Higher proportion of intact exon 9 CFTR mRNA in nasal epithelium compared with vas deferens
Higher proportion of intact exon 9 CFTR mRNA in nasal epithelium compared with vas deferensVictor Mak1,2,4, Keith A. Jarvi1,4, Julian Zielenski2, Peter Durie3,4,5 and Lap-Chee Tsui2,4,6,*
1Division of Urology, Department of Surgery, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5, Canada, 2Department of Genetics and 3Division of Gastroenterology and Nutrition, The Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada, 4Institute of Medical Science, 5Department of Pediatrics, and 6Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
Received July 1, 1997;Revised and Accepted August 27, 1997
The 5-thymidine (5T) variant of the cystic fibrosis transmembrane conductance regulator (CFTR) intron 8 polypyrimidine tract (IVS8-T tract) is the most frequent CFTR gene alteration identified in men with congenital bilateral absence of vas deferens (CBAVD). This alternative splicing variant gives rise to two transcripts, one normal with exon 9 intact and the other with in-frame deletion of exon 9. That CBAVD men usually have none of the other clinical signs of classical cystic fibrosis (CF) suggests less functional CFTR is produced in the reproductive tract than in other CF-associated organs. Nasal epithelia and segments of vas deferens were obtained from healthy, previously vasectomized men who presented for vasectomy reversal. Quantitative RT-PCR was performed on these specimens, with the region of CFTR cDNA spanning exon 9 amplified. For both nasal and vasal tissues, a strong positive correlation was found between the length of the IVS8-T tract and the proportion of mRNA with exon 9 intact. In addition, within the same subject, a significantly higher level of transcripts lacking exon 9 was found in vas deferens than nasal epithelia, regardless of the IVS8-T genotype. These findings suggest that the splicing of CFTR precursor mRNA is less efficient in vasal epithelia compared with respiratory epithelia. Thus, differential splicing efficiency between the various tissues which express CFTR provides one possible explanation for the reproductive tract abnormalities observed in infertile men with CFTR gene alterations but without other clinical manifestations of CF.
Cystic fibrosis (CF) is the most common fatal autosomal recessive disorder in the white population, with an incidence of ~1/2500 live births and a carrier frequency of 1/25 persons among populations of Northern European descent. The gene responsible for CF, called the cystic fibrosis transmembrane conductance regulator (CFTR), encodes the cyclic adenosine monophosphate (cAMP) regulated chloride channel found in the apical membrane of secretory epithelial cells (1 ). The classic form of CF is characterized by abnormalities of electrolyte, fluid and macromolecule secretion of exocrine glands. Clinical hallmarks of CF include chronic pulmonary obstruction and infections, exocrine pancreatic insufficiency, neonatal meconium ileus, elevated sweat electrolytes and male infertility (1 ). Studies have shown that >95% of men with CF are azoospermic (absence of spermatozoa in ejaculate) due to abnormalities in the structures derived from the Wolffian duct (2 -5 ). The body and tail of the epididymis, vas deferens, seminal vesicles and ejaculatory ducts are atrophic, fibrotic or completely absent. Spermatogenesis is present on testicular histology. The pathogenesis probably involves intrauterine obstruction of the Wolffian duct with inspissated secretions, which results from decreased or abnormal CFTR chloride channel function, and gives rise to a dehydrated intraluminal fluid content with secondary obliteration of the Wolffian duct (5 ).
