Hum Genet (1997) 100 : 365–377
O R I G I N A L I N V E S T I G AT I O N
Thilo Dörk · Bernd Dworniczak · Christa Aulehla-Scholz ·
Dagmar Wieczorek · Ingolf Böhm · Antonia Mayerova ·
Hans H. Seydewitz · Eberhard Nieschlag ·
Dieter Meschede · Jürgen Horst · Hans-Jürgen Pander ·
Herbert Sperling · Felix Ratjen · Eberhard Passarge ·
Jörg Schmidtke · Manfred Stuhrmann
Distinct spectrum of CFTR gene mutations
in congenital absence of vas deferens
Received: 15 April 1997 / Accepted: 29 April 1997
Abstract Congenital absence of the vas deferens (CAVD)
is a frequent cause for obstructive azoospermia and ac-
counts for 1%–2% of male infertility. A high incidence of
mutations of the cystic fibrosis transmembrane conduc-
tance regulator (CFTR) gene has recently been reported in
males with CAVD. We have investigated a cohort of 106
German patients with congenital bilateral or unilateral ab-
sence of the vas deferens for mutations in the coding re-
gion, flanking intron regions and promotor sequences of
T. Dörk (Y) · J. Schmidtke · M. Stuhrmann
Institut für Humangenetik, OE 6300,
Medizinische Hochschule Hannover,
D-30625 Hannover, Germany
Tel.: +49-511-5323876; Fax: +49-511-5325865
B. Dworniczak · D. Meschede · J. Horst
Institut für Humangenetik, Universität Münster,
Münster, Germany
C. Aulehla-Scholz · H.-J. Pander
Abteilung für Klinische Genetik der Frauenklinik Berg,
Stuttgart, Germany
D. Wieczorek · E. Passarge
Institut für Humangenetik, Universitätsklinikum Essen,
Essen, Germany
I. Böhm
Genetisches Labor Dr Waldenmaier, München, Germany
A. Mayerova
Institut für Humangenetik der Universitätskinderklinik,
Universität Freiburg, Freiburg, Germany
H. H. Seydewitz
Klinisch-chemisches Labor der Universitätskinderklinik,
Universität Freiburg, Freiburg, Germany
E. Nieschlag
Institut für Reproduktionsmedizin, Universität Münster,
Münster, Germany
H. Sperling
Klinik für Urologie, Universitätsklinikum Essen, Germany
F. Ratjen
Zentrum für Kinderheilkunde, Universitätsklinikum Essen,
Essen, Germany
© Springer-Verlag 1997
the CFTR gene. Of the CAVD patients, 75% carried CFTR
mutations or disease-associated CFTR variants, such as
the “5T” allele, on both chromosomes. The distribution of
mutation genotypes clearly differed from that observed in
cystic fibrosis. None of the CAVD patients was homozy-
gous for ∆F508 and none was compound heterozygous for
∆F508 and a nonsense or frameshift mutation. Instead,
homozygosity was found for a few mild missense or splic-
ing mutations, and the majority of CAVD mutations were
missense substitutions. Twenty-one German CAVD pa-
tients were compound heterozygous for ∆F508 and
R117H, which was the most frequent CAVD genotype in
our study group. Haplotype analysis indicated a common
origin for R117H in our population, whereas another fre-
quent CAVD mutation, viz. the “5T allele” was a recur-
rent mutation on different intragenic haplotypes and mul-
tiple ethnic backgrounds. We identified a total of 46 dif-
ferent mutations and variants, of which 15 mutations have
not previously been reported. Thirteen novel missense mu-
tations and one unique amino-acid insertion may be con-
fined to the CAVD phenotype. A few splice or missense
variants, such as F508C or 1716 G→A, are proposed here
as possible candidate CAVD mutations with an apparently
reduced penetrance. Clinical examination of patients with
CFTR mutations on both chromosomes revealed elevated
sweat chloride concentrations and discrete symptoms of
respiratory disease in a subset of patients. Thus, our col-
laborative study shows that CAVD without renal malfor-
mation is a primary genital form of cystic fibrosis in the
vast majority of German patients and links the particular
expression of clinical symptoms in CAVD with a distinct
subset of CFTR mutation genotypes.
Introduction
Cystic fibrosis (CF) is a fatal autosomal recessive ex-
ocrinopathy that affects approximately one in 2000 indi-
viduals in Caucasian populations (Welsh et al. 1995). The
physiological basis of the disease is characterized by ab-
normal secretion of electrolytes and fluid across the ep-
366
ithelial membranes of most exocrine organs. Clinical hall-
marks of CF include chronic infections and pulmonary
obstruction, pancreatic insufficiency, neonatal meconium
ileus, failure to thrive, cholestasis, diabetes mellitus, ele-
vated sweat electrolytes and male infertility (Welsh et al.
1995). CF, in its classic form, is usually diagnosed by two
or more of these symptoms during the first few years of
life and is associated with a mean life expectancy of cur-
rently about 30–35 years (FitzSimmons 1993). More than
95% of adult CF males are infertile because of obstructive
azoospermia. The spectrum of observed Wolffian duct ab-
normalities in CF includes the absence or atrophy of the
epididymal body and tail, seminal vesicles and the ejacu-
latory ducts and, in many cases, the congenital bilateral
absence of the vas deferens (CBAVD; Kaplan et al. 1968;
Taussig et al. 1972; Stern et al. 1982; Heaton and Pryor
1990; Wilschanski et al. 1996).
CBAVD (McKusick 277180) has long been noted as a
frequent cause of male infertility that is inherited in an au-
tosomal recessive fashion (Nelson 1950; Schellen and van
Stratten 1980); it occurs in 1%–2% of men presenting
with infertility at urological departments and may have an
incidence of up to 1:1000 in Caucasian males (Charny and
Gillenwater 1965; Dubin and Amelar 1971; Holsclaw et
al. 1971; Jequier et al. 1985; Oates and Amos 1994; Mak
and Jarvi 1996). Although isolated CBAVD is considered
a distinct clinical and genetic entity, it was suggested as
long ago as 1971 that some males with CBAVD may have
an unusually mild form of CF (Holsclaw et al. 1971). This
assumption has been corroborated following the identifi-
cation of the cystic fibrosis transmembrane conductance
regulator (CFTR) gene, which is the causative defective
in CF (Rommens et al. 1989; Riordan et al. 1989; Kerem
et al. 1989). The observation that many men presenting
with CBAVD have mutations in their CFTR genes has led
to the proposal that CBAVD may be a primary genital
form of CF (Dumur et al. 1990; Anguiano et al. 1992).
Subsequent reports have confirmed the high incidence
of CFTR gene mutations in otherwise healthy men with
CBAVD (Patrizio et al. 1993; Oates and Amos 1994; Au-
garten et al. 1994; Casals et al. 1995; Mercier et al. 1995).
However, the initial work also revealed that many investi-
gated CBAVD patients did not seem to carry mutations on
both copies of the gene. Two explanations were found for
this apparent lack of CFTR mutations in this large sub-
group of patients. First, CBAVD concomitant with con-
genital renal malformations is thought to represent a dif-
ferent clinical and embryological aetiology that is unre-
lated to the expression of CFTR (Rubin 1975; Augarten et
al. 1994; Oates and Amos 1994; Dumur et al. 1996). Sec-
ond, a partially penetrant CFTR splice variant termed the
“5T” allele had initially not been recognized as a disease-
causing mutation but turned out to be associated with the
phenotype of CBAVD in more recent studies (Chillón et
al. 1995; Costes et al. 1995; Zielenski et al. 1995).
In the present study, we have investigated the propor-
tion and distribution of CFTR gene mutations in a large
cohort of 106 German males who presented at various
urological centres with congenital absence of the vas def-
erens (CAVD). We describe the results of this collabora-
tive effort with particular emphasis on novel mutations
that appear to be specific for CAVD and on the differences
of CFTR genotypes between isolated CAVD and classic
CF. This approach should improve our understanding of
genotype-phenotype relationships for both of these com-
mon diseases.
Materials and methods
Patients
Our study group comprised 106 infertile men who were diagnosed
as having obstructive azoospermia at various urology departments.
All patients were found to have either bilateral (n = 101) or unilat-
eral (n = 5) absence of the vas deferens by scrotal exploration and
the clinical observation of impalpable vasa. Absence of seminal
vesicles was confirmed in some cases by semen analysis (low fruc-
tose, low volume, low pH). Histological examinations were per-
formed in most cases and revealed reduced spermatogenesis in a
subset of patients. Unilateral renal agenesis was found in 9 men by
abdominal ultrasonography. The patients were 25–38 years of age
and most of them had parents of German descent. We only in-
cluded patients who were apparently unrelated. In one family, two
brothers were ascertained as having CBAVD and the CFTR geno-
type ∆F508/L375F.
Mutation analysis:
All patients underwent a genetic evaluation for the most common
CFTR gene mutations at one of the participating centres in Han-
nover, Münster, Freiburg, Stuttgart, Essen or Munich. At this first
step of mutation screening, 18 CFTR gene mutations known to be
frequent in Germany were tested directly by allele-specific means,
such as restriction enzyme analysis or heteroduplex analysis. This
initial screening included the mutations ∆F508, G542X, R553X,
G551D, N1303K, 1717–1 G→A, 3272–26 A→G, Y1092X,
2143delT, R347P, R347H, R334W, I336K, R117H, R117C,
2789+5 G→A, 3849+10kB C→T and the “5T” allele, the latter
two splice variants being tested according to the instructions of
Highsmith et al. (1994) and Chillón et al. (1995).
We next investigated those patients with only one or no identi-
fied mutations and with no known renal malformations for novel
and rare mutations of the CFTR gene by single-strand conforma-
tion analysis (SSCP) of all 27 exons and flanking intron and pro-
motor sequences. Samples with an abnormal migration pattern in
the SSCP analysis were directly sequenced by using the Sequenase
2.0 polymerase chain reaction (PCR) product sequencing kit
(Amersham/USB). Primer sequences and SSCP conditions were as
described elsewhere and had previously been optimized to detect
more than 95% of mutations in 350 German CF families and 100%
of mutations in 63 Austrian CF families (Dörk et al. 1994b; M.
Stuhrmann et al. in preparation). However, we exchanged one up-
stream primer for the amplification of exon 23, as we had observed
a frequent G/A polymorphism located within the primer binding
site of the commonly used 23i-5 primer (Zielenski et al. 1991a),
which under stringent conditions could result in an amplification
bias from heterozygote samples. Our new primer 23i-5(II) had the
sequence 5′-AGAAGTACTGGTGATTCTAC-3′ and could be used
together with primer 23i-3 at an annealing temperature of 58° C. In
addition to the coding region and flanking intron sequences, a 512-
bp portion containing the 5′UTR and minimum promotor region of
the CFTR gene was amplified by primers 5′-GTCCTCCAG CGT-
TGCCAACTGG-3′ (forward) and 5′-CAACGCTGGCCTTTTCC-
AGAGG-3′ (reverse) under standard PCR conditions with anneal-
ing at 66°C. These products were also screened for mutations by
SSCP analysis but, except for polymorphism 125 G→C, no varia-
tions could be detected in that region.

