ABCMdb

PMID: 25042876

Sharma H, Jollivet Souchet M, Callebaut I, Prasad R, Becq F
Function, pharmacological correction and maturation of new Indian CFTR gene mutations.
J Cyst Fibros. 2015 Jan;14(1):34-41. doi: 10.1016/j.jcf.2014.06.008. Epub 2014 Jul 16., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Ser549Asn ABCC7 p.Leu69His ABCC7 p.Gly126Ser ABCC7 p.Phe157Cys ABCC7 p.Phe87Ile ABCC7 p.Tyr852Phe ABCC7 p.Glu543Ala ABCC7 p.Asp1270Glu ABCC7 p.His139Gln ABCC7 p.Ser118Pro ABCC7 p.Phe494Leu Methods: We used Western blot, pharmacology and iodide efflux to study CFTR maturation and chloride transport in BHK cells expressing pEGFP-CFTR constructs for L69H, F87I, S118P, G126S, H139Q, F157C, F494L, E543A, S549N, Y852F and D1270E. Login to comment 5 ABCC7 p.Leu69His Results: Among these CFTR mutants, only L69H is not processed as a c-band and not functional at 37 &#b0;C. Login to comment 6 ABCC7 p.Ser549Asn ABCC7 p.Leu69His ABCC7 p.Leu69His However, the functions of L69H and S549N and the maturation of L69H are corrected at 27 &#b0;C and by the investigational drug VX809. Login to comment 8 ABCC7 p.Leu69His We identified L69H as a novel class II CF mutation. Login to comment 10 ABCC7 p.Ser549Asn ABCC7 p.Leu69His Keywords: Missense CF mutations; India; L69H-CFTR; S549N-CFTR; Low temperature; VX809 1. Login to comment 33 ABCC7 p.Ser549Asn ABCC7 p.Leu69His ABCC7 p.Gly126Ser ABCC7 p.Phe157Cys ABCC7 p.Phe87Ile ABCC7 p.Tyr852Phe ABCC7 p.Glu543Ala ABCC7 p.Asp1270Glu ABCC7 p.His139Gln ABCC7 p.Ser118Pro ABCC7 p.Phe494Leu Because the cellular and functional data on these mutations can improve CF genetic counseling, we examined here the functional and cellular consequences of eleven rare missense mutations, L69H, F87I, S118P, G126S, H139Q, F157C, F494L, E543A, S549N, Y852F and D1270E present in CFTR gene from both classical CF patients and CBAVD patients, which have been detected during molecular diagnosis of Indian CF patients (Fig. 1). Login to comment 38 ABCC7 p.Leu69His Except for L69H and F508del, the profiles of core-glycosylated and mature glycosylated forms were similar to that of WT-CFTR protein. Login to comment 39 ABCC7 p.Leu69His Mature glycosylated c-band was absent in L69H mutant expressing cells. Login to comment 44 ABCC7 p.Gly126Ser ABCC7 p.Phe157Cys ABCC7 p.Phe87Ile ABCC7 p.Tyr852Phe ABCC7 p.Glu543Ala ABCC7 p.Asp1270Glu ABCC7 p.His139Gln ABCC7 p.Ser118Pro ABCC7 p.Phe494Leu The activation of nine CFTR mutants F87I, S118P, G126S, H139Q, F157C, F494L, E543A, Y852F and D1270E was not significantly different from WT-CFTR (Fig. 3A for example of traces and Fig. 3B for a summary). Login to comment 45 ABCC7 p.Ser549Asn The response to agonists of S549N-CFTR mutant was reduced (Fig. 3A) compared to WT-CFTR (Fig. 3B). Login to comment 46 ABCC7 p.Leu69His The most dramatic effect was observed with the L69H-CFTR mutant, whose response to F + G was null (Fig. 3A) and not significantly different from F508del-CFTR (Fig. 3B). Login to comment 47 ABCC7 p.Leu69His This result is in agreement with the Western blot data showing lack of mature fully glycosylated c-band for L69H- and