ABCMdb

ABCC7 p.Leu346Pro

ClinVar:	c.1037T>C , p.Leu346Pro ? , not provided
CF databases:	c.1037T>C , p.Leu346Pro (CFTR1) ? ,
Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (85%), K: D (95%), M: D (91%), N: D (95%), P: D (75%), Q: D (91%), R: D (95%), S: D (95%), T: D (95%), V: D (75%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: N, 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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Publications
[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

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413 L346P destabilized the local hydrophobic character and therefore was sufficient for marking CFTR as a non-native protein to the ER quality control, with accompanying deleterious consequences to global protein folding events [199]. Login to comment

[hide] Destabilization of the transmembrane domain induce... J Biol Chem. 2005 Feb 11;280(6):4968-74. Epub 2004 Nov 10. Choi MY, Partridge AW, Daniels C, Du K, Lukacs GL, Deber CM
Destabilization of the transmembrane domain induces misfolding in a phenotypic mutant of cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2005 Feb 11;280(6):4968-74. Epub 2004 Nov 10., 2005-02-11 [PMID:15537638]

Abstract [show]
Two phenotypic missense mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel pore (L346P and R347P in transmembrane (TM) segment 6) involve gain of a proline residue, but only L346P represents a significant loss of segment hydropathy. We show here that, for synthetic peptides corresponding to sequences of CFTR TM6 segments, circular dichroism spectra of wild type and R347P TM6 in membrane mimetic environments are virtually identical, but L346P loses approximately 50% helicity, implying a membrane insertion defect in the latter mutant. A similar defect was observed in the corresponding double-spanning ("hairpin") TM5/6-L346P synthetic peptide. Examination of the biogenesis of CFTR revealed that the full-length protein harboring the L346P mutation is rapidly degraded at the endoplasmic reticulum (ER), whereas the wild type and the R347P protein process normally. Furthermore, a second site mutation (R347I) that restores in vitro membrane insertion and folding of the TM5/6-L346P peptide also rescues the folding and cell surface chloride channel function of full-length L346P CFTR. The correlated in vitro/in vivo results demonstrate that destabilizing local hydrophobic character represents a sufficient signal for marking CFTR as a non-native protein by the ER quality control, with accompanying deleterious consequences to global protein folding events.

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0 Destabilization of the Transmembrane Domain Induces Misfolding in a Phenotypic Mutant of Cystic Fibrosis Transmembrane Conductance Regulator* Received for publication, September 1, 2004, and in revised form, November 8, 2004 Published, JBC Papers in Press, November 10, 2004, DOI 10.1074/jbc.M410069200 Mei Y. Choi‡§¶, Anthony W. Partridge‡§ʈ, Craig Daniels**‡‡, Kai Du**‡‡, Gergely L. Lukacs**‡‡§§, and Charles M. Deber‡§ §§ From the ‡Division of Structural Biology and Biochemistry and **Program in Cell and Lung Biology, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8 and the Departments of §Biochemistry and ‡‡Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada Two phenotypic missense mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel pore (L346P and R347P in transmembrane (TM) segment 6) involve gain of a proline residue, but only L346P represents a significant loss of segment hydropathy. Login to comment
1 We show here that, for synthetic peptides corresponding to sequences of CFTR TM6 segments, circular dichroism spectra of wild type and R347P TM6 in membrane mimetic environments are virtually identical, but L346P loses ϳ50% helicity, implying a membrane insertion defect in the latter mutant. Login to comment
2 A similar defect was observed in the corresponding double-spanning ("hairpin") TM5/6-L346P synthetic peptide. Login to comment
3 Examination of the biogenesis of CFTR revealed that the full-length protein harboring the L346P mutation is rapidly degraded at the endoplasmic reticulum (ER), whereas the wild type and the R347P protein process normally. Login to comment
4 Furthermore, a second site mutation (R347I) that restores in vitro membrane insertion and folding of the TM5/6-L346P peptide also rescues the folding and cell surface chloride channel function of full-length L346P CFTR. Login to comment
15 A number of mutations occurring in TM5/6 have been found to cause mild (usually pancreatic sufficient) forms of CF, two of which involve introduction of a proline residue: L346P, a mutation that was identified in two unrelated Cypriot patients in 1994 (10); and a second sequentially adjacent CF-phenotypic mutant, R347P (11). Login to comment
35 Furthermore, in CFTR mutant L346P, the loss of Leu significantly increases the local hydrophilicity of this TM segment, i.e. on the Liu-Deber hydropathy index where values are scaled between ϩ5 and -5, Leu ranks third (ϩ4.76), whereas Pro ranks 19th (-4.92) out of the 20 commonly occurring amino acids (19). Login to comment
37 In the present work, we have used solid-phase peptide synthesis to prepare sequences corresponding to TM6 segments of wild type (WT) and mutants L346P and R347P of CFTR, along with some corresponding double-spanning TM5/6 peptides for comparative structural analyses. Login to comment
38 In parallel, we examined the relative effects of L346P versus R347P on cellular processing of full-length CFTR. Login to comment
58 Construction and Expression of CFTR Variants in Mammalian Cells-The L346P, R347P, and R347H CFTR mutants were constructed FIG. 1. Login to comment
60 A, wild type sequence; B, TM5/6-L346P; C, TM5/6-R347P; and D, TM5/6-L346P-R347I. Login to comment
70 Baby hamster kidney (BHK) cells were stably transfected with the pNUT expression plasmids, containing the wild type (WT), L346P, or R347P CFTR, harboring an HA-epitope in the C-terminal tail of CFTR (CFTR-CintHA) (21). Login to comment
84 RESULTS Hydrophobicity Threshold of CF-phenotypic Mutant TM Segments-Although both L346P and R347P represent a gain of a Pro residue in CFTR TM6, the resulting local 346/347 diads (PR and LP, respectively) differ significantly in hydrophobic character. Login to comment
86 The outputs for the wild type (WT) and L346P CFTR sequences support an initial hypothesis that the L346P mutation decreases the average net hydrophobicity of the original TM6 segment sufficiently to prevent the proper membrane insertion of the full TM6 segment (Fig. 1, A and B). Login to comment
88 In contrast, the output for the L346P mutant sequence (residues 330-341) fails to predict a sufficiently long stretch of amino acids that are above the threshold hydrophobicity required for a TM helix (27, 28). Login to comment
89 However, unlike L346P, the R347P mutation doesn`t involve a significant change in hydrophobicity, and TM Finder predicts that the R347P mutant has the same membrane-inserted amino acid stretch (residues 330-349) as the WT TM6 sequence (Fig. 1C). Login to comment
93 A, CD spectra of TM6-WT, TM6-L346P, and TM6-R347P peptides in lysophosphatidylcholine (LPC) detergent micelles. B, CD spectra for TM5/ 6-WT and TM5/6-L346P peptides in LPC micelles. Login to comment
94 The TM5/6-L346P displays a decrease in the helical content when compared with the TM5/6-WT spectrum. Login to comment
97 D, Trp fluorescence spectra for the unlabeled and labeled TM5/6 and L346P peptides in LPC micelles. FRET measurements were performed using peptides labeled with dansyl chloride as the acceptor fluorophore with the Trp residue in TM6 serving as a donor fluorophore. Login to comment
99 synthesized Lys-tagged versions of the TM6-WT and the two mutant (L346P and R347P) sequences (Table I). Login to comment
102 Although the TM6-WT peptide adopted an ␣-helical structure in the presence of LPC micelles, the TM6-L346P peptide displays only ϳ50% of the helicity observed for the TM6-WT sequence. Login to comment
104 Because the L346 locus appeared to be most affected by the Pro mutation, we further examined the effect of the L346P mutation in an expanded context by synthesizing the helix-loop-helix TM5/6-WT and TM5/6-L346P peptides. Login to comment
106 CD spectra indicate that the TM5/6-L346P construct exhibits a 25% decrease in helicity when compared with the TM5/6-WT construct in SDS micelles, consistent with a 50% decrease in one TM helix (Fig. 2B). Login to comment
107 Fluorescence Studies of CFTR Single TM6 and Double TM5/6 Peptides-The proposition that the L346P TM segment is only partially inserted into micellar membranes was further examined by fluorescence experiments. Login to comment
109 Characteristic Trp fluorescence spectra of these two peptides in detergent micelles are presented in Fig. 2C. Noting that a membrane-embedded Trp residue will typically display increased fluorescence intensity with a blue-shifted position versus an aqueous-located counterpart, the data indicate that the Trp residue in the WT species resides in an apolar environment (maximum near 320 nm) whereas the Trp in the L346P peptide is largely aqueous exposed (shoulder near 340 nm), supporting the notion that the apolar-to-polar mutation prevents proper TM6 insertion. Login to comment
116 WT, L346P, R347P, L346P/R347I, and R347H CFTR expression was assayed by immunoblotting, using the mouse monoclonal anti-HA Ab. Login to comment
120 C, biosynthetic processing of the WT and L346P CFTR was monitored in stably transfected BHK cells by the metabolic pulse-chase technique as described under "Experimental Procedures." Login to comment
124 D, relative translational rate of the full-length L346P CFTR. Login to comment
126 To avoid clonal variations, COS-1 cells were transiently transfected with the WT and L346P CFTR. Login to comment
129 The radioactivity associated with the WT and L346P CFTR was normalized for cellular protein and was not more than 4% variant in two experiments. Login to comment
133 The FRET results suggest that (i) the WT sequence is likely folded into a helical hairpin and (ii) a substantial increase in fluorophore separation occurs between the N and C termini of the L346P mutant hairpin compared with the WT sequence. Login to comment
134 The L346P Mutation Impairs the Folding of CFTR in Vivo- ER-retained, core-glycosylated (or incompletely folded) CFTR can be readily distinguished from the mature, complex-glycosylated (or folded) CFTR by immunoblot analysis, based on the faster electrophoretic mobility of the core-glycosylated form compared with the complex-glycosylated CFTR. Login to comment
135 Because impaired post-translational folding of the CFTR usually causes its biosynthetic processing arrest, we examined the processing of full-length L346P- and R347P-CFTR by immunoblotting and pulse-chase analysis of BHK cells, which stably express CFTR. Login to comment
137 As shown in Fig. 3A, immunoblot analysis of equal amounts of cell extracts demonstrated that the L346P, but not the R347P, mutation, prevented the expression of the complex-glycosylated CFTR. Login to comment
139 Similar results were obtained in transiently transfected COS-1 cells, indicating that the cellular phenotype of the L346P CFTR is independent of the expression system used (Fig. 3B). Login to comment
140 Impaired steady-state expression of L346P-CFTR could be a consequence of its rapid degradation at the ER and/or of accelerated removal of the channel from post-Golgi compartments (32). Login to comment
141 To distinguish between these scenarios, the biogenesis of L346P CFTR was monitored in BHK cells by the metabolic pulse-chase technique. Login to comment
142 Although the accumulation of the complex-glycosylated WT-CFTR was obvious after 2 h of chase, the L346P mutation prevented the appearance of the complex-glycosylated form as shown by autoradiography (Fig. 3C). Login to comment
143 These results suggest that the L346P mutation imposes a folding defect on CFTR, leading to the retention and degradation of the mutant at the ER. Login to comment
144 On the other hand, the L346P mutation does not appear to cause premature translational termination or failure of the TM5-6 or TM7-8 segments to insert into the ER, because similar amounts of radioactively labeled core-glycosylated WT and L346P CFTR were accumulated during a 10-min radioactive labeling (Fig. 3D). Login to comment
145 A Second Site Mutation in TM6 Restores Biosynthetic Processing of CFTR-To test the assumption that destabilization of local TM5/6 hairpin formation may inhibit post-translational folding in the context of full-length CFTR, we searched for a second site mutation that could re-establish the stability of the L346P TM5/6. Login to comment
146 Consideration of amino acid replacements in the vicinity of the L346P mutation identified a second site mutation (R347I) that restored the hydrophobicity of the TM6 L346P-containing segment to the threshold level that ensured membrane insertion according to TM Finder (Fig. 1D) (26). Login to comment
147 The L346P/R347I single spanning TM6 peptide was first synthesized, and analysis of its CD spectrum confirmed that this mutation restored the ␣-helical content of this TM6 double mutant to its WT counterpart (Fig. 4A). Login to comment
148 We then assessed whether the R347I mutation could restore hairpin formation of the TM5/6-L346P polypeptide by the FRET assay. Login to comment
149 Using a TM5/6-L346P/R347I peptide in which a Trp residue was inserted near the C terminus (Table I), and in which the N terminus was labeled with a dansyl group, we found that the donor fluorescence quench in the double mutant was now similar to that of the WT TM5/6 (Fig. 4B). Login to comment
150 If destabilization of the TM5/6 hairpin accounts for the processing defect of the L346P CFTR, introducing the second site mutation should correspondingly restore the folding and biosynthetic processing of full-length CFTR. Login to comment
152 Immunoblot analysis demonstrated the appearance of the complex-glycosylated L346P/R347I CFTR in both transiently transfected COS-1 and stably transfected BHK cells, whereas no detectable amount of L346P CFTR was present (Figs. Login to comment
154 Functional assessment of the plasma membrane protein kinase A-activated halide conductance confirmed the partial reversion of the processing defect by demonstrating that the cAMP-stimulated iodide release of the L346P CFTR (6.3 Ϯ 0.2 nmol/min) was increased by 3-fold in the presence of the second site mutation (18.4 Ϯ 03 nmol/min) (Fig. 5B). Login to comment
155 The detection of L346P CFTR by functional assay, but not by immunoblotting, is conceivable due to the higher sensitivity of the iodide efflux assay. Login to comment
160 A, CD spectra of TM6-WT and TM6-L346P/R347I in LPC micelles. B, Trp fluorescence spectra for the unlabeled and labeled TM5/ 6-WT and TM5/6-L346P/R347I peptides in LPC micelles. FRET measurements were performed using peptides labeled with dansyl chloride as the acceptor fluorophore with the Trp residue in TM6 serving as a donor fluorophore. Login to comment
164 Thus, despite the similarity of the two mutations and their adjacent positions in the sequence, the TM6-L346P peptide displayed only ϳ50% of the helicity observed for the TM6-WT sequence, whereas the TM6-R347P retained WT character, indicating that the ability of the L346P peptide to properly insert into the apolar milieu has been significantly compromised (Fig. 2A). Login to comment
167 To address this situation, we synthesized and compared helix-loop-helix peptides corresponding to the CFTR TM5/6-WT and the TM5/6-L346P sequence. Login to comment
169 Fluorescence resonance energy transfer experiments on labeled TM5/6 constructs further suggested that L346P prevents formation of proper TM6 topology, because FRET effects were significantly reduced in the TM5/ 6-L346P peptide versus the corresponding WT construct. Login to comment
176 Thus, a mutation in TM6 such as L346P may further reduce the efficiency of proper membrane anchoring of the protein. Login to comment
177 Consistent with these considerations, we found that full-length CFTR protein harboring the L346P mutation is subjected to core glycosylation but was unable to fold and was rapidly degraded in vivo. Login to comment
179 Because the full-length L346P protein is indeed synthesized, an additional possibility is that the protein is able to compensate, at least in part, for the topological defect at the TM5/6 locus via TM-packing interactions with the second (TM7-12) CFTR TM domain. Login to comment
180 Based on our study, it appears that L346P may affect local CFTR TM5/6 structure to such an extent that the ER-associated quality-control mechanism recognizes the mutant as non-native and marks it for degradation. Login to comment
181 As a result, escape from the ER and cell surface delivery of L346P CFTR is severely compromised FIG. 5. Login to comment
182 The effect of a second site mutation on the expression and function of L346P CFTR. Login to comment
186 B, cAMP-stimulated iodide efflux of BHK cells expressing wild-type (wt), L346P, or L346P/ R347I CFTR. Login to comment
190 Schematic model of the possible topological consequences of the L346P mutation in CFTR. Login to comment
200 The partial reversion of the L346P CFTR processing defect by a second site mutation (R347I) (Fig. 3B), which restores full-length TM6 insertion potential (Fig. 1D), suggests that segment hydrophobicity is prominent among the factors that play determining roles in the post-translational folding of CFTR. Login to comment
203 Because the resulting TM6 is now too short to span the cellular bilayers, a compensatory "pull" on the residues within the TM5/6-L346P loop region may also occur (Fig. 6B). Login to comment
208 Based on our results, impaired post-translational folding of CFTR is the primary defect in the case of the L346P mutation. Login to comment
212 Note that this salt bridge would similarly be abolished in the L346P/R347I mutant, perhaps explaining, in part, why this "rescue mutant" is not fully functional. Login to comment

