EXPRESSION OF CFTR mRNA IN NASAL EPITHELIUM AND VAS DEFERENS Victor Mak, M.D. A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Institute of Medical Science University of Toronto O Copyright by Victor Mak 1997
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EXPRESSION OF CFTR mRNA IN NASAL EPITHELIUM AND VAS DEFERENS · University of Toronto The gene responsible for cystic fibrosis (CF), called the cystic fibrosis transmembrane conductance
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EXPRESSION OF CFTR mRNA
IN NASAL EPITHELIUM
AND VAS DEFERENS
Victor Mak, M.D.
A thesis submitted in conformity with
the requirements for the degree of
Master of Science
Graduate Department of Institute of Medical Science
University of Toronto
O Copyright by Victor Mak 1997
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University of Toronto
The gene responsible for cystic fibrosis (CF), called the cystic fibrosis
transmembrane conductance regulator (CFTR), encodes the CAMP-regulated chloride
channel found in the apical membrane of secretory epithelial cells. It has been well
established that almost al1 males with CF are azoospermie due to atrophy or absence of
structures derived fiom the Wolffian duct. Interestingly, a higher than expected
fiequency of mutations in the CFTR gene has been identified in men with congenital
absence of vas deferens and men with epididymal obstruction. In particular, these
individuals have been found to have a significantly higher incidence of the 5-thymidine
(5T) variant of the CFTR intron 8 polypyrimidine tract (IVS8-T tract) compared to
normal or CF populations. The 5T variant results in less efficient splicing of CFTR exon
9 compared to the more common 7T and 9T variants and therefore produces less normal,
full-length CFTR mRNA. The protein produced by the CFTR transcript lacking exon 9
fails to fùnction as a CAMP-dependent chloride channel. The fact that these infertile
males have no other clinical signs of classical CF suggests that the epithelia of the male
reproductive tract may have the highest requirement for CFTR fûnction or, altematively,
splicing of CFTR mRNA in the reproductive tract is less efficient than the other CF-
associated organs. Nasal epithelia and segments of vas deferens were obtained fiom 24
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 TVS8-T tract and the proportion of
&A 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 NS8-T genotype. These findings suggest that the splicing of CFTR
precursor mRNA is less efficient in vasal epithelia compared to respiratory epitheiia.
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 manifestations of CF.
The author wishes to express sincere appreciation
to Prof. Lap-Chee Tsui, Dr. Keith Jarvi, and
Dr. Johanna Rornmens for their assistance in the
preparation of this manuscript.
.......................................................................................................................... List of Tables iv
.......................................................................................................................... List of Figures v
Abbreviations .......................................................................................................................... vi
.............................................................................................. Attribution of Labor and Data 1
........................................................................................ 1.A.l .b.i i. The testis 5 .. ......................................................................... LA.1 .b.ii. a. Leydig cells 5
.......................................................... I.A. 1 .b.ii. b. Seminiferous tubules 6 . . I.A. 1 .b.ii. c. Spermatogenesis ................................................................ -7
......................................... I.A. 1 .b.ii i. Hormonal control of spermatogenesis 7
.................................... I.A. 1 .b.i v. Sperm maturation, storage, and transport 8
LA . 1 .b. v. Fertilization ................................................................................... -9 . . .................................*.......,................... LA 1 c. Clinical Aspects of Male Infertility 9
