Thorax 1999;54:278–281
Clinical presentation of exclusive cystic fibrosislung disease Inez Bronsveld, Jan Bijman, Frauke Mekus, Manfred Ballmann, Henk J Veeze,Burkhard Tümmler Abstract
child was born preterm with meconium ileus The diagnosis of cystic fibrosis (CF) is
and died at day 10. The second child died dur- based on the occurrence of two mutations
in the cystic fibrosis transmembrane con-
suspected at necropsy. Our patient suVered ductance regulator (CFTR) gene and on
from chronic nasal polyposis which had led to assays that measure the basic defect of
13 polypectomies between the age of five and abnormal chloride transport in the af-
23 years. By the age of 15 years she was referred fected organs. However, in cases of atypi-
to a chest physician because of shortness of cal CF not all diagnostic tests may be
breath during sports activity. Clubbing, sub- positive. We present a patient with an
normal lung function, and decreased exercise atypical CF phenotype in whom the only
tolerance were noted. The diagnosis of CF was presenting symptom was severe CF-like
proposed because of the typical pulmonary lung disease substantiated by an abnormal
manifestations but was discarded when normal nasal potential diVerence. Genetic analy-
sweat electrolyte concentrations were found.
sis showed that the patient was a sympto-
The diVerential diagnosis of allergy as the matic heterozygote, which suggests that
underlying disease was excluded by normal IgE one lesion in the CFTR gene may be suY-
skin prick tests and normal serum levels of spe- cient to cause CF-like lung disease.
cific IgE. Immotile cilia syndrome was ex- (Thorax 1999;54:278–281)
cluded by the normal microscopic appearanceof biopsy specimens of nasal cilia. Since Keywords: cystic fibrosis; atypical cystic fibrosis; nasal adolescence she had been underweight (below 3rd percentile) and had recurrent lower airway Departments of
Paediatrics and Cell

infections. Over the years increased sputum Biology, Erasmus
Typical cystic fibrosis (CF) is caused by two production and chronic cough became a clini- University Rotterdam,
lesions in the cystic fibrosis transmembrane cal problem. A lung biopsy specimen in 1984 Rotterdam,
conductance regulator (CFTR) gene which showed a histological pattern consistent with The Netherlands
give rise to a generalised exocrine disease of the CF (bronchiectasis with localised purulent respiratory, gastrointestinal, reproductive, and bronchitis and surrounding fibrosis). Cultures hepatobiliary tracts.1 The protein product of of throat swabs or sputum were often positive Department of
the CFTR gene is a chloride channel expressed for Staphylococcus aureus (>90% of samples) Neonatology, Sophia
in the apical membrane of epithelial cells2 so but never for Pseudomonas aeruginosa. How- Children’s Hospital,
diagnostic tests that measure the chloride con- ever, the specific anti-P aeruginosa oprF IgG Rotterdam,
ductance in exocrine epithelia—for example, titre was positive, indicating that she had been The Netherlands
H J Veeze
the pilocarpine iontophoresis sweat test,3 intes- exposed to P aeruginosa. Bronchodilators and tinal current measurement (ICM),4 and nasal intermittent antibiotics were prescribed but the Department of
potential diVerence (NPD)5 6—have abnormal patient generally discontinued medication after Paediatrics and the
values in cases of typical CF. Atypical cases of a few days, even during acute respiratory tract Clinical CF Research
CF have a diVerent clinical presentation with infections. At the age of 23 her lung function Group, Medizinische
pancreatic suYciency, mild bronchitis, nasal was reduced (vital capacity 1.5 l (48% pre- Hochschule Hannover,
D-30623 Hannover,

polyposis, congenital bilateral absence of the dicted)), her chest radiograph had a Chrispin- vas deferens (CBAVD), a borderline sweat test, Norman score of 20, grade 3 (range 0–38), her or ICM values which point to low residual height was at the 25th percentile and weight chloride secretion in intestinal tissue either by below the 3rd percentile. However, her pancre- CFTR or an alternative chloride channel.7–10 atic function was suYcient as shown by normal We present a case characterised exclusively by chymotrypsin levels in 1986, 1992, 1995, nor- severe lung disease in whom other organs typi- mal stool elastase levels (386 mg/g) in 1995, cally involved in CF were not aVected and who and by ultrasound investigation of the pan- failed to show a defect in chloride transport.
