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Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. The 5T allele in intron 8 (IVS8)
causes abnormal splicing in the CFTR gene, and is associated with
lung disease when it occurs in cis with a missense mutation in the
CFTR gene, R117H. However, the 5T variant alone has not been reported to cause lung disease. We describe two adult female patients with CF-like lung disease associated with the 5T allele. One
patient's genotype is 5T-TG12-M470V/5T-TG12-M470V, and the other is 
F508/5T-TG12-M470V; full sequencing of the CFTR gene revealed no other mutation on the same allele as the 5T variant. The levels of full-length CFTR mRNA in respiratory epithelia were very low in these patients (11 and 6%, respectively, of total CFTR
mRNA expression). Both patients had defective CFTR-mediated chloride conductance in the sweat ductal and/or acinar epithelia (sweat chloride, mmol/L, mean ± SEM: 40.0 ± 5.0 [n = 8 samples] and 80.0 ± 3.5 [n = 6 samples]) and airway epithelia (mV, mean ± SEM CFTR-mediated Cl
conductance of 1.2 ± 2.2 [n = 5 studies]
and 
6.75 ± 8.1 [n = 4 studies]). These data suggest that the 5T
polythymidine tract sequence on specific haplotype backgrounds
(TG12 and M470V) may cause a low level of full-length functional
CFTR protein and CF-like lung disease.
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The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene consists of 250 kb of genomic sequence, with 27 exons (1), with over 900 CFTR mutations associated with clinical disease described (see http://www.genet.sickkids.on.ca/cftr/) (2). Phenotypic expression of CF varies from severe, progressive pulmonary disease with pancreatic insufficiency (PI), to mild pulmonary disease with pancreatic sufficiency (PS) depending upon the site of the mutation and the relative impairment of the function of the CFTR chloride channel (2). Single-organ disease, such as congenital bilateral absence of the vas deferens (CBAVD) in males without lung disease, is associated with mutations in the CFTR gene, including the 5T allele of the polythimidine tract in the intron 8 (IVS8) (3, 4). Of the three alleles that have been identified in IVS8 (5T, 7T, and 9T), the 9T allele is associated with the most efficient usage of the intron 8 splice acceptor site (5). This efficiency decreases with shorter polythymidine tracts (5T and 7T), which results in a lower than normal level of full-length CFTR mRNA and presumably a decrease in mature, functional CFTR protein. Previously, the 5T and 7T alleles have been described as polymorphisms responsible for the variable expression of the mild CFTR gene mutation R117H. For example, an R117H-bearing allele in cis with a 7T allele may result in CBAVD, whereas when associated with the 5T allele, the phenotypic expression may be associated with mild CF lung disease and pancreatic sufficiency. Other haplotype backgrounds, such as the TG12 sequence and the M470V polymorphism, may influence CFTR splicing and function (6, 7).
This report describes two female patients with lung disease associated with the 5T sequence, with no other CF gene mutations on the same allele. To address the link between the 5T allele and disease at the molecular, cellular, and organ level, we performed detailed evaluations on both patients.
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Patients
Two female patients were referred to the University of North Carolina Hospitals because of unexplained chronic respiratory symptoms. We performed standard medical evaluations, including spirometry, radiographs of the chest, and tests of digestive function. All studies were carried out under the auspices of the Committee for the Protection on the Rights of Human Subjects at UNC-Chapel Hill.
Evaluation of CFTR Physiological Function in Airway Epithelia
Bilateral sweat chloride testing was performed in both patients, including tests at referring hospitals and at UNC (8, 9). We tested sweat acinar function with an intradermal injection of isoproterenol and aminophylline (to stimulate cAMP-mediated sweating) and atropine (to block sweating via cholinergic mechanisms) (10, 11). Non-CF individuals sweat briskly in response to the mixture, whereas CF patients do not sweat because of abnormalities of the cAMP CFTR-mediated pathway. Ion transport physiology across airway epithelia was tested by performing nasal potential difference (PD) measurements in both patients (12).