................................................... I.A. 1 .c .i. Evaluation of the infertile male 10
................................................................. I.A. l .c.i. a. Semen analysis 1 0
................................... l.A.l .c.i i. Etiologic classification of male infertility 11
l.A.l . c i i. Treatment of male infertility .................................................... 11
follows: 94OC for 2 min; denaturation at 94OC for 20 S. annealing starting at 60°C then
auto-down OS°C/s over 20 s, and extension at 72OC for 30 s, for 20 cycles; followed by
denaturation at 94OC for 20 s, annealing at 50°C for 20 s, and extension at 72OC for 30 s,
for 15 cycles; followed by one final cycle of denaturation at 94OC for 20 s, annealing at
50°C for 20 s, and extension at 72OC for 7 min. The reaction mixture contained 5 pl of
PCR buffer (10 mM Tris-HC1 (pH 8.3), 50 rnM KCl, 1.75 mM MgC12, 0.001% gelatin), 5
pl of dNTP's (2 m M each of deoxyadenine triphosphate, deoxycytidine triphosphate,
deoxyguanosine triphosphate, and deoxyrhymidine triphosphate; Pharmacia); 50 ng of
each primer; and 0.5 unit of Taq DNA polymerase (Perkin Elmer/Cetus) in a final volume
of 50 pl, containing 50-100 ng of genomic DNA. Al1 PCR amplifications were
performed with the GeneAmp PCR System 9600 (Perkin ElmerlCetus). Ten pl of PCR
arnplified DNA was denatured and vacuum-blotted to nylon membrane (Hybond-N+;
Amersharn). The following allele specific oligonucleotides were used: ST (5 ' -
GTGTGmAACAGG-3') , 7T (5'-GTGTGTTTTTTTAACAG-3'), and 9T (5'-
TOTGmAAC AG-3'). Each oligonucleotide was labeled with 3 2 ~ by terminal
transferase (Pharmacia), and hybridization was performed ovemight at 42OC and the
washings were done once with 3xSSC/O.l%SDS at room temperature for 20 min, and
twice with 0.2xSSC at 36OC for 20 min. Amplified DNA fiom three individuals
previously s h o w to have the 5T/7T, 7T/7T, or 9T/9T genotype were used to control for
oligonucleotide specificity [Jarvi et al., 19951.
For reconstruction studies, a segment of exon 9+ cDNA was obtained fiom a
plasmid containing full-length CFTR cDNA [Rommens et al., 19911 following two
rounds of nested PCR. First round PCR was performed with 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;
60°C, 20 s; 72OC, 30 s) (Fig. 3A). The reaction mixture contained 5 pl of PCR buffer (10
rnM Tris-HCl (pH 8.3), 50 mM KCl, 1.75 m M MgCI2, 0.001% gelatin), 5 pl of dNTP's
(2 rnM of each of the four nucleotides triphosphate; Pharmacia); 200 ng of each primer;
and 0.5 unit of Taq DNA polymerase (Perkin ElmerKetus) in a final volume of 50 pl,
containing 400 ng of plasmid wild-type CFTR cDNA. Nested PCR was performed on 115
of the reaction product fiom 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. The PCR products were
size fiactionated on 1% agarose gel in 1 X TAE (40 mM Tris-acetate; 1 mM EDTA, pH
8.0). The exon 9+ cDNA fhgments were extracted fiom the agarose gel and purified
according to the manufacturer's protocol (QIAquick Gel Extraction KitlQLAGEN). Exon
9- cDNA fragments were isolated fkom commercially-obtained hurnan lung total RNA
(Clontech) after conversion to cDNA and amplification by two rounds of nested PCR as
described above, except the exon 9- hgments were extracted fiom the agarose gel and
purified (Fig. 3B).
POLYMERASE CHAIN REACTION
Nasal epithelial cells were collected by centrifugation and lysed in 800 pl of
TRIzot Reagent (Gibco/BRL). Vas deferens sarnples were homogenized with a rotor-
stator homogenizer (PRO 200 Handheld Homogenizer, PRO Scientific Inc.) in 800 pl of
TNzol Reagent. Total RNA was extracted fiom the lysates according to the
manufacturer's instructions (Gibco/BRL). First strand cDNA was synthesized fiom total
RNA using Superscript II RNase H- reverse transcriptase (RT) and random
hexanucleotide pnmers. The reaction (21 pl) contained up to 1 1 pl of total RNA, 2 pl
lOxPCR buffer (100 mM Tns-HCl (pH 8.4), 500 rnM K I ) , 2 pl MgCl2 (25mM), 1 pl 10
m M dNTP mix, 2 pl DTT (0.1 M), 1 pl random hexamers (5Ong/pL), 1 pl Superscnpt II
RT (200 unitslpl), and 1 pl E. coli RNase H (2 unitdpl), with al1 components obtained
comrnercially fiom Gibco/BRL.
