1 Department of Medical Biology and 2 Department of Urology, Faculty of Medicine, Hacettepe University, Sihhiye, 06100 Ankara, Turkey and 3 Clinics of Obstetrics and Gynecology, Medical School Hannover, Podbielskistrasse 380, D-30659 Hannover, Germany
4 To whom correspondence should be addressed: e-mail: didayan{at}hacettepe.edu.tr or doerk.thilo{at}mh-hannover.de
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Abstract |
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Key words: congenital absence of vas deferens/cystic fibrosis/genotypephenotype correlation/male infertility/splicing mutation
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Introduction |
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Little is known about the spectrum and frequency of CFTR gene mutations in Turkey. Classic cystic fibrosis is less common in the Turkish population than in Central Europe, with an estimated incidence of about 1:10 000 newborns, but other entities such as CBAVD have not been investigated yet. Two recent studies have provided evidence for an extensive allelic heterogeneity in Turkish patients with classic CF (Onay et al., 2001; Kilinç et al., 2002
). We here report the results of a detailed investigation of CFTR mutation genotypes in 51 Turkish CBAVD patients. We have identified by direct sequencing a high frequency of different CFTR gene alterations and a mutational spectrum that is distinct from other populations.
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Materials and methods |
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Mutation analysis
Genomic DNA was extracted from peripheral blood leukocytes by routine procedures. All 51 samples were first screened for the F508 and 1677delTA mutations in exon 10 with PCR followed by polyacrylamide gel electrophoresis. We next screened for six further CFTR gene mutations of the coding region and flanking intron sequences by previously described restriction-enzyme based methods: G85E, D110H, R347H, 2789+5G
A, D1152H, N1303K (Dörk et al., 1994a
, 1997). One deep intronic mutation 3849+10kBC
T was tested by restriction enzyme analysis (Highsmith et al., 1994
) and four large genomic deletions CFTRdele2(5kB), CFTRdele2(ins186), CFTRdele2,3(21kB) and 3120+1kBdel8.6kB were screened using specific primer pairs (Lerer et al., 1999
; Dörk et al., 2000a
,b). Missense polymorphism M470V in exon 10 was typed by HphI restriction enzyme analysis and the exon 9 and its flanking intron regions were sequenced in all samples to determine the status of polyvariant alleles that modulate the splicing and maturation of CFTR (Cuppens et al., 1998
). The 33 samples with no or only one clearly pathogenic mutation identified after this initial screening were then subjected to direct sequencing of the entire coding sequence and all intron/exon boundaries of the CFTR gene using dye terminator chemistry and capillary electrophoresis on an ABI 310 genetic analyser (Applied Biosystems). We used published primer pairs for PCR and sequencing of most of the 27 exons (Zielenski et al., 1991
) except for the following primers:
5'-GGTCTTTGGCATTAGGAGCTTG-3' and 5'-TCAGTTGCA AGTAGATGTGGC-3' (exon 1, annealing 59°C); 5'-GGAA GATGAAATTGTGTGTACCTTG-3' and CCACTACCATAATG CTTGGGAG-3' (exon 14b, annealing 58°C); 5'-CTACCCCAT GGTTGAAAAGCTG-3' and 5'-GCAGGAACTATCACATGTGAG-3' (exon 23, annealing 58°C); 5'-CTGACCTGCCTTCTGTCCCAG-3' and 5'-CTGAGGCAGAGGTAACTGTTCC-3' (exon 24, annealing 61°C). Finally, the 5'-UTR and minimum promoter region were amplified and sequenced using the primer pair 5'-GTCCTC CAGCGTTGCCAACTGG-3' and 5'-CAACGCTGGCCTTTTCC AGAGG-3' (annealing at 62°C).