Molecular Studies
Detection of CF mutations and IVS8 polythimidine tract polymorphisms. DNA was extracted from peripheral lymphocytes, and 32 CF-causing mutations, and 5T, 7T, and 9T alleles were tested as described (13). Additional mutations were sought by sequencing of each exon of the CFTR gene including the flanking splice sites after amplification by polymerase chain reaction (PCR). PCR products were purified (QIAquick PCR purification kit; Qiagen, Valencia, CA) and sequenced (BigDye Terminator Cycle Sequencing kit; Perkin-Elmer, Applied Biosystem, Foster City, CA). Fluorescence-labeled DNA fragments were analyzed on an ABI Prism 310 (Perkin Elmer).
Nasal scrape biopsy and RNA extraction. Airway epithelial cells were obtained by scraping the inferior surface of the inferior turbinate with a plastic curette (Rhinoprobe, Arlington Scientific Inc., Arlington, TX). The cells were immediately washed in F12 medium, and lysed in 1 ml of TRIzol reagent for RNA extraction (GIBCO BRL, Bethesda, MD).
cDNA synthesis. The first strand of cDNA was synthesized by using oligo(dT)12-18 and SuperScript II RNase H reverse transcriptase
(GIBCO BRL) according to the manufacturer's protocol. Approximately 1 µg of the total nasal epithelial RNA was used in a 20 µl reaction. The reaction mixture was incubated at 42° C for 1 h.
Quantitation of CFTR mRNA Expression by Hot PCR
To estimate the relative amounts of the normally spliced CFTR mRNA
(exon 9+) expressed from the non-F508 allele, the CFTR cDNA was
selectively amplified using an allele-specific reverse primer located in
exon 10 (1669R: 5'-CATAGGAAACACCAAAGATGA) and a forward primer located in exon 6a (781F: 5'-GAGTTGTTACAGGCGTCTGCCTTC). A 50-µl reaction mix of each sample included 5 µl of
10× buffer (200 mM Tris-HCl, 500 mM KCl, pH 8.4; GIBCO BRL),
2 µl of 50 mM MgCl2, 0.25 µl of 5 U/µl Taq DNA polymerase (GIBCO
BRL), 1 µl of dNTP mix (2.5 mM of each), 1 µl of [
-32P]dCTP (3,000 µCi/mmol; ICN), 2 µl of each primer (10 pmol/µl), 2 µl of cDNA, 5 µl
of 60% sucrose with 0.04% Cresol Red (Sigma), and 30 µl of H2O.
The PCR mix was then divided into two tubes equally (25 µl each) for
a 24-cycle reaction and a 27-cycle reaction. Of the end PCR products
20 µl was subjected to electrophoresis on a 6% acrylamide gel (29:1)
containing 1× TBE and 4% glycerol. The gel was vacuum dried and
exposed to the film (Kodak BioMax MR film, Rochester, NY) at
70° C for 2 h to overnight. The exon 9+ and exon 9 fragments on
the dried gel were quantified by using a PhosphoImager (Molecular
Dynamics, Sunnyvale, CA). The molar ratio of the exon 9+ fragment
(889 bp) and the exon 9 fragment (706 bp) was adjusted in the final
calculation according to the fragment size.
Analysis of DNA Polymorphisms in or Near the CFTR Gene
Extragenic loci, XV2c, KM-19, and JG3c, were evaluated as previously described (16, 17). Intragenic polymorphisms were examined mainly by DNA sequencing.
Studies of Uniparental Disomy (UPD) for Chromosme 7
Methylation-specific PCR was used to evaluate patient 1 for uniparental disomy for chromosome 7. Bisulfite treatment of DNA and PCR were performed by methods described previously (18, 19). Both maternal and paternal allele-specific reactions were performed to evaluate parental inheritance.