To ensure that total RNA was successfidly extracted and synthesized into cDNA,
rnRNA of the human housekeeping gene glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) [Tokunaga et al., 19871 fiom each sample was amplified using the PCR
conditions described below. Primers used in the reaction were S'primer GAPDHSB (5'-
GGTCGGAGTCAACGGATTTGGTCG-3') and 3' primer GAPDH3B (5' -
CCTCCG ACGCCTGCTTCACCAC-3 '). The ampli fied products (78 8 bp) were size
fractionated by agarose gel (1.5%) electrophoresis, transferred to Hybond N+ nylon
membrane (Amersham) by the rnethod of Southem [Southem, 19751 and evaluated with a
32~-labeled human GAPDH cDNA probe (GAPDH 5A; 5'-
CCAATATGATTCCACCCATG-3') interna1 to the amplified region. The resultant
3 5 SoftwareProtein Databases Inc).
AAer being adjusted with GAPDH mRNA transcnpt level for each individual
sample, transcripts of the CFTR gene were amplified by PCR. The oligonucleotide
primers included 5 ' primer X8-5 (5 '-ACGACTACAGAAGTAGTGATGGAG-3 ' , in
exon 8) and 3' primer C 16D (5'-GTT'GGCATGCTITGATGACGCTTC-3', in exon 10)
(Fig. SA), and amplification was performed as follows: 94OC for 2 min; denahiration at
94OC for 20 s, annealing at 5S°C for 20 s, and extension at 72OC for 30 s, for 30 cycles;
followed by one final cycle of denaturation at 94OC for 20 s, mealing at 50°C for 20 s,
and extension at 72OC for 7 min. The PCR products were blotted to nylon membrane and
hybridized with a CFTR cDNA oligonucleotide probe derived fiom exon 10, which
would anneal to both exon 9+ and exon 9- fragments (C16B; 5'-
GT'ITTCCTGGATTATGCCTGGCAC-3') (Fig. 5B). The expected size of the exon 9+
and exon 9- products were 4 16 bp and 233 bp, respectively. To ensure that the 4 1 6 bp
and 233 bp fragments respectively represented exon 9+ and exon 9-, two oligonucleotide
probes were designed: 9i-5s (5'-ACAGGGATITGGGGAATTATTTG-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 (Fig. SB).
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 with CFTR primers, and subjected to Southem analysis using a '*P-
labeled exon 10 probe (C 16B) as described in Section H.D. The resulting autoradiograph
was analyzed by scanning densitometry, and the proportion of exon 9+ transcripts, as a
percentage of total CFTR transcripts, was denved by using the total densitometric units of
both transcripts as 100%.
As standards for quantification of exon 9+ and exon 9- mRNA species, the
isolated exon 9+ and exon 9- cDNA fragments (see Section II.C.) were serially mixed in
varying, known quantities and subjected to the same PCR conditions as for the nasal and
vas deferens samples as outlined in Section U.D. Quantification of exon 9+ and exon 9-
was then determined as described above. The results were plotted and graphed. Al1
experimental proportions of exon 9+ and exon 9- CFTR transcripts were accordingly
adjusted, based on this graph.
1I.G. ANALYSIS OF DATA
Differences in IVS8-T allele frequencies between study subjects and the general
population were compared by the X2 statistic or Fisher's exact test. The mean proportions
of CFTR exon 9+ mRNA transcripts associated with the different WS8-T genotypes were
compared uing analysis of variance (ANOVA). Post-hoc pairwise comparisons between
the different means were evaluated by the Bonferroni test. The correlation between the
proportion of exon 9+ transcripts and length of the IVS8-T tract was analyzed by the
Spearman rank correlation. Intra-individual nasal and vas deferens levels of exon 9+
cornparisons and those less than 0.05 were considered to indicate statistical significance.
Al1 statisticai analyses were performed using SPSS for Windows 1994.
