J Korean Med Sci.  2010 Jan;25(1):166-171. 10.3346/jkms.2010.25.1.166.

The L441P Mutation of Cystic Fibrosis Transmembrane conductance Regulator and its Molecular Pathogenic Mechanisms in a Korean Patient with Cystic Fibrosis

• Gee HY
• Kim CK
• Kim SW
• Lee JH
• Kim JH
• Kim KH
• Lee MG

• KMID: 1713852
• DOI: http://doi.org/10.3346/jkms.2010.25.1.166

Abstract

Cystic fibrosis (CF) is an autosomal recessive disorder usually found in populations of white Caucasian descent. CF is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. A 5-yr-old Korean girl was admitted complaining of coughing and greenish sputum. Chest radiographs and computed tomographic (CT) scan revealed diffuse bronchiectasis in both lungs. The patient had chronic diarrhea and poor weight gain, and the abdominal pancreaticobiliary CT scan revealed atrophy of the pancreas. Finally, CF was confirmed by the repeated analysis of the quantitative pilocarpine iontophoresis test. The chloride concentration of sweat samples taken from both forearms of the pateint was an average of 88.7 mM/L (normal value <40 mM/L). After a comprehensive search for mutations in the CFTR gene, the patient was found to carry the non-synonymous L441P mutation in one allele. Molecular physiologic analysis of the L441P mutation of CFTR revealed that the L441P mutation completely abolished the CFTR Cl- channel activity by disrupting proper protein folding and membrane trafficking of CFTR protein. These results confirmed the pathogenicity of the L441P mutation of CFTR circulating in the Korean population. The possibility of CF should be suspected in patients with chronic bronchiectasis, although the frequency of CF is relatively rare in East Asia.

Keyword

Bronchiectasis; Cl- channel; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Korea

MeSH Terms

Amino Acid Substitution
Base Sequence
Cell Line
Child, Preschool
Cystic Fibrosis/diagnosis/*genetics
Cystic Fibrosis Transmembrane Conductance Regulator/*genetics/metabolism
Female
Humans
Lung/radiography
*Mutation
Patch-Clamp Techniques
Republic of Korea
Tomography, X-Ray Computed
Cystic Fibrosis Transmembrane Conductance Regulator

Figure

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  Fig. 1 Chest radiographs on admission. (A) Chest plain radiograph shows diffuse reticulonodular densities in both central lung areas symmetrically. (B) On computed tomographic image with lung window setting, diffuse bronchiectasis is seen in both lungs. There are hyperlucent areas in the lung parenchyma due to peripheral bronchial obstruction.

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  Fig. 2 Denaturing gradient gel electrophoresis (DGGE) and nucleotide sequencing. (A) DGGE on CFTR exon 9 using the DNA sample from patient shows a typical heterozygote banding pattern containing heteroduplex bands (see reference no. 10). (B) Nucleotide sequencing shows that the patient's CFTR gene contains a mutation changed from T nucleotide at 1454 to C (heterozygous for L441P, CTG:Leu → CCG; Pro).

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  Fig. 3 Immunoblotting and surface biotinylation of L441P mutant CFTR protein. HEK 293 cells were transfected with plasmids for wild type CFTR or CFTR carrying the L441P mutation and protein samples were blotted with anti-CFTR M3A7 antibody (Cell Signaling Technology, Danvers, MA). (A) Most of the wild type CFTR protein was detected as the fully glycosylated mature form (band C), whereas virtually all of the L441P mutant protein appeared as the core-glycosylated form of around 150 kDa (band B). Surface proteins were collected using the avidin-biotin interaction. Surface proteins were labeled with EZ-Link biotin-LC-Hydrazide (Pierce, Rockford, IL) and the biotinylated proteins were pelleted with the UltraLink Immobilized NeutrAvidin beads (10%, Pierce). (B) The ER-localized protein calnexin was not detected in the biotinlyated fractions, and demonstrating that biotin conjugates were specifically from the cell-surface proteins.

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  Fig. 4 Immunocytochemistry of L441P mutant CFTR. HEK 293 cells were transfected with plasmids for wild type CFTR or CFTR carrying the L441P mutation, immunostained with anti-CFTR 24-1 antibody (R&D Systems) and fluorescein isothiocyante-conjugated secondary antibodies. The calnexin protein was stained with anti-calexin antibodies (Abcam) and rhodamine-conjugated secondary antibodies. Images were collected with a Zeiss LSM510 confocal microscope.

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  Fig. 5 cAMP-activated Cl- channel activity of L441P mutant CFTR. HEK 293 cells were transfected with plasmids for wild type CFTR or CFTR carrying the L441P mutation and the cAMP-activated Cl- channel activity was measured in the whole cell configuration. (A) Cells were stimulated with forskolin (5 µM) and the currents were measured at a -30 mV holding potential. Mean currents were normalized as current densities (pA/pF, n=6 for wild type CFTR and n=10 for L441P mutant CFTR). (B) The I-V relationships were obtained with a step pulse from -120 mV to +120 mV applied at peak current.

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