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Clinical Data (Table 1)
Patient 1 (white female) reported frequent respiratory infections
as an adolescent and young adult, undergoing sinus surgery on
three occasions. Sinus and lower airway have been culture
positive for mucoid and nonmucoid Pseudomonas aeruginosa,
Alcaligenes xylosoxidans, Staphylococcus aureus, and Aspergillus flavus. A bronchoscopy (at age 46) showed normal airways, mucoid P. aeruginosa, and a positive smear (4+) and culture for Mycobacterium avium complex (MAC). She was
treated with intravenous antipseudomonal antibiotics with improvement. She has only had one subsequent positive smear for MAC (January 2000). Recently, she has required intravenous antipseudomonal antibiotics ~ 4 times per year. There is
no family history of CF, and no consanguinity has been identified. The patient was small: her height was 145 cm (4 ft 9 in.)
and her weight was 33 kg (~ 73 lb). Bilateral crackles were
noted. A high-resolution chest computed tomography (HRCT)
showed diffuse bronchiectasis with a marked right upper and
right lower lobe predominance. Images of the pancreas were
normal, with no fatty replacement typical of CF. Sinus CT
scans showed complete opacification of the sinuses. Lung function showed mild obstructive airways disease (FEV1 1.71 L,
76% predicted, and FVC 2 L, 75%, respectively). Serum trypsinogen was normal (34 ng/ml, normal 10-57). A 3-d stool fat
quantitation test showed normal fat absorption (12% of stool
was excreted as fat). Other causes of bronchiectasis were excluded: allergic bronchopulmonary aspergillosis (no history of
asthma [serum IgE; 72 IU/ml, normal range 0-158], and negative precipitins to A. fumigatus), primary ciliary dyskinesia
(normal ciliary ultrastructure and function), hypogammaglobulinemia (normal immunoglobulin and subclass levels), and
serum 1-antitrypsin levels were normal (139 mg/dl, normal 89-178).
Patient 2 (white female) experienced recurrent respiratory
infections during adolescence and adulthood, with a cough productive of purulent sputum, and sinus infections. Sputum and
sinus samples have repeatedly cultured mucoid and nonmucoid
Pseudomonas aeruginosa. She has no symptoms of pancreatic
insufficiency. Her family history is strongly positive for CF: a
daughter had CF (the husband is heterozygote for F508), and
two first cousins had CF. On examination, the nasal mucosa
was inflamed, with crackles audible at the left lung base. The
chest CT scan showed patchy densities in the left upper and
right middle lobe, and both lower lobes. Sinus CT scans show
opacification of the maxillary sinuses. Lung function was normal: FEV1 2.80 (115% predicted), FVC 3.77 (125%). Blood
work showed normal serum trypsinogen (34 ng/dl, normal 10-
57) and IgE levels (104 IU/dl, normal 10-180). Other causes of
chronic lung disease were excluded, such as primary ciliary dyskinesia (normal ciliary activity and ultrastructure) and hypogammaglobulinemia (normal serum immunoglobulins).
In Vivo Respiratory Epithelial Cell and Sweat Gland Physiological Data (Table 2)
Sweat chloride results (bilateral tests) for patient 1 demonstrated "borderline" values on three separate occasions (40 and 51, 40 and 53, and 51 and 48 mmol/L) and normal values (18 and 19 mmol/L) on a fourth occasion. Sweat chloride testing in patient 2 was abnormal on three occasions: 86 and 83 mmol/L in 1995, 85 and 88 mmol/L, and 70 mmol/L and 68 mmol/L in 1996. In both patients, there was complete absence of sweating in response to the intradermal injection of aminophylline, atropine, and ioproterenol, signifying defective sweat acinar CFTR function.
The mean basal PD for both patients was normal, although
patient 2 had a raised (CF-like) maximal PD of 56.5 mV.