F508del-CFTR proteins. Login to comment 49 ABCC7 p.Ser549Asn ABCC7 p.Leu69His ABCC7 p.Gly126Ser ABCC7 p.Phe157Cys ABCC7 p.Phe87Ile ABCC7 p.Glu543Ala ABCC7 p.His139Gln ABCC7 p.Ser118Pro ABCC7 p.Phe494Leu Mutation Nucleotide change Location in CFTR Patient phenotype CFTR dysfunction L69H T to A at 338 N-terminal Patient 1: Pancreatic insufficient, sweat chloride N 60 mEq/L, S. aureus positive; Patient 2: CBAVD Defective CFTR maturation and channel activity, class-II CF mutation F87I T to A at 391 MSD1 CBAVD No dysfunction S118P T to C at 484 MSD1 CBAVD No dysfunction G126S G to A at 508 MSD1 CBAVD No dysfunction H139Q C to G at 549 MSD1 CBAVD No dysfunction F157C T to G at 602 MSD1 CBAVD No dysfunction F494L T to C at 1612 NBD1 CBAVD No dysfunction E543A A to C at 1760 NBD1 CBAVD No dysfunction S549N G to A at 1778 NBD1 Patient 1: Frequent respiratory infection. Login to comment 50 ABCC7 p.Tyr852Phe ABCC7 p.Asp1270Glu Patient 2: CBAVD Defective channel activity, class III mutation Y852F A to T at 2687 Cytoplasmic region between regulatory domain and MSD2 CBAVD No dysfunction D1270E T to A at 3942 NBD2 CBAVD No dysfunction MSD: membrane spanning domain, NBD: nucleotide binding domain, CBAVD: congenital bilateral absence of the vas deferens. Login to comment 57 ABCC7 p.Ser549Asn ABCC7 p.Leu69His Rescue by low temperature of L69H, F508del and S549N-CFTR mutants F508del protein is temperature sensitive, that is the mutant can be rescued from its abnormal ER location to the plasma membrane by lowering the temperature of the cells from 37 &#b0;C to 27 &#b0;C [10]. Login to comment 58 ABCC7 p.Ser549Asn ABCC7 p.Leu69His We studied the effect of low-temperature with cells expressing L69H and S549N compared to F508del-CFTR. Login to comment 61 ABCC7 p.Leu69His Immunoblots confirmed for L69H that at 27 &#b0;C the proteins acquired a mature state as c-band can be detected (Fig. 4C, lane 6) but not at 37 &#b0;C (Fig. 4C, lane 4). Login to comment 64 ABCC7 p.Leu69His ABCC7 p.Leu69His Rescue by the investigational drug VX809 of L69H and F508del The pharmacological corrector VX809 [11] was evaluated on L69H and compared to F508del. Login to comment 65 ABCC7 p.Leu69His The activation of L69H and F508del were both significantly corrected when cells were treated with VX809 (24 h, 10 bc;M, Fig. 4B). Login to comment 66 ABCC7 p.Ser549Asn ABCC7 p.Leu69His We also performed Western blot with L69H and F508del in the presence of VX809 but not with S549N because it is already mature (Fig. 2A). Login to comment 67 ABCC7 p.Leu69His Remarkably we detected mature c-band for L69H (Fig. 4C, lane 6) as with the low-temperature protocol. Login to comment 70 ABCC7 p.Ser549Asn ABCC7 p.Leu69His ABCC7 p.Gly126Ser ABCC7 p.Phe157Cys ABCC7 p.Phe87Ile ABCC7 p.Tyr852Phe ABCC7 p.Glu543Ala ABCC7 p.Asp1270Glu ABCC7 p.His139Gln ABCC7 p.Ser118Pro ABCC7 p.Phe494Leu Discussion The present study investigated the potential deleterious functional consequence of novel rare missense mutations 0 2 4 6 8 0.0 0.1 0.2 0.3 WT F87I S118P H139Q F157C NT Time (min) k (min -1 ) 0 2 4 6 8 0.0 0.1 0.2 0.3 G126S S549N Y852F WT F508del L69H Time (min) k (min -1 ) 0 2 4 6 8 0.0 0.1 0.2 0.3 WT F508del F494L D1270E NT E543A Time (min) k (min -1 ) W T F 8 7 I S 1 1 8 P G 1 2 6 S H 1 3 9 Q F 1 5 7 C F 4 9 4 L E 5 4 3 A Y 8 5 2 F D 1 2 7 0 E S 5 4 9 N L 6 9 H F 5 0 8 d e l 0.0 0.5 1.0 1.5 2.0 ns *** *** *** *** ns (k peak - k basal) mutant / (k peak - k basal) WT A B Fig. 3. Login to comment 72 ABCC7 p.Ser549Asn ABCC7 p.Leu69His ABCC7 p.Gly126Ser ABCC7 p.Phe157Cys ABCC7 p.Phe87Ile ABCC7 p.Tyr852Phe ABCC7 p.Glu543Ala ABCC7 p.Asp1270Glu ABCC7 p.His139Gln ABCC7 p.Ser118Pro ABCC7 p.Phe494Leu Iodide efflux experiments in transfected BHK-21 cells, WT-CFTR, L69H, F87I, S118P, G126S, H139Q, F157C, F494L, E543A, S549N, Y852F and D1270E. Login to comment 80 ABCC7 p.Ser549Asn ABCC7 p.Leu69His Functional rescue of L69H, F508del and S549N by low temperature and VX809. Login to comment 82 ABCC7 p.Ser549Asn ABCC7 p.Leu69His Iodide efflux experiments with BHK-21 cells transfected with L69H, S549N and F508del-CFTR as indicated. Login to comment 88 ABCC7 p.Leu69His ABCC7 p.Leu69His ABCC7 p.Leu69His Each experimental condition is indicated on each lane with WT-CFTR (lane 1), F508del (lane 2), F508del + VX809 (lane 3), L69H (lane 4), L69H + VX809 (lane 5) and L69H at 27 &#b0;C (lane 6). Login to comment 94 ABCC7 p.Leu69His Global view (a) of the model of the 3D structure of human CFTR (open channel), based on the experimental 3D structure of Sav1866, in an outward-facing conformation according to Mornon et al.[15], on which the mutation L69H is located (yellow square) MSD1 and 2 are membrane-spanning domains 1 and 2 (in blue and red, respectively). Login to comment 100 ABCC7 p.Ser549Asn ABCC7 p.Leu69His ABCC7 p.Gly126Ser ABCC7 p.Phe157Cys ABCC7 p.Phe87Ile ABCC7 p.Tyr852Phe ABCC7 p.Glu543Ala ABCC7 p.Asp1270Glu ABCC7 p.His139Gln ABCC7 p.Ser118Pro ABCC7 p.Phe494Leu In our study, the eleven CFTR mutants i.e. L69H, F87I, S118P, G126S, H139Q, F157C, F494L, E543A, S549N, Y852F and D1270E produced different results. Login to comment 101 ABCC7 p.Leu69His The first salient result is the fact that we identified L69H as a novel class II CF mutation. Login to comment 102 ABCC7 p.Leu69His The trafficking to the plasma membrane of L69H-CFTR is abnormal as corroborated by our Western blot analysis, which revealed only the presence of b-band. Login to comment 103 ABCC7 p.Leu69His Further confocal microscopy imaging showed the abundance of L69H mutated CFTR proteins in the ER and absence on the plasma membrane (data not shown). Login to comment 108 ABCC7 p.Leu69His Our results clearly indicated that the L69H mutant is responsive to VX809 as well as to rescue following low temperature incubation of cells. Login to comment 110 ABCC7 p.Leu69His We propose thus to categorize L69H as a class II form of CF mutation. Login to comment 111 ABCC7 p.Leu69His Interestingly, using the structural information provided by the model of the 3D structure of CFTR, based on the Sav1866 experimental 3D structure [15] (Fig. 4Ea), we evaluated the possible impact of L69H on CFTR (Fig. 4Eb). Login to comment 115 ABCC7 p.Leu69His The network formed by these hydrophobic amino acids in