1II.A. IVSS-T TRACT GENOTYPE
Leukocyte genomic DNA was isolated and amplified by PCR using CFTR
primers which encompass the IVS8-T tract and exon 9 region (Fig. 2A). Subsequent
ethidium bromide staining and gel electrophoresis confirmed successfùl amplification
(Fig. 2B). Slot blot analysis using allele specific oligonucleotides for the 5T, 7T, and 9T
variants revealed that, of the 24 patients, 8 were 7Tl9T heterozygotes, 14 were 7Tl7T
homozygotes, and the remaining 2 were 5Tl7T heterozygotes (Fig. 2C). As shown in
Table 3, the fiequencies of the 9T, 7T, and 5T alleles were not significantly different
between individuals in the present study and the general population [Kiesewetter et al.,
1993; Cuppens et al., 1994; Dork et al., 1994; Chillon et al., 19951 (9T: 16.7% vs.
1 1.5%, p=0.28; 7T: 79.2% vs. 83.3%, e . 4 6 ; 5T: 4.2% vs. 5.2%, p=0.75).
1II.B. CONVERSION OF EXPERIMENTAL TO ACTUAL PROPORTIONS OF EXON 9+ CFTR mRNA
To establish accurate measurement of the proportion of exon 9+ CFTR
transcnpts, it was fmt necessary to establish a standard c u v e using known concentrations
of isolated fiagments of exon 9+ and exon 9- transcripts. A plasmid containing Ml-
length CFTR cDNA served as the source of exon 9+ transcnpts pomrnens et al., 19911.
M e r two rounds of nested PCR using primers as shown in Fig. 3A, the exon 9+
fragments were extracted fiom agarose gel and purified. Exon 9- CFTR trmscnpts were
similarly obtained, except iung total RNA served as the source (Fig. 3B). Analysis by
electrophoresis and ethidium bromide staining revealed that a greater concentration of
. -
was then serially diluted and mixed with a constant quantity of the latter, and analyzed by
Southern transfer and scanning densitometry. This demonstrated that the concentration of
the isolated exon 9+ species was higher than that of exon 9- (Fig. 3D). Therefore, the
former was diluted to obtain equivalent concentrations of exon 9+ and exon 9-
transcripts. We then verified our calibration and the purity of the isolated exon 9+ and
exon 9- fragments by subjecting them to Southern analysis and hybridization with both
specific and cornmon radiolabeled oligonucleotide probes (Fig. 3E). Hybridization with
the probe comrnon to both transcripts (C16B) and subsequent densitomeûic anaiysis
confmed that we had accwately obtained equivaient concentrations of the exon 9+ and
exon 9- fiagments (Fig. 3F). Furthemore, hybridization with probes unique for exon 9+
or exon 9- rnRNA, validated the purity of the exon 9+ and exon 9- isolates, as the exon
9+ specific probe (9i-5s) and the exon 9- specific probe (ex8/10) annealed only to the
exon 9+ and exon 9- cDNA hgrnents, respectively (Fig. 3F).
Next, the exon 9+ and exon 9- fiagments were serially mixed in known
proportions and subjected to the identical PCR conditions and quantification protocol as
for the nasal and vas deferens specirnens (Fig. 3G-1). The experimental proportions of
exon 9+ and exon 9- transcripts were derived from densitometric scanning, taking the
surn of densitometric units for the exon 9+ and exon 9- bands as 100%. The
corresponding actual and experirnental proportions of exon 9+ transcripts were then
recorded graphically (Fig. 35). Consequently, al1 experhental proportions of exon 9+
transcripts from our tissue samples were adjusted according to this graph.