Evaluation of CF symptoms:
All patients with CAVD were offered genetic counselling and an
investigation of other phenotypic features consistent with CF. Sev-
eral males refused any further clinical evaluation. Those who par-
ticipated were asked for a history of respiratory disease, gastroin-
testinal or hepatobiliary manifestations or other disease symptoms.
Sweat electrolytes were then measured in some cases by pilo-
carpine iontophoresis according to established methods (Gibson
and Cooke 1959).
Results
Mutation screening
Direct analysis of the most common CFTR mutations was
performed on 106 male German individuals presenting
with CAVD: 92 had CBAVD without renal agenesis, 9
had CBAVD with concomitant unilateral renal malforma-
tion and 5 had unilateral absence of the vas deferens. Two
CF mutations (∆F508 and R117H) and one splicing vari-
ant (the “5T” allele) were found to be particularly com-
mon in these patients. The 3-bp deletion ∆F508 in exon
10 was found on 57 chromosomes (26%) and was the
most frequent mutation, although its incidence was much
lower than among German CF patients (allele frequency
of 72%). Missense substitution R117H was identified on
24 chromosomes (11%), which is an approximately 30-
fold increase in allele frequency compared with German
CF chromosomes (Dörk et al. 1994b). This frequency of
R117H seems to be the highest number reported so far in
a patient subpopulation and confirms a particular associa-
tion of R117H with the CAVD phenotype in Mid-Europe
(Gervais et al. 1993). All the R117H alleles were from
patients of German or Austrian descent and carried the
same diagnostic marker haplotype composed of four in-
tragenic dimorphisms and the 7T allele (Table 1). This
haplotype is otherwise rare on normal or CF chromo-
somes and accounts for less than 0.5% in the German
population, suggesting a common origin of the R117H
mutation, which occurs at a CpG dinucleotide, in Mid-Eu-
ropean populations. The third frequent gene variant in our
CAVD cohort, collectively termed the “5T” allele, re-
flects at least three different alleles that can be distin-
guished by their different intragenic backgrounds. The
“5T” reduction of the polythymidine tract in intron 8 was
preceded by 13, 12 or 11 GT dinucleotides, respectively,
and was associated with each of the three most common
intragenic marker haplotypes (Table 1), indicating recurrent
mutational events. In further support of recurrence, the
“5T” variant was present in patients of German, Turkish,
Lebanese, Vietnamese, Greek or Austrian descent. It is in-
teresting to note that the most common “5T” allele in
CAVD, viz. (TG)125T, is located on the most frequent
dimorphic marker haplotype usually associated with
(TG)117T in intron 8. Similarly, dimorphic marker analy-
sis indicated that the (TG)135T allele resides on the most
frequent (TG)127T haplotype, whereas the (TG)115T al-
lele results from a (TG)107T haplotype. This suggests a
367
recurrent mutational mechanism that involves nucleotide
misincorporation rather than deletion/insertion mutagen-
esis.
Screening for the three mutations ∆F508, R117H and
“5T” together allowed the identification of some 40% of
mutations in our CAVD cohort. Only four other common
CF mutations were found in more than one family by this
initial screening: 2789+5 G→A on 4 alleles, R347H on 3
alleles, G542X and 3272–26 A→G each on 2 alleles
(Table 1). No mutation was found in the nine CBAVD pa-
tients with known renal involvement.
We then searched for additional novel and rare CFTR
gene mutations by SSCP analyses of all exons and flank-
ing intron regions in samples from those individuals in
whom CFTR mutations had not been found on both alle-
les and renal malformation had not been documented. The
46 different mutations and variants that we were finally
able to identify by using this approach are listed in Table
1. Fifteen mutations were uncovered that had never been
observed previously on normal or CF chromosomes. In-
terestingly, thirteen of these are new missense substitu-
tions, some of which may be subtle changes specific for
the CAVD phenotype, and one mutation is a unique
amino-acid insertion within the first transmembrane re-
gion. Missense mutations were also the predominant type
of lesion on the other CAVD chromosomes where a
CFTR mutation could be found, most of these missense
mutations occurring within the CFTR transmembrane do-
mains (Fig. 1). In contrast, only 8 alleles with nonsense or
frameshift mutations were identified. The incidence of
CFTR “null alleles” is therefore about three-fold lower in
CAVD males than in German CF patients.
New mutations
Four novel missense mutations affect charged amino
acids in the amino terminal transmembrane domain
(TMD) of the CFTR protein. The E56K mutation was
found in a German CBAVD patient who carried ∆F508 on
the maternal allele and E56K on the paternal allele. The
D58N mutation was uncovered in a Lebanese CBAVD pa-
tient who had inherited D58N from his father and a “5T”
allele from his mother. The R297W mutation was identi-
fied on a Q1352H chromosome in a Vietnamese CBAVD
patient (see below). The R334L mutation occurs at a posi-
tion at which another mutation, R334W, has been described
in mild to moderate CF and has been found to affect chloride
channel conductivity significantly (Gasparini et al. 1991;
Sheppard et al. 1993). The German CBAVD patient in our
study was compound heterozygous for R334L and for the
missense mutation I336K in the same exon. Six other new
missense mutations were located in the carboxyterminal
transmembrane domain, particularly in the third cytoplas-
mic loop (R933S, V938G, L973F, D979A). The R933S
substitution was found together with the R75Q allele in a
heterozygous CBAVD patient who also carried the ∆F508
deletion. The V938G substitution was identified in two
unrelated patients, one homozygote with unilateral ab-
368
Table 1A Frequency distribution and haplotypes of CFTR mutations in 106 CAVD patients
Mutationa Nucleotide changesb Locationc Frequencyd Haplotypee Referencef

