Pro841Arg ABCC7 p.Leu102Pro ABCC7 p.Leu167Arg ABCC7 p.Lys696Arg K696R and P841R amino acid substitutions affect CFTR channel activity. Login to comment 194 ABCC7 p.Pro574Ser This observation is sustained by confocal microscopic imaging clearly showing different staining patterns for P574S- CFTR and the ER resident calreticulin. Login to comment 195 ABCC7 p.Pro574Ser Taken together, these results strengthen the idea that a small part of P574S could reach post-Golgi compartments and, thus, the plasma membrane. Login to comment 197 ABCC7 p.Asn1303Lys ABCC7 p.Pro574Ser His genotype was p.[Asn1303Lys] ϩ [Pro574Ser], and the allele in cis is considered a CF-causing mutation.21 Five sweat test results were negative, and this patient is now a healthy 3-year-old girl, asymptomatic of CF disease. Login to comment 199 ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Val562Ile ABCC7 p.Pro574Ser A B C V562I actin mergeV562I actin merge % of maximal activation ns 0 50 100 WT V562I % of maximal activation ns 0 50 100 WT V562I % of maximal activation ns 0 50 100 WT V562I B CFTR NaKATPase C WT F508del V562I B CFTR NaKATPase C WT F508del V562I Figure 4. Login to comment 200 ABCC7 p.Val562Ile ABCC7 p.Pro574Ser The V562I amino acid substitution does not alter CFTR phenotype. Login to comment 201 ABCC7 p.Pro574Ser However, if the results of our Western blot analysis indicate a mild phenotype, P574S-CFTR-impaired channel activation would suggest a severe CF phenotype. Login to comment 203 ABCC7 p.Pro574Ser Obviously, patch-clamp analysis would be useful to measure the real impact of P574S in CFTR channel gating. Login to comment 205 ABCC7 p.Pro841Arg ABCC7 p.Lys696Arg Our results suggest a mild phenotype for the K696R and P841R CFTR mutants: the trafficking and maturation rates appear normal, whereas activation is approximately half reduced. Login to comment 207 ABCC7 p.Pro574Ser ABCC7 p.Lys696Arg Obviously, patch-clamp analysis would be useful to measure the real impact of P574S in CFTR channel gating. Login to comment 209 ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg ABCC7 p.Lys696Arg Our results suggest a mild phenotype for the K696R and P841R CFTR mutants: the trafficking and maturation rates appear normal, whereas activation is approximately half reduced. Login to comment 210 ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp ABCC7 p.Pro841Arg His genotype was p.[Pro841Arg] ϩ [Arg74Trp;Val201Met; Asp1270Asn]. Login to comment 211 ABCC7 p.Pro841Arg ABCC7 p.Lys696Arg The variant K696R was isolated from a familial study, in which we did not find any other CFTR mutation. Login to comment 212 ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg Therefore, in the case of ICSI, it was decided by a pluridisciplinary antenatal staff that, if the resulting fetuses carried P841R and F508del alleles, this couple would be offered a therapeutic abortion, given the high risk of CF for the evaluated offspring.18 Our biological data are not fully in accordance with this conclusion because the P841R variant seems to not cause a severe CF phenotype because the CFTR protein could reach the plasma membrane and could be activated. Login to comment 213 ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg The P841R variant was carried by a patient with CBAVD, referred for pre-ICSI assessment; his spouse was heterozygous compound for p.[Phe508del]. Login to comment 214 ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp ABCC7 p.Val562Ile ABCC7 p.Pro841Arg His genotype was p.[Pro841Arg] ϩ [Arg74Trp;Val201Met; Asp1270Asn]. Login to comment 215 ABCC7 p.Pro841Arg The complex CFTR allele harboring three substitutions is considered responsible for a mild phenotype because a case of homozygosis has been reported for a patient exhibiting CBAVD, without severe CF features.22 Because the genotype of the patient results in CBAVD, we can deduce that P841R is either a mild or a severe mutation. Login to comment 216 ABCC7 p.Pro841Arg ABCC7 p.Pro841Arg Therefore, in the case of ICSI, it was decided by a pluridisciplinary antenatal staff that, if the resulting fetuses carried P841R and F508del alleles, this couple would be offered a therapeutic abortion, given the high risk of CF for the evaluated offspring.18 Our biological data are not fully in accordance with this conclusion because the P841R variant seems to not cause a severe CF phenotype because the CFTR protein could reach the plasma membrane and could be activated. Login to comment 217 ABCC7 p.Pro841Arg Taken together, these data strongly suggest that P841R is more likely to be a mild than a severe CF mutation. Login to comment 218 ABCC7 p.Val562Ile We found the V562I missense variation in a case of male infertility (genotype previously given). Login to comment 220 ABCC7 p.Val562Ile Our results are concordant with those previously obtained in baby hamster kidney cells overexpressing CFTR23 (by using Western blot analysis, iodide effluxes, and inside-out patch-clamp experiments in which the V562I mutant behaves like WT-CFTR). Login to comment 221 ABCC7 p.Val562Ile ABCC7 p.Val562Ile However, V562I, which was first reported as a polymorphism,24 has been reassigned as a non-CF-causing mutation, notably implicated in the CBAVD phenotype.20 Because the V562I-CFTR protein appears unaffected, the mutation could only elicit dysfunction at the RNA level. Login to comment 224 ABCC7 p.Val562Ile ABCC7 p.Pro574Ser Our results are concordant with those previously obtained in baby hamster kidney cells overexpressing CFTR23 (by using Western blot analysis, iodide effluxes, and inside-out patch-clamp experiments in which the V562I mutant behaves like WT CFTR). Login to comment 225 ABCC7 p.Val562Ile ABCC7 p.Val562Ile However, V562I, which was first reported as a polymorphism,24 has been reassigned as a non-CF-causing mutation, notably implicated in the CBAVD phenotype.20 Because the V562I-CFTR protein appears unaffected, the mutation could only elicit dysfunction at the RNA level. Login to comment 228 ABCC7 p.Pro574Ser Moreover, it appears that P574S, an allele exhibiting a lower severity for two of three criteria, is not a CF-causing mutation, despite strongly decreased estimated channel activity. Login to comment