Total RNA was extracted fiom nasal and vas deferens specimens, converted to
cDNA, arnplified initially with primers for the transcript of the ubiquitously expressed
GAPDH gene, and analyzed by Southem hybridization. Quantification of the
autoradiographic bands by scanning densitometry showed differences in the yield of RNA
extraction and cDNA synthesis (Fig. 4). This information was then employed to estimate
the volume of cDNA to be used for PCR amplification with CFTR primers. For
example, Fig. 4 illustrates that cDNA obtained fiom the vas of patient 5 was -3.4 times
less than that of patient 1, hence 3.4 times the amount of cDNA fiom patient 5 was used
for the CFTR transcript analysis. These steps minimized inter-sample variability in RNA
extraction and cDNA synthesis, thus allowing cornparison of the proportion of exon 9+
CFTR transcripts within and between subjects.
Evaluation of CFTR mRNA transcripts fiom nasal epithelial and vas deferens
cells in the region encompassing exons 8 to 10 d e r conversion to cDNA and PCR
amplification revealed two different kgments. The difference in size between these two
bands (416 bp and 233 bp) corresponded to the size of exon 9 (183 bp) (Fig. 5A).
Southem analysis with an exon 10 specific probe (C16B) showed that both fragments
contained exon 10 sequences (Fig. 33; Fig. SC, lane 1). However, Southern analysis with
the exon 9+ specific probe (9i-5s) annealeci to the larger fiagrnent only (Fig. 5B; Fig. SC,
lane 2), while the exon 9- specific probe (ex8/10) detected only the smaller transcript
(Fig. 5B; Fig. SC, lane 3). 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-fi-ame exon 9 deleted transcript.
1ll.U. VUAN'I'Ik'1LA171VN OF EXVfl Y+ CFI'K mKRA
Al1 proportions of exon 9+ transcripts reported hereafter refer to the corrected,
actual proportions, based on Fig. 35. The proportion of exon 9+ nasal transcripts were
93&2%, 82t3%, and 74&1% (memSD) for the 7T/9T, 7T/7T, and 5T/7T genotypes,
respectively, while in the vas deferens sarnples, the proportion of exon 9+ transcripts
were 88&3%, 76k3%, and 64k4% for the 7T/9T, 7T/7T, and 5W7T groups, respectively
(Fig. 6 & 7; Table 4).
1XI.E. RELATIONSHIP BETWEEN LEVEL OF EXON 9+ CFTR mRNA AND THE IVSS-T TRACT
For both the nasal and vas deferens samples, significant differences were noted in
the mean exon 9+ levels for the different IVS8-T genotypes (p<0.0001, ANOVA; p<O.O5,
Bonferroni). In addition, a strong positive correlation was found between the length of
the IVS8-T tract and the proportion of exon 9+ &anscripts in nasal epithelia ( ~ 0 . 8 8 ,
p<0.001, Spearman) and in vas deferens ( ~ 0 . 8 7 , p<0.001, Spearman) (Fig. 7).
1II.F. COMPARISON OF THE PROPORTION OF EXON 9+ CFTR mRNA IN NASAL EPITHELIUM TO VAS DEFERENS
The mean levels of nasal and vasal exon 9+ transcripts were 85I7% and 79I8%
(meankSD), respectively. When evaluating the proportions of exon 9+ transcripts for
these two different CFTR expressing tissues fiom the same subject as paired samples, a
significantly higher proportion of exon 9+ transct-ipts was found within the nasd
epitheliurn compared to the vas deferens (p<0.001, paired t-test) (Fig. 8).
1V.A. SPLICING EFFICIENCY OF CFTR EXON 9 IS RELATED TO THE IVSS- T TRACT LENGTH
In the present study, we found a relationship between the WS8-T genotype and
the proportion of normal, full-length CFTR mRNA ûmscripts (exon 9+) in nasal
epithelia and vas deferens. It appears that the longer the WS8-T tract, the greater the
proportion of normal CFTR transcripts in nasal epithelia and vas deferens cells (Fig. 7;
Table 4). This fmding is consistent with that reported by Chu et al., who also
demonstrated a positive correlation between the proportion of exon 9+ transcripts in
branchial epitheliai cells and the length of the NS8-T tract [Chu et al., 19931. It is likely
that this relationship holds true for other CFTR-expressing tissues as well.