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Results |
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In summary, this study led to the identification of 27 CFTR gene mutations and presumably pathogenic alterations on 72.5% of chromosomes from 51 Turkish CBAVD patients. Two mutant alleles were identified in 34 patients (66.7%). One mutant allele was identified in six patients (11.7%), of whom two carried clearly pathogenic mutations (F508 and R347H, respectively) and four harboured unclassified variants. No mutations were found in 11 patients (21.6%), including the three patients with known renal malformations.
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Discussion |
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The results of this study reflect the high allelic heterogeneity of CFTR gene mutations, although two mutations, IVS8-5T and D1152H, were found to be very common in Turkish CBAVD patients. First, the IVS8-5T allele was observed on 19.6% of all chromosomes. This polythymidine tract deletion is known as a splicing mutation with reduced penetrance (Chillón et al., 1995; Costes et al., 1995; Zielenski et al., 1995
) and is found at an allele frequency of only 35% in the general Turkish population (Onay et al., 2001
; Kilinç et al., 2002
). Consistent with previous reports (Costes et al., 1995
; Dörk et al., 1997
; Cuppens et al., 1998
), we find that the IVS8-5T allele in CBAVD patients is exclusively associated in cis with long IVS8-(TG)1213 tracts which have been shown to increase the extent of exon 9 skipping induced by the 5T variant (Cuppens et al., 1998; Niksic et al., 1999; Buratti et al., 2001
). It has previously been found that IVS8-5T is a recurrent mutation on different ethnic backgrounds, thus our study corroborates the suggestion that it may contribute substantially to the etiology of CBAVD worldwide (Dörk et al., 1997
; Lissens et al., 1999
). Secondly, the D1152H missense substitution was uncovered on 14.7% of all chromosomes. This finding was unexpected because this mutation has not been detected in Turkish CF patients before (Onay et al., 2001
; Kilinç et al., 2002
). It has, however, been previously observed in CBAVD patients of diverse ethnicities at low frequencies (Dörk et al., 1997
; Kerem et al., 1997
; Claustres et al., 2000
) and was initially reported as a mild cystic fibrosis mutation. In vitro studies have shown that the D1152H substitution does not interfere with the maturation of the CFTR protein but strongly reduces its cAMP-activated chloride conductance (Vankeerberghen et al., 1998
) which may provide the basis for a mild expression of disease. Our results implicate D1152H as a common missense mutation in Turkey which appears to be specifically associated with CBAVD, perhaps comparable to the role of the R117H missense substitution in Central Europe (Gervais et al., 1993
; Dörk et al., 1997
). All D1152H alleles appeared to be linked with the same haplotype comprising the IVS8-7T(TG)11 and Val470 alleles (Table II). Screening for the IVS8-5T and D1152H mutations together led to the identification of more than one-third of alleles in Turkish CBAVD males.
Most of the less frequent mutations were also of the missense or splicing type and, beyond the IVS8-5T allele, four other putative splicing mutations have been identified that also target a polypyrimidine tract. The IVS8-6T allele constitutes a new member of the growing family of polyT variants in intron 8 and may have clinical implications. Although the 6T allele has first been observed in an apparently healthy female carrier seeking assisted reproduction (Dörk et al., 2001), it could nevertheless be a CBAVD causing mutation. With the four alleles that have previously been identified in intron 8 of the CFTR gene (T9, T7, T5 and T3), the efficiency of the splice acceptor site usage consistently decreases with shorter polythymidine tracts which results in a lower than normal level of full-length CFTR mRNA (Chu et al., 1993
; Mak et al., 1997
; Teng et al., 1997
; Larriba et al., 1998
; Buratti et al., 2001
). The 6T allele can be predicted to affect splicing to a degree between the 5T and 7T alleles and thus it could represent a low-penetrance mutation, at the border between health and mild disease. The other polypyrimidine tract variants 2752-15C
G, 3041-15T
G, and 3041-13del7 all reduce the maximum entropy score of the respective acceptor splice sites (Yeo and Burge 2004;
http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq_acc.html), but the quantitative impact of these mutations on the accuracy and efficiency of splicing remains to be determined.