the cytosolic N-terminal extension (L69), in the intracellular loop ICL1 (F191) and in the MSD1-NBD1 linker (I368) might thus play an important role for MSD1 folding and stabilization of this domain at the membrane and, accordingly, the L69H may perturb these mechanisms. Login to comment 116 ABCC7 p.Leu206Trp ABCC7 p.Pro67Leu Supporting this hypothesis is the fact that VX-809 has been shown to act on MSD1 folding and that it also rescues functional defects in CFTR caused by disease-related mutations in the vicinity of the three amino acids highlighted here (P67L, L206W) [16]. Login to comment 118 ABCC7 p.Ser549Asn S549N causes reduction in Cl-channel activity classifying it as a class III CF mutation [17]. Login to comment 119 ABCC7 p.Ser549Asn Normal maturation of S549N-CFTR protein was observed in our Western blot analysis as reported by others [17]. Login to comment 120 ABCC7 p.Ser549Asn It is noteworthy here that S549N defective channel activity was nevertheless potentiated after incubating cells at 27 &#b0;C or VX809, a result which on the contrary was not anticipated. Login to comment 121 ABCC7 p.Gly126Ser ABCC7 p.Phe157Cys ABCC7 p.Phe87Ile ABCC7 p.Tyr852Phe ABCC7 p.Glu543Ala ABCC7 p.Asp1270Glu ABCC7 p.His139Gln ABCC7 p.Ser118Pro ABCC7 p.Phe494Leu The functional characterization of nine other novel mutations associated with CBAVD viz., F87I, S118P, G126S, H139Q, F157C, F494L, E543A, Y852F and D1270E revealed that these mutants did not cause any effect on normal CFTR maturation process and Cl-channel activity. Login to comment 129 ABCC7 p.Ser549Asn ABCC7 p.Leu69His ABCC7 p.Gly126Ser ABCC7 p.Phe157Cys ABCC7 p.Phe87Ile ABCC7 p.Tyr852Phe ABCC7 p.Glu543Ala ABCC7 p.Asp1270Glu ABCC7 p.His139Gln ABCC7 p.Ser118Pro ABCC7 p.Phe494Leu Patients profile Eleven rare missense mutations i.e. L69H, F87I, S118P, G126S, H139Q, F157C, F494L, E543A, S549N, Y852F, and D1270E were characterized by using single stranded conformation polymorphism and subsequently by DNA sequencing in Indian infertile CBAVD male patients [7,8]. Login to comment 131 ABCC7 p.Ser549Asn All the detected mutations except S549N are novel, rare mutations identified only from the Indian population, however, the exact frequency is still not known [7,8]. Login to comment 132 ABCC7 p.Ser549Asn ABCC7 p.Leu69His Additionally L69H and S549N mutations were also observed in Indian patients diagnosed with classical CF [7]. Login to comment 133 ABCC7 p.Leu69His The L69H missense mutation was identified in three-month-old child having classical phenotype like elevated level of sweat chloride and lung Staphylococcus aureus infection with respiratory distress. Login to comment 135 ABCC7 p.Ser549Asn Moreover S549N missense mutation was also observed in two patients. Login to comment 138 ABCC7 p.Gly126Ser ABCC7 p.Phe157Cys ABCC7 p.Phe87Ile ABCC7 p.Tyr852Phe ABCC7 p.Glu543Ala ABCC7 p.Asp1270Glu ABCC7 p.His139Gln ABCC7 p.Ser118Pro ABCC7 p.Phe494Leu The remaining all nine mutations viz., G126S, Y852F, F87I, S118P, H139Q, F157C, F494L, E543A, and D1270E were identified in Indian infertile males diagnosed with only CBAVD. Login to comment