As demonstrated by DNA mutation analyses, 50-82% of men with congenital bilateral absence of vas deferens (CBAVD) have at least one detectable CFTR gene mutation, and ~15% have two detectable CFTR mutations (6 -17 ). At least one of the two CFTR mutations appears to be of a mild form, usually specific for CBAVD. Recent reports show that the 5-thymidine (5T) variant of the CFTR intron 8 polypyrimidine tract (IVS8-T tract) may confer the absence of vas deferens phenotype. In fact, the frequency of the 5T variant in these patients is six times (30%) that of the general (5%) population (12 ,13 ). Azoospermic men with idiopathic epididymal obstruction have also been shown to have a higher frequency (47%) of CFTR mutations and the 5T variant (17 ). The 5T variant results in less efficient splicing of CFTR exon 9 compared with the more common 7T and 9T variants and therefore produces less normal, full-length CFTR mRNA (exon 9+) (18 -20 ). The protein product of the CFTR transcript lacking exon 9 (exon 9-), if made, is apparently devoid of cAMP-activated chloride conductance (21 ,22 ).
An intriguing question which comes from these observations pertains to the mechanism by which alterations in CFTR expression results in reproductive tract abnormalities in the absence of pathologic changes in other CF-associated organs (e.g., lung, nasal sinus, pancreas, liver). It is possible that the reproductive tract is more sensitive to CFTR dysfunction than the other tissues. Alternatively, the level of functional CFTR may vary between different tissues, with the lowest expression in the Wolffian structures. In order to elucidate this problem, we studied the levels of CFTR exon 9+ mRNA obtained from different CFTR expressing tissues (i.e., nasal epithelium and vas deferens) and compared them within the same individual.
With respect to the polythymidine tract at the splice acceptor site of intron 8 of CFTR(IVS8-T tract), of the 24 patients, eight were 7T/9T heterozygotes, 14 were 7T/7T homozygotes, and the remaining two were 5T/7T heterozygotes (Table 1 ). The frequencies of the 9T, 7T, and 5T alleles were not significantly different between individuals in the present study and the general population (12 ,23 -25 ; our unpublished data).
Evaluation of CFTR mRNA transcripts from nasal epithelial and vas deferens cells in the region encompassing exons 8-10 after conversion to cDNA and PCR amplification revealed two different fragments. The difference in size between these two bands (416 and 233 bp) was identical to the size of exon 9 (183 bp). Southern analysis with an exon 10 specific probe (C16B) showed that both fragments contained exon 10 sequences (data not shown). However, Southern analysis with the exon 9+ specific probe (9i-5s) annealed to the larger fragment only, while the exon 9- specific probe (ex8/10) detected only the smaller transcript (data not shown). These results confirm that the larger transcript represented the normal, full-length transcript with exon 9 intact, and that the smaller transcript represented the in-frame exon 9 deleted transcript.
In order to quantitate the proportion of exon 9+ CFTR mRNA transcripts accurately, it was first necessary to establish a standard curve using known concentrations of isolated fragments of exon 9+ and exon 9- transcripts (Fig. 1 ). Accordingly, the exon 9+ and exon 9- fragments were serially mixed in varying proportions and subjected to the identical PCR conditions and quantification protocol as for the nasal and vas deferens specimens (Fig. 2 A). The experimental proportions of exon 9+ and exon 9- transcripts were derived from densitometric scanning, taking the sum of optical density measurements for the exon 9+ and exon 9- bands as 100%. The corresponding actual and experimental proportions of exon 9+ transcripts were then recorded graphically (Fig. 2 B). Consequently, all experimental proportions of exon 9+ transcripts from our tissue samples were adjusted according to this graph.
All proportions of exon 9+ transcripts reported hereafter refer to the corrected, actual proportions, based on Figure 2 B. The proportions of exon 9+ nasal transcripts were 93±2%, 82±3%, and 74±1% (mean±SD) for the 7T/9T, 7T/7T, and 5T/7T genotypes, respectively, while in the vas deferens samples, the proportions of exon 9+ transcripts were 88±3%, 76±3%, and 64±4% (mean±SD) for the 7T/9T, 7T/7T, and 5T/7T groups, respectively (Figs 3 and 4 ; Table 1 ).