The observation that almost every Turkish CBAVD patient with identified mutation genotype harboured a missense or splicing mutation on at least one allele is in line with previous reports of such a distinction between mutation genotypes in CF and CBAVD (Dörk et al., 1997; Claustres et al., 2000
). The first exception is one patient in our study who is a compound heterozygote for the two truncating mutations 1677delTA and E831X. Although early reports have suggested that CF patients with two null alleles may have milder lung disease compared with
F508 homozygotes (Gasparini et al., 1992
; Wine, 1992
), such patients are generally pancreatic insufficient and previous studies of patients with isolated CBAVD have not revealed any patient carrying two truncating mutations, thus far (Dörk et al., 1997
; Claustres et al., 2000
). It may thus be the particular genotype of our single case that confers an unusually mild phenotype. Exon 14a is the subject of alternative splicing (Hull et al., 1994
; Bienvenu et al., 1996
) and the mutation E831X at the first base of this exon may increase its in-frame skipping. Such a by-pass of a premature termination codon has been shown to ameliorate the disease phenotype in other clinical conditions (Morisaki et al., 1993
; Ginjaar et al., 2000
; Su et al., 2000
).
Our study detected CFTR gene mutations on both alleles in two-thirds of the Turkish CBAVD males. This is comparable with thorough studies of European CBAVD patients as well as with the detection rate obtained for Turkish CF patients. Eleven patients, including three with confirmed renal agenesis, remained with no mutation identified after sequencing all exons. The reasons for the incomplete detection could not be fully established but may be related to some heterogeneity in the etiology of CBAVD (Rave-Harel et al., 1995; McCallum et al., 2001
). There is evidence that the prevalence of CFTR mutations is significantly reduced in patients who have CBAVD accompanied by renal agenesis (Augarten et al., 1994
; Schlegel et al., 1996
; Dörk et al., 1997
; Casals et al., 2000
; McCallum et al., 2001
). It is also possible that some unknown mutations outside of the analysed regions account for a proportion of unidentified alleles in our study and in Turkish CF patients (Onay et al., 2001
; Kilinç et al., 2002
). Despite these limitations, we believe that direct sequencing is the most sensitive of the presently available methods for a whole CFTR mutation genotyping in Turkish CBAVD patients. Any of todays commercially distributed kits would miss the majority of the mutant alleles in our patient cohort. For instance, the analysis of the 31 most common cystic fibrosis mutations found within the white population would have detected three mutations, accounting for 8 of the 102 alleles in the Turkish CBAVD patients (8%, compared with 31% in Canadian CBAVD males; Mak et al., 1999
). The ACMG 25 mutation panel that includes core mutations recommended for general population CF carrier screening, would also have detected three mutations on 7 of the 102 alleles in our cohort (7%; Grody et al., 2001
). An extension of this panel to 100 CF mutations together with mass spectrometry analysis would have detected 7 mutations on 12 of the 102 alleles (12%; Wang et al., 2002
).
In conclusion, none of the currently used routine mutation panels is well-suited for Turkish couples seeking genetic testing before assisted reproduction. It would be desirable to have more population-specific mutation panels which could help to overcome the underdiagnosis of cystic fibrosis and other CFTR-related diseases in those countries where the mutational distribution does not match that of Central Europe. In this regard, a population-specific mutation panel including the D1152H mutation and the IVS8-5T allele should be highly recommended for Turkish CBAVD patients. Our data add to a growing body of evidence that CFTR mutations can have a substantial clinical impact also in populations where cystic fibrosis in its classic form may be infrequent. The spectrum of mutations and genotypes that we have presented here, including the identification of a common missense mutation, should be useful to refine the diagnosis of CBAVD or other CFTR-related diseases in patients of Turkish descent.
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Acknowledgements |
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Submitted on October 13, 2003; accepted on February 24, 2004.