This article has been cited by other articles in PMC. Abstract Background Cystic fibrosis CF is the most common genetic disorder with autosomal recessive inheritance among Caucasian populations.
Methods We tested 27 patients admitted to the medical genetics laboratory of Kermanshah University of Medical Sciences. Conclusion Three disease-causing mutations in CF patients in the present study account for approximately Introduction Cystic fibrosis CF; MIM is the most common lethal autosomal recessive disorder in Caucasian population; it affects 1 in newborns among Caucasians 1 , 2.
The importance of study Adequate and accurate information of CF mutations in particular populations provides information for CF prevention programs applicable via heterozygote screening and prenatal diagnosis 6. Table 1 Primers and PCR conditions.
Open in a separate window. Results Clinical profile and laboratory findings A total of 27 unrelated patients 13 males and 14 females aged two months to 19 years originating from Kermanshah province were analyzed in this study.
Figure 1. Table 2 CFTR gene mutations identified as a result of the study exon 10 and exon 17a. Gene Location Nucleotide change Mutation type No. Fdel 1 6 Global Exon 10 C to G at p. TI - 1 Iran. SX mutation This mutation is the most widespread one among Iran's proportional mutations and in contrast to other reported mutations has remarkable percentage in the country.
TI mutations In exon 17a, TI mutation is reported as a heterozygote for the second time in the world. MV polymorphism Polymorphism of MV is a silent polymorphism which changes one nucleotide and converts the methionine amino acid to valine in the place of Acknowledgement The authors are thankful of the patients and their families for their participation in this study. Conflict of Interest The authors declare no conflict of interest. References 1.
Lung biology in health and disease series. Cystic fibrosis; pp. Geborek A, Hjelte L. Association between genotype and pulmonary phenotype in cystic fibrosis patients with severe mutations.
J Cyst Fibros. Cystic fibrosis: recent structural insights. Rommens JM. Geographic distribution and regional origin of cystic fibrosis mutations in European populations. Hum Mutat. The geographic distribution of cystic fibrosis mutations gives clues about population origins. Eur J Pediatr. Cystic fibrosis: defining a disease under-diagnosed in Pakistan. Trop Med Int Health. Analysis of the CFTR gene in Iranian cystic fibrosis patients: identification of eight novel mutations.
A haplotype framework for cystic fibrosis mutations in Iran. J Mol Diagn. Alibakhshi R, Zamani M. Iran J Allergy Asthma Immunol. Wikipedia, the free encyclopedia. Wikimedia Foundation, Inc; Jan 10, Kurdish people [Internet] [updated Aug 14; cited Aug 20]. A simple salting out procedure for extracting DNA from human nucleated cells.
Nucleic Acids Res. Can a place of origin of the main cystic fibrosis mutations be identified? Am J Hum Genet. CFTR mutations in the Algerian population. Cystic fibrosis: a worldwide analysis of CFTR mutations--correlation with incidence data and application to screening. For the early diagnosis in the neonates presenting with a suspected digestive pathology, where the sweat test is very difficult to be carried out, 2.
For the confirmation of a doubtful diagnosis with some clinical signs and a borderline or negative sweat chloride test, 3. For the patient follow-up, as there is a correlation between the seriousness of respiratory involvement measured by FEV1 study forced expiratory volume in first second and the value of bioelectrical potential difference.
The method is simple, less expensive and accessible. CFTR gene, which is responsible for this disorder, contains 27 exons spreading over kb of chromosome 7 7q31 and encodes an mRNA of 6.
The early hypotheses regarding the function of CFTR protein revolved around two possibilities. This hypothesis was compatible with the defect in permeability of Cl- ions at the CF epithelial apical membranes. The other proposed that the CFTR protein is not an ionic channel but it plays a role in the regulation of Cl- channel either by associating with them, or by transporting a regulatory factor for Cl- channels in or out of the cell.
The molecular anomalies have variable effects on the CFTR protein and its functions. Class 1 : mutations altering the production of the protein.
These mutations result in the total or partial absence of the protein. This class includes the nonsense mutations and those that produce a premature stop codon anomalies of splicing and frameshift mutations.
In other cases, the abnormal protein produced will probably be unstable and degrade rapidly. This is what produces the truncated protein or the protein containing the aberrant sequence anomalies of splicing or the frame shift. Functionally, these mutants are characterized by a loss of conductance of Cl- channel in the affected epithelia.