enzymes or vitamin supplements. Serum levels Patient history
of vitamins (vitamin A 370 µg/l, vitamin E The 34 year old patient was the third child of 11 mg/l), bilirubin and liver enzymes were Caucasian first generation cousins. The first Exclusive cystic fibrosis lung disease gel electrophoresis, blotting and hybridisationfollowed protocols 1, 5, 7, 8 and 9 of Bautsch etal.14 The intragenic CFTR haplotype was deter-mined for the sequence alterations T854T andM470V,15 16 the splice site polymorphism inintron 8 (TG) T ,11 17 and the microsatellites NASAL POTENTIAL DIFFERENCE (NPD)NPD was measured using an adaptation of themethod described previously.5 In short, the nasalturbinate was superfused (1.7 ml/min) for peri-ods of three minutes with the following solutions(mol/l): saline to measure baseline PD; amilo-ride (10–4) to block Na+ channels; gluconate withamiloride to assess spontaneous Cl– conduct-ance; isoprenaline (10–4) in gluconate with ami- Autoradiogram of genomic ApaI digestions probed with CFTR cDNA (exon 7–24). No anomalous loride to evaluate the presence of CFTR, and band pattern is seen for the sample from the patient (lane ATP (10–3) in isoprenaline/gluconate+amiloride 1). For comparison, lane 4 shows the probe reactive solution to open non-CFTR Cl– channels. To fragments from a specimen which carries a deletion in oneCFTR allele. ApaI cleaved fragments were separated in a access the submucosal space a small needle (25 CHEF-DRTMII cell at 5.6 V/cm in 1% agarose gels (0.5 gauge) was inserted into the forearm and filled TBE buVer, 10°C). Pulse times were linearly increased in with saline. Both the needle and superfusion two ramps from 5 to 20 s in 18 hours and from 5 to 90 s in20 hours. catheter were connected to a high inputresistance voltage measuring device via salt repetitive pilocarpine iontophoresis sweat tests bridges and Ag/AgCl electrodes. The saline performed since she was 11 years old the sweat solution consisted of (mmol/l): NaCl (120), Na electrolyte levels were always normal (range (0.4), Na HPO (2.4). In the gluconate solution NaCl was replaced by Na gluconate (145). Allsolutions were adjusted to pH 7.4.
This study was approved by the medical ethical
committees of the Dutch and German partici-
pating hospitals and informed consent was obtained from the patient. All chemicals were described previously.4 Freshly obtained rectal biopsy specimens were mounted in saline solu- tion in the Ussing chamber (aperture 1.2 mm).
After stabilising the basal short circuit current(I ) the tissue was exposed to the following inhibitors and secretagogues (mol/l) to the The promoter (–3.8 to –0.3 kb) and coding mucosal (M) or serosal (S) side: glucose (10–2, regions and the exon flanking intron sequences M+S); amiloride (10–4, M); indomethacin of the CFTR gene were screened for mutations (10–5, M+S) to inhibit basal Cl– secretion by by single strand conformation polymorphism inhibiting the endogenous prostaglandin for- (SSCP) analysis11 12 and, in the case of the mation; carbachol (10–4, S), a Ca2+ linked appearance of non-wild type band patterns in secretagogue; forskolin (10–5, M+S) + 8-bromo the high resolution SSCP gel, by subsequent cyclic adenosine monophosphate (cAMP, 10–3, sequencing.13 Deletions in the CFTR gene were M+S) to open Cl– channels by activation of a sought by repeated Southern hybridisations of cAMP-dependent protein kinase and phospho- rylation of specific membrane proteins; 4,4'- probes encoding exon 1, exons 7–24, or the sec- diisothiocyanatostilbene-2,2'-disulphonic acid ond nucleotide binding fold (codons 1202– (DIDS, 2.10–4, M) to inhibit Ca2+ dependent 1422). Preparation of unsheared genomic DNA Cl– secretion; and histamine (5.10–4, S) to reac- from fresh blood, complete restriction digestions with ApaI, EagI, FspI, SalI or XhoI, pulsed field Electrophysiological results for the study patient. Mean (SD) values for a control group and a group of patients with CF are given for comparison CFTR GENETICSDespite the familial consanguinity, the patient was heterozygous at the CFTR locus which is shown by the diVerent number of (TA) repeats (n = 30, 34) for the microsatellite at the locus IVS17bTA and her heterozygosity for a rare the TG repeat with the T allele and the V470 CFTR isoform are known to decrease signifi- cantly the amount and chloride channel activ- ity of CFTR.16 After all CFTR exons and flank- ing intron sequences and most of the promoter decrease of 40%. Superfusion of gluconate in the presence of amiloride resulted in a net response of –3 mV, indicating subnormal Cl–conductance. With isoprenaline, which opens CFTR Cl– channels, only a small response of –4 mV was obtained which suggested that fewCFTR channels are present. No ATP response, which is usually indicative of the presence of alternative Cl– channels,20 was seen in the In previous ICM studies carbachol has pro- voked a negative reversed I in rectal tissue of patients with CF, sometimes followed by a positive residual response indicating the pres- ence of residual Cl– secretion. In our patient the Nasal potential diVerence (PD) measurements ICM showed a Cl– secretory current in the of the patient (x) and mean (SD) PD values of 25 controls normal range on addition of carbachol (fig 3).