[hide] Segregation analysis in cystic fibrosis at-risk fa... Prenat Diagn. 2004 Dec 15;24(12):981-3. D'Apice MR, Gambardella S, Russo S, Lucidi V, Nardone AM, Pietropolli A, Novelli G
Segregation analysis in cystic fibrosis at-risk family demonstrates that the M348K CFTR mutation is a rare innocuous polymorphism.
Prenat Diagn. 2004 Dec 15;24(12):981-3., 2004-12-15 [PMID:15614862]

Abstract [show]
OBJECTIVE: Cystic fibrosis (CF; OMIM# 219700) is caused by mutation in the CF transmembrane regulator (CFTR) gene. We investigate whether the (paternal) M348K mutation is a benign polymorphism or a disease-causing mutation in a patient clinically affected with CF, with the second (maternal) CFTR allele identified as N1303K. METHODS: The patient and his father were studied for the presence of mutations in the CFTR gene using the DHPLC system to analyze all CFTR exons. Amplicons showing an abnormal elution profile were sequenced. RESULTS: The CFTR gene from the healthy father has two mutations, M348K and G1244E. The affected son inherited only the G1244E paternal mutation from his father, and hence the two paternal mutations are trans and do not occur in the same CFTR gene. The patient's genotype is G1244E(paternal)/N1303K(maternal). This information was used to study an ongoing pregnancy of the couple, where the fetus inherited the same genotype as the affected proband and therefore is affected. CONCLUSION: M348K in the CFTR gene is not a mutation causing CF, but a rare polymorphism. These data are important for genetic counseling and prenatal diagnosis and illustrate the importance of full sequence data when studying rare mutations.

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41 However, in another report, Deltas et al. (1996) described a Cypriot individual without symptoms of CF who is compound heterozygous for L346P/M348K. Login to comment

[hide] Pharmacological induction of CFTR function in pati... Pediatr Pulmonol. 2005 Sep;40(3):183-96. Kerem E
Pharmacological induction of CFTR function in patients with cystic fibrosis: mutation-specific therapy.
Pediatr Pulmonol. 2005 Sep;40(3):183-96., [PMID:15880796]

Abstract [show]
CFTR mutations cause defects of CFTR protein production and function by different molecular mechanisms. Mutations can be classified according to the mechanisms by which they disrupt CFTR function. This understanding of the different molecular mechanisms of CFTR dysfunction provides the scientific basis for the development of targeted drugs for mutation-specific therapy of cystic fibrosis (CF). Class I mutations are nonsense mutations that result in the presence of a premature stop codon that leads to the production of unstable mRNA, or the release from the ribosome of a short, truncated protein that is not functional. Aminoglycoside antibiotics can suppress premature termination codons by disrupting translational fidelity and allowing the incorporation of an amino acid, thus permitting translation to continue to the normal termination of the transcript. Class II mutations cause impairment of CFTR processing and folding in the Golgi. As a result, the mutant CFTR is retained in the endoplasmic reticulum (ER) and eventually targeted for degradation by the quality control mechanisms. Chemical and molecular chaperones such as sodium-4-phenylbutyrate can stabilize protein structure, and allow it to escape from degradation in the ER and be transported to the cell membrane. Class III mutations disrupt the function of the regulatory domain. CFTR is resistant to phosphorylation or adenosine tri-phosphate (ATP) binding. CFTR activators such as alkylxanthines (CPX) and the flavonoid genistein can overcome affected ATP binding through direct binding to a nucleotide binding fold. In patients carrying class IV mutations, phosphorylation of CFTR results in reduced chloride transport. Increases in the overall cell surface content of these mutants might overcome the relative reduction in conductance. Alternatively, restoring native chloride pore characteristics pharmacologically might be effective. Activators of CFTR at the plasma membrane may function by promoting CFTR phosphorylation, by blocking CFTR dephosphorylation, by interacting directly with CFTR, and/or by modulation of CFTR protein-protein interactions. Class V mutations affect the splicing machinery and generate both aberrantly and correctly spliced transcripts, the levels of which vary among different patients and among different organs of the same patient. Splicing factors that promote exon inclusion or factors that promote exon skipping can promote increases of correctly spliced transcripts, depending on the molecular defect. Inconsistent results were reported regarding the required level of corrected or mutated CFTR that had to be reached in order to achieve normal function.

