Download Detection of cystic fibrosis transmembrane conductance regulator

Human Reproduction Vol.22, No.5 pp. 1285–1291, 2007
doi:10.1093/humrep/dem024
Advance Access publication February 28, 2007
Detection of cystic fibrosis transmembrane conductance
regulator (CFTR) gene rearrangements enriches the
mutation spectrum in congenital bilateral absence of the
vas deferens and impacts on genetic counselling
Ilham Ratbi1, Marie Legendre1, Florence Niel1, Josiane Martin1, Jean-Claude Soufir2, Vincent
Izard3, Bruno Costes1, Catherine Costa1, Michel Goossens1 and Emmanuelle Girodon1,4
1
Service de Biochimie et Génétique Moléculaire, AP-HP et INSERM U841, IMRB, eq 21, Hôpital Henri-Mondor, Créteil, France,
Service de Biologie de la Reproduction et du développement and 3Service d’Urologie, AP-HP, Hôpital de Bicêtre, Le Kremlin-Bicêtre,
France
2
4
To whom correspondence should be addressed at: Service de Biochimie et Génétique Moléculaire, AP-HP et INSERM U654, Hôpital
Henri-Mondor, 94010 Créteil, France. Tel.: þ33 1 49 81 28 57; Fax: þ33 1 49 81 28 42; E-mail: [email protected]
BACKGROUND: Mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene have been
widely detected in infertile men with congenital bilateral absence of the vas deferens (CBAVD). Despite extensive
analysis of the CFTR gene using varied screening methods, a number of cases remain unsolved and could be attributable to the presence of large gene rearrangements, as recently shown for CF patients. METHODS: We carried out a
complete CFTR gene study in a group of 222 CBAVD patients with strict diagnosis criteria and without renal anomaly,
and searched for rearrangements using a semi-quantitative assay in a subgroup of 61 patients. RESULTS: The overall
mutation detection rate was 87.8%, and 82% of patients carried two mutations. Ten out of the 99 different mutations
accounted for 74.6% of identified alleles. Four large rearrangements were found in patients who already carried a
mild mutation: two known partial deletions (exons 17a to 18 and 22 to 23), a complete deletion and a new partial
duplication (exons 11 to 13). The rearrangements accounted for 7% of the previously unknown alleles and 1% of
all identified alleles. CONCLUSIONS: Screening for rearrangements should be part of comprehensive CFTR gene
studies in CBAVD patients and may have impacts on genetic counselling for the patients and their families.
Key words: congenital bilateral absence of the vas deferens/cystic fibrosis transmembrane conductance regulator mutations/deletion/
duplication/gene rearrangement
Introduction
Cystic fibrosis (CF) (MIM#219700) is one of the most common
autosomal recessive diseases in Caucasians (Welsh et al.,
2001). It affects about one neonate in 2500 and approximately
one individual in 25 is a heterozygote, with marked regional
variations (Estivill et al., 1997; Bobadilla et al., 2002)
(www.genet.sickkids.on.ca/cftr). The disease is caused by
mutations in the CF transmembrane conductance regulator
(CFTR or ABCC7) gene (MIM*602421). Up to 1500 CFTR
sequence variations have been described so far, and these are
also involved in a broad spectrum of phenotypes, including
male infertility by congenital bilateral absence of the vas
deferens (CBAVD) (Dumur et al., 1990; Chillon et al.,
1995a; Costes et al., 1995; Bienvenu et al., 1997; Dörk
et al., 1997), disseminated bronchiectasis (Pignatti et al.,
1995; Girodon et al., 1997) and chronic pancreatitis (Cohn
et al., 1998; Sharer et al., 1998). Since the first identification
of CFTR mutations in CBAVD in the 1990s (Dumur et al.,
1990; Anguiano et al., 1992), extensive studies have shown
that 45– 80% of CBAVD patients carry two CFTR mutations,
mostly in compound heterozygosity, these mutations accounting for 60– 86% of disease alleles (Chillon et al., 1995a;
Costes et al., 1995; Dörk et al., 1997; Casals et al., 2000;
Claustres et al., 2000; Grangeia et al., 2004). All mutation
types can be encountered, with effects ranging from mild to
severe. The most frequent are the main CF-associated defect,
F508del (21 – 40% of alleles in CBAVD patients, depending
on studies), and two mild defects, the T(5) variant of the
polypyrimidin tract of the intron 8 (IVS8) acceptor splice site
(19 – 37%) and R117H (3 – 14%). Unlike genotypes found in
CF patients, the majority of those identified in CBAVD patients
combine either a severe CF mutation and a mild mutation or
# The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
All rights reserved. For Permissions, please email: [email protected]
1285
I.Ratbi et al.
two mutations with a mild or variable effect (Claustres et al.,
2000; Stuhrmann and Dörk, 2000). Nonetheless, in 20– 55%
of patients, only one or no CFTR mutation could be found.