N.B. HlGHER PROPORTION OF NORMAL, CFTR mRNA IN NASAL EPITHELIUM THAN VAS DEFERlENS
Our study aisu showed that there is a greater proportion of the normal, Ml-length
CFTR message in nasal epithelia than in vas deferens fiom the same subject, regardless of
the NS8-T genotype @<0.001, paired t-test) (Fig. 8). In other words, the precise excision
of intron 8 with in-£&ne joining of exon 8 and exon 9 of CFTR *A may be less
efficient in epithelia of the reproductive tract compared to those of the respiratory tract.
Two recent reports corroborate our findings. Teng et al. found that, for the same NS8-T
tract genotype, the proportion of exon 9+ transcripts was lower in a series of vas deferens
samples obtained fiom vasectornized men than in a series of nasal biopsies obtained fiom
different men and women with chronic nasal obstruction or sinusitis [Teng et al., 19971.
Rave-Harel et al. documented that three men with CBAVD had an increased level of exon
19971. Taken together, these and our observations support the hypothesis that splicing
efficiency varies between the different tissues affected in CF.
W.C. THE CFTR WSS-T TRACT MODIFIES PHENOTYPE BY VARIABLE SPLICING OF EXON 9 IN DIFFERENT TISSUES
The discovery of differential splicing effrciency between the various tissues which
express CFTR provides important insights into the relationship between tevels of normal
CFTR and phenotypic variation. For instance, the R117H mutation is associated with
pancreatic-sufficient CF (CF-PS) mst id i s et al., 19921. CF-PS patients with this
mutation have pulmonary dysfunction but do not have pancreatic exocrine insufficiency
and their sweat chloride measurements are only modestly elevated [Harnosh et al., 19941.
Not surprisingly, the rnild R117H mutation has been identified in otherwise healthy males
with CBAVD. However, M e r 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., AF508, G55 1 D) developed lung
disease characteristic of CF, whereas the RI 17H mutation found in CBAVD men is
associated exclusively with the more efficient splice acceptor 7T (Le., R117H and 7T on
the same chromosome) [Kiesewetter et al., 19931. It is also important to note that the
R117H mutation gives rise to a partially functional CFTR protein [Sheppard et al., 19931.
Therefore, the R117W5T 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 R117W7T allele, although producing a sufficient level of
partially functional CFTR in the lung to prevent disease, the lower level in the genital
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.
1V.D. PROPORTION OF NORMAL CFTR mRNA PRODUCED BY THE IVSS-T ALLELES
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 wesewetter et ai.,
1993; Cuppens et al., 1994; Dork et al., 1994; Chillon et al., 19951 (Table 3). 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 5TET genotype is likely a
consequence of the relatively small sarnple size. Despite the absence of these groups,
based on our subjects with the 7T/9T, 7Tl7T and 5T/7T genotypes (Table 4) and the
assumption that each of the two CFTR alleles contributes equally to the total arnount of
CFTR transcripts, it is inferred that the 7T ailele produces -41% exon 9+ transcripts (ie.,
-41% of the total arnount of CFTR transcripts is exon Pt) 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 ailele produces -49.5% and 4 9 % , exon 9+ CFTR mRNA
in nasal epithelial and vas deferens cells, respectively (Table 5). Therefore, although the
present study does not consist of any individuals with the 9T/9T, 5T/9T, or 5T/5T
genotypes, it c m 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 6).
phenotype and the amount of normal CFTR message (Fig. 9). A phenotypically normal,
male CF carrier typically has the 7T variant on one chromosome and a severe CFTR gene
mutation (e.g., AF508) 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 f ict ion, but
-26% in the reproductive tract, an insufficient level to confer a normal genital duct
phenotype. However, the occurrence of fertile males with the severe CFTR mutation6T
genotype, such as fathers of some CF patients [Chillon et al., 19951, suggests that other
genetic factors (e.g., expression of dternative chloride channels) andfor environmental
influences can ameliorate the unfavorable eflects of certain CFTR gene sequence
alterations. Alternatively, since a range of exon 9+ mRNA level does exist for the same
ïVS8-T genotype (Fig. 7; Table 4), these heaithy, fertile men with a severe CFTR
mutation and the 5T allele may produce a level of normal CFTR fkom the 5T
chromosome that exceeds a minimal essential threshold. Furthermore, CBAVD men with
the severe CFTR mutatiod5T genotype may harbor additional mutations not detectable
by our curent DNA mutation screening methods (e.g., mutations within the promoter
region or introns of the CFTR gene).