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58 C-D565G II DF508 D1507 S549R S549I S549N S549R S945D S945L H1054D G1061R L1065P R1066C R1066M L1077P H1085R N1303K G85E III G551D S492F V520F R553G R560T R560S Y569D IV R117H, R117C, R117P, R117L D1152H, L88S, G91R, E92K, Q98R, P205S, L206W, L227R, F311L, G314E, R334W, R334Q, I336K, T338I, L346P, R347C, R347H, R347L, R347P, L927P, R1070W, R1070Q V 3849 þ 10 kb C ! Login to comment

[hide] Misfolding of the cystic fibrosis transmembrane co... Biochemistry. 2008 Feb 12;47(6):1465-73. Epub 2008 Jan 15. Cheung JC, Deber CM
Misfolding of the cystic fibrosis transmembrane conductance regulator and disease.
Biochemistry. 2008 Feb 12;47(6):1465-73. Epub 2008 Jan 15., 2008-02-12 [PMID:18193900]

Abstract [show]
Understanding the structural basis for defects in protein function that underlie protein-based genetic diseases is the fundamental requirement for development of therapies. This situation is epitomized by the cystic fibrosis transmembrane conductance regulator (CFTR)-the gene product known to be defective in CF patients-that appears particularly susceptible to misfolding when its biogenesis is hampered by mutations at critical loci. While the primary CF-related defect in CFTR has been localized to deletion of nucleotide binding fold (NBD1) residue Phe508, an increasing number of mutations (now ca. 1,500) are being associated with CF disease of varying severity. Hundreds of these mutations occur in the CFTR transmembrane domain, the site of the protein's chloride channel. This report summarizes our current knowledge on how mutation-dependent misfolding of the CFTR protein is recognized on the cellular level; how specific types of mutations can contribute to the misfolding process; and describes experimental approaches to detecting and elucidating the structural consequences of CF-phenotypic mutations.

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115 WT, L346P, R347P, L346P/R347I, and R347H CFTR expression was assayed by immunoblotting, using the mouse monoclonal anti-HA Ab. Equal loading of proteins was verified by visualizing the Na+/K+-ATPase (lower panel). Login to comment
135 We therefore examined two CF-phenotypic missense mutations in the CFTR channel pore [L346P and R347P in TM6] that involve gain of a Pro residue, but where only the nonconservative mutation L346P represents a significant loss of segment hydropathy. Login to comment
137 When the biogenesis of corresponding full-length CFTR mutants was examined in this context, the protein harboring the L346P mutation was found to be unstable, while the wild type, R347P, along with the R347H mutant protein, processed normally (Figure 4A). Login to comment
138 The defect could be rescued in part by restoring hydrophobicity to TM6 via the double mutant L346P/R347I (Figure 4B). Login to comment

[hide] Atypical cystic fibrosis and CFTR-related diseases... Clin Rev Allergy Immunol. 2008 Dec;35(3):116-23. Paranjape SM, Zeitlin PL
Atypical cystic fibrosis and CFTR-related diseases.
Clin Rev Allergy Immunol. 2008 Dec;35(3):116-23., [PMID:18493878]

Abstract [show]
Cystic fibrosis (CF), which is among the most common life-shortening recessive illnesses, is caused by mutations of the CF transmembrane conductance regulator (CFTR) and typically involves chronic infection and progressive obstruction of the respiratory tract as well as pancreatic exocrine insufficiency. Disease severity, to some extent, correlates with organ sensitivity to CFTR dysfunction and to the amount of functional protein, which is influenced by the type of mutation. Atypical CF represents approximately 2% of affected individuals, and includes cases presenting in adolescence or adulthood with pancreatic exocrine sufficiency, normal or borderline sweat chloride concentrations, or with a single predominant clinical feature. This review briefly describes diagnostic methods and phenotypic characteristics of classic and atypical CF, as well as CFTR-related diseases, conditions in which mutated CFTR may contribute to the pathogenesis but do not strictly fit established diagnostic criteria.

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64 Determination of the transepithelial nasal potential difference has been beneficial in establishing a CF Table 1 Mutations, sites, and molecular consequences associated with either an atypical presentation of CF respiratory disease or pancreatic sufficiency or late-onset pancreatic insufficiency (http:// www.genet.sickkids.on.ca) Mutation Site Consequence Atypical presentation M1210I Exon 19 Met to Ile at 1210 S1455X Exon 24 Ser to Stop at 1455 1811+18G→A Intron 11 mRNA splicing defect L346P Exon 7 Leu to Pro at 346 Y161D Exon 4 Tyr to Asp at 161 R31C Exon 2 Arg to Cys at 31 I752S Exon 13 Ile to Ser at 752 2811G/T Exon 15 Sequence variation Pancreatic sufficiency or late-onset pancreatic insufficiency R600G Exon 13 Arg to Gly at 600 D1152H Exon 18 Asp to His at 1152 Y89C Exon 3 Tyr to Cys at 89 R117H Exon 4 Arg to His at 117 D110E Exon 4 Asp to Glu at 110 296 + 3insT Intron 2 mRNA splicing defect E217G Exon 6a Glu to Gly at 217 V392G Exon 8 Val to Gly at 392 N1088D Exon 17b Asn to Asp at 1088 S737F Exon 13 Missense 1716+1G→A Intron 10 mRNA splicing defect R334W Exon 7 Arg to Trp at 334 R347P Exon 7 Arg to Pro at 347 A455E Exon 9 Ala to Glu at 455 P574H Exon 12 Pro to His at 574 3850-3T→G Intron 19 mRNA splicing defect diagnosis in many atypical cases. Login to comment

[hide] Cooperative assembly and misfolding of CFTR domain... Mol Biol Cell. 2009 Apr;20(7):1903-15. Epub 2009 Jan 28. Du K, Lukacs GL
Cooperative assembly and misfolding of CFTR domains in vivo.
Mol Biol Cell. 2009 Apr;20(7):1903-15. Epub 2009 Jan 28., [PMID:19176754]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) architecture consists of two membrane spanning domains (MSD1 and -2), two nucleotide binding domains (NBD1 and -2), and a regulatory (R) domain. Several point mutations lead to the channel misprocessing, with limited structural perturbation of the mutant domain. To gain more insight into the basis of CFTR folding defect, the contribution of domain-wise and cooperative domain folding was assessed by determining 1) the minimal domain combination that is recognized as native and can efficiently escape the endoplasmic reticulum (ER) retention and 2) the impact of mutation on the conformational coupling among domains. One-, two-, three-, and most of the four-domain assemblies were retained at the ER. Solubilization mutations, however, rescued the NBD1 processing defect conceivably by thermodynamic stabilization. The smallest folding unit that traversed the secretory pathway was composed of MSD1-NBD1-R-MSD2 as a linear or split polypeptide. Cystic fibrosis-causing missense mutations in the MSD1, NBD1, MSD2, and NBD2 caused conformational defect in multiple domains. We propose that cooperative posttranslational folding is required for domain stabilization and provides a plausible explanation for the global misfolding caused by point mutations dispersed along the full-length CFTR.

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No. Sentence Comment
37 MATERIALS AND METHODS Cell Lines Baby hamster kidney (BHK) cells, stably expressing the wt and mutant (G91R, L346P, L1093P, N1303K, ⌬F508, 4D, 1218X, 1158X, and 823X) CFTR with three tandem hemagglutinin (HA)-epitope (3HA) inserted into the fourth extracellular loop, were isolated and maintained as described previously (Sharma et al., 2004; Du et al., 2005). Login to comment
252 Four CF mutations, G91R (Xiong et al., 1997), L346P (Choi et al., 2005), L1093P (Seibert et al., 1996), and N1303K (Gregory et al., 1991), localized to the transmembrane (TM) 1 and TM6 in MSD1, the cytosolic loop (CL) 4, and in the NBD2, respectively (Figure 7a). Login to comment
274 Remarkably, the G91R, L346P, ⌬F508, 4D, and L1093P mutations, regardless of their location, profoundly augmented the protease susceptibility of the NBD2 (ϳ30 kDa), probed with the M3A7 Ab (Figure 7g and Supplemental Figure S7c). Login to comment
301 One of the most important observations of this study is that the NBD2 conformational stability was dramatically impaired regardless the localization of mutations (G91R, L346P, ⌬F508, 4D, and L1093P) in the NBD1, TM1, TM6, or CL4 (Figure 6e). Login to comment
314 Cooperative misfolding is predicted to amplify relatively modest and localized structural defects caused by point mutations (e.g., L346P and ⌬F508). Login to comment
315 The L346P mutation destabilizes the TM5/6 hairpin in vitro by decreasing intrahelical Hϩ bondings and the TM6 helix net hydrophobicity that could be reverted by a suppressor mutation (Choi et al., 2005). Login to comment

[hide] Membrane-integration characteristics of two ABC tr... J Mol Biol. 2009 Apr 17;387(5):1153-64. Epub 2009 Feb 21. Enquist K, Fransson M, Boekel C, Bengtsson I, Geiger K, Lang L, Pettersson A, Johansson S, von Heijne G, Nilsson I
Membrane-integration characteristics of two ABC transporters, CFTR and P-glycoprotein.
J Mol Biol. 2009 Apr 17;387(5):1153-64. Epub 2009 Feb 21., [PMID:19236881]

Abstract [show]
To what extent do corresponding transmembrane helices in related integral membrane proteins have different membrane-insertion characteristics? Here, we compare, side-by-side, the membrane insertion characteristics of the 12 transmembrane helices in the adenosine triphosphate-binding cassette (ABC) transporters, P-glycoprotein (P-gp) and the cystic fibrosis transmembrane conductance regulator (CFTR). Our results show that 10 of the 12 CFTR transmembrane segments can insert independently into the ER membrane. In contrast, only three of the P-gp transmembrane segments are independently stable in the membrane, while the majority depend on the presence of neighboring loops and/or transmembrane segments for efficient insertion. Membrane-insertion characteristics can thus vary widely between related proteins.