174delA deletion of A at 174 exon 1 1 D3 This study

E56K G→A at 298 exon 3 1 B3 This study
D58N G→A at 304 exon 3 1 C2 This study
D110H G→A at 460 exon 4 2 C2 Dean et al. (1990)
R117H G→A at 482 exon 4 24 B6 Dean et al. (1990)
A120T G→A at 490 exon 4 1 n.p. Chillón et al. (1994)
hL138 insertion of CTA after 546 exon 4 1 A2 This study
L206W T→G at 749 exon 6a 1 B8 Claustres et al. (1993)
M265R T→G at 926 exon 6b 1 A2 Schwarz et al. (pers. comm.)
R297W C→T at 1021 exon 7 1 C2 This study
1078delT deletion of T at 1078 exon 7 1 C2 Claustres et al. (1992)
R334W C→T at 1132 exon 7 1 B1 Gasparini et al. (1991)
R334L G→T at 1133 exon 7 1 D3 This study
I336K T→A at 1139 exon 7 1 A2 Cuppens et al. (1993)
R347H G→A at 1172 exon 7 3 D1 Cremonesi et al. (1992)
L375F A→C at 1257 exon 8 1 B3 Jézéquel et al. (1996)
∆F508 deletion of 3 bp
between 1652–1655 exon 10 57 B1 Kerem et al. (1989)
G542X G→T at 1756 exon 11 2 B1 Kerem et al. (1990)
R553X C→T at 1789 exon 11 1 A4 Cutting et al. (1990)
L568F G→T at 1836 exon 12 1 B3 This study
2184insA insertion of A at 2184 exon 13 1 D3 Dörk et al. (1994b)
2789+5 G→A G→A at 2789+5 intron 14b 4 D3 Highsmith et al. (1997)
R933S A→T at 2931 exon 15 1 n.p. This study
V938G T→G at 2945 exon 15 3 D3 This study
L973F T→A at 3048
and C→T at 3049 exon 16 1 D3 This study
D979A A→C at 3068 exon 16 1 n.p. This study
L997F G→C at 3123 exon 17a 1 A2 Fanen et al. (1992)
Y1032C A→G at 3227 exon 17a 1 B3 This study
3272-26 A→G A→G at 3272-26 intron 17a 2 D3 Fanen et al. (1992)
D1152H G→C at 3586 exon 18 3 C2, A2 Highsmith et al. (pers. comm.)
V1153E T→A at 3590 exon 18 1 B3 This study
3659delC deletion of C at 3659 exon 19 1 C2 Kerem et al. (1990)
W1282X G→A at 3978 exon 20 1 D3 Vidaud et al. (1991)
N1303K C→G at 4041 exon 21 1 B1 Osborne et al. (1991)
K1351E A→G at 4183 exon 22 1 A2 This study
D1377H G→C at 4261 exon 22 1 C1 Costes et al. (1995)
L1388Q T→A at 4295 exon 23 1 n.p. This study
Variants:
R75Qg G→A at 356 exon 3 2 A2 Zielenski et al. (1991b)
T351S C→G at 1184 exon 7 1 C4 Mercier et al. (1993)
5Th reduction of oligoT tract
to 5T at 1342-12 intron 8 26 C2, A4, D3, A2 Chu et al. (1991)
F508C T→G at 1655 exon 10 3 C2 Kobayashi et al. (1990)
1716 G→A G→A at 1716 exon 10 3 D3 Kerem et al. (1990)
G576Ai G→C at 1859 exon 12 2 D3 Fanen et al. (1992)
R668Ci C→T at 2134 exon 13 2 D3 Fanen et al. (1992)
S1235R T→G at 3837 exon 19 1 n.p. Cuppens et al. (1993)
Q1352Hh G→C at 4188 exon 22 2 C2 Nukiwa and Seyama (pers. comm.)
a The nomenclature of mutations follows Beaudet and Tsui (1993) by M. Schwarz, A. Haworth and G. Malone, D1152H by W. E.
The symbol “h” is used to designate an amino-acid insertion Highsmith jr., L. Burch, K. J. Friedman, B. M. Wood, A. Spock, L.
b Nucleotides are numbered according to the cDNA sequence of M. Silverman and M. R. Knowles, Q1352H by T. Nukiwa and K.
Riordan et al. (1989) Seyama are indicated
c Exons and introns are numbered according to Zielenski et al. g Missense substitutions R933S and R75Q occurred together in a