Various studies have reported different mean values for the proportion of exon 9+
transcripts produced fiom the various IVSS-T tract genotypes. For example, with respect
ro me 1 i I I i genorype, u u er ai. reporcea a mean proporcion or exon Y+ nasal epitheliai
transcripts to be 75% [Chu et al., 19931 while Teng et al. docurnented 86% [Teng et al.,
19971. Although these estimates are not significantly different fiom ours (82%), the
slight discrepancy may be partly explained by the different methods used in the
quantitative RT-PCR analysis, such as the nurnber of cycles and nested rounds of PCR
employed, which pairs of primers were used in the PCR reaction, whether oligo-
deoxythymidine or random primers were utilized in the first strand cDNA synthesis, etc.
in addition, these studies employed a differential RT-PCR which may lead to preferential
amplification of the smaller exon 9- cDNA [Walsh et ai., 19923. Recently, Rave-Harel et
al. designed nondifferential RT-PCR reactions in which both exon 9+ and exon 9- cDNA
products were of the sarne size [Rave-Harel et al., 19971. However, this necessitated the
use of different primers and oligonucleotide probes. The former may result in different
amplification efficiencies while the latter will require deprobing and rehybridization
procedures which may lead to inadequate stripping of the previous probe andor some
loss of membrane-bound PCR products. In our approach, we minimize the number of
PCR cycles and introduce a standardization curve which should more accurately reflect
the actual proportion of exon 9+ transcripts (Fig. 3). The methodology involved is simple
and may be applied broadly to other similar quantitative RT-PCR analyses.
N.E. MOLECULAR MECNANISM OF DIFFERENTIAL SPLICING EFFICIENCY OF CFTR EXON 9
Several consensus sequences are found within introns of higher organisms that are
important for the efficient splicing of nuclear pre-mRNA. These include the s'-GU splice
donor, 3'-AG splice acceptor, branch-point A at -20 to 50 bases fiom the 3' splice site,
consensus has eleven consecutive nucleotides consisting of thymidine andfor cytosine
[Krainer and Maniatis. 19881. Extensive polypyrimidine tracts can make these sites more
cornpetitive as splice acceptor sites [Helfman and Ricci, 1989; Smith and Nadal-Ginard,
19891, while deletions in the polypyrimidine tract has been s h o w to inhibit the
S'cleavage reaction [Frendeway and Keller, 1985; Reed and Maniatis, 1986; Ruskin and
Green, 19851, binding of the splicing factors U2A.F and U2snRNP [Ruskin et al, 1 9881,
and assembly of the splicing complex [Frendeway and Keller, 19851. Furthemore, the
observation that identical pre-mRNA transcripts are processed into alternatively spliced
forms in a cell-type specific manner strongly suggests that the splicing environment of
different cells varies for these transcripts [Breibart and Nadal-Ginard, 1987; Wieczorek et
al., 19881. This tissue-specific difference in the splicing environment may be due to the
presence of specific alternative splicing factors or variations in the activities or levels of
constitutive splicing factors. Thus, the short polypyrimidine tract in CFTR intron 8,
especially with ST, likely makes it underutilized as a splice acceptor site, resulting in
reduced efficiency of exon 9 splicing. This effect is presumably more pronounced in the
reproductive tract than in the other CF-associated organs.
1V.F. StJMMAFtY
In sumrnary, o u study confums the hypothesis that splicing efficiency of CFTR
pre-mRNA varies between the different organs affected in CF. Specifically, depending
on the IVS8-T tract background, the splicing efficiency of CFTR exon 9 may be poor in
the genital tract but adequate in other tissues, thus explaining some cases of the CAVD or
- A . - . - . . . . - - - - - - - - - .