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113 For CFTR, we chose mutations located in TM1CFTR (F87L, G91R), TM3CFTR (P205S, L206W), TM4CFTR (C225R), TM5CFTR (DF311, G314E), TM6CFTR (R334L/W, I336K/R/D, I340N/S, L346P, R347L/H), TM8CFTR (S909I, S912L), TM9CFTR (I1005R, A1006E), TM10CFTR (Y1032N), and TM12CFTR (M1137R, ΔM1140, M1140K), or close to the TM region of TM1CFTR (R74W, L102R/P), TMF2CFTR (R117P/L, L137P), and TM11CFTR (M1101K/R). Login to comment
115 As seen in Supplementary Data Table S2, only a few of the tested mutations in the 19 residue long CFTR TM segments alter the insertion efficiency significantly: TM4CFTR (C225R) (decrease from 88% to 34%), TM6CFTR (I336K/R/D) (decrease from 55% to 34%, 36% and 35%, respectively), TM6CFTR (I340N/S) (decrease from 55% to 32% and 35%, respectively), TM6CFTR (L346P) (decrease from 55% to 14%,), and TM12CFTR (ΔM1140) (decrease from 34% to 7%). Login to comment
116 One mutation, TM6CFTR (R334L), increased the insertion efficiency from 55% to 85%, as expected for a charged-to-hydrophobic replacement. Login to comment
120 Mutations TM6VCFTR (I336K), TM6VCFTR (I340N), and TM6VCFTR (L346P) still caused a large decrease in membrane insertion (from 81% to 39%, 35% and 14%, respectively). Login to comment
121 Addition of flanking segments did not rescue the insertion of TMF6CFTR (I336K) or TMF6CFTR (L346P) (decrease from 87% to 52% and 21%, respectively), nor did the addition of TMF5CFTR and the intervening loop to TMF6CFTR (L346P) (decrease from 86% to 36%). Login to comment
133 In particular, neither TM1P-gp nor TM2P-gp have been found capable of independent integration into the lipid bilayer.37 Also, TM3P-gp has been found not to be able to re-initiate translocation.36,40 Both TM3CFTR and TM4CFTR have been found to exhibit weak signal-anchor activities, and the translocation of the second extra cellular loop of the protein was found to be efficient when both segments were present.34-36 Interestingly, very recent results show that the poorly inserting TM8CFTR stays close to the translocon for an extended period of time before integrating fully into the membrane, and that this retention depends on an acidic residue, Asp924, located near the center of the TM segment.64 Three of the non-conservative mutations in CFTR and P-gp that we have studied strongly reduce the insertion of a transmembrane helix even when flanking residues are included: TMF6CFTR (I336K), TMF6CFTR (L346P), and TMF1P-gp (L70P). Login to comment
134 For TMF6CFTR (L346P), it has been observed that the mutation compromises the insertion of a synthetic peptide into a membrane-mimetic environment and causes rapid degradation of the mutated full-length protein in the endoplasmic reticulum in vivo.65 CFTR and P-gp both arose from gene duplication and the two halves share similarities in their overall topology. Login to comment
109 For CFTR, we chose mutations located in TM1CFTR (F87L, G91R), TM3CFTR (P205S, L206W), TM4CFTR (C225R), TM5CFTR (DF311, G314E), TM6CFTR (R334L/W, I336K/R/D, I340N/S, L346P, R347L/H), TM8CFTR (S909I, S912L), TM9CFTR (I1005R, A1006E), TM10CFTR (Y1032N), and TM12CFTR (M1137R, ƊM1140, M1140K), or close to the TM region of TM1CFTR (R74W, L102R/P), TMF2CFTR (R117P/L, L137P), and TM11CFTR (M1101K/R). Login to comment
111 As seen in Supplementary Data Table S2, only a few of the tested mutations in the 19 residue long CFTR TM segments alter the insertion efficiency significantly: TM4CFTR (C225R) (decrease from 88% to 34%), TM6CFTR (I336K/R/D) (decrease from 55% to 34%, 36% and 35%, respectively), TM6CFTR (I340N/S) (decrease from 55% to 32% and 35%, respectively), TM6CFTR (L346P) (decrease from 55% to 14%,), and TM12CFTR (ƊM1140) (decrease from 34% to 7%). Login to comment
117 Addition of flanking segments did not rescue the insertion of TMF6CFTR (I336K) or TMF6CFTR (L346P) (decrease from 87% to 52% and 21%, respectively), nor did the addition of TMF5CFTR and the intervening loop to TMF6CFTR (L346P) (decrease from 86% to 36%). Login to comment
129 In particular, neither TM1P-gp nor TM2P-gp have been found capable of independent integration into the lipid bilayer.37 Also, TM3P-gp has been found not to be able to re-initiate translocation.36,40 Both TM3CFTR and TM4CFTR have been found to exhibit weak signal-anchor activities, and the translocation of the second extra cellular loop of the protein was found to be efficient when both segments were present.34-36 Interestingly, very recent results show that the poorly inserting TM8CFTR stays close to the translocon for an extended period of time before integrating fully into the membrane, and that this retention depends on an acidic residue, Asp924, located near the center of the TM segment.64 Three of the non-conservative mutations in CFTR and P-gp that we have studied strongly reduce the insertion of a transmembrane helix even when flanking residues are included: TMF6CFTR (I336K), TMF6CFTR (L346P), and TMF1P-gp (L70P). Login to comment
130 For TMF6CFTR (L346P), it has been observed that the mutation compromises the insertion of a synthetic peptide into a membrane-mimetic environment and causes rapid degradation of the mutated full-length protein in the endoplasmic reticulum in vivo.65 CFTR and P-gp both arose from gene duplication and the two halves share similarities in their overall topology. Login to comment

[hide] Homozygous CFTR mutation M348K in a boy with respi... Eur J Pediatr. 2012 Jul;171(7):1039-46. doi: 10.1007/s00431-012-1672-1. Epub 2012 Jan 25. Hentschel J, Riesener G, Nelle H, Stuhrmann M, Schoner A, Sommerburg O, Fritzsching E, Mall MA, von Eggeling F, Mainz JG
Homozygous CFTR mutation M348K in a boy with respiratory symptoms and failure to thrive. Disease-causing mutation or benign alteration?
Eur J Pediatr. 2012 Jul;171(7):1039-46. doi: 10.1007/s00431-012-1672-1. Epub 2012 Jan 25., [PMID:22274833]

Abstract [show]
We report on a 6-month-old premature boy from consanguineous parents. He presented with respiratory distress, necrotizing enterocolitis and hyperbilirubinemia shortly after birth. Persisting respiratory symptoms and failure to thrive prompted cystic fibrosis diagnostics, which showed the lack of wild-type signal for the mutation R347P suggesting a homozygous deletion or an alteration different from the known mutation at this position. Sequencing of this region revealed the homozygous substitution 1175 T > A (HGVS: c.1043 T > A) in exon 7 resulting in the homozygous amino acid change M348K. This mutation has never been reported in homozygosity before. Computational analysis tools classified M348K as 'presumably disease causing.' In our patient, sweat testing and electrophysiological assessment of CFTR function in native rectal epithelium demonstrated normal Cl(-) secretion. Conclusion: We assume that the homozygous alteration M348K is a harmless variant rather than a CF-causing mutation.

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22 In another publication, M348K was found together with the mutation L346P in a 48-year-old unaffected individual [6]. Login to comment
23 L346P in combination with F508del or 1677delTA (c.1545_1546delTA) leads to a mild or atypical phenotype. Login to comment
24 Therefore, the authors suggest that either L346P is dominant over M348K or that M348K is a J. Hentschel (*) :G. Riesener :H. Nelle :F. von Eggeling Institute of Human Genetics, Jena University Hospital, Kollegiengasse 10, 07743 Jena, Germany e-mail: julia.hentschel@mti.uni-jena.de J. Hentschel :J. Login to comment

[hide] Genotyping microarray for the detection of more th... J Mol Diagn. 2005 Aug;7(3):375-87. Schrijver I, Oitmaa E, Metspalu A, Gardner P
Genotyping microarray for the detection of more than 200 CFTR mutations in ethnically diverse populations.
J Mol Diagn. 2005 Aug;7(3):375-87., [PMID:16049310]

Abstract [show]
Cystic fibrosis (CF), which is due to mutations in the cystic fibrosis transmembrane conductance regulator gene, is a common life-shortening disease. Although CF occurs with the highest incidence in Caucasians, it also occurs in other ethnicities with variable frequency. Recent national guidelines suggest that all couples contemplating pregnancy should be informed of molecular screening for CF carrier status for purposes of genetic counseling. Commercially available CF carrier screening panels offer a limited panel of mutations, however, making them insufficiently sensitive for certain groups within an ethnically diverse population. This discrepancy is even more pronounced when such carrier screening panels are used for diagnostic purposes. By means of arrayed primer extension technology, we have designed a genotyping microarray with 204 probe sites for CF transmembrane conductance regulator gene mutation detection. The arrayed primer extension array, based on a platform technology for disease detection with multiple applications, is a robust, cost-effective, and easily modifiable assay suitable for CF carrier screening and disease detection.