In the present study, we found a relationship between the IVS8-T genotype and the proportion of normal, full-length CFTR mRNA transcripts (exon 9+) in nasal epithelia and vas deferens. It appears that the longer the IVS8-T tract, the greater the proportion of normal CFTR transcripts in nasal epithelia and in vas deferens cells (Fig. 4 ; Table 1 ). This finding is consistent with that reported by Chu et al. (20 ), who also demonstrated a positive correlation between the proportion of exon 9+ transcripts in bronchial epithelial cells and the length of the IVS8-T tract. It is likely that this relationship holds true for other CFTR-expressing tissues as well.
We also showed a higher proportion of the normal, full-length CFTR message in nasal epithelia than in vas deferens from the same subject, regardless of the IVS8-T genotype (p <0.001, paired t-test) (Fig. 5 ). In other words, the precise excision of intron 8 with in-frame joining of exon 8 and exon 9 of CFTR mRNA may be less efficient in epithelia of the reproductive tract compared with those of the respiratory tract. Two recent reports corroborate our findings. Teng et al. (26 ) found that, for the same IVS8-T tract genotype, the proportion of exon 9+ transcripts was lower in a series of vas deferens samples obtained from vasectomized men than in a series of nasal biopsies obtained from different men and women with chronic nasal obstruction or sinusitis. Rave-Harel et al. (27 ) documented that three men with CBAVD had an increased level of exon 9+ transcripts in nasal epithelia compared with epididymal samples. Taken together, these and our observations support the hypothesis that splicing efficiency varies between the different tissues affected in CF.
The discovery of differential splicing efficiency between the various tissues which express CFTR provides important insights into the relationship between levels of normal CFTR and phenotypic variation. For instance, the R117H mutation is associated with pancreatic-sufficient CF (CF-PS) (28 ). CF-PS patients with this mutation have pulmonary dysfunction but do not have pancreatic exocrine insufficiency and their sweat chloride measurements are only modestly increased (29 ). Not surprisingly, the mild R117H mutation has been identified in otherwise healthy males with CBAVD. However, further genetic analysis uncovered that individuals heterozygous for the R117H mutation on a 5T background (i.e., R117H and 5T on the same chromosome) and a `severe' CFTR mutation (e.g., [Delta]F508, G551D) developed lung disease characteristic of CF, whereas the R117H mutation found in CBAVD men is associated exclusively with the more efficient splice acceptor 7T (i.e., R117H and 7T on the same chromosome) (23 ). It is also important to note that the R117H mutation gives rise to a partially functional CFTR protein (30 ). Therefore, the R117H/5T allele results in a low enough level of partially functioning CFTR in the lung and an even lower level in the reproductive tract such that both organs are affected. In contrast, the R117H/7T allele, although producing a sufficient level of partially functional CFTR in the lung to prevent disease, the lower level in the genital ducts results in characteristic pathologic changes. These observations coupled with our results strongly suggest that the specific IVS8-T tract background on which a CFTR mutation resides can modulate disease severity in a tissue-specific manner.
The frequencies of the 9T, 7T, and 5T alleles of the IVS8-T tract of the CFTR gene in our study sample are similar to those in the general population (12 ,23 -25 ; our unpublished data). This finding is not unexpected as both groups consist of normal, healthy subjects. The fact that our study lacked subjects with the 9T/9T, 5T/9T, or 5T/5T genotype is likely a consequence of the relatively small sample size. Despite the absence of these groups, based on our subjects with the 7T/9T, 7T/7T and 5T/7T genotypes (Table 1 ) and the assumption that each of the two CFTR alleles contributes equally to the total amount of CFTR transcripts, it is inferred that the 7T allele produces ~41% exon 9+ transcripts (i.e., ~41% of the total amount of CFTR transcripts are exon 9+) in nasal epithelium and ~38% exon 9+ transcripts in vasal epithelium. It follows, then, that the 5T allele produces ~32% and ~26.5%, and the 9T allele produces ~49.5% and ~49%, exon 9+ CFTR mRNA in nasal epithelial and vas deferens cells, respectively (Table 2 ). Therefore, although the present study does not consist of any individuals with the 9T/9T, 5T/9T, or 5T/5T genotypes, it can be deduced that they would have 99 and 98%, 82 and 76%, and 64 and 53%, exon 9+ CFTR transcripts in nasal epithelia and vas deferens, respectively (Table 3 ).