(1991a) ∆F508 heterozygous patient

d Allele frequency is given as number of chromosomes h Q1352H is associated with 5T and R297W, respectively

e Haplotypes were defined as listed in B below. Minor haplotypes i Missense substiutions G576A and R668C are linked on the same

are shown in italics; (n.p.) phase unknown allele in both CBAVD patients
f Personal communications to the Cystic Fibrosis Genetic Analysis

Consortium (http://www.genet.sickkids.on.ca/cftr.html): M265R

 369

Table 1B Dimorphic marker

haplotypes. Haplotypes were Extragenic XV-2c KM.19 Intragenic (GATT)n M470V T854 TUB18
defined following a previous
study of CF chromosomes A 1 1 1 6 1 1 1
(Dörk et al. 1994b) by using B 1 2 2 7 2 1 1
the dimorphic extragenic C 2 1 3 7 1 2 2
RFLPs XV2c and KM.19 and D 2 2 4 6 1 2 1
the four intragenic markers 5 7 1 1 1
(GATT), M470V, T854,
6 7 1 2 1
TUB18 as previously pub-
lished (Estivill et al. 1987; 7 6 1 1 2
Dörk et al. 1992) 8 8 1 2 2

Fig. 1 Distribution of CAVD

missense mutations in the
transmembrane domains
(TMD) of CFTR. Missense
mutations were frequent in the
sixth transmembrane helix of
the amino terminal TMD (top)
and throughout the first intra-
cytoplasmic loop of the car-
boxyterminal TMD (bottom).
New amino-acid substitutions
are indicated in bold, whereas
missense variants of uncertain
significance are shown in ital-
ics. The membrane topology of
the CFTR protein was adapted
from the original report and
has been confirmed in vitro by
glycosylation site mutagenesis
(Riordan et al. 1989; Chang et
al. 1994)