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51 Complete List of Mutations Detectable with the CF APEX Assay CFTR location Amino acid change Nucleotide change 1 E 1 Frameshift 175delC 2 E 2,3 Frameshift del E2, E3 3 E 2 W19C 189 GϾT 4 E 2 Q39X 247 CϾT 5 IVS 2 Possible splicing defect 296 ϩ 12 TϾC 6 E 3 Frameshift 359insT 7 E 3 Frameshift 394delTT 8 E 3 W57X (TAG) 302GϾA 9 E 3 W57X (TGA) 303GϾA 10 E 3 E60X 310GϾT 11 E 3 P67L 332CϾT 12 E 3 R74Q 353GϾA 13 E 3 R75X 355CϾT 14 E 3 G85E 386GϾA 15 E 3 G91R 403GϾA 16 IVS 3 Splicing defect 405 ϩ 1GϾA 17 IVS 3 Possible splicing defect 405 ϩ 3AϾC 18 IVS 3 Splicing defect 406 - 1GϾA 19 E 4 E92X 406GϾT 20 E 4 E92K 406GϾA 21 E 4 Q98R 425AϾG 22 E 4 Q98P 425AϾC 23 E 4 Frameshift 444delA 24 E 4 Frameshift 457TATϾG 25 E 4 R117C 481CϾT 26 E 4 R117H 482GϾA 27 E 4 R117P 482GϾC 28 E 4 R117L 482GϾT 29 E 4 Y122X 498TϾA 30 E 4 Frameshift 574delA 31 E 4 I148T 575TϾC 32 E 4 Splicing defect 621GϾA 33 IVS 4 Splicing defect 621 ϩ 1GϾT 34 IVS 4 Splicing defect 621 ϩ 3AϾG 35 E 5 Frameshift 624delT 36 E 5 Frameshift 663delT 37 E 5 G178R 664GϾA 38 E 5 Q179K 667CϾA 39 IVS 5 Splicing defect 711 ϩ 1GϾT 40 IVS 5 Splicing defect 711 ϩ 1GϾA 41 IVS 5 Splicing defect 712 - 1GϾT 42 E 6a H199Y 727CϾT 43 E 6a P205S 745CϾT 44 E 6a L206W 749TϾG 45 E 6a Q220X 790CϾT 46 E 6b Frameshift 935delA 47 E 6b Frameshift 936delTA 48 E 6b N287Y 991AϾT 49 IVS 6b Splicing defect 1002 - 3TϾG 50 E 7 ⌬F311 3-bp del between nucleotides 1059 and 1069 51 E 7 Frameshift 1078delT 52 E 7 Frameshift 1119delA 53 E 7 G330X 1120GϾT 54 E 7 R334W 1132CϾT 55 E 7 I336K 1139TϾA 56 E 7 T338I 1145CϾT 57 E 7 Frameshift 1154insTC 58 E 7 Frameshift 1161delC 59 E 7 L346P 1169TϾC 60 E 7 R347H 1172GϾA 61 E 7 R347P 1172GϾC 62 E 7 R347L 1172GϾT 63 E 7 R352Q 1187GϾA 64 E 7 Q359K/T360K 1207CϾA and 1211CϾA 65 E 7 S364P 1222TϾC 66 E 8 Frameshift 1259insA 67 E 8 W401X (TAG) 1334GϾA 68 E 8 W401X (TGA) 1335GϾA 69 IVS 8 Splicing changes 1342 - 6 poly(T) variants 5T/7T/9T 70 IVS 8 Splicing defect 1342 - 2AϾC Table 1. Continued CFTR location Amino acid change Nucleotide change 71 E 9 A455E 1496CϾA 72 E 9 Frameshift 1504delG 73 E 10 G480C 1570GϾT 74 E 10 Q493X 1609CϾT 75 E 10 Frameshift 1609delCA 76 E 10 ⌬I507 3-bp del between nucleotides 1648 and 1653 77 E 10 ⌬F508 3-bp del between nucleotides 1652 and 1655 78 E 10 Frameshift 1677delTA 79 E 10 V520F 1690GϾT 80 E 10 C524X 1704CϾA 81 IVS 10 Possible splicing defect 1717 - 8GϾA 82 IVS 10 Splicing defect 1717 - 1GϾA 83 E 11 G542X 1756GϾT 84 E 11 G551D 1784GϾA 85 E 11 Frameshift 1784delG 86 E 11 S549R (AϾC) 1777AϾC 87 E 11 S549I 1778GϾT 88 E 11 S549N 1778GϾA 89 E 11 S549R (TϾG) 1779TϾG 90 E 11 Q552X 1786CϾT 91 E 11 R553X 1789CϾT 92 E 11 R553G 1789CϾG 93 E 11 R553Q 1790GϾA 94 E 11 L558S 1805TϾC 95 E 11 A559T 1807GϾA 96 E 11 R560T 1811GϾC 97 E 11 R560K 1811GϾA 98 IVS 11 Splicing defect 1811 ϩ 1.6 kb AϾG 99 IVS 11 Splicing defect 1812 - 1GϾA 100 E 12 Y563D 1819TϾG 101 E 12 Y563N 1819TϾA 102 E 12 Frameshift 1833delT 103 E 12 D572N 1846GϾA 104 E 12 P574H 1853CϾA 105 E 12 T582R 1877CϾG 106 E 12 E585X 1885GϾT 107 IVS 12 Splicing defect 1898 ϩ 5GϾT 108 IVS 12 Splicing defect 1898 ϩ 1GϾA 109 IVS 12 Splicing defect 1898 ϩ 1GϾC 110 IVS 12 Splicing defect 1898 ϩ 1GϾT 111 E 13 Frameshift 1924del7 112 E 13 del of 28 amino acids 1949del84 113 E 13 I618T 1985TϾC 114 E 13 Frameshift 2183AAϾG 115 E 13 Frameshift 2043delG 116 E 13 Frameshift 2055del9ϾA 117 E 13 D648V 2075TϾA 118 E 13 Frameshift 2105-2117 del13insAGAA 119 E 13 Frameshift 2108delA 120 E 13 R668C 2134CϾT 121 E 13 Frameshift 2143delT 122 E 13 Frameshift 2176insC 123 E 13 Frameshift 2184delA 124 E 13 Frameshift 2184insA 125 E 13 Q685X 2185CϾT 126 E 13 R709X 2257CϾT 127 E 13 K710X 2260AϾT 128 E 13 Frameshift 2307insA 129 E 13 V754M 2392GϾA 130 E 13 R764X 2422CϾT 131 E 14a W846X 2670GϾA 132 E 14a Frameshift 2734delGinsAT 133 E 14b Frameshift 2766del8 134 IVS 14b Splicing defect 2789 ϩ 5GϾA 135 IVS 14b Splicing defect 2790 - 2AϾG 136 E 15 Q890X 2800CϾT 137 E 15 Frameshift 2869insG 138 E 15 S945L 2966CϾT 139 E 15 Frameshift 2991del32 140 E 16 Splicing defect 3120GϾA interrogation: ACCAACATGTTTTCTTTGATCTTAC 3121-2A3G,T S; 5Ј-ACCAACATGTTTTCTTTGATCTTAC A GTTGTTATTAATTGTGATTGGAGCTATAG-3Ј; CAACAA- TAATTAACACTAACCTCGA 3121-2A3G,T AS. Login to comment
150 Primers Generated to Create Synthetic Templates That Serve As Positive Mutation Controls Primer name Sense strand 5Ј 3 3Ј Name Antisense strand 5Ј 3 3Ј 175delC synt F T(15)ATTTTTTTCAGGTGAGAAGGTGGCCA 175delC synt R T(15)ATTTGGAGACAACGCTGGCCTTTTCC W19C synt F T(15)TACCAGACCAATTTTGAGGAAAGGAT W19C synt R T(15)ACAGCTAAAATAAAGAGAGGAGGAAC Q39X synt F T(15)TAAATCCCTTCTGTTGATTCTGCTGA Q39X synt R T(15)AGTATATGTCTGACAATTCCAGGCGC 296 ϩ 12TϾC synt F T(15)CACATTGTTTAGTTGAAGAGAGAAAT 296 ϩ 12TϾC synt R T(15)GCATGAACATACCTTTCCAATTTTTC 359insT synt F T(15)TTTTTTTCTGGAGATTTATGTTCTAT 359insT synt R T(15)AAAAAAACATCGCCGAAGGGCATTAA E60X synt F T(15)TAGCTGGCTTCAAAGAAAAATCCTAA E60X synt R T(15)ATCTATCCCATTCTCTGCAAAAGAAT P67L synt F T(15)TTAAACTCATTAATGCCCTTCGGCGA P67L synt R T(15)AGATTTTTCTTTGAAGCCAGCTCTCT R74Q synt F T(15)AGCGATGTTTTTTCTGGAGATTTATG R74Q synt R T(15)TGAAGGGCATTAATGAGTTTAGGATT R75X synt F T(15)TGATGTTTTTTCTGGAGATTTATGTT R75X synt R T(15)ACCGAAGGGCATTAATGAGTTTAGGA W57X(TAG) synt F T(15)AGGATAGAGAGCTGGCTTCAAAGAAA W57X(TAG) synt R T(15)TATTCTCTGCAAAAGAATAAAAAGTG W57X(TGA) synt F T(15)AGATAGAGAGCTGGCTTCAAAGAAAA W57X(TGA) synt R T(15)TCATTCTCTGCAAAAGAATAAAAAGT G91R synt F T(15)AGGGTAAGGATCTCATTTGTACATTC G91R synt R T(15)TTAAATATAAAAAGATTCCATAGAAC 405 ϩ 1GϾA synt F T(15)ATAAGGATCTCATTTGTACATTCATT 405 ϩ 1GϾA synt R T(15)TCCCTAAATATAAAAAGATTCCATAG 405 ϩ 3AϾC synt F T(15)CAGGATCTCATTTGTACATTCATTAT 405 ϩ 3AϾC synt R T(15)GACCCCTAAATATAAAAAGATTCCAT 406 - 1GϾA synt F T(15)AGAAGTCACCAAAGCAGTACAGCCTC 406 - 1GϾA synt R T(15)TTACAAAAGGGGAAAAACAGAGAAAT E92X synt F T(15)TAAGTCACCAAAGCAGTACAGCCTCT E92X synt R T(15)ACTACAAAAGGGGAAAAACAGAGAAA E92K synt F T(15)AAAGTCACCAAAGCAGTACAGCCTCT E92K synt R T(15)TCTACAAAAGGGGAAAAACAGAGAAA 444delA synt F T(15)GATCATAGCTTCCTATGACCCGGATA 444delA synt R T(15)ATCTTCCCAGTAAGAGAGGCTGTACT 574delA synt F T(15)CTTGGAATGCAGATGAGAATAGCTAT 574delA synt R T(15)AGTGATGAAGGCCAAAAATGGCTGGG 621GϾA synt F T(15)AGTAATACTTCCTTGCACAGGCCCCA 621GϾA synt R T(15)TTTCTTATAAATCAAACTAAACATAG Q98P synt F T(15)CGCCTCTCTTACTGGGAAGAATCATA Q98P synt R T(15)GGTACTGCTTTGGTGACTTCCTACAA 457TATϾG synt F T(15)GGACCCGGATAACAAGGAGGAACGCT 457TATϾG synt R T(15)CGGAAGCTATGATTCTTCCCAGTAAG I148T synt F T(15)CTGGAATGCAGATGAGAATAGCTATG I148T synt R T(15)GTGTGATGAAGGCCAAAAATGGCTGG 624delT synt F T(15)CTTAAAGCTGTCAAGCCGTGTTCTAG 624delT synt R T(15)TAAGTCTAAAAGAAAAATGGAAAGTT 663delT synt F T(15)ATGGACAACTTGTTAGTCTCCTTTCC 663delT synt R T(15)CATACTTATTTTATCTAGAACACGGC G178R synt F T(15)AGACAACTTGTTAGTCTCCTTTCCAA G178R synt R T(15)TAATACTTATTTTATCTAGAACACGG Q179K synt F T(15)AAACTTGTTAGTCTCCTTTCCAACAA Q179K synt R T(15)TTCCAATACTTATTTTATCTAGAACA 711 ϩ 5GϾA synt F T(15)ATACCTATTGATTTAATCTTTTAGGC 711 ϩ 5GϾA synt R T(15)TTATACTTCATCAAATTTGTTCAGGT 712 - 1GϾT synt F T(15)TGGACTTGCATTGGCACATTTCGTGT 712 - 1GϾT synt R T(15)TATGGAAAATAAAAGCACAGCAAAAAC H199Y synt F T(15)TATTTCGTGTGGATCGCTCCTTTGCA H199Y synt R T(15)TATGCCAATGCTAGTCCCTGGAAAATA P205S synt F T(15)TCTTTGCAAGTGGCACTCCTCATGGG P205S synt R T(15)TAAGCGATCCACACGAAATGTGCCAAT L206W synt F T(15)GGCAAGTGGCACTCCTCATGGGGCTA L206W synt R T(15)TCAAGGAGCGATCCACACGAAATGTGC Q220X synt F T(15)TAGGCGTCTGCTTTCTGTGGACTTGG Q220X synt R T(15)TATAACAACTCCCAGATTAGCCCCATG 936delTA synt F T(15)AATCCAATCTGTTAAGGCATACTGCT 936delTA synt R T(15)TGATTTTCAATCATTTCTGAGGTAATC 935delA synt F T(15)GAAATATCCAATCTGTTAAGGCATAC 935delA synt R T(15)TATTTCAATCATTTCTGAGGTAATCAC N287Y synt F T(15)TACTTAAGACAGTAAGTTGTTCCAAT N287Y synt R T(15)TATTCAATCATTTTTTCCATTGCTTCT 1002 - 3TϾG synt F T(15)GAGAACAGAACTGAAACTGACTCGGA 1002 - 3TϾG synt R T(15)TCTAAAAAACAATAACAATAAAATTCA 1154insTC syntwt F T(15)ATCTCATTCTGCATTGTTCTGCGCAT 1154insTC syntwt R T(15)TTGAGATGGTGGTGAATATTTTCCGGA 1154insTC syntmt F T(15)TCTCTCATTCTGCATTGTTCTGCGCAT 1154insTC syntmt R T(15)TAGAGATGGTGGTGAATATTTTCCGGA DF311 mt syntV1 F T(15)CCTTCTTCTCAGGGTTCTTTGTGGTG dF311 mt syntV1 R T(15)GAGAAGAAGGCTGAGCTATTGAAGTATC G330X synt F T(15)TGAATCATCCTCCGGAAAATATTCAC G330X synt R T(15)ATTTGATTAGTGCATAGGGAAGCACA S364P synt F T(15)CCTCTTGGAGCAATAAACAAAATACA S364P synt R T(15)GGTCATACCATGTTTGTACAGCCCAG Q359K/T360K mt synt F T(15)AAAAAATGGTATGACTCTCTTGGAGC Q359K/T360K mt synt R T(15)TTTTTTACAGCCCAGGGAAATTGCCG 1078delT synt F T(15)CTTGTGGTGTTTTTATCTGTGCTTCC 1078delT synt R T(15)CAAGAACCCTGAGAAGAAGAAGGCTG 1119delA synt F T(15)CAAGGAATCATCCTCCGGAAAATATT 1119delA synt R T(15)CTTGATTAGTGCATAGGGAAGCACAG 1161delC synt F T(15)GATTGTTCTGCGCATGGCGGTCACTC 1161delC synt R T(15)TCAGAATGAGATGGTGGTGAATATTT T338I synt F T(15)TCACCATCTCATTCTGCATTGTTCTG T338I synt R T(15)ATGAATATTTTCCGGAGGATGATTCC R352Q synt F T(15)AGCAATTTCCCTGGGCTGTACAAACA R352Q synt R T(15)TGAGTGACCGCCATGCGCAGAACAAT L346P synt F T(15)CGCGCATGGCGGTCACTCGGCAATTT L346P synt R