. Estimated proportion of CFTR exon 9+ transcripts produced by the IVS8-T tract genotypesa
Proportion of exon 9+ (%)
IVS8-T allele
Nasal epithelium
Vas deferens
9T/9T
99
98
7T/9T
93
88
7T/7T
82
76
5T/9T
82
76
5T/7T
74
64
5T/5T
64
53
aValues represent corrected proportions of exon 9+ transcripts, according to Figure 2B.
These observations shed light on the relationship between the CBAVD or CF carrier phenotype and the amount of normal CFTR message (Fig. 6 ). A phenotypically normal, male CF carrier typically has the 7T variant on one chromosome and a severe CFTR gene mutation (e.g., [Delta]F508) on the other. The latter will result in absent or non-functional CFTR protein while the 7T variant may give rise to ~41% normal CFTR in respiratory tract and ~38% normal CFTR in reproductive tract, enough to sustain a normal phenotype in these tissues. On the other hand, a typical CBAVD patient may harbor the 5T variant on one chromosome and a severe CFTR mutation on the other. He may produce ~32% normal CFTR in the lung, which is adequate to sustain normal pulmonary function, but ~26% in the reproductive tract, an insufficient level to confer normal genital duct phenotype. However, the occurrence of fertile males with the severe CFTR mutation/5T genotype, such as fathers of some CF patients (12 ), suggests that other genetic factors (e.g., expression of alternative chloride channels) and/or environmental influences can ameliorate the unfavorable effects of certain CFTR gene sequence alterations. Alternatively, since a range of exon 9+ mRNA level does exist for the same IVS8-T genotype (Fig. 4 ; Table 1 ), these healthy, fertile men with a severe CFTR mutation and the 5T allele may produce a level of normal CFTR from the 5T chromosome that exceeds a minimal essential threshold. Furthermore, CBAVD men with the severe CFTR mutation/5T genotype may harbor additional mutations not detectable by our current DNA mutation screening methods (e.g., mutations within the promoter region or introns of the CFTR gene).
Twenty-four healthy men with a previous vasectomy presenting to a male infertility clinic for sterilization reversal were recruited for the study which was approved by the Human Subjects Review Committee of the University of Toronto, and informed consent was obtained from each subject. The following were obtained from each patient: peripheral venous blood for analysis of the IVS8-T tract genotype, nasal epithelia and a small piece of vas deferens (~5-10 mm, which would ordinarily be discarded) for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.
Genomic DNA was isolated from peripheral blood lymphocytes according to standard protocols (43 ). Exon 9 including the IVS8-T tract was PCR-amplified (44 )with primers 9i-5 and 9i-3, located in introns flanking exon 9 (45 ), and evaluated by allele-specific oligonucleotide hybridization, as previously described (23 ).
For reconstruction studies, a segment of exon 9+ cDNA was obtained from a plasmid containing full-length CFTR cDNA (46 ) following two rounds of nested PCR. First round PCR was performed with 400 ng of plasmid wild-type CFTR cDNA, 5' primer X5-5 (5'-GCTGTCAAGCCGTGTTCTAG-3', in exon 5) and 3' primer 13i-3sA (5'-TGGTCGAAAGAATCACATCC-3', in exon 13) for 35 cycles (94oC, 20 s; 60oC, 20 s; 72oC, 30 s) (Fig. 1 A). Nested PCR was performed on 1/5 of the reaction product from the first round under identical PCR conditions, except that 5' primer 7i-5s (5'-TTCAATAGCTCAGCCTTC-3', in exon 7) and 3' primer X12-3 (5'-GTTAAAACATCTAGGTATCC-3', in exon 12) were used (Fig. 1 A). The PCR products were size fractionated on 1% agarose gel in 1* TAE (40 mM Tris-acetate; 1 mM ethylenediaminetetraacetate, pH 8.0). The exon 9+ cDNA fragments were extracted from the agarose gel and purified according to the manufacturer's protocol (QIAquick Gel Extraction Kit/QIAGEN). Exon 9- cDNA fragments were isolated from commercially-obtained human lung total RNA (Clontech) after conversion to cDNA and amplification by two rounds of nested PCR as described above, except the exon 9- fragments were extracted from the agarose gel and purified (Fig. 1 B).