Class 2 : mutations altering the cellular maturation of the protein. A number of mutations alter the maturation of the protein and thus the transport of these proteins to the plasma membrane. In this way, the protein is either absent from the plasma membrane or present in a very small quantity.
The mutations of this class represent the majority of CF alleles DF Class 3 : mutations disturbing the regulation of Cl- channel. Class 4 : mutations altering the conduction of Cl- channel. This varied from 11 to 17 dinucleotide units and appeared to bear an inverse relationship to that of the TA repeats. These repeats were considered to be useful in genetic linkage studies, in counseling CF families with unknown mutations, and in tracing the origins of various mutant CF alleles.
Morral et al. The significance of the inverse correlation between the lengths of the 2 repeat regions was not investigated; length compensation may be involved and may have functional importance. By use of PCR, it was possible to detect such ectopic transcription as in the case of other genes such as those for dystrophin and factor VIII Fonknechten et al.
Ferrie et al. Cutting et al. They found a cluster of 4 mutations in a bp region of exon Three of the mutations caused amino acid substitutions at residues that are highly conserved among the CFTR protein, the multiple-drug-resistance proteins, and ATP-binding membrane-associated transport proteins.
The fourth mutation created a premature termination signal. To explore the molecular mechanisms responsible for defective chloride transport in patients with CF, Yang et al. They concluded that the molecular pathology of GD is explained by an abnormality in channel activity, while the defect in delF is a combination of mislocalization and instability of the protein in addition to partial defects in channel function.
Some of their observations suggested the possibility of pharmacologic therapies for CF based on activating latent CFTR. Not only is there heterogeneity in the mutations causing cystic fibrosis, but the pathogenetic mechanisms also vary. Deletion of phenylalanine appears to cause disease by abrogating normal biosynthetic processing and thereby resulting in retention and degradation of the mutant protein within the endoplasmic reticulum. Other mutations, such as the relatively common glyto-asp mutation, appear to be normally processed and, therefore, must cause disease through some other mechanism.
These results implicated defective ATP-binding as the pathogenic mechanism of a relatively common mutation leading to CF and suggested that structural integrity of a highly conserved region present in over 30 prokaryotic and eukaryotic nucleotide-binding domains may be critical for normal nucleotide binding.
There is a polymorphic string of thymidines at the end of intron 8 of the CFTR gene; 3 different alleles can be found depending on the number of thymidines 5, 7, or 9 present at this site Chu et al. The number of thymidines determines the efficiency by which the intron 8 splice acceptor site is used. The efficiency decreases when a shorter stretch of thymidine residues is found. A higher proportion of CFTR transcripts that lack exon 9 sequences, which encode part of the functionally important first nucleotide-binding domain, will therefore be found when a shorter stretch of thymidine residues is present Chu et al.
Teng et al. Such exon 9-deficient CFTR transcripts are known to be translated into CFTR proteins that will not mature, and will therefore not function as chloride channels in the apical membrane of epithelial cells. Among CBAVD patients, the frequency of this T5 allele is 4- to 6-fold higher than in the control population see Alternative splicing of exon 9, which had been known to occur in nasal epithelial cells, also occurred in vas deferens cells.
The extent of this alternative splicing was determined by the allele present at the Tn locus at the end of intron 8 of the CFTR gene. However, the proportion of transcripts lacking exon 9 sequences was increased in vas deferens cells compared with nasal epithelial cells, independent of the Tn genotype.
Thus, Teng et al. Cuppens et al. On a T7 background, the TG 11 allele gave a 2. Such polyvalent mutant genes could explain why apparently normal CFTR genes cause disease. Moreover, they might be responsible for variation in the phenotypic expression of CFTR mutations. This study suggested that genetic and functional studies of polymorphisms in relation to genetic diseases will become of major interest, in relation both to monogenic disorders and complex traits. The results confirmed, at the molecular genetic level, a clinical connection between CF and one obstructive pulmonary disease, disseminated bronchiectasis of unknown origin.
Similarly, Girodon et al. Six of these mutations, which had previously been reported as CF defects, were found in 9 alleles. Four patients were compound heterozygotes; 6 were heterozygous for a mutation. Girodon et al. It has been proposed that in heterozygous state mutations of the CFTR gene provide increased resistance to infectious diseases, thereby maintaining mutant CFTR alleles at high levels in selected populations.
