ABCMdb

ABCC7 p.Leu69His

CF databases:	c.206T>G , p.Leu69Arg (CFTR1) ? , The above mutation was identified by SSCP analysis and characterized by direct DNA sequencing. The mutation was not found on 100 non-CF chromosomes.
Predicted by SNAP2:	A: D (63%), C: N (53%), D: D (85%), E: D (80%), F: D (63%), G: D (80%), H: D (75%), I: N (57%), K: D (75%), M: N (53%), N: D (75%), P: D (85%), Q: D (71%), R: D (75%), S: D (71%), T: D (71%), V: N (61%), W: D (75%), Y: D (71%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: N, K: D, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D,

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[hide] Identification and characterization of CFTR gene m... Ann Hum Genet. 2009 Jan;73(1):26-33. Epub 2008 Sep 8. Sharma N, Singh M, Kaur G, Thapa BR, Prasad R
Identification and characterization of CFTR gene mutations in Indian CF patients.
Ann Hum Genet. 2009 Jan;73(1):26-33. Epub 2008 Sep 8., [PMID:18782298]

Abstract [show]
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This study was performed on Indian CF patients (n = 50) to investigate the spectrum of mutations in the CFTR gene and their association with intragenic and extragenic marker haplotypes. We report identification of 14 previously known and eight novel mutations, namely 3986-3987delC, 876-6del4, 1792InsA, L69H, S158N, Q493L, I530L and E1329Q. The frequency of delta F508 was found to be 27%. Absolute linkage between delta F508 and the KM.19-GATT-TUB9-M470V-T854T haplotype (2-2-1-1-1) predicts a relatively recent appearance of delta F508 in Indian CF patients. Low frequency of delta F508 mutation and detection of eight novel and thirteen rare mutations reflect a heterogeneous spectrum of mutations in Indian CF patients. Failure to detect mutations in 34% of alleles indicates the possible presence of gross deletions involving one or more exons or may indicate the location of the molecular defects in either the noncoding parts of the gene or in the promoter region, which warrants analysis of those regions.

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2 We report identification of 14 previously known and eight novel mutations, namely 3986-3987delC, 876-6del4, 1792InsA, L69H, S158N, Q493L, I530L and E1329Q. Login to comment
67 They included nine missense mutations (L69H, S158N, Q493L, Y517C, V520F, I530L, S549N, E1329Q, and Y1381H), one insertion mutation (1792insA), three splice site mutations (876-6del4, 1525-1G-A, 3120+1G-A), two deletion mutations (1161delC, 3986delC), and 1 nonsense mutation (L218X). Login to comment
73 Novel Mutations and Phenotypic Features Output prediction scores were assessed for five novel mutations; they were >0.5 for L69H & Q493L and <0.5 for S158N, I530L & E1329Q (Table 3). Login to comment
74 L69H L69H was identified on one allele in a 3 month old baby with a history of vomiting and coughs for the last month. Login to comment
96 Table 2 Genotypes of CF subjects (n=50) Genotype Number of subjects Delta F508/Delta F508 5 Delta F508/3849+10kb C-T 1 Delta F508/S549N 2 Delta F508/S158N 1 Delta F508/Y1381H 1 Delta F508/1525-1 G-A 2 V520F/R117H 1 I530L/I530L 1 876-6del4/876-6del4 1 1792ins A/1792insA 1 3986-3987delC/3986-3987delC 1 Delta F508/U 10 1161 delC/U 2 L69H/U 1 R117H/U 1 Q493L/U 1 Y517C/U 1 S549N/U 3 G551D/U 1 E1329Q/U 1 N1303K/U 1 Y1381H/U 1 L218X/U 1 R553X/U 1 1525-1G-A/U 3 3120+1G-A/U 2 3849+10kb C-T/U 2 U/U 1 U-unidentified Table 3 Outcome prediction scores of novel substitution mutations identified in Indian CF patients Wild type Mutant Position Output Reliablity Prediction L H 69 0.5210 0 Pathological S N 158 0.3304 3 Neutral Q L 493 0.7784 5 Pathological I L 530 0.0591 8 Neutral E Q 1329 0.1018 7 Neutral Molecular Modelling and Bioinformatics (MMB) program (http://mmb.pcb.ub.es/PMut/) was used for pathological predictions of novel sequence variants. Login to comment
113 We first identified five of the mutations by ARMS (Delta F508, R117H, R553X, N1303K & G551D) and one by restriction digestion (3849+10kbC-T) and later identified by SSCP eight known (Y517C, V520F, S549N, Y1381H, 1525-1G-A, 3120+1G-A, 1161delC and L218X) and eight previously unreported mutations (L69H, S158N, Q493L, I530L, E1329Q, 876-6del4, 1792insA and 3986-3987delC). Login to comment
133 Output prediction scores deduced using molecular modeling and a bioinformatics program (http://mmb.pcb.ub.es/PMut/) revealed that among the novel mutations, L69H and Q493L are pathologic but S158N, I530L and E1329Q are neutral. Login to comment