sence of the vas deferens (CUAVD) and one heterozygote
with CBAVD. The homozygous case may classify V938G
as the first “pure” CUAVD mutation, whereas the hetero-
zygous CBAVD patient carries a potentially severe muta-
tion, the new frameshift deletion 174delA, on the other
chromosome. The amino-acid substitution L973F results
from a tandem nucleotide substitution in exon 16; this
gives rise to a silent change in codon 972 and to the
phenylalanine for leucine substitution at codon 973. Mu-
tation Y1032C is an amino-acid substitution encoded by
exon 17a and is predicted to reside within the tenth trans-
membrane helix of CFTR; it was found in a German
CBAVD patient heterozygous for Y1032C and for ∆F508.
D979A and V1153E are non-conservative amino-acid
changes in exons 16 and 18, respectively, at positions ad-
jacent to other known CBAVD mutations, viz. I980K and
D1152H (Bienvenu et al. 1996; W. E. Highsmith et al.
personal communication). Three other new missense sub-
stitutions, viz. L568F, K1351E and L1388Q, are located
within or flanking the CFTR nucleotide-binding domains
(NBD). L568F was detected in a CBAVD male of Turkish
descent. This mutation targets the conserved “Walker B”
ATP-binding motif of NBD1 at a crucial position (Higgins
1992). K1351E is located at a consensus motif of NBD2,
which is essential for the signature of ATP-binding cas-
sette transporters and which is thus a frequent site of CF
mutations. Our finding of a German CBAVD patient het-
erozygous for ∆F508 and K1351E adds evidence to previ-
ous indications that ABC signature mutations have less
severe consequences in the second than in the first CFTR
nucleotide-binding fold (e.g. the CF mutation pair G551D/
G1349D). Missense mutation L1388Q flanks NBD2 at its
carboxyterminal side and was found in a CBAVD male
heterozygous for ∆F508 and L1388Q. This finding indi-
cates that even substitutions within the final part of the
CFTR protein exert subtle effects on CFTR function. An-
other novel mutation was a 3-bp insertion, termed hL138,
within exon 4 of CFTR. This mutation was uncovered in
a CBAVD male who is a compound heterozygote for the
insertion and for a “5T” allele, from Southern Germany.
His father had inherited the hL138 mutation and was of
Polish descent; his ancestors had lived in the region of
former Eastern Prussia for centuries. The hL138 muta-
tion is predicted to add a fourth leucine residue to the sec-
ond transmembrane helix at the cytosolic mouth of the
channel pore. The molecular and pathological conse-
quences of this unusual mutation, the first amino-acid in-
sertion known so far in CFTR, remain to be defined.
CFTR variants
Several patients carried no typical CF mutation but a po-
tentially disease-associated CFTR variant on at least one
chromosome (Table 1). Splicing variants, such as “5T” or
1716 G→A, and missense variants, such as F508C or the
370
Table 2 Frequency distribution of CFTR variants in different sub-
groups of individuals. Allele frequencies of six CFTR variants
were compared in randomly selected anonymous blood donors vis-
iting the Medical School, Hannover, in the parents of CF patients
Variant Allele frequency n (% of chromosomes)
Random donors Non-CF
125G→C 15/178 (8.5%) n.d.
R75Q 4/188 (2.2%) 3/130 (2.1%)
5T 9/186 (4.8%) 2/65 (2.9%)
F508C 0/188 n.d.
1716G→A 5/188 (2.6%) 3/212 (1.5%)
G576A-R668C 0/188 n.d.
a One CF allele with R75X and 125G→C
b One CBAVD allele with R75Q and R933S
c One CBAVD allele with 5T and Q1352H
d Two CF alleles with F508C and S1251N
double mutant allele G576A and R668C, have previously
been reported to be benign but no other mutation could be
detected on these alleles in our patients after scanning the
whole coding region, except for one case where the R75Q
and R933S mutations were found together in a ∆F508 het-
erozygote. In order to investigate whether these variants
are preferentially associated with the CAVD phenotype,
we compared their frequencies in males with CAVD
with the frequencies in a random panel of healthy German
blood donors and in CF families (Table 2). The promotor
polymorphism 125 G→C is unlikely to be a disease-asso-
ciated substitution, as it is present, on two different haplo-
types, in some 8.5% of chromosomes from the general
population and has been seen only twice in our CAVD
cohort. Conversely, the “5T” variant has an incidence of
some 5% in the random donors but reaches a significantly
increased allele frequency of 12.3% in our CAVD study
group. The association of the “5T” allele with this partic-
ular disease phenotype is explained by the extensive skip-
ping of exon 9 in epithelial CFTR mRNA transcripts (Chu
et al. 1993; Chillón et al. 1995; Teng et al. 1997).
The next two most frequent variants were the splice
site substitution 1716 G→A and missense substitution
R75Q. Splicing variant 1716 G→A was identified in three
of our CBAVD males. This variant affects the final nu-
cleotide of exon 10 and results in extensive exon skipping
in carrier lymphocyte mRNA (data not shown). However,
no specific association with CAVD was found in the
present data set and we observed two proven fathers with
the compound heterozygous genotypes ∆F508/1716 G→A
or G551D/1716 G→A, respectively, in our cohort of CF
families. On the other hand, we saw the compound het-
erozygous genotype ∆F508/1716 G→A twice in our
CAVD cohort, which is 20-fold more often than that ex-
pected from the allele frequencies of each change in the
general population. Interestingly, some CF patients had
an unusually mild form of the disease and were compound
heterozygous for ∆F508 and the splicing variant 1716
G→A, with no other CFTR mutation being detected, de-
spite exhaustive searches (Cuppens et al. 1993; our un-
(non-CF chromosomes), in our CAVD cohort and in a large cohort
of CF patients. The latter group includes and extends that of a pre-
vious study (Dörk et al. 1994b). Complex alleles are indicated
CBAVD CF
2/212 (0.9%) 1/1000 (0.1%)a
2/212 (0.9%)b 1/1000 (0.1%)
26/212 (12.3%)c 3/1000 (0.3%)
3/212 (1.4%) 2/1000 (0.2%)d
3/212 (1.4%) 2/1000 (0.2%)e
2/212 (0.9%)f 3/1000 (0.3%)f
e One CF allele with 1716G→A and L619S
f G576A and R668C were linked on two CBAVD and three CF al-
leles, whereas two additional CF alleles carried R668C together
with the 3849+10kB C→T mutation (Dörk and Stuhrmann 1995)
published data). Therefore, exclusion of the possibility
that splicing variant 1716 G→A has the potential to result
in very mild disease is premature and future investigations
should examine possible genetic factors that may modify
the penetrance of this variant.
Similar considerations may apply to the five missense
substitutions that have been described as polymorphic or
rare variants on normal Caucasian chromosomes. R75Q is
a candidate mutation but there is no evidence of a specific
association with CAVD. We have identified, in a set of
65 CF families, two proven fathers with the compound
heterozygous genotype ∆F508/R75Q and additional mu-
tations have been detected here and elsewhere in patients
with R75Q alleles (Zielenski and Tsui 1995; D. Hughes,
personal communication). However, these complex alle-
les do not explain all cases and a final classification of
R75Q requires further examination. The less frequent
F508C variant was initially reported to be benign
(Kobayashi et al. 1990) but is present in three of our
CBAVD males who are all compound heterozygous for
∆F508 and F508C with no other detected mutation (one
patient has been reported previously by Meschede et al.
1993). The missense substitutions G576A and R668C oc-
curred in cis on the same allele in two of our CBAVD
males and in three further patients with very mild CF
(data not shown); it is thus difficult to decide whether one
or both of these changes are required to predispose to-
wards mild disease (Anguiano et al. 1992; Fanen et al.
1992; Chillón et al. 1995).
Two other double mutant alleles were identified in a
CBAVD patient of Vietnamese descent. This male was
homozygous for a missense mutation, Q1352H, in exon
22 of the CFTR gene, but heterozygous for a “5T” allele
and for the novel R297W missense mutation. Missense
mutation Q1352H, located in the ABC signature of the
NBD2 of CFTR, has so far only been observed in the
heterozygous state in 1 out of 18 Japanese patients with
diffuse panbronchiolitis (K. Seyama, personal communi-
cation). The Q1352H mutation may be insufficient to
cause CBAVD but the additional occurrence of one “5T”
 371
Table 3 CFTR mutation genotypes in 106 males with CAVD
Genotype PolyT Frequency Ethnic descent Diagnosis