Pier et al. This disease is initiated when Salmonella typhi enters gastrointestinal epithelial cells for submucosal translocation. They found that S. Monoclonal antibodies and synthetic peptides containing a sequence corresponding to the first predicted extracellular domain of CFTR inhibited uptake of S. The Cftr genotype had no effect on the translocation of S. Van de Vosse et al. They genotyped patients and controls in a typhoid endemic area in Indonesia for 2 highly polymorphic markers in CFTR and the most common CF mutation, Fdel.
Consistent with the apparently very low incidence of CF in Indonesia, the Fdel mutation was not present in any patients or controls. However, they found significant association between a common polymorphism in intron 8 16 or 17 CA repeats and selective advantage against typhoid.
Sharer et al. They also determined the length of the noncoding sequence of thymidines in intron 8, since the shorter the sequence, the lower the proportion of normal CFTR mRNA. None of the patients had a mutation on both copies of the CFTR gene. Eighteen patients A total of None had the combination of sinopulmonary disease, high sweat electrolyte concentrations, and low nasal potential-difference values that is diagnostic of cystic fibrosis.
Similarly, Cohn et al. Cohn et al. Eight CFTR mutations were detected. In 3 patients both alleles were affected. These 3 patients did not have lung disease typical of cystic fibrosis on the basis of sweat testing, spirometry, or base-line nasal potential-difference measurements. Nonetheless, each had abnormal nasal cyclic AMP-mediated chloride transport.
These genotypes do not typically cause lung disease. To test whether the number of TG repeats adjacent to 5T influences disease penetrance, Groman et al. Those individuals with 5T adjacent to either 12 or 13 TG repeats were substantially more likely to exhibit an abnormal phenotype than those with 5T adjacent to 11 TG repeats odds ratio Thus, determination of TG repeat number will allow for more accurate prediction of benign versus pathogenic 5T alleles.
Lee et al. Several haplotypes, especially those with QH The common MV polymorphism The T5-V haplotype showed higher disease association than T5-M, but the QH mutation in a V background showed the strongest disease association. The results revealed that interactions between multiple genetic variants in cis affected the final function of the gene products. Buratti et al. They suggested that their results provide a mechanistic explanation for the association data of Groman et al.
Individual and tissue-specific variability in the concentration of this inhibitory splicing factor may even determine whether an individual will develop multisystemic non-classic CF or monosymptomatic CBAVD disease. Audrezet et al. A total of 18 mutant alleles were identified in 14 patients None of the 4 compound heterozygotes were found to have unrecognized CF-related pulmonary symptoms following reevaluation.
However, a sweat test done retrospectively was positive in 2 of them. The 5T allele of the polymorphic string of thymidines at the end of intron 8 of the CFTR gene was present in 7 of the 36 patients tested, an allele frequency 9. The molecular pathogenesis of cystic fibrosis has been investigated by analysis of delF CFTR in different heterologous systems, revealing an abrogation of CFTR expression by defective protein maturation.
Mutant CFTR was found arrested in an early wildtype intermediate, unable to adopt a protease-resistant mature conformation Cheng et al. Reduction of temperature Denning et al.
However, at the cell surface, the chloride channel formed therefrom showed a decreased half-life and reduced open probability and sensitivity to stimulation with cAMP agonists. Kalin et al. CFTR expression was detected at the luminal surface of reabsorptive sweat ducts and airway submucosal glands, at the apex of ciliated cells in pseudostratified respiratory epithelia and of isolated cells of the villi of duodenum and jejunum, and within intracellular compartments of intestinal goblet cells.
In delF homozygous patients, expression of the mutant protein proved to be tissue specific. Whereas delF CFTR was undetectable in sweat glands, the expression in the respiratory and intestinal tracts could not be distinguished from the wildtype by signal intensity or localization.
Welsh and Smith provided a classification of the mechanisms by which mutations in CFTR cause cystic fibrosis. The grouping of mutations into 5 classes was based on their functional effect: I defective protein production; II defective protein processing; III defective protein regulation; IV defective protein conductance; and V reduced amounts of functional CFTR protein.