T(15)GGAACAATGCAGAATGAGATGGTGGT 1259insA synt F T(15)AAAAAGCAAGAATATAAGACATTGGA 1259insA synt R T(15)TTTTTGTAAGAAATCCTATTTATAAA W401X(TAG)mtsynt F T(15)AGGAGGAGGTCAGAATTTTTAAAAAA W401X(TAG)mtsynt R T(15)TAGAAGGCTGTTACATTCTCCATCAC W401X(TGA) synt F T(15)AGAGGAGGTCAGAATTTTTAAAAAAT W401X(TGA) synt R T(15)TCAGAAGGCTGTTACATTCTCCATCA 1342 - 2AϾC synt F T(15)CGGGATTTGGGGAATTATTTGAGAAA 1342 - 2AϾC synt R T(15)GGTTAAAAAAACACACACACACACAC 1504delG synt F T(15)TGATCCACTGTAGCAGGCAAGGTAGT 1504delG synt R T(15)TCAGCAACCGCCAACAACTGTCCTCT G480C synt F T(15)TGTAAAATTAAGCACAGTGGAAGAAT G480C synt R T(15)ACTCTGAAGGCTCCAGTTCTCCCATA C524X synt F T(15)ACAACTAGAAGAGGTAAGAAACTATG C524X synt R T(15)TCATGCTTTGATGACGCTTCTGTATC V520F synt F T(15)TTCATCAAAGCAAGCCAACTAGAAGA V520F synt R T(15)AGCTTCTGTATCTATATTCATCATAG 1609delCA synt F T(15)TGTTTTCCTGGATTATGCCTGGCACC 1609delCA synt R T(15)CAGAACAGAATGAAATTCTTCCACTG 1717 - 8GϾA synt F T(15)AGTAATAGGACATCTCCAAGTTTGCA 1717 - 8GϾA synt R T(15)TAAAAATAGAAAATTAGAGAGTCACT 1784delG synt F T(15)AGTCAACGAGCAAGAATTTCTTTAGC 1784delG synt R T(15)ACTCCACTCAGTGTGATTCCACCTTC A559T synt F T(15)ACAAGGTGAATAACTAATTATTGGTC A559T synt R T(15)TTAAAGAAATTCTTGCTCGTTGACCT Q552X synt F T(15)TAACGAGCAAGAATTTCTTTAGCAAG Q552X synt R T(15)AACCTCCACTCAGTGTGATTCCACCT S549R(AϾC) synt F T(15)CGTGGAGGTCAACGAGCAAGAATTTC S549R(AϾC) synt R T(15)GCAGTGTGATTCTACCTTCTCCAAGA S549R(TϾG) synt F T(15)GGGAGGTCAACGAGCAAGTATTTC S549R(TϾG) synt R T(15)CCTCAGTGTGATTCCACCTTCTCCAA L558S synt F T(15)CAGCAAGGTGAATAACTAATTATTGG L558S synt R T(15)GAAGAAATTCTCGCTCGTTGACCTCC 1811 ϩ 1.6 kb AϾG synt F T(15)GTAAGTAAGGTTACTATCAATCACAC 1811 ϩ 1.6 kb AϾG synt R T(15)CATCTCAAGTACATAGGATTCTCTGT 1812 - 1GϾA synt F T(15)AAGCAGTATACAAAGATGCTGATTTG 1812 - 1GϾA synt R T(15)TTAAAAAGAAAATGGAAATTAAATTA D572N synt F T(15)AACTCTCCTTTTGGATACCTAGATGT D572N synt R T(15)TTAATAAATACAAATCAGCATCTTTG P574H synt F T(15)ATTTTGGATACCTAGATGTTTTAACA P574H synt R T(15)TGAGAGTCTAATAAATACAAATCAGC 1833delT synt F T(15)ATTGTATTTATTAGACTCTCCTTTTG 1833delT synt R T(15)CAATCAGCATCTTTGTATACTGCTCT Table 4. Continued Primer name Sense strand 5Ј 3 3Ј Name Antisense strand 5Ј 3 3Ј Y563D synt F T(15)GACAAAGATGCTGATTTGTATTTATT Y563D synt R T(15)CTACTGCTCTAAAAAGAAAATGGAAA T582R synt F T(15)GAGAAAAAGAAATATTTGAAAGGTAT T582R synt R T(15)CTTAAAACATCTAGGTATCCAAAAGG E585X synt F T(15)TAAATATTTGAAAGGTATGTTCTTTG E585X synt R T(15)ATTTTTCTGTTAAAACATCTAGGTAT 1898 ϩ 5GϾT synt F T(15)TTTCTTTGAATACCTTACTTATATTG 1898 ϩ 5GϾT synt R T(15)AATACCTTTCAAATATTTCTTTTTCT 1924del7 synt F T(15)CAGGATTTTGGTCACTTCTAAAATGG 1924del7 synt R T(15)CTGTTAGCCATCAGTTTACAGACACA 2055del9ϾA synt F T(15)ACATGGGATGTGATTCTTTCGACCAA 2055del9ϾA synt R T(15)TCTAAAGTCTGGCTGTAGATTTTGGA D648V synt F T(15)TTTCTTTCGACCAATTTAGTGCAGAA D648V synt R T(15)ACACATCCCATGAGTTTTGAGCTAAA K710X synt F T(15)TAATTTTCCATTGTGCAAAAGACTCC K710X synt R T(15)ATCGTATAGAGTTGATTGGATTGAGA I618T synt F T(15)CTTTGCATGAAGGTAGCAGCTATTTT I618T synt R T(15)GTTAATATTTTGTCAGCTTTCTTTAA R764X synt F T(15)TGAAGGAGGCAGTCTGTCCTGAACCT R764X synt R T(15)ATGCCTGAAGCGTGGGGCCAGTGCTG Q685X synt F T(15)TAATCTTTTAAACAGACTGGAGAGTT Q685X synt R T(15)ATTTTTTTGTTTCTGTCCAGGAGACA R709X synt F T(15)TGAAAATTTTCCATTGTGCAAAAGAC R709X synt R T(15)ATATAGAGTTGATTGGATTGAGAATA V754M synt F T(15)ATGATCAGCACTGGCCCCACGCTTCA V754M synt R T(15)TGCTGATGCGAGGCAGTATCGCCTCT 1949del84 synt F T(15)AAAAATCTACAGCCAGACTTTATCTC 1949del84 synt R T(15)TTTTTAGAAGTGACCAAAATCCTAGT 2108delA synt F T(15)GAATTCAATCCTAACTGAGACCTTAC 2108delA synt R T(15)ATTCTTCTTTCTGCACTAAATTGGTC 2176insC synt F T(15)CCAAAAAAACAATCTTTTAAACAGACTGGAGAG 2176insC synt R T(15)GGTTTCTGTCCAGGAGACAGGAGCAT 2184delA synt F T(15)CAAAAAACAATCTTTTAAACAGACTGG 2184delA synt R T(15)GTTTTTTGTTTCTGTCCAGGAGACAG 2105-2117 del13 synt F T(15)AAACTGAGACCTTACACCGTTTCTCA 2105-2117 del13 synt R T(15)TTTCTTTCTGCACTAAATTGGTCGAA 2307insA synt F T(15)AAAGAGGATTCTGATGAGCCTTTAGA 2307insA synt R T(15)TTTCGATGCCATTCATTTGTAAGGGA W846X synt F T(15)AAACACATACCTTCGATATATTACTGTCCAC W846X synt R T(15)TCATGTAGTCACTGCTGGTATGCTCT 2734G/AT synt F T(15)TTAATTTTTCTGGCAGAGGTAAGAAT 2734G/AT synt R T(15)TTAAGCACCAAATTAGCACAAAAATT 2766del8 synt F T(15)GGTGGCTCCTTGGAAAGTGAGTATTC 2766del8 synt R T(15)CACCAAAGAAGCAGCCACCTGGAATGG 2790 - 2AϾG synt F T(15)GGCACTCCTCTTCAAGACAAAGGGAA 2790 - 2AϾG synt R T(15)CGTAAAGCAAATAGGAAATCGTTAAT 2991del32 synt F T(15)TTCAACACGTCGAAAGCAGGTACTTT 2991del32 synt R T(15)AAACATTTTGTGGTGTAAAATTTTCG Q890X synt F T(15)TAAGACAAAGGGAATAGTACTCATAG Q890X synt R T(15)AAAGAGGAGTGCTGTAAAGCAAATAG 2869insG synt F T(15)GATTATGTGTTTTACATTTACGTGGG 2869insG synt R T(15)CACGAACTGGTGCTGGTGATAATCAC 3120GϾA synt F T(15)AGTATGTAAAAATAAGTACCGTTAAG 3120GϾA synt R T(15)TTGGATGAAGTCAAATATGGTAAGAG 3121 - 2AϾT synt F T(15)TGTTGTTATTAATTGTGATTGGAGCT 3121 - 2AϾT synt R T(15)AGTAAGATCAAAGAAAACATGTTGGT 3132delTG synt F T(15)TTGATTGGAGCCATAGCAGTTGTCGC 3132delTG synt R T(15)AATTAATAACAACTGTAAGATCAAAG 3271delGG synt F T(15)ATATGACAGTGAATGTGCGATACTCA 3271delGG synt R T(15)ATTCAGATTCCAGTTGTTTGAGTTGC 3171delC synt F T(15)ACCTACATCTTTGTTGCAACAGTGCC 3171delC synt R T(15)AGGTTGTAAAACTGCGACAACTGCTA 3171insC synt F T(15)CCCCTACATCTTTGTTGCTACAGTGC 3171insC synt R T(15)GGGGTTGTAAAACTGCGACAACTGCT 3199del6 synt F T(15)GAGTGGCTTTTATTATGTTGAGAGCATAT 3199del6 synt R T(15)CCACTGGCACTGTTGCAACAAAGATG M1101K synt F T(15)AGAGAATAGAAATGATTTTTGTCATC M1101K synt R T(15)TTTTGGAACCAGCGCAGTGTTGACAG G1061R synt F T(15)CGACTATGGACACTTCGTGCCTTCGG G1061R synt R T(15)GTTTTAAGCTTGTAACAAGATGAGTG R1066L synt F T(15)TTGCCTTCGGACGGCAGCCTTACTTT R1066L synt R T(15)AGAAGTGTCCATAGTCCTTTTAAGCT R1070P synt F T(15)CGCAGCCTTACTTTGAAACTCTGTTC R1070P synt R T(15)GGTCCGAAGGCACGAAGTGTCCATAG L1077P synt F T(15)CGTTCCACAAAGCTCTGAATTTACAT L1077P synt R T(15)GGAGTTTCAAAGTAAGGCTGCCGTCC W1089X synt F T(15)AGTTCTTGTACCTGTCAACACTGCGC W1089X synt R T(15)TAGTTGGCAGTATGTAAATTCAGAGC L1093P synt F T(15)CGTCAACACTGCGCTGGTTCCAAATG L1093P synt R T(15)GGGTACAAGAACCAGTTGGCAGTATG W1098R synt F T(15)CGGTTCCAAATGAGAATAGAAATGAT W1098R synt R T(15)GGCGCAGTGTTGACAGGTACAAGAAC Q1100P synt F T(15)CAATGAGAATAGAAATGATTTTTGTC Q1100P synt R T(15)GGGAACCAGCGCAGTGTTGACAGGTA D1152H synt F T(15)CATGTGGATAGCTTGGTAAGTCTTAT D1152H synt R T(15)GTATGCTGGAGTTTACAGCCCACTGC R1158X synt F T(15)TGATCTGTGAGCCGAGTCTTTAAGTT R1158X synt R T(15)ACATCTGAAATAAAAATAACAACATT S1196X synt F T(15)GACACGTGAAGAAAGATGACATCTGG S1196X synt R T(15)CAATTCTCAATAATCATAACTTTCGA 3732delA synt F T(15)GGAGATGACATCTGGCCCTCAGGGGG 3732delA synt R T(15)CTCCTTCACGTGTGAATTCTCAATAA 3791delC synt F T(15)AAGAAGGTGGAAATGCCATATTAGAG 3791delC synt R T(15)TTGTATTTTGCTGTGAGATCTTTGAC 3821delT synt F T(15)ATTCCTTCTCAATAAGTCCTGGCCAG 3821delT synt R T(15)GAATGTTCTCTAATATGGCATTTCCA Q1238X synt F T(15)TAGAGGGTGAGATTTGAACACTGCTT Q1238X synt R T(15)AGCCAGGACTTATTGAGAAGGAAATG S1255X (ex19)synt F T(15)GTCTGGCCCTCAGGGGGCCAAATGAC S1255X (ex19) synt R T(15)CGTCATCTTTCTTCACGTGTGAATTC S1255X;L synt F T(15)AAGCTTTTTTGAGACTACTGAACACT S1255X;L synt R T(15)TATAACAAAGTAATCTTCCCTGATCC 3849 ϩ 4AϾG synt F T(15)GGATTTGAACACTGCTTGCTTTGTTA 3849 ϩ 4AϾG synt R T(15)CCACCCTCTGGCCAGGACTTATTGAG 3850 - 1GϾA synt F T(15)AGTGGGCCTCTTGGGAAGAACTGGAT 3850 - 1GϾA synt R T(15)TTATAAGGTAAAAGTGATGGGATCAC 3905insT synt F T(15)TTTTTTTGAGACTACTGAACACTGAA 3905insT synt R T(15)AAAAAAAGCTGATAACAAAGTACTCT 3876delA synt F T(15)CGGGAAGAGTACTTTGTTATCAGCTT 3876delA synt R T(15)CGATCCAGTTCTTCCCAAGAGGCCCA G1244V synt F T(15)TAAGAACTGGATCAGGGAAGAGTACT G1244V synt R T(15)ACCAAGAGGCCCACCTATAAGGTAAA G1249E synt F T(15)AGAAGAGTACTTTGTTATCAGCTTTT G1249E synt R T(15)TCTGATCCAGTTCTTCCCAAGAGGCC S1251N synt F T(15)ATACTTTGTTATCAGCTTTTTTGAGACTACTG S1251N synt R T(15)TTCTTCCCTGATCCAGTTCTTCCCAA S1252P synt F T(15)CCTTTGTTATCAGCTTTTTTGAGACT S1252P synt R T(15)GACTCTTCCCTGATCCAGTTCTTCCC D1270N synt F T(15)AATGGTGTGTCTTGGGATTCAATAAC D1270N synt R T(15)TGATCTGGATTTCTCCTTCAGTGTTC W1282R synt F T(15)CGGAGGAAAGCCTTTGGAGTGATACC W1282R synt R T(15)GCTGTTGCAAAGTTATTGAATCCCAA R1283K synt F T(15)AGAAAGCCTTTGGAGTGATACCACAG R1283K synt R T(15)TTCCACTGTTGCAAAGTTATTGAATC 4005 ϩ 1GϾA synt F T(15)ATGAGCAAAAGGACTTAGCCAGAAAA 4005 ϩ 1GϾA synt R T(15)TCTGTGGTATCACTCCAAAGGCTTTC 4010del4 synt F T(15)GTATTTTTTCTGGAACATTTAGAAAAAACTTGG 4010del4 synt R T(15)AAAATACTTTCTATAGCAAAAAAGAAAAGAAGAA 4016insT synt F T(15)TTTTTTTCTGGAACATTTAGAAAAAACTTGG 4016insT synt R T(15)AAAAAAATAAATACTTTCTATAGCAAAAAAGAAAAGAAGA CFTRdele21 synt F T(15)TAGGTAAGGCTGCTAACTGAAATGAT CFTRdele21 synt R T(15)CCTATAGCAAAAAAGAAAAGAAGAAGAAAGTATG 4382delA synt F T(15)GAGAGAACAAAGTGCGGCAGTACGAT 4382delA synt R T(15)CTCTATGACCTATGGAAATGGCTGTT Bold, mutation allele of interest; bold and italicized, modified nucleotide. 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[hide] Description of a symptomless cystic fibrosis L346P... Mol Cell Probes. 1996 Aug;10(4):315-8. Deltas CC, Boteva K, Georgiou A, Papageorgiou E, Georgiou C
Description of a symptomless cystic fibrosis L346P/M348K compound heterozygous Cypriot individual.
Mol Cell Probes. 1996 Aug;10(4):315-8., [PMID:8865181]