(L) and 23 patients with CF ( ) following superfusion DIDS, which inhibits the alternative pathway with saline solution, amiloride (10–4 M) in saline solution,Cl– free solution with amiloride, isoprenaline (10–4 M) in of Ca2+ activated Cl– currents, did not influence Cl– free solution with amiloride, and ATP (10–3 M) in Cl– the Cl– current, suggesting the presence of nor- free solution with amiloride and isoprenaline. mal CFTR mediated chloride conductance inthe intestine.
The clinical and diagnostic features presented
by this case are conflicting in terms of the expression of the basic defect in diVerent tissues. In typical CF defective electrolyte transport in the sweat gland, intestinal and air- way epithelium is shown by a pathological out- come of the sweat test, ICM and NPD. In ourcase, however, the sweat chloride concentrationand ion flow in the intestine were normal while the NPD values were abnormally high. Patho-logically raised NPD values have so far only been found in CF and not in any other lung Measurements of intestinal current expressed as disease with related clinical manifestations changes in short circuit current (I ) of the patient (x) and mean (SD) I of 50 controls (L) and 51 patients with CF syndrome, or congenital bronchiectasis.21 As ( ) following the addition of amiloride, carbachol, andexposure to 4,4'-diisothiocyanatostilbene-2,2'-disulphonic well as a high baseline nasal PD, the patient’s gluconate and isoprenaline response were alsoin the CF range. However, they do point to the area up to –4 kb had been screened by SSCP, presence of little residual CFTR Cl– permeabil- one molecular lesion (the splice site consensus ity in her respiratory epithelium. The clinical transition 1898+3 A->G) was identified. A symptoms of our patient, such as progressive pancreatic insuYcient child with CF at our obstructive bronchitis and bronchiectasis, spu- clinic is homozygous for this condition and tum production, and pathological bacterial exhibited the typical pulmonary and gastro- flora, are typical of CF and she has been intestinal manifestations of CF indicating that permanently on oxygen since the age of 30.
1898+3 A->G is a CF-causing lesion. No This could explain her anorexic status since anomalous bands were seen in autoradiograms there is no sign of malabsorption or gastro- of macrorestriction blots probed with CFTR intestinal disease, as confirmed by normal vita- cDNAs, indicating that the two CFTR alleles of min A and E levels, pancreatic suYciency, and our patient do not carry any major genomic a normal ICM. This highly discordant expres- sion of the basic defect in diVerent tissues isdescribed here for the first time: CFTR function was unaVected in gastrointestinal tis- On the day of electrophysiological investigation sue whereas in airway tissue the abnormally at the age of 32 she was underweight (below the low Cl– conductance can only be accounted for by defective epithelial Cl– transport. Other 17 kg/m2) and her lung function was severely cases of CF with normal sweat test results and pulmonary disease, as described for patients 1.23 l (38% predicted)). At this time she was treated with continuous oxygen and showed mutation,7 8 22 23 can clearly be diagnosed by an severe clubbing. Electrophysiological measure- abnormal ICM even when there are few or no clinical signs of gastrointestinal involvement.10 presence of chloride conductance in her airway The CFTR gene was screened for disease and intestinal epithelium (table 1). The basal causing lesions in all exons and flanking intron NPD was –52 mV (fig 2). In the presence of sequences and on one chromosome a sequence amiloride the PD depolarised to –31 mV, a alteration in a donor splice site was found Exclusive cystic fibrosis lung disease (1898+3 A->G). This sequence alteration was 4 Veeze HJ, Sinaasappel M, Bijman J, et al. Ion transport first described in a compound heterozygous abnormalities in rectal suction biopsies from children with
cystic fibrosis. Gastroenterology 1991;101:398–403.
pancreatic suYcient patient (Ferrari et al, per- 5 Knowles MR, Carson JL, Collier AM, et al. Measurements sonal communication) and was found also in a of nasal transepithelial electrical potential diVerences innormal human subjects in vivo. Am Rev Respir Dis pancreatic insuYcient CF patient homozygous 1981;124:484–90.
for this condition (see above). According to 6 Knowles MR, Paradiso AM, Boucher RC. In vivo nasal potential diVerence: techniques and protocols for assessing CFTR mutation analysis and the family anam- eYcacy of gene transfer in cystic fibrosis. Hum Gene Ther nesis, our patient is a carrier for one CFTR 1995;6:445–55.
mutation and hence should not express any CF 7 Gan KH, Veeze HJ, van den Ouweland AM, et al. A cystic fibrosis mutation associated with mild lung disease. N Engl symptoms. However, she carries an unfavour- J Med 1995;333:95–9.
able combination of common intragenic poly- 8 Gilbert F, Li Z, Arzimanoglou I, et al. Clinical spectrum in homozygotes and compound heterozygotes inheriting morphisms. She is homozygous TG T for the cystic fibrosis mutation 3849 + 10kb C > T: significance for geneticists. Am J Med Genet 1995;58:356–9.