Class I, II, and III mutations have been associated with typical severe multiorgan disease on the basis of clinical studies. Haardt et al. They suggested that the mutations can be grouped into 2 major categories. The first group includes those mutants that are unable to accumulate at the cell surface, either because of impaired biosynthesis class I and class V , or because of defective folding at the endoplasmic reticulum class II. Mutants that belong to the second category are expressed at the cell surface but fail to translocate chloride ions because of a defect in activation class IV or channel conductance class III.
Because the biosynthetic processing and macroscopic chloride channel function of some of the truncated CFTR constructs appear to be normal but the biologic stability of their mature, complex-glycosylated form is dramatically reduced, Haardt et al.
To study the consequences that disease-causing mutations have on the regulatory function of CFTR, Mickle et al. However, CFTR that had disease-associated mutations in other domains retained both functions, regardless of the associated phenotype. Bronsveld et al. In intestinal tissue, 4,4-prime-diisothiocyanatostilbene-2,2-prime-disulfonic acid DIDS -insensitive chloride secretion, which is indicative of functional CFTR channels, correlated with a milder phenotype, whereas DIDS-sensitive chloride secretion was observed mainly in more severely affected patients.
Bobadilla et al. Although wide mutational heterogeneity was found throughout the world, characterization of the most common mutations in most populations was possible. A significant positive correlation was found between delta-F frequency and the CF incidence of regional populations. Primary sclerosing cholangitis PSC; see , a slowly progressive cholestatic liver disease characterized by fibroobliterative inflammation of the biliary tract, leads to cirrhosis and portal hypertension and is a major indication for liver transplantation.
Sheth et al. They found an increased prevalence of CFTR abnormalities in heterozygous state in PSC as demonstrated by molecular and functional analyses, and concluded that these abnormalities may contribute to the development of PSC in a subset of patients with IBD. Eighty-nine percent of PSC patients carried genotypes containing the G variant CFTR chloride channel function assessed by nasal potential difference testing demonstrated a reduced median isoproterenol response in PSC patients compared with disease controls and healthy controls.
Pagani et al. This was the case in 2 natural mutations of which was gly to ala GA; This phenomenon was due to the interference with a regulatory element, which the authors named composite exonic regulatory element of splicing CERES.
The effect of single-nucleotide substitutions at CERES could not be predicted by either serine-arginine-rich SR matrices or enhancer identification. Aznarez et al. Both mutations caused aberrant splicing in a predicted manner, supporting a role for the putative ESE sequence in pre-mRNA splicing. In addition, 3 mutations, including DV The breakpoints of these 5 mutations were precisely determined.
Although nonhomologous recombination may be invoked to explain all 5 complex lesions, each mutation appeared to have arisen through a different mechanism. Dinucleotide repeats are ubiquitous features of eukaryotic genomes. The highly variable nature of dinucleotide repeats makes them particularly interesting candidates for modifiers of RNA splicing when they are found near splicing signals. An example of a variable dinucleotide repeat that affects splicing is a TG repeat located in the splice acceptor of exon 9 of the CFTR gene.
Higher repeat numbers result in reduced exon 9 splicing efficiency and, in some instances, the reduction in full-length transcript is sufficient to cause male infertility due to congenital bilateral absence of the vas deferens or nonclassic cystic fibrosis. Placement of the TG dinucleotide tract in the minigene with random sequence abolished splicing of exon 9. Replacement of the TG tract with sequences that can self-basepair suggested that the formation of an RNA secondary structure was associated with efficient splicing; however, splicing efficiency was inversely correlated with the predicted thermodynamic stability of such structures, demonstrating that intermediate stability was optimal.
Finally, substitution with TA repeats of differing length confirmed that stability of the RNA secondary structure, not sequence content, correlated with splicing efficiency. Wong et al. The child had 2 different null mutations, glu7 to ter The mutations identified were different from those usually observed in Western countries. The T5 allele with 12 or 13 TG repeats was significantly associated with earlier age at onset in patients with ICP, although the frequency of this allele did not differ between patients and controls.
Sun et al. Patients selected for this study were positive for both the 5T variant and the major cystic fibrosis mutation, delta-F Therefore, it is possible to determine the haplotype of the 5T variant in trans. Of the 49 cases for which they had clinical information, Sun et al. The authors also evaluated 12 males affected with congenital bilateral absence of vas deferens and positive for the 5T variant; 10 of 12 had the 12TG-5TV haplotype. Alonso et al. In addition to their antimicrobial activity, aminoglycoside antibiotics can suppress premature termination codons by allowing an amino acid to be incorporated in place of the stop codon, thus permitting translation to continue to the normal end of the transcript.