Total RNA was extracted from nasal epithelial and vas deferens specimens using TRIzol, according to the manufacturer's instructions (Gibco-BRL). First strand cDNA was synthesized from total RNA using Superscript II RNase H- reverse transcriptase and random hexanucleotide primers, according to the manufacturer's protocol (Gibco-BRL). Transcripts of the CFTR gene were then PCR amplified by using 5' primer X8-5 (5'-ACGACTACAGAAGTAGTGATGGAG-3', in exon 8) and 3' primer C16D (5'-GTTGGCATGCTTTGATGACGCTTC-3', in exon 10), and amplification was performed for 30 cycles (94oC, 20 s; 55oC, 20 s; 72oC, 30 s). The amplified products were size fractionated by agarose gel (1.5%) electrophoresis, transferred to Hybond N+ nylon membrane (Amersham) by the method of Southern (47 ), and hybridized with a CFTR cDNA probe derived from exon 10, which would anneal to both exon 9+ and exon 9- fragments (C16B; 5'-GTTTTCCTGGATTATGCCTGGCAC-3'). The expected size of the exon 9+ and exon 9- products were 416 and 233 bp, respectively. To ensure that the 416 and 233 bp fragments respectively represented exon 9+ and exon 9-, two oligonucleotide probes were designed: 9i-5s (5'-ACAGGGATTTGGGGAATTATTTG-3'), a sequence within exon 9, and ex8/10 (5'-TGGGAGGAGACTTCACTT-3'), a sequence which spans the 3' end of exon 8 and the 5' end of exon 10.
The absence of contaminants in RT-PCR assays was regularly assessed by controls that did not contain any cDNA template, starting RNA, or RT enzyme.
Nasal epithelial and vas deferens total RNA were extracted, converted to cDNA, amplified by PCR and subjected to Southern analysis using a 32P-labeled exon 10 probe (C16B) as described above. The resulting autoradiograph was analyzed by scanning densitometry (PDI Model DNA 35 Software/Protein Databases Inc.), and the proportion of exon 9+, as a percentage of total CFTR transcripts, was derived.
As standards for quantification of exon 9+ and exon 9- mRNA species, the isolated exon 9+ and exon 9- cDNA fragments were serially mixed in varying, known quantities and subjected to the same PCR conditions as for the nasal and vas deferens samples as described above. Quantification of exon 9+ and exon 9- was then determined as described above. The results were plotted and graphed. All experimental proportions of exon 9+ and exon 9- CFTR transcripts were accordingly adjusted, based on this graph (Fig. 2 B).
We thank Theresa Longley for her assistance in the collection of specimens. This work was supported by grants from the National Institutes of Health, USA (DK49096-03), the Canadian Cystic Fibrosis Foundation (CCFF) and the Canadian Genetic Diseases Network. V.M. is recipient of the Kinsmen Fellowship from CCFF, and K.A.J., Roscoe-Reid Graham Scholarship in Surgical Science from the Canadian Urology Association. L.-C.T. is Senior Scientist of the Medical Research Council of Canada, International Scholar of the Howard Hughes Medical Institute, USA, and the Sellers Chair of Cystic Fibrosis Research at the Hospital for Sick Children, Toronto.
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*To whom correspondence should be addressed. Tel: +1 416 813 6015; Fax: +1 416 813 4931; Email: lctsui@genet.sickkids.on.ca
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