∆F508/R117H 9/7 21 German, Austrian 20 CBAVD, 1 CUAVD

∆F508/5T 9/5 9 German, Austrian 8 CBAVD, 1 CUAVD
∆F508/F508C 9/7 3 German CBAVD
∆F508/R347H 9/9 2 German CBAVD
∆F508/1716 G→A 9/7 2 German CBAVD
∆F508/3272-26 A→G 9/7 2 German CBAVD
∆F508/E56K 9/7 1 German CBAVD
∆F508/M265R 9/7 1 German-Portuguese CBAVD
∆F508/R334W 9/9 1 German CBAVD
∆F508/T351S 9/9 1 German CBAVD
∆F508/L375F 9/7 1 Volga German CBAVD
∆F508/G576A & R668C 9/7 1 German CBAVD
∆F508/R933S 9/7 1 German CBAVD
∆F508/L997F 9/9 1 German CBAVD
∆F508/Y1032C 9/7 1 German CBAVD
∆F508/D1152H 9/7 1 German CBAVD
∆F508/K1351E 9/7 1 German CBAVD
∆F508/D1377H 9/7 1 Portuguese CBAVD
∆F508/L1388Q 9/7 1 German CBAVD
∆F508/unknown 9/7 4 German 3 CBAVD, 1 CUAVD
5T/5T 5/5 2 German CBAVD
5T/G542X 5/9 2 German, Turkish CBAVD
5T/D58N 5/7 1 Lebanese CBAVD
5T/hL138 5/7 1 German-Polish CBAVD
5T/1078delT 5/7 1 German CBAVD
5T/R553X 5/7 1 German CBAVD
5T/2184insA 5/7 1 Turkish CBAVD
5T/D979A 5/7 1 Vietnamese CBAVD
5T/D1152H 5/7 1 Turkish CBAVD
5T/3659delC 5/7 1 German CBAVD
5T/S1235R 5/7 1 Greek CBAVD
5T/W1282X 5/7 1 German CBAVD
5T & Q1352H/
R297W & Q1352H 5/7 1 Vietnamese CBAVD
5T/unknown 5/7 1 German CBAVD
R117H/L206W 7/9 1 German CBAVD
R117H/2789+5 G→A 7/7 1 German CBAVD
R117H/unknown 7/7 1 German CBAVD
2789+5 G→A/2789+5 G→A 7/7 1 Lebanese CBAVD
2789+5 G→A/L973F 7/7 1 German CBAVD
V938G/V938G 7/7 1 Greek CBAVD
V938G/174delA 7/7 1 German CBAVD
D110H/D110H 7/7 1 Turkish CBAVD
R334L/I336K 7/7 1 German CBAVD
R347H/N1303K 9/9 1 German CBAVD
L568F/D1152H 7/7 1 Turkish CBAVD
3272-26 A→G/V1153E 7/7 1 German CBAVD
R75Q/unknown 7/7 1 German CBAVD
A120T/unknown 9/7 1 German CBAVD
1716G→A/unknown 7/7 1 German CBAVD
G576A & R668C/unknown 7/7 1 German CBAVD
2752-15 C→G/unknown 7/7 1 Iranian CBAVD
Unknown/unknown 17 German, Turkish 7 CBAVD and 1 CUAVD without observed renal
agenesis, 9 CBAVD with renal agenesis
372

Fig. 2 Spectrum of CFTR mutation genotypes in CF patients (left)
and in patients with congenital absence of the vas deferens (right).
Mutation genotypes were classified according to the nature of the
CFTR mutation (premature termination mutations, missense sub-
stitutions or splice site mutations) to allow better comparison be-
tween the two patient populations. Note that the two most common
genotype classes in CF (∆F508/∆F508 and ∆F508/termination)
were absent in the males with isolated congenital absence of the
vas deferens
allele and the R297W mutation on a homozygous Q1352H
background may then reduce CFTR function to a disease-
causing level.
In summary, whereas all these variants are candidates
for disease-associated mutations with a reduced pene-
trance, further studies are required to establish their phys-
iological significance. A firm association with CAVD has
only been established for the “5T” variant.

Table 4 Clinical symptoms in 26 CAVD males. Patients are com- pendent measurements by pilocarpine iontophoresis (40–60 mM
piled for whom sweat chloride was elevated and/or CF-like symp- borderline, > 60 mM pathological; n.d. not determined). Lung
toms were recorded. Height and weight parameters were in the function tests indicated initial pulmonary deterioration in a few
normal range, or even indicated overweight in a few individuals. cases (FEV1 forced expiratory volume in 1 s, given as percent pre-
Sweat chloride values are given as the mean of two or three inde- dicted)
Subject Age Genotype Height Weight Sweat C1– Symptoms
(years) (cm) (kg) (mM)

1 33 ∆F508/R117H 172 75 46 Dyspnoe

2 37 ∆F508/R117H 178 83 31 Nasal polyposis
3 31 ∆F508/R117H 181 91 n.d. Nasal polyposis
4 32 R117H/unknown 164 70 33 Recurrent infections
5 33 ∆F508/E56K 193 100 85 Sinusitis, recurrent bronchitis
6 31 ∆F508/M265R 192 112 59 Recurrent infections, pancreatitis
7 33 ∆F508/R334W 182 78 n.d. Recurrent infections, pneumonia
8 28 ∆F508/R347H n.d. n.d. n.d. Recurrent infections
9 32 ∆F508/F508C 192 98 32 Pneumonia
10 34 ∆F508/Y1032C n.d. n.d. n.d. Recurrent bronchitis
11 33 ∆F508/3272-26 A→G 172 82 125 Recurrent infections, maldigestion, FEVI 73%
12 28 ∆F508/unknown 185 95 n.d. Maldigestion
13 25 5T/D58N 184 99 55 –
14 34 5T/hL138 177 80 53 –
15 33 5T/1078delT 187 87 56 Recurrent bronchitis
16 31 5T/G542X 181 85 79 –
17 31 5T/2184insA n.d. n.d. 60 Borderline pancreatic sufficiency
18 31 5T/D979A n.d. n.d. 55 Recurrent infections, FEVI 76%
19 29 5T/D1152H n.d. n.d. 57 –
20 32 5T/W1282X 180 76 n.d. Recurrent infections, nasal polyposis
21 37 5T/unknown 180 74 n.d. Nasal polyposis
22 28 D110H/D110H 175 80 n.d Asthma bronchiale, obstipation
23 33 R334L/I336K 170 65 n.d. Recurrent infections, nasal polyposis, maldigestion,
salt depletion episodes
24 35 N1303K/R347H 167 77 93 –
25 30 V938G/174delA n.d. n.d. 42 –
26 29 V938G/V938G 197 115 n.d. Asthma bronchiale