The mechanism translation termination is highly conserved among most organisms and is almost always signaled by an amber UAG , ochre UAA , or opal UGA termination codon. The nucleotide sequence surrounding the termination codon has an important role in determining the efficiency of translation termination.
Aminoglycoside antibiotics can reduce the fidelity of translation, predominantly by inhibiting ribosomal 'proofreading,' a mechanism to exclude poorly matched aminoacyl-tRNA from becoming incorporated into the polypeptide chain.
In this way aminoglycosides increase the frequency of erroneous insertions at the nonsense codon and permit translation to continue to the end of the gene, as has been shown in eukaryotic cells Burke and Mogg, , including human fibroblasts Buchanan et al. Howard et al. Others demonstrated this effect in cultured cells bearing CFTR nonsense mutations and in connection with stop mutations in muscular dystrophy in mice and in vitro in Hurler syndrome , cystinosis , and other disorders.
Wilschanski et al. Nasal potential difference was measured at baseline and after each treatment. Gentamicin treatment caused a significant reduction in basal potential difference in 19 patients carrying stop mutations and a significant response to chloride-free isoproterenol solution.
This effect of gentamicin on nasal potential difference occurred both in patients who were homozygous for stop mutations and in those who were heterozygous, but not in patients who were homozygous for delta-F After gentamicin treatment, a significant increase in peripheral and surface staining for CFTR was observed in the nasal epithelial cells of patients carrying stop mutations.
Tata et al. McCombie et al. They suggested that C. The same approach was applied even more extensively by Waterston et al. Zeiher et al. Dorin et al. To understand the pathophysiology of the disease and to evaluate new therapies, Zeiher et al. They could show that affected epithelia from homozygous Fdel mice lacked CFTR in the apical membrane and were chloride ion-impermeable.
Forty percent of homozygous animals survived into adulthood and displayed several abnormalities found in human disease and in CFTR null mice. Van Doorninck et al. In this model, the intron structure was not disturbed, in contrast to similar models Zeiher et al. French et al. However, the mutant protein was expressed as functional chloride channels in the plasma membrane of cells cultured at reduced temperature.
Furthermore, they could show that the electrophysiologic characteristics of the mouse phedel-CFTR channels were indistinguishable from normal. In homozygous mutant mice they did not observe a significant effect of genetic background on the level of residual chloride channel activity. The data showed that like its human homolog, the mouse mutant CFTR is a temperature-sensitive processing mutant, and therefore an authentic model for study of pathophysiology and therapy.
Dickinson et al. The GC cystic fibrosis mouse model expressed the GC mutant transcript at a level comparable to that of wildtype Cftr. The homozygous mutant mice were fertile and had normal survival, weight, tooth color, and no evidence of cecal blockage, despite mild goblet cell hypertrophy in the intestine.
The bioelectric phenotype of these mice revealed organ-specific electrophysiological effects. In contrast to delta-F 'hit-and-run' homozygotes, the classic defect of forskolin-induced chloride ion transport was not replicated in the cecum, but the response to low chloride in the nose was clearly defective in the GC mutant animals. Of importance to any gene-replacement strategy for treatment of CF is the identification of the cell type s within the lung milieu that need to be corrected and an indication whether this is sufficient to restore a normal inflammatory response and bacterial clearance.
Oceandy et al. Following chronic pulmonary infection with Pseudomonas aeruginosa, CF mice with epithelial-expressed but not macrophage-specific CFTR showed an improvement in pathogen clearance and inflammatory markers compared with control CF animals.
The authors concluded that there may be a role for CFTR-mediated events in epithelial cells in response of the lung to bacterial pathogens.
Di et al. Curcumin, a major component of the curry spice turmeric, is a nontoxic calcium-adenosine triphosphatase pump inhibitor that can be administered to humans safely.
Egan et al. These effects were not observed in mice homozygous for a complete knockout of the CFTR gene. Curcumin also induced the functional appearance of delta-F CFTR protein in the plasma membranes of transfected baby hamster kidney cells. However, delayed puberty has been reported as a feature of CF even in the setting of good nutritional and clinical status Johannesson et al. This finding, along with evidence that Cftr is expressed in rat brain, human hypothalamus, and a gonadotropin-releasing hormone secreting line, raised the possibility that some of the pubertal delay in cystic fibrosis could stem directly from alterations in Cftr function that affects the hypothalamic-pituitary-gonadal axis.