The mean CFTR-mediated Cl conductance (mV) for both
patients was reduced below normal, though not as low as patients with pancreatic insufficient CF (see Table 2). The mean
CFTR-mediated Cl conductance for both patients falls into
the range of pancreatic sufficient CF, and indicates reduced
CFTR function. An adequate chloride conductance was present in response to the triphosphate nucleotide UTP in both
patients (ranging from 5.5 to 26.5 mV in patient 1 and 4
to 11 mV in patient 2) suggesting normal epithelial integrity
for the studies.
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Molecular Data: CFTR Genotyping
Initially, 32 CF-causing mutations and the polythymidine polymorphism in intron 8 (5T, 7T, and 9T) were tested in the two patients (14). Patient 1 was found to be homozygous for the 5T allele. Patient 2 was found to be heterozygous for the
F508 mutation and heterozygous for the 5T and 9T alleles in
intron 8. Because F508 is almost always found in cis to a 9T,
the 5T allele may be assigned to the opposite chromosome
(20). Thus, patient 2 is a compound heterozygote for two abnormalities in the CFTR gene, one of which is the 5T allele
alone. No additional mutations were detected after sequencing all 27 exons of the CFTR gene including the flanking splice
sites. Given the reported influence of the number of TG repeats (TG10, 11, 12, 13) adjacent to the poly-T tract to reduce
the splicing efficiency of full-length CFTR, both patients were
tested for the polymorphic TG sequence (6, 7). Both patients
were tested for the M470V polymorphism, as the 5T allele has
been reported in preferential association with this exon 10 sequence polymorphism (21). This latter variant, although
not linked with any pathology, is associated with a reduced (~ 60%) chloride channel capacity (6). Patient 1 is homozygous for the M470V polymorphism and the TG12 repeat sequence, whereas patient 2 has M470V and the TG12 sequence
in cis with the 5T allele. In addition, RT-PCR products encompassing all 27 exons were examined for the presence of abnormal splicing. No aberrant fragments, other than the exon 9
fragment, were seen, suggesting no intronic splice mutations.
Quantitative Analysis of CFTR mRNA
Two CFTR mRNA transcripts result from variants in the
poly-T tract: exon 9+ and exon 9 (22). The exon 9+ transcript
is the normal "full-length" CFTR mRNA, whereas the exon
9 is the aberrant transcript. We estimated the levels of normally spliced CFTR mRNA (exon 9+) expressed from the 5T
alleles in nasal epithelial cells. We compared the two patients
with healthy F508 carriers with either a 7T or a 9T on the
other allele (n = 4), and healthy control subjects with no
mutations in CFTR (n = 9, 7T/7T) (Figure 1). We considered
7T/7T individuals as the best control subjects for the study, as
this variant comprises the largest proportion of individuals in
the general populations (~ 70%) (20). To exclude the CFTR
mRNA expressed from the F508 allele in patient 2, allele-specific PCR was performed. Only the cDNA representing the
CFTR mRNA expressed from the non-F508 allele was amplified and the exon 9+ and exon 9 products were analyzed
for quantification. The levels of normal full-length CFTR
mRNA (exon 9+) expressed from the 5T alleles were severely
reduced in the patients as compared with the mRNA expressed from the 7T or 9T alleles in the control group (Figure
1). The relative amount of the exon 9+ CFTR mRNA in patient 1 (5T/5T) was also compared with healthy individuals homozygous for 7T or 9T by Southern hybridization (data not
shown). The entire coding region of the CFTR cDNA (4.5 kb)
was amplified to minimize unequal amplification due to the size difference (183 bp) between the exon 9+ and exon 9 fragments. The results were compatible with the quantitative analysis depicted in Figure 1. In patient 1, the exon 9+ CFTR
mRNA associated with the 5T alleles was 11-12% of total. Finally, to exclude the possibility of mutations in the promoter regions, we compared the amounts of CFTR mRNA expressed from the 5, 7, and 9T alleles relative to the F508 allele. The amount of CFTR mRNA from the 5T allele relative
to the F508 allele was comparable with the amounts from the
7T and 9T alleles in the healthy F508 carriers (data not
shown). This suggests no mutations in the promoter region, as
there was no change in CFTR mRNA expression arising from
the 5T allele in patient 2.