Abstract [show]
During the past few years we have been testing the hypothesis that Cyprus may have been spared many severe cystic fibrosis (CF) cases but not cystic fibrosis transmembrane conductance regulator (CFTR) mutations. We have been analysing by molecular methods patients with atypical mild phenotypes where CF enters the differential diagnosis. With this approach we identified a mutation, L346P, which in association with the severe mutation delta F508 or 1677delTA, confers a mild and atypical presentation. Recently, we identified another entirely symptomless 48-year-old individual, with genotype L346P/M348K. The fact that M348K was initially identified in a severely affected Italian patient strengthens the hypothesis that L346P, a putative mild mutation, is dominant over severe ones. One other explanation is that M348K is not a causative defect but a rare polymorphism. These findings have important implications for genetic counselling, especially when the counselling is sought by concerned couples for prenatal diagnostic purposes.

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No. Sentence Comment
0 Molecular and Cellular Probes (1996) 10, 315-318 Short Communication Description of a symptomless cystic fibrosis L346P/ M348K compound heterozygous Cypriot individual C. Constantinou Deltas,1 * Kalina Boteva,1 Andreas Georgiou,2 Elena Papageorgiou1 and Christina Georgiou3 1 The Cyprus Institute of Neurology and Genetics, 2 Nicosia General Hospital, and 3 Archbishop Makarios III Hospital, Nicosia, Cyprus (Received 12 January, Accepted 6 February 1996) During the past few years we have been testing the hypothesis that Cyprus may have been spared many severe cystic fibrosis (CF) cases but not cystic fibrosis transmembrane conductance regulator (CFTR) mutations. Login to comment
2 With this approach we identified a mutation, L346P, which in association with the severe mutation F508 or 1677delTA, confers a mild and atypical presentation. Login to comment
3 Recently, we identified another entirely symptomless 48-year-old individual, with genotype L346P/M348K. Login to comment
4 The fact that M348K was initially identified in a severely affected Italian patient strengthens the hypothesis that L346P, a putative mild mutation, is dominant over severe ones. Login to comment
17 The second mutation in the three Cypriot patients who carry L346P is F508,is that Cyprus might have been spared many severe CF cases, but not CFTR mutations. Login to comment
34 pothesis that suggests that severe mutations are recessive over mild mutations.8,10,13 In the three CypriotOur patient (III-7, see Fig. 1) had inherited the M348K mutation from his mother who belongs to a patients, the same L346P mutation, which is present in exon 7, in one of the transmembrane domains offamily of nine siblings. Login to comment
38 Mutation R347P,14 which is right next toa carrier of another mutation, L346P, that she inherited from her mother. Login to comment
39 She passed one of each mutations L346P, was also found to be associated with mild symptomatology. Login to comment
42 She aminoacid further, was initially described in a severely affected patient.7 It is quite interesting that the twohas no symptoms whatsoever, and there has never been any suspicion that she might be suffering from patients with genotypes L346P/ F508 and L346P/ 1677delTA did have some symptoms, althougha major inherited disorder. Login to comment
43 She is rather short, but her sister II-11 is also short and is only a carrier of one mild,8 whereas the most recent patient with genotype L346P/M348K has almost no symptomatology at themutation. Login to comment
49 Her physical examination and laboratory examinations were nor- Finally, our findings provide further evidence that mutation L346P might be responsible for the mildmal, including one sweat test. Login to comment
50 Mutation L346P was initially identified in two other symptomatology of CF patients who are also carriers of mutations that are known to seriously affect theunrelated Cypriot patients. Login to comment
52 However, onemajor findings other than failure to thrive, occasional electrolyte disturbances and some chest infections in other likely explanation for the symptomless status of the 48-year-old lady described in this report is thatone of the patients.8 So far we are not aware of additional patients from other populations carrying mutation M348K does not really affect the function L346P/M348K CF symptomless individual 317 293 255 38 118 17 NlaIII NlaIII1 428 Abolished by the M348K mutation L346P M348K [M348K/N] L346P/N M348K/N M348K/N M348K/N M348K/N N/N N/N 1 2 3 4 5 6 7 8 9 10 11 12 1 2 1 2 3 4 5 6 7 M348K/NM348K/N L346P/N N/N M348K/? Login to comment
56 The proband was individual III-7, and his maternal aunt II-2 was found to have inherited mutations M348K and L346P. Login to comment
58 L346P was tested by DNA amplification of exon 7, followed by digestion with BstUI. Login to comment