To examine this hypothesis, Jin et al. Homozygous knockout mice, which have chronic inflammation and gastrointestinal disease, grew more slowly and had later onset of puberty than wildtype animals. Jin et al. They found, however, that these mice had earlier onset of puberty, as assessed by vaginal opening VO , than wildtype. These findings were confirmed in a second independent model of CF engineered to generate the delta-F mutation in mice.
Again the homozygotes displayed later pubertal timing, and the heterozygotes displayed earlier VO than the wildtype animals. These data provided further evidence that Cftr can directly modulate the reproductive endocrine axis and raised the possibility that heterozygote mutation carriers may have a reproductive advantage.
To investigate the abnormalities that impair elimination when a bacterium lands on the pristine surface of a newborn CF airway, Pezzulo et al. As a model, they studied CF pigs, which spontaneously develop hallmark features of CF lung disease. The data indicated that the patient has the heterozygous deletion of c. Figure 3a and b shows the original chromatograms of the patient A and the control B. This finding confirmed the genomic deletion spanning exons 16, 17a and 17b in one allele.
Genomic rearrangements were not detected in the other allele. Quantitative fragment analysis by MLPA. The signals corresponding to exons 16 The signals corresponding to exon 1 Long PCR with the No. PCR of the fragments spanning exons 4—24 No. The smaller-size bands detected by No. To examine the CFTR transcript from the other allele without exons 16—17b deletion , PCR was carried out by setting the reverse primer on exon 16 Figure 4c.
PCR of the fragments spanning exons 1—16 No. PCR of the fragments spanning exons 2—16 No. However, the bands from the patient P sample were weak but of the same size. PCR of the fragments spanning exons 18—24 No. The density of the bands was similar. These data suggest that there is a deletion or skipping of exon 1 in the other allele.
Mouchel et al. Two weak bands were detected in the control C , but not in the patient P; Figure 4c. The data suggest that the CFTR transcript from the other allele without exons 16—17b deletion of the patient is not the reported splicing variant. Cloning and sequence analysis of the CFTR transcripts from nasal swab revealed a heterozygous deletion spanning exons 16, 17a and 17b.
None of the CF-causing mutations was detected by conventional PCR, and direct sequence of all 27 exons, their boundaries and promoter region. The deletion was confirmed by MLPA and the direct sequence of the junction fragment obtained from the genomic DNA by primer walking, which revealed the mutation spanning intron 15 and intron 17b, c. A similar massive deletion spanning exon 16—17b: c.
We also identified a novel splicing defect in the CFTR transcript from the other allele. Although exon 2 was conserved in the transcript, the exact breakpoint remains undetermined. Five different splicing mutations in intron 1 have been reported to the Cystic Fibrosis Mutation Database, but they were not detected in the present study.
Such splicing variants have not been reported in CF patients. We detected a small amount of the splicing variant lacking exon 1 in nasal swab from a control subject. However, the transcript lacking exon 1 from the patient was not this type of splicing variant.
Though the exact breakpoint was unknown, the skipping of exon 1 would cause translation to start downstream, most likely utilizing an ATG in exon 4 of the CFTR transcript, which leads to the generation of the CFTR protein lacking the N-terminus. CF is rare in Japan and only 80 cases have been counted since by the Research Committee of Intractable Pancreatic Diseases data not published.
The median survival time is about 18 years. Early diagnosis and treatment is important. The pilocarpine iontophoresis is the standard method to stimulate sweat-gland secretion in the forearm or back. However, it requires experienced personnel and a quality-controlled laboratory, 15 which is hardly expected in Japan. Yoshimura and Anzai have found CFTRdele16—17b mutation the breakpoint was not examined in three patients all in homozygosis , although most of the other detected CFTR mutations are of rare types and many of them have unknown or uncertain clinical relevance Research Committee of Intractable Pancreatic Diseases, data not published.
Thus, we need to explore the presence of heterozygous deletion of 16—17b in Japanese CF patients. This test requires only conventional PCR technique and can be included in the first-step analysis. Mutations in the regulatory elements located in sites distant from the coding regions are probably present in many of these cases, but they cannot be identified by currently available techniques.
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