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Haplotype Analysis and Uniparental Disomy Studies of Patient 1
Patient 1 is of short stature, thus the possibility is raised of a variant of Russell Silver Syndrome, a condition associated with maternal uniparental disomy (UPD) for chromosome 7, which is the cytogenetic basis for CF in individuals who received both copies of their mutation-bearing chromosomes from one parent (23). Because no parental or sibling DNA was available because all were deceased, evidence for UPD 7 was sought by methylation-sensitive PCR of the DNA of patient 1 to distinguish the differentially methylated parental alleles of the imprinted gene, MEST, which, along with CFTR, maps to 7q32. Both maternal and paternal alleles were detected in patient 1, suggesting that she does not have UPD 7 (data not shown).
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This report provides evidence for a link between the 5T allele
of the polythymidine tract of intron 8, occurring with the TG12 and the M470V haplotype background, and low-level
expression of full length of CFTR mRNA, defective CFTR-mediated Cl conductance, and CF-type lung disease. At a
clinical level, both of the patients in this report demonstrate
characteristics consistent with a mild variant of cystic fibrosis.
Each patient had defective CFTR function on sweat testing
and sweat gland stimulation and reduced CFTR-mediated Cl
conductance across respiratory epithelia (12). There were no other established mutations in CFTR in cis to the 5T allele after full sequencing of the CFTR gene. This suggests that the
5T allele, perhaps with influence from the TG12 repeat sequence and the M470V polymorphism, is responsible for the
defective CFTR expression and function in these patients,
leading to clinical disease. To strengthen this hypothesis, the
expression of full-length CFTR mRNA (exon 9+) in respiratory epithelia was found to be reduced as compared with
healthy individuals.
Other reports have suggested a link between the 5T allele
and lung disease. Chillon and colleagues studied patients with
CBAVD, and found three adults with CBAVD and mild lung
disease who were compound heterozygotes for the F508 allele and the 5T allele (3). Three other (younger) patients were
compound heterozygotes for E858X/5T and K710X/5T, with
episodes of dehydration and abnormal sweat chlorides, but no
other clinical features of CF. Unfortunately, other gene variant and CFTR expression data, and further clinical details,
were not available for these six patients. In another report, the
5T allele was found to occur more frequently than expected in
a population of patients with diffuse bronchiectasis of unknown origin (5/15, 31%) (26). None of these patients had CF
as assessed by clinical and molecular methods. Therefore, the clinical spectrum of disease and sweat gland CFTR function of these previous reports differs from the patients in this report. Finally, Kerem and coworkers reported a series of patients
with CF, variant CF, and chronic lung disease, and found that
the incidence of the 5T variant was above that expected for
the population (Jewish/Arab) (27). However, the patients had
a very variable expression of disease, and the clinical and
physiological data were gathered retrospectively from a number of centers. Interestingly, the three patients homozygous
for the 5T variant did not have lung disease. These data suggested, at least on a population basis, that the 5T variant is associated with lung disease in some patients.
Another clinical disease recently linked to mutations in the CFTR gene is idiopathic pancreatitis (28, 29). Although those published studies initially did not demonstrate an increase in the prevalence of the 5T allele in that population of patients with nonpulmonary disease, further analyses subsequently indicated a higher than expected incidence of 5T compound heterozygotes and homozygotes (30). Neither of the patients in this current report has a history of pancreatitis resembling those patients with CFTR-related pancreatitis. This may relate in part to different levels of CFTR expression, and the threshold level required for the presence or absence of pancreatic disease. Alternatively, it may relate to tissue-specific normal and abnormal splicing of CFTR mRNA, or the influence of epigenetic or environmental factors.