[hide] Novel cystic fibrosis mutation associated with mil... Hum Genet. 1994 May;93(5):529-32. Boteva K, Papageorgiou E, Georgiou C, Angastiniotis M, Middleton LT, Constantinou-Deltas CD
Novel cystic fibrosis mutation associated with mild disease in Cypriot patients.
Hum Genet. 1994 May;93(5):529-32., [PMID:7513296]

Abstract [show]
Cyprus is an island in the eastern Mediterranean basin inhabited by people of Caucasian extraction, mostly Greek-Cypriots. The most common inherited disease among Caucasians is cystic fibrosis (CF). Although no careful scientific study had ever been done the impression was that CF was extremely rare among the Greek-Cypriots, with an incidence estimated at around 1:30,000. About 2 years ago, we introduced molecular diagnostic methodology in an effort to assist clinicians in safer diagnosis of patients presenting with atypical CF symptomatology, and also for testing the hypothesis that mutations that cause milder phenotypes might be responsible for misdiagnosis or for missing entirely some cases of CF. Initial screening for delta F508 revealed that it is indeed rare in the general population. Further screening of suspected CF patients revealed a novel mutation that converted leucine at position 346 to proline (L346P) in two unrelated families. The second CF mutation was delta F508 and 1677delTA in the two families respectively, both reportedly associated with severe phenotypes. Yet our patients did not present with typical CF pictures possibly because of the dominant nature of this novel mild mutation in exon 7. Symptoms included failure to thrive, chest infections and electrolyte disturbances. These findings raise the possibility that Cyprus might have been spared very severe CF phenotypes but not cystic fibrosis transmembrane conductance regulator (CFTR) mutations.

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21 Here we report on the identification of a novel mutation, L346P, in two unrelated Cypriot patients with mild symptomatology. Login to comment
42 Symptoms of characterized patients Patient 4457 Patient 5292 (L346P/AF508) (L346P/1677delTA) - No meconium ileus at birth - No gastrointestinalproblems - No pancreatic insufficiency - Sweat tests of 55 and 78 MEq/1 - Occasional electrolyte disturbances (mostly in summer as dehydration) - Some chest infectionsa - Failure to thrive - No meconium ileus at birth - One incidence of gastrointestinalproblems (vomiting, diarrhoea) - No pancreatic insufficiency - Negative sweat test - Occasional electrolyte disturbances - No lung involvement - Slight physical growth delay aNot unusuallyfrequent consideringthat this patient was born with transposition of the great arteries, for which he was operated on in the neonatal period Fig. 1. Login to comment
44 Members of the families that carry the L346P mutation are shown. Login to comment
54 This transition results in the substitution of proline for leucine at residue 346 (L346P), and it introduces a new restriction site for BstUI. Login to comment
65 Patient with DNA no. 5292 was first identified as a carrier of 1677delTA, Here it is shown that patients 4457 and 5292 are both also heterozygous for L346P. Login to comment
70 Detection of mutation L346P by PCR-restriction digestion. Login to comment
78 Also shown is that the sister of 4457 in family 2801 (4460), is heterozygous for L346P population (Constantinou-Deltas et al. 1992), whereas in mainland Greece one expects 1 AF508 chromosome in 110 (Balassopoulou et al. 1990). Login to comment
89 Hence, in our patients this is probably the case, with L346P dominant to AF508 and 1677delTA. Login to comment
90 It is interesting that L346P was found in two unrelated families, and this raises the possibility that other mildly affected patients carry this Cypriot mutation. Login to comment