[hide] Heterogenous spectrum of CFTR gene mutations in In... Hum Reprod. 2009 May;24(5):1229-36. Epub 2009 Jan 30. Sharma N, Acharya N, Singh SK, Singh M, Sharma U, Prasad R
Heterogenous spectrum of CFTR gene mutations in Indian patients with congenital absence of vas deferens.
Hum Reprod. 2009 May;24(5):1229-36. Epub 2009 Jan 30., [PMID:19181743]

Abstract [show]
BACKGROUND: Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene can cause congenital bilateral absence of vas deferens. Yet, the spectrum and frequency of CFTR mutations in Indian males with congenital absence of vas deferens (CAVD) is unknown. METHODS: We investigated 50 Indian males, diagnosed with unilateral or bilateral absence of vas deferens at the PGIMER, Chandigarh, for the presence of the most common CFTR gene mutations as well as unknown mutations by single-strand conformation polymorphism followed by sequence analysis. RESULTS: This study led to the identification of 12 CFTR gene mutations on 48% of 100 Indian CAVD chromosomes. CFTR mutations were identified on both alleles in 11 patients (22%) and on one allele in 26 patients (52%). Novel CFTR mutations identified were L69H, F87I, G126S, F157C, E543A, Y852F and D1270E. The T5 allele (25%) and F508del (11%) were the most common mutations identified. The most common intragenic marker haplotype for F508del was 2111 (GATT, TUB9, M470V and T854T). No mutations could be detected in 13 CAVD patients (26%), including 4 with renal malformations. CONCLUSIONS: This study confirms the molecular heterogeneity of CFTR mutations in CAVD. Although the mutation detection rate is indeed lower in Indian CAVD patients, 74% of the patients tested had at least one CFTR mutation. CAVD alleles with no mutations suggest that other changes may be located at the non-screened sites that require extensive search by direct sequencing. Furthermore, the novel CFTR mutations identified require functional studies in a cell-based system.