9 Kerem E, Rave-Harel N, Augarten A, et al. A cystic fibrosis acceptor site, and the TG repeat together with transmembrane conductance regulator splice variant with the T stretch places the branch point nucle- partial penetrance associated with variable cystic fibrosis presentations. Am J Respir Crit Care Med 1997; otide in an unfavourable position for splicing 155:1914–
leading to 30% of exon 9− CFTR transcript 10 Veeze HJ, Halley DJ, Bijman J, et al. Determinants of mild clinical symptoms in cystic fibrosis patients. J Clin Invest
proteins that do not mature.16 24 Moreover, she 11 Dörk T, Mekus F, Schmidt K, et al. More than 50 diVerent is homozygous for the V470 allele in exon 10.
CFTR mutations in a large group of German cystic fibrosis
patients. Hum Genet 1994;94:533–42.
V470 CFTR proteins have a 1.7 fold decreased 12 Ravnik-Glavcak M, Glavcak D, Dean M. Sensitivity of sin- intrinsic chloride channel activity compared gle strand conformation polymorphism and heteroduplex
method for mutation detection. Hum Mol Genet 1994;3:
with M470 CFTR proteins.16 These predispos- ing polymorphisms in the intragenic back- 13 Zielenski J, Rozmahel R, Bozon D, et al. Genomic sequence of the CFTR gene. Genomics 1991;10:214–28.
ground decrease the expression and function of 14 Bautsch W, Römling U, Schmidt KD, et al. Long-range CFTR. Yet, they are present in both rectal and restriction mapping of genomic DNA. In: Dear PH, ed.
Genome mapping—a practical approach. Oxford: OxfordUniversity Press, 1997: 281–313.
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fibrosis families. Hum Genet 1992;88:417–25.
absence of Cl– conductance was found. How- 16 Cuppens H, Lin W, Jaspers M, et al. Polyvariant mutant ever, the patient’s consanguineous descent genes. J Clin Invest 1998;101:487–96.
leads to a genome-wide overrepresentation of 17 Teng H, Jorissen M, van Poppel H, et al. Increased homozygous genotypes which may lead to fur- proportion of exon 9 alternatively spliced CFTR transcriptin vas deferens compared with nasal epithelial cells. Hum ther unfavourable combinations of factors that Mol Genet 1997;6:85–90.
modify CFTR expression or lung function. As 18 Morral N, Estivill X. Multiplex amplification of three gastrointestinal disease is not present, we microsatellites within the CFTR gene. Genomics 1992;13:
propose that the genetic background predis- 19 Mekus F, Dörk T, Deufel T, et al. Analysis of microsatellites poses to severe lung disease caused by tissue by direct blotting electrophoresis and chemiluminescence
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specific regulatory elements which lead to the 20 Knowles MR, Clarke LL, Boucher RC. Activation by extra- loss of CFTR function exclusively in the respi- cellular nucleotides of chloride secretion in the airway epi-thelia of patients with cystic fibrosis. N Engl J Med 1991;325:533–8.
21 Knowles MR, Gatzy J, Boucher RC. Increased bioelectrical potential diVerence across respiratory epithelia in cystic This work was supported by the Deutsche Forschungsgemein- fibrosis. N Engl J Med 1981;305:1489–95.
22 Dreyfus DH, Bethel R, Gelfand EW. Cystic fibrosis 3849+10kb C > T mutation associated with severe pulmo- 1 Welsh MJ, Tsui LC, Boat TF, et al. Cystic fibrosis. In: Scriver nary disease and male fertility. Am J Respir Crit Care Med CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic and 1996;153:858–60.
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24 Strong TV, Wilkinson DJ, Mansoura MK, et al. Expression of an abundant alternatively spliced form of the cystic 3 Gibson LE, Cooke RE. A test for concentration of fibrosis transmembrane conductance regulator (CFTR) electrolytes in sweat in cystic fibrosis of the pancreas utiliz- gene is not associated with a cAMP-activated chloride con- ing pilocarpine by iontophoresis. Pediatrics 1959;23:545–9.
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