The cell physiological data from the patients in this report
may provide insights into the relationship between CFTR defects and clinical expression of disease. Classic CF is associated with a high basal PD, and absent CFTR-mediated Cl
conductance (12). Both of the patients in this report had basal PDs lower than classic CF, but higher than normal, though
both had maximal PDs compatible with that seen in CF.
CFTR-mediated Cl conductance, on the other hand, was reduced into the CF range in both patients (12). These PD values are similar to the pattern of bioelectric properties reported
in men with CBAVD but no lung disease (31). It is unknown
whether these young males with CBAVD may eventually develop CFTR-related lung disease. The observation in this report that both patients presented with relatively late onset disease is of particular interest, suggesting the more subtle way
that this genetic abnormality influences the phenotypic expression of disease. The spectrum of disease in both patients
resembles that of "variant" pancreatic sufficient CF, with an
adult presentation and late diagnosis (32, 33).
At a molecular level, the data are congruent with the notion that the length of the polythymidine tract bears a direct
relationship to normal splicing of CFTR
the 9T/9T allele resulting in the highest expression of normal CFTR mRNA and
the 5T/5T resulting in ~ 15% of normal CFTR mRNA (3).
The threshold level of CFTR mRNA expression is theorized
to relate to the expression of disease, CBAVD being on the
threshold because development of the vas deferens is exquisitely sensitive to reduced levels of CFTR function, and pancreatic insufficient CF is at the end of the spectrum with lowest
levels of full-length CFTR. Subsequent data from Kerem and
coworkers support this scenario: there is a direct correlation
between disease expression and the level of correctly spliced
mRNA from the nose and the epididymis in 11 patients (9 males, 2 females) who carried at least one copy of the 5T variant (34). Patients with the least amount of correctly spliced
CFTR mRNA (6-14%) in respiratory epithelial cells had reduced lung function (FEV1 < 80% predicted). It is important to note that the patients in this current report have, in addition to the 5T allele, the TG12 sequence on the same allele, which may further reduce the splicing efficiency of CFTR mRNA,
and the M470V polymorphism, which may reduce CFTR function at the apical membrane (6).
In summary, variations in the IVS8 polythymidine tract alone, with the added influence of the TG12 repeat sequence and the M470V polymorphism, are associated with defective CFTR function in sweat glands and airway epithelia, and late onset lung disease resembling that seen in CF. Whereas the 5T allele has a frequency of ~ 5% among white individuals, only ~ 20% of these are in combination with the TG12 repeat as seen in the patients in this report. The other, shorter, TG sequences may not be associated with a pulmonary disease phenotype. Approximately 1 in 25 whites are carriers for a mutation in CFTR, and approximately 2% of white individuals are carriers of the 5T-TG12 allele. That is, as many as ~ 1:10,000 white individuals are homozygous for the 5T-TG12 allele, and 1:2,500 may be a compound heterozygote for a mutation in CFTR and the 5T-TG12 allele. Consequently, under the influence of modifier genes or accompanying polymorphisms, as many as 1:2,000 white individuals (or ~ 115,000 of the U.S. population) may at some point in their lives be at risk for some or all of the many manifestations of this variant in the CFTR gene. If the 5T- TG12 allele plays a role in the sinopulmonary and urogenital health of the number of people as these calculations suggest, it is essential that we understand fully the impact of this variant.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Peadar G. Noone, Pulmonary Division, CB #7248, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7248. E-mail: pnoone{at}med.unc.edu
(Received in original form March 29, 2000 and in revised form July 14, 2000).
Acknowledgments: The authors thank Vicky LeGrys, Ph.D., for helpful discussions regarding the sweat tests and interpretation. P. G. Noone and C. A. Pue contributed equally to this work.
Supported in part by grants from the National Institutes of Health (NIH 34332, RR00046) and The Cystic Fibrosis Foundation (CFF L543).
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References |
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