Spectrum of CFTR genotypes and associated phenotypes
Some intriguing aspects of genotype-phenotype relation-
ships for CF and CAVD arise from the analysis of the
complete CFTR mutation genotypes in our CAVD co-
hort, as the genotype distribution and frequencies are
markedly different from those in CF (Table 3, Fig.2).
Whereas none of the males with isolated CAVD was ho-
mozygous for ∆F508, homozygosity was observed for
four other mutations: one Lebanese and one Turkish male
with CBAVD were homozygous for the mutations D110H
and 2789+5 G→A, respectively, both of which had previ-
ously been identified as very mild CF mutations (Dean et
al. 1990; Highsmith et al. 1997). As mentioned above, one
further male presenting with unilateral absence of the vas
deferens as the only clinical symptom was found to be ho-
mozygous for missense mutation V938G. Finally, two
CBAVD patients were homozygous for the “5T” variant,
one being compound heterozygous (TG)135T/(TG)125T
and the other being homozygous for (TG)125T. Interest-
ingly, none of the CAVD patients was compound het-
erozygous for ∆F508 and a nonsense or frameshift muta-
tion or for any two other CF mutations that had previously
been classified as “severe” with regard to pancreatic func-
tion, indicating that the correlation between mutation
genotype and CAVD phenotype is remarkably strict. By
far the most frequent CAVD genotype in our population
was the ∆F508/R117H genotype that was present in every
fifth German male presenting with CBAVD. A few other
genotypes overlapped with those previously observed in
some CF adults with mild or very mild disease, e.g. com-
pound heterozygosity for ∆F508 and mutations R334W,
R347H, 3272–26 A→G or D1152H. This partial overlap
of clinical spectra prompted us to examine retrospectively
whether additional disease symptoms were recorded or
observable in males with these CF-like mutation geno-
types. Cases with positive findings were compiled if pa-
tient history and clinical records were informative (Table
4). Sweat chloride measurements were initiated in a series
of patients and yielded borderline or even pathological
values in several cases. On closer examination, a sub-
group of males presenting with CAVD had episodes of
further clinical symptoms that most frequently involved
the upper respiratory tract (Table 4).
Discussion
CF and CAVD are such different phenotypes that they ap-
pear to be distinct clinical entities. CF is a life-threatening
disease that is usually diagnosed by pediatricians in chil-
dren during their first few years, because of gastrointesti-
nal and pulmonary manifestations and failure to thrive.
CAVD is diagnosed by urologists in adult males pre-
senting with an infertility problem; some of these patients
have additional renal malformations, which is not a symp-
tom of CF. However, despite these differences, a flurry of
recent work indicates a genetic commonality for both dis-
eases in the majority of patients. From the results of our
373
study, the type of mutation and the genotype of the CFTR
gene appear to be pivotal to the differences in the clinical
and tissue-specific expression of disease in these individ-
uals.
CAVD is a genetically heterogeneous disorder, in-
volving CFTR gene mutations and variants in over 80%
of our cases; 75% of our patients are compound heterozy-
gous for two CFTR gene mutations or variants. Nine out
of the 17 males with no CFTR mutation detected had uni-
lateral renal agenesis or malformations concomitant with
Wolffian duct abnormalities. This indicates a develop-
mental insult to the mesonephric duct before weeks 6–7 of
gestation and thus represents a distinct clinical aetiology
and perhaps the involvement of other genes (Augarten et
al. 1994; Oates and Amos 1994). However, the eight other
males of our study who had neither detectable CFTR mu-
tations nor an apparent renal maldevelopment did not ap-
pear to fall into this category. A similar proportion of 10–
30% of CBAVD males with no detectable CFTR mutation
has been noted by others (Chillón et al. 1995; Costes et al.
1995; Zielenski et al. 1995) and one of these studies did
not include patients with renal agenesis (Costes et al.
1995). This could indicate a failure to detect all CFTR
mutations within the investigated portions of the gene (al-
though it would be unusual to have so many patients un-
resolved on both alleles) or the presence of subtle forms
of kidney malformation. Alternatively, these males may
belong to a minor entity of CAVD that is neither caused
by CFTR gene mutations nor defined by mesonephric duct
maldevelopment. Further clinical and molecular studies
are required to address this issue.
Recent work on CFTR-knockout mice suggests that
CF-associated male infertility is directly related to an im-
pairment of chloride transport, the cardinal function of
CFTR (Leung et al. 1996). In this regard, it is interesting
that the vast majority of CAVD-associated mutations in
our study are missense substitutions, many of which target
transmembrane regions known to regulate chloride per-
meability. Six different amino-acid substitutions are lo-
cated within the sixth transmembrane domain and two
others flank the transmembrane helix 12. These two he-
lices appear to contribute most to the anion conductance
of CFTR (McDonough et al. 1994; Cheung and Akabas
1996). A cluster of four novel missense substitutions, in-
cluding the “ultramild” V938G mutation, target the third
intracytoplasmic loop. This portion of CFTR is thought to
be involved in channel gating (Seibert et al. 1996). Two
further CAVD missense mutations, viz. R117H and
R347H, have been thoroughly studied in vitro and both
were shown to result in a pH-sensitive decrease of chlo-
ride conductance (Sheppard et al. 1993; Tabcharani et al.
1993). We can conclude that this type of mutation, which
alters but does not abolish the CFTR channel conductiv-
ity, is particularly frequent in men with isolated CAVD.
A different class of mutation associated with CAVD
may be represented by the frequent “5T” variant, which is
thought to be a partially penetrant and “leaky” splicing
mutation (Chillón et al. 1995; Costes et al. 1995; Zielen-
ski et al. 1995). Homozygotes for “5T” have been de-
374
scribed who are phenotypically normal, although the “5T”
variant induces the skipping of the essential exon 9 in ap-
proximately 90% of their CFTR mRNA (Chu et al. 1992,
1993). Our study indicates that homozygosity for “5T”
can be sufficient to cause disease and adds further weight
to the association of the “5T” variant with CAVD in an-
other large cohort of individuals. It is noteworthy that the
“5T” variant recurs on diverse ethnic backgrounds, such
as German, Turkish, Lebanese or Vietnamese. This sug-
gests that the CFTR gene is involved in many cases of
male infertility in populations where CF is rare and ∆F508
is absent. Indeed, given their total allele frequency of
some 5% in most investigated populations, the “5T” vari-
ants collectively appear to represent the most common
disease-causing mutations of the CFTR gene worldwide.
“5T” alleles have also been noted in some CF patients
with very mild symptoms, perhaps defining the other end
of the clinical spectrum associated with “5T” (Dörk et al.
1994a; Chillón et al. 1995). Severe disease results when
additional mild mutations such as R117H occur in cis on
a “5T” allele in certain populations (Kiesewetter et al.
1993) and the joint effect of both mutations strongly sup-
ports the view of CBAVD as representing a partial form of
CF.
Partial penetrance could be a feature not only of the
“5T” variant, but also of other CFTR variants, which have
initially been described as benign polymorphisms. The
two linked missense substitutions G576A and R668C, for
example, have previously been classified as polymor-
phisms on normal chromosomes (Fanen et al. 1992), as
polymorphisms on CBAVD alleles (Osborne et al. 1993;
Culard et al. 1994) or as separate disease-causing muta-
tions in CBAVD (Anguiano et al. 1992; Chillón et al.
1995; Mercier et al. 1995). The penetrance of a CBAVD
allele probably depends on the severity of the other allele
in trans (Deltas et al. 1996). In addition, the expression of
a clinical phenotype can be influenced by modifying
genes, as has recently been observed in CF mice (Rozmahel
et al. 1996). Therefore, missense variants, such as F508C
or G576A, or splicing variants, such as 1716 G→A, de-
serve closer examination with regard to what extent they
can impair CFTR function in an epithelial tissue, such as
the vas deferens. The concept of partial penetrance may
finally explain some observations of discordant brothers
with and without CBAVD and who share the same CFTR
genotype, although these cases could be equally well ac-
commodated on the assumption of a different causative
gene (Mercier et al. 1995; Rave-Harel et al. 1995).
Striking CFTR genotypic differences are observed be-
tween our CAVD cohort and German patients with CF.
Homozygosity for ∆F508 or compound heterozygosity for
∆F508 and a nonsense or frameshift mutation account for
most cases with classic CF but are missing among the
males presenting with isolated CAVD. Previous studies
have reported few homozygous CBAVD patients for mu-
tations R117H, D1152H and “5T” (Costes et al. 1995;
Rave-Harel et al. 1995; Zielenski et al. 1995). Our analy-
sis adds mutations D110H, 2789+5 G→A and V938G to
the growing list of CFTR mutations that, in the homozy-
gous state, can result in a restricted and primarily genital
expression of disease. Compound heterozygosity of these
mutations with a “severe” CF allele, such as ∆F508, either
leads to CBAVD or extends this phenotype to a pancreatic
sufficient form of CF, which concurs with the proposed
overlap between different CFTR-related phenotypes (Es-
tivill 1996; Stern 1997). It will be interesting to determine
whether these genotype-phenotype relationships hold true
for the other congenital Wolffian duct abnormalities that
have now been associated with compound heterozygosity
for mild CFTR gene mutations, such as unilateral absence
of the vas deferens or idiopathic epididymal obstruction
(Mickle et al. 1995; Jarvi et al. 1995), and for possible fe-
male equivalents, such as hypofertility with thick cervical
mucus (Gervais et al. 1996).
The high frequency of mild or very mild CFTR muta-
tions in patients with CAVD suggests that the vas deferens
is one of the tissues most susceptible to the effect of
changes in CFTR activity. However, the most common
mild CF mutation in German CF adults with borderline
sweat chloride and pancreatic sufficiency, viz. splicing
mutation 3849+10kB C→T (Highsmith et al. 1994), has
not been found in any of our 106 CAVD males. Interest-
ingly, this mutation has repeatedly been observed in the
few fertile male CF patients (Highsmith et al. 1994; Stern
et al. 1995; Dörk and Stuhrmann 1995; Dreyfus et al.
1996). Thus, there seem to be two classes of mild CFTR
mutations in males: those that primarily target the male
genital tract (e.g. R117H, “5T”, D1152H) and those that
may leave the vas deferens patent but exert deleterious ef-
fects at a more advanced age and lead to late-onset disease
of the pulmonary tract, as is often observed for the
3849+10kb C→T mutation.
Because some 75% of the CAVD males are homozy-
gous or compound heterozygous for two CFTR muta-
tions, some of them can be expected to have elevated
sweat chloride levels or additional symptoms consistent
with a mild form of CF, as seen here. Similar observations
have been documented in a few initial studies (Costes et
al. 1995; Durieu et al. 1995; Colin et al. 1996; Dumur et
al. 1996). Whether these CAVD males should be treated
as CF patients is doubtful. Few means exist to prevent
CF-type symptoms, such as chronic infections, and it is
not known whether these males would benefit from
presymptomatic therapy. Instead, learning to cope with
“having CF” in addition to infertility may raise anxiety in
some patients or anger in others who feel healthy. Never-
theless, knowledge of their predisposition could prove
helpful, if complications occur later in life, and may guide
important clinical decisions, e.g. regarding the manage-
ment of pulmonary disease. We believe that CAVD pa-
tients with two confirmed CFTR mutations should be
counselled with all appropriate caution and should be of-
fered clinical follow-up to ascertain potential complica-
tions as soon as possible. With experience gained from fu-
ture prospective studies, mutation analysis of the CFTR
gene may become of prognostic value in estimating the
life-time risk for CF-like symptoms in men presenting
with CAVD.