Unidentified CFTR mutations may lie in introns or in
regulatory regions, which are not routinely investigated, or
may correspond to gene rearrangements such as large deletions
at the heterozygous state which escape detection using current
PCR-based techniques. Recent sudies on CF patients have
shown that 14 –23% of the alleles that remain unknown after
extensive analysis of the CFTR gene using scanning techniques
consist of large deletions, insertions and duplications (Audrézet
et al., 2004; Niel et al., 2004; Bombieri et al., 2005; Ferec
et al., 2006; Hantash et al., 2006b). Only one study has reported
on screening for rearrangements in CBAVD patients: of 48
patients with one or no identified CFTR mutation, one, heterozygous for the IVS8(T)5 variant, was found to carry a deletion
that removed exons 22 to 24 (Hantash et al., 2006a).
Investigation for the presence of such large rearrangements
in 61 CBAVD patients using a semi-quantitative fluorescent
PCR assay developed in our laboratory (Niel et al., 2004)
led us to identify four rearrangements. Here we discuss these
findings together with the results of comprehensive CFTR
gene studies in a group of 222 CBAVD patients.
Materials and methods
Patients
A group of 222 unrelated infertile patients with isolated CBAVD and
from diverse ethnic and geographic origins were investigated. They
were recruited between 1992 and 2006 from different French clinics.
Preliminary data have already been reported for a number of them
(Costes et al., 1995; Claustres et al., 2000). Diagnosis of CBAVD was
assessed by the clinicians on the following criteria: (i) absence of palpable vas deferens at physical examination; (ii) azoospermia with low
seminal fluid volume (,2.0 ml); (iii) typical biochemical changes:
pH , 7.2, absence or decreased level of fructose and alpha1–4 glucosidase (markers of seminal vesicles and epididymal secretions, respectively) and (iv) anomalies at transrectal ultrasound: absence of the vas
deferens and of the distal part of the epididymis, globus major and different degrees of hypoplasia of the seminal vesicles. These anomalies were
also confirmed at surgical sperm retrieval whenever patients and their
partners requested assisted reproduction. Patients with renal ultrasound
anomalies were not included, as a number of studies failed to show a significantly increased frequency of CFTR mutations in patients with
CAVD and renal ultrasound anomalies, which may thus represent a distinct clinical entity (Stuhrmann and Dörk, 2000). Plasma FSH, LH and
testosterone levels were normal in all subjects. No other CF symptom
was evidenced in the patients from questioning or clinical examination
at referral. However, follow-up data were not always available and
occurrence of late CF symptoms cannot be ruled out. Sweat chloride
tests were not performed systematically.
According to French legislation and the recommendations of the
local ethics committee, written consent to the genetic study were
obtained from all patients.
CFTR gene analysis
Genomic DNAs were extracted from whole blood samples collected
on EDTA using varied protocols, mostly a phenol chloroform reference protocol or a commercial kit (Nucleon, BACC3, Amersham Biosciences, Saclay, France). DNA concentration and quality were
determined for each sample.
1286
Screening for point mutations
CFTR gene analyses included: (i) screening for frequent mutations by
means of diverse commercial assays; (ii) scanning of the 27 exons and
their boundaries using denaturing gradient gel electrophoresis
(DGGE) (Fanen et al., 1992; Costes et al., 1993) or denaturing highperformance liquid phase chromatography (Le Marechal et al., 2001),
followed by sequencing to characterize the variants and (iii) screening
for the intronic splicing 1811 þ 1.6 kbA.G mutation (Chillon et al.,
1995b). The (TG)m(T)n haplotype of the IVS8 acceptor splice site was
analysed by combination of DGGE and DNA sequencing (Costes
et al., 1995).
Mutation names were those reported to the international consortium
mutation database (www.genet.sickkids.on.ca/cftr). For nucleotide
changes, the A of the ATG translation start codon was numbered as
þ133, in accordance with the current CFTR gene numbering based
on cDNA sequence (GenBank NM_000492.2).
Screening for large CFTR rearrangements
We used a semi-quantitative fluorescent multiplex PCR (QFM-PCR)
assay recently developed in our laboratory, which enabled detection
of rearrangements in 20% of the previously unidentified alleles in
CF patients (Niel et al., 2004). Briefly, the assay is based on comparison of fluorescent patterns of multiplex PCR fragments obtained from
patients and control samples, the amplification being stopped in the
exponential phase. This procedure allows the detection of heterozygous deletions (2-fold reduction in fluorescence intensity) and
heterozygous duplications (1.5-fold increase).