However, the existence of fertile males with the 5T genotype [Chillon et al., 19951 imply
that other genetic factors (e.g., expression of alternative chloride channels) and
environmental influences contribute to the overall phenotype. Studies such as ours may
have important future implications for CF gene therapy, which will involve delivery of
the appropriate level of normal CFTR into affected cells. Moreover, the study of the
molecular pathogenesis underlying CF-associated male infertility is especially relevant
given that the barrier to conception for men with CF, CAVD, or epididymal obstruction
has been overcome by advances made in the assisted reproductive technologies, thus
raising the concem of passing deleterious genetic traits ont0 subsequent generations [Mak
and Jarvi, 19961.
Alternative splicing variants other than exon 9- have been detected in CFTR
transcripts by RT-PCR [Bremer et al., 1992; Delaney et al., 19931. including ones which
involve the 5' end, portions of TM1, portions of NBD1, or the 3' end of the CFTR gene.
To date, these variants have not been dernonstrated to result in functional CFTR isoforms.
These patterns of alternative RNA splicing have been identified for the CFTR gene in a
variety of tissues, including lung, pancreas, intestine, and testis; however, similar
experiments have not been conducted in Wolffian-derived tissues. Since the latter
structures are highly sensitive to aberrant splicing as our study suggests, it would be
reasonable to assume that altematively spliced transcripts may occur with greater
fiequency in the reproductive tract. Therefore, we hypothesize that aberrantly spliced
transcripts other than exon 9-, previously discovered or novel, occur in the vas deferens
andor epididyrnis.
The tissue-specific difference in the splicing environment between the various
organs which express CFTR may be due to the presence of specific alternative splicing
factors. The identification and characterization of alternative splicing factors can be
approached by developing in viho splicing systems able to accurately reproduce cell-
specific splicing patterns. This should be possible either by using nuclear extracts fiom
the different ce11 types, or by complementing HeLa nuclear extracts [Smith et al., 1989;
Hodges and Bernstein, 19941. Other strategies for the isolation of factors include co-
injection of nuclei or nuclear extracts into Xenopur oocytes, dong with the experimental
transcnpt [Kelley and Perry, 1986; Kedes and Steitz, 19881. This system would also
vilru. With these approaches, alternative splicing factors which act on CFTR rnay be
identified and their mode of action subsequently characterized. The elucidation of
alternative splicing mechanisms could play an important role in development of novel
therapeutic strategies for CF. For example, it may be possible to design phmacologic
agents which upregulate the level andlor activity of constitutive splicing factors, or which
downregulate that of alternative splicing factors as means of augmenting levels of the
normal CFTR gene product.
Our findings may have a more immediate application to ow study population in
tems of providing prognostic information. Although too early in the post-operative
period for us to address this issue critically, it would be interesting to see whether a
correlation exists (after controlling for variables such as patient age, interval between
vasectomy and vasectorny reversal, and CF carrier statu) between the length of the IVS8-
T tract and the presence of sperm in the ejaculate pst-vasectomy reversal. Indeed, it
appears that azoospemiic men with idiopathic epididymal obstruction (who have a
significantly higher fiequency of the 5T variant (29%) compared to the general population
(5%) [Jarvi et al., 19951) have a lower rate of surgical success (def5ned as presence of
sperm in the ejaculate pst-vasoepididymostomy) compared to men with acquired
epididymal or vasal obstruction [Jarvi, K., unpublished data]. Although the difficulty of
surgery and the actual site of anastomosis are paramount in prognosticating post-
operative success, inadequate CFTR chloride function in the former group may contribute
to re-stenosis of the excurrent genital ducts.
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Organization, 19871
Semen volume:
Spenn count:
Percentage motility:
Percentage nonnal morphology:
White blood ce11 count:
'L'able 2. CFI'K gene mutations or vanations ana cHAvu
CFTR genotype 1 No. of CBAVD men (%y 1 Percentage of CBAVD mena