Acknowledgements We cordially thank all the men with CAVD
and our CF families who agreed to participate in this study. We are
indebted to our colleagues B. Fasselt, T. Dirksen, B. Albrecht, G.
Gillessen-Kaesbach (Essen), J. Denil, S. Morlot (Hannover), L.
Bispink (Bad Münder), I. Weber, G. Utermann (Innsbruck), D.
Emmerich, B. Schmieders, G. Wolff (Freiburg), H. Schindler
(Baden-Baden), W. Weidner (Gießen), M. Claßen (Bremen), M.
Pruggmayer (Peine), S. Palm (Köln), W. Ihring (Leinefelde), H.L.
Zienau (Darmstadt), R. and D.J. Ziegler (Ottersweier), E. Schröder
(Düsseldorf), A. Freksa (Mainz), D. Wöhrle (Neu-Ulm) and H.
Theile (Leipzig) for their contribution of patients and support of
this study. Special thanks are due to Anja Schridde, Hildegard
Frye, Andrea Korte, Andrea Trefilov, Kathrin Rommel, Wolfram
Antonin, Alexander Stegh, Britta Skawran, Margit Ebhardt and
Anneke Loos for their experimental contributions to haplotype
analysis and mutation screening, and to Christoph Lanfer for his
help with the manuscript. We gratefully acknowledge the collabo-
ration with members of the Cystic Fibrosis Genetic Analysis Con-
sortium (directed by Prof. Lap-Chee Tsui, Toronto). Part of this
work was supported by a grant from the Deutsche Forschungsge-
meinschaft.
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