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6 Novel CFTR mutations identified were L69H, F87I, G126S, F157C, E543A, Y852F and D1270E. Login to comment
74 SSCP analysis performed in patients with only one or no mutation revealed nine further mutations on one allele each including seven new sequence alterations: L69H, F87I, G126S, F157C, E543A, Y852F and D1270E (Table I). Login to comment
78 Pathological predictions and multiple sequence alignments of novel substitution mutations The output prediction scores (http://blocks.fhrc.org/sift/SIFT.html) for L69H, E543A and D1270E were less than the 0.05 (threshold for pathological mutations) (Table II). Login to comment
79 Pathological predictions confirmed by another computer algorithm (http://genetics.bwh.harvard.edu/pph) revealed L69H, E543 and D1270E as deleterious mutations and other four mutations, F87I, G126S, F157C and Y852F, as benign sequence alterations (Table II). Login to comment
107 of alleles T5 Reduction of oligo T tract to 5T at 1342-6 Aberrant splicing Intron 8 25 F508del Deletion of 3 bp (CTT or TTT) between 1652 and 1655 Deletion of phenylalanine at 508 Exon 10 11 L69Ha T to A at 338 Leucine to histidine at 69 Exon 3 1 F87Ia T to A at 391 Phenylalanine to isoleucine Exon 3 1 R117H G to A at 482 Arginine to histidine at 117 Exon 4 3 G126Sa G to A at 508 Glycine to serine at 126 Exon 4 1 F157Ca T to G at 602 Phenylalanine to cystine at 157 Exon 4 1 E543Aa A to C at 1760 Glutamate to alanine at 543 Exon 11 1 Y852Fa A to T at 2687 Tyrosine to phenylalanine at 852 Exon 14a 1 3120 þ 1 G-A G to A 3120 þ 1 Aberrant splicing Intron 16 1 P1021S C to T at 3193 Proline to serine at 1021 Exon 17a 1 D1270Ea T to A at 3942 Aspartate to glutamate at 1270 Exon 20 1 Total chromosomes: 100; known mutations: 48%; unknown mutations: 52%. Login to comment
121 Intriguingly, among the seven novel substitution mutations identified, L69H, E543A and D1270E were predicted to be damaging, whereas F87I, G126S, F157C and Y852F were possibly neutral (http://blocks.fhcrc.org/sift/SIFT.html and http://genetics.bwh. Login to comment
123 It is noteworthy that L69H has been previously identified on one allele of the Indian classic CF patient (Sharma et al., 2009). Login to comment
132 In CBAVD patients, a high frequency of compound heterozygosity with severe/mild or mild/mild mutations has been reported Figure 1 Multiple alignments of CFTR amino acid sequences from different species (human, rhesus monkey, bovine, sheep, pig and mouse) and seven novel substitution mutations (L69H, F87I, G126S, F157C, E543A, Y852A and D1270E) identified in Indian CAVD patients. Login to comment
146 9 (18) (TG)10T9/(TG)12T7 2/2 1/1 1/1 1/1 (3), 1/2 (1) 4 (8) 2/2 1/1 1/1 2/2 1 (2) 2/2(1), 2/1(1) 1/1 1/1 1/1 (1), 1/2 (1) 2 (4) (TG)10T7/(TG11)T9 2/2 1/1 1/1 2/2 1 (2) (TG)10T9/(TG)13T7 2/2 1/2 1/1 2/2 1 (2) L69H/? Login to comment

[hide] Molecular basis of cystic fibrosis disease: an Ind... Indian J Clin Biochem. 2010 Oct;25(4):335-41. Epub 2010 Nov 19. Prasad R, Sharma H, Kaur G
Molecular basis of cystic fibrosis disease: an Indian perspective.
Indian J Clin Biochem. 2010 Oct;25(4):335-41. Epub 2010 Nov 19., [PMID:21966101]

Abstract [show]
Cystic fibrosis is a common autosomal recessive disorder usually found in population of white Caucasian descent. Now it is well documented the presence of CF disease in India with the advancement of laboratory testing. As once it was thought non existence of this disease in our population. Most of the phenotype of CF disease was in accordance of western population. Genetic analysis of CFTR gene in Indian CF patients revealed that most common mutation was delta F508 mutation. However, it was less than Caucasian population. CFTR mutations are also a causative factor in the pathogenesis of male infertility due to obstructive azoospermia. There are two most common mutation viz. IVS8-T5 and delta F508 which are responsible for congenital absence of vas deferens in male infertility patients. Elevated levels of sweat chloride at two occasions along with the presence of two mutations in CFTR gene was gold standard method for diagnosis of CF disease. It is noteworthy here that due to magnitude of Indian population, the total CF disease load would be more than many European countries. Clinical data demonstrate the prevalence of both classical and genetic form of CF in India.

