Lung Disease Associated with the IVS8 5T Allele of the CFTR Gene

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1 Lung Disease Associated with the IVS8 5T Allele of the CFTR Gene PEADAR G. NOONE, CHARLES A. PUE, ZHAOQING ZHOU, KENNETH J. FRIEDMAN, EMMA L. WAKELING, MUTTIAH GANESHANANTHAN, RICHARD H. SIMON, LAWRENCE M. SILVERMAN, and MICHAEL R. KNOWLES Cystic Fibrosis/Pulmonary Research and Treatment Center, Departments of Medicine, and Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Institute of Child Health, University of London, London, United Kingdom; Pinnacle Health at Polyclinic Hospital, Harrisburg, Pennsylvania; and University of Michigan, Ann Arbor, Michigan 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: [n 8 samples] and [n 6 samples]) and airway epithelia (mv, mean SEM CFTR-mediated Cl conductance of [n 5 studies] and [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. 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 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 R117Hbearing allele in cis with a 7T allele may result in CBAVD, (Received in original form March 29, 2000 and in revised form July 14, 2000) Supported in part by grants from the National Institutes of Health (NIH 34332, RR00046) and The Cystic Fibrosis Foundation (CFF L543). 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 pnoone@med.unc.edu Am J Respir Crit Care Med Vol 162. pp , 2000 Internet address: 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. METHODS 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 CFcausing mutations, and 5T, 7T, and 9T alleles were tested as described (13 15). 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) and SuperScript II RNase H reverse transcriptase (GIBCO BRL) according to the manufacturer s protocol. Approximately 1 g of the total nasal epithelial RNA was used in a 20 l reaction. The reaction mixture was incubated at 42 C for 1 h.

2 1920 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 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 -GAGTTGTTACAGGCG- TCTGCCTTC). A 50- l reaction mix of each sample included 5 l of 10 buffer (200 mm Tris HCl, 500 mm KCl, ph 8.4; GIBCO BRL), 2 l of 50 mm MgCl 2, 0.25 l of 5 U/ l Taq DNA polymerase (GIBCO BRL), 1 l of dntp mix (2.5 mm of each), 1 l of [ - 32 P]dCTP (3,000 Ci/mmol; ICN), 2 l of each primer (10 pmol/ l), 2 l of cdna, 5 l of 60% sucrose with 0.04% Cresol Red (Sigma), and 30 l of H 2 O. The PCR mix was then divided into two tubes equally (25 l each) for a 24-cycle reaction and a 27-cycle reaction. Of the end PCR products 20 l 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. RESULTS 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 (FEV 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 ). 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: FEV (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 ). 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 TABLE 1 CLINICAL DATA FOR TWO PATIENTS WITH CHRONIC LUNG AND SINUS DISEASE ASSOCIATED WITH THE IVS8 5T ALLELE IN THE CFTR GENE Patient 1 (5T-TG12-M470V homozygote) Patient 2 ( F508/5T-TG12-M470V) Age at diagnosis Smoker Nonsmoker Nonsmoker Respiratory symptoms Cough/sputum since childhood Several pneumonias in childhood Cough/sputum since age 60 Sinus symptoms Since age 23 Since age 50 CXR/CT Bilateral right upper lobe predominant Right middle lobe infiltrate/nodule bronchiectasis Upper lobe scarring Sinus radiographs Opacification of maxillary, sphenoid, and Opacification of maxillary sinuses, frontal sinuses with air fluid levels mucosal thickening of ethmoid sinuses Sputum bacteriology P. aeruginosa (mucoid and nonmucoid), P. aeruginosa (mucoid) S. aureus, Mycobacterium avium complex FEV 1, L (% pred) 1.7 (76%) 2.8 (115%) Definition of abbreviation: CFTR cystic fibrosis transmembrane regulator.

3 Noone, Pue, Zhou, et al.: The 5T Variant in Intron 8 Causes CF-type Lung Disease 1921 TABLE 2 SWEAT CHLORIDE CONCENTRATIONS (meq/l), SWEAT SECRETION TESTS (see text), AND NASAL EPITHELIAL PD DATA FROM THE TWO PATIENTS WITH ABNORMAL IVS8 CFTR ALLELES* Sweat Chloride (meq/l) Sweat Stimulation Mean Basal /Maximal PD (mv) Amiloride Response (% inhibition) CFTR-Mediated Cl Conductance (mv) UTP Response (mv) Patient NS 24.8/ Patient 2** NS 31.6/ Definition of abbreviations: CFTR cystic fibrosis transmembrane regulator; PD potential difference; UTP uridine triphosphate. * CFTR-mediated Cl conductance is calculated as the sum of the response to Cl -free solution and isoproterenol. Normal range 60 mmol/l. Mean basal PD from five sites under the turbinate, normal range 30 mv. Results of three studies in patient 1, two studies in patient 2. Delta PD, normal values mv, classic (pancreatic insufficient) CF values mv, CF with pancreatic sufficient mutations Mean SEM data derived from four separate bilateral sweat tests and five separate PD studies. NS no sweating visible after stimulation of CFTR-mediated sweat acinar epithelia. ** Mean SEM data derived from three separate bilateral sweat tests and four separate PD studies. 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 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. 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 Figure 1. Quantitative estimation of CFTR mrna in two patients with the 5T variant in the CFTR gene as compared with healthy control subjects. (A) Autoradiograph of a dried gel showing the 32 P-labeled RT- PCR products of the exon 9 and exon 9 CFTR mrna expressed from the non- F508 alleles in the patients and control subjects. The quantitative estimation of CFTR mrna represent patient 1 (5T/5T, lane 1) and patient 2 ( F508/5T, lane 2) and healthy carriers of F508/7T (lanes 3 and 4) and F508/9T (lanes 5 and 6). (B) Relative levels of total functional CFTR mrna derived from the RT-PCR autoradiograph above (lanes 1 6). Each column reflects the relative amount of functional CFTR mrna (exon 9 mrna expressed from the non- F508 allele) in patient 1 (5T/5T; 11%), patient 2 (5T/ F508; 6%), four healthy carriers of F508 (7T/ F508 and 9T/ F508; 41 48%), and nine healthy normal subjects with no CFTR mutations (n 9, 7T/7T; % SEM: , autoradiograph not shown).

4 1922 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL the general populations ( 70%) (20). To exclude the CFTR mrna expressed from the F508 allele in patient 2, allelespecific 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. 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 25). 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). DISCUSSION 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 CFTRmediated 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 pancre-

5 Noone, Pue, Zhou, et al.: The 5T Variant in Intron 8 Causes CF-type Lung Disease 1923 atic 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 (FEV 1 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. Acknowledgment : 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. References 1. Stutts MJ, Boucher RC. Cystic fibrosis gene and functions of CFTR. In: Yankaskas JR, Knowles MR, editors. Cystic fibrosis in adults. Philadelphia: Lippincott-Raven; p Knowles MR, Friedman KJ, Silverman LM. Genetics, diagnosis and clinical phenotype. In: Yankaskas JR, Knowles MR, editors. Cystic fibrosis in adults. 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