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98 25 mutation Table 2 CFTR mutation identified in Indian population with classical CF [25] Genotype No. of subjects Delta F508/Delta F508 5 Delta F508/3849?10kb C-T 1 Delta F508/S549 2 Delta F508/Y138H 1 Delta F508/15251G-A 1 V520F/R117H 2 1530L/1530L 1 876-6del4/876-6del4 1 1792insA/1792insA 1 3986-3987delC/3986-3987delC 1 Delta F508/U 10 1161delC/U 2 L69H/U 1 R117H/U 1 Q493L/U 1 Y517C/U 1 S549N/U 3 G551D/U 1 E1329Q/U 1 N1303K/U 1 Y1381H/U 1 L218X/U 1 R553X/U 1 1525-1G-A/U 3 3120?1G-A/U 2 3849?10kbC-T/U 2 U/U 1 U unidentified panel were detected in our population at a combined frequency of (10%). Login to comment
99 The other seven known but rare mutations (1161delC, Y517C, V520F, S549N, Y1381H, L218X and 1525-1G-A) were identified at a combined frequency of (17%), and eight new mutations (3986delC, 1792InsA, L69H, S158N, Q493L, I530L, E1329Q and 876-8del4) identified in our CF population represented (15%) of the total CF alleles analyzed. Login to comment
127 SSCP analysis performed in patients with only one or no mutation revealed nine further mutations on one allele each including seven new sequence alterations: L69H, F87I, G126S, F157C, E543A, Y852F and D1270E (Table 3). Login to comment
130 Table 3 CFTR mutations identified and characterized in the Indian patients with CAVD [12] Mutation Nucleotide change No. of alleles T5 Reduction of oligo T tract to 5T at 1342-6 25 F508del Deletion of 3 bp(CTT or TTT) between 1652 and 1655 11 L69H T to A at 338 1 F87I T to A at 391 1 R117H G to A at 482 3 G126S G to A at 508 1 F157C T to G at 602 1 E543A A to C at 1760 1 Y852F A toT at 2687 1 3120?1G-A G to A 3120?1 1 P1021S CtoT at 3193 1 D1270E T to A at 3942 1 We documented NBD1 and NBD2 as the hotspot identified in the CFTR protein in Indian CF population, whereas the regions known to alter chloride permeability (transmembrane regions) and delta F508 mutation in NBD1 are the hot spot for mutation identification in our genital form of CF cases (obstructive azoospermia). Login to comment

[hide] Function, pharmacological correction and maturatio... J Cyst Fibros. 2015 Jan;14(1):34-41. doi: 10.1016/j.jcf.2014.06.008. Epub 2014 Jul 16. 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., [PMID:25042876]

Abstract [show]
BACKGROUND: Cystic fibrosis (CF) is rare in India. Most CF mutations identified are not yet functionally characterized. Hence, genetic counseling and adoption of therapeutic approach are particularly difficult. Our aim was to study the function and maturation of a spectrum of eleven Indian CFTR mutations from classical CF and infertile male patients with CBAVD. 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. RESULTS: Among these CFTR mutants, only L69H is not processed as a c-band and not functional at 37 degrees C. However, the functions of L69H and S549N and the maturation of L69H are corrected at 27 degrees C and by the investigational drug VX809. CONCLUSION: These data should help in developing counseling and therapeutic approaches in India. We identified L69H as a novel class II CF mutation.

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4 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 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 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 We identified L69H as a novel class II CF mutation. Login to comment
10 Keywords: Missense CF mutations; India; L69H-CFTR; S549N-CFTR; Low temperature; VX809 1. Login to comment
33 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 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 Mature glycosylated c-band was absent in L69H mutant expressing cells. Login to comment
46 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 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 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
57 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 We studied the effect of low-temperature with cells expressing L69H and S549N compared to F508del-CFTR. Login to comment
61 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 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 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 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 Remarkably we detected mature c-band for L69H (Fig. 4C, lane 6) as with the low-temperature protocol. Login to comment
70 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 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 Functional rescue of L69H, F508del and S549N by low temperature and VX809. Login to comment
82 Iodide efflux experiments with BHK-21 cells transfected with L69H, S549N and F508del-CFTR as indicated. Login to comment
88 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 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 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 The first salient result is the fact that we identified L69H as a novel class II CF mutation. Login to comment
102 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 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 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 We propose thus to categorize L69H as a class II form of CF mutation. Login to comment
111 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 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
129 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
132 Additionally L69H and S549N mutations were also observed in Indian patients diagnosed with classical CF [7]. Login to comment
133 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