All abnormal patterns were confirmed by at least one other QFMPCR experiment, preferably using different sets of primers. When a
pattern was compatible with an already described deletion, identification was performed using specific primers [available upon request
and adapted from Lerer et al. (1999) and Audrézet et al. (2004)].
Results
First screening for point mutations in the group of 222 CBAVD
patients led to the identification of 95 different mutations, scattered over the whole gene, of all types and with effects ranging
from mild to severe. Apart from the IVS8(T)5 variant,
sequence variations which are reported as neutral because of
segregation analysis in CF families or their frequency in the
general population (Bombieri et al., 2000) (such as
356G.A (R75Q), 1540A.G (M470V) or 1716G.A) were
not considered as disease-causing when found in isolation.
CFTR mutations were detected in 387 out of 444 alleles
(87.2%), most of them being previously described in patients
with CF of varying severity, CBAVD or other CFTR diseases:
45% of identified alleles consisted of severe CF mutations (e.g.
F508del, W1282X, 2183AA.G); 13.8% of mild or variable
CF mutations (e.g. L206W, 3272-26A.G, R117H, D1152H);
36.7% of mild CFTR defects which are currently not considered CF-causing (e.g. IVS8(T)5, Q1352H, the complex
alleles [D443Y;G576A;R668C] and [R74W;D1270N]) and
4.5% of rare missense mutations whose effect is difficult
to predict (e.g. A959V, G1069R, V1153E). Twenty-nine
mutations were found more than once and 10 were found at a
frequency above 1% among the identified mutations (Table I).
Among the 112 IVS8(T)5 identified alleles, all in the
heterozygous state, 16 (14.3%) were (TG)13(T)5, 82 (73.2%)
were (TG)12(T)5 and 14 (12.5%) were (TG)11(T)5. In five
out of the 14 patients carrying the (TG)11(T)5 allele, V562I
CFTR gene rearrangements in CBAVD
Table I. Frequent cystic fibrosis transmembrane conductance regulator
(CFTR) defects found in congenital bilateral absence of the vas deferens
(CBAVD) patients (above 1% among the identified alleles)
Table II. CFTR genotypes of CBAVD patients investigated for the presence
of large CFTR gene rearrangements
Genotypes
Mutation
No. of alleles
% of the 390
identified alleles
F508dela
IVS8(T)5a,b
(TG)12(T)5
(TG)13(T)5
(TG)11(T)5b
R117Ha
R668C
[D443Y;G576A;R668C]
[G576A;R668C]
R668C
L206W
D1152H
W1282Xa
[V562I;(TG)11(T)5]
[R74W;D1270 N]
[R74W;D1270 N]
[R74W;V201M;D1270 N]
Q1352H(G . C)
119
107
82
16
9
25
9
6
2
1
7
6
5
5
4
3
1
4
30.5
27.4
Total
291
74.6
6.4
2.3
1.8
1.5
1.3
1.3
1.0
1.0
a
Mutations that are part of commercial kit panels.
Do not include the [V562I;(TG)11(T)5] complex alleles that are indicated
below. Details regarding possible complex alleles are indicated in italics.
b
was also identified, probably in cis as documented in two of
these cases and in other patients who do not have CBAVD
(Girodon et al., unpublished data).
With a view to segregation analysis, family studies were
performed, especially when rare missense mutations were
detected. In several instances, however, patients were reluctant
to disclose their medical status to their relatives and opposed
our request to perform genetic analysis in their family.
Compound heterozygous genotypes were suggested, based
upon literature data and linkage of F508del with the neutral
IVS8(TG)10(T)9 variant.
According to the results of the first screening, the patients
fell into three groups: 179 (80.6%) had two CFTR defects,
including the IVS8(T)5 variant, 29 (13.1%) had one detectable
CFTR mutation and in the 14 remaining patients (6.3%), no
CFTR defect was detected.
Sixty-one patients were then investigated for the presence of
large rearrangements by QFM-PCR (Table II): (i) the
14 patients with no identified mutation (28 unidentified
alleles); (ii) the 29 patients with one identified mutation, of
whom 16 carried a mild or variable mutation, 10 carried a
severe CF mutation and three carried a rare missense mutation
whose effect was difficult to predict (29 unidentified alleles)
and (iii) 15 patients with two mutations of mild or unknown
effect and three who were apparently homozygous for a mild
mutation (0 – 18 unidentified alleles). Three heterozygous
deletions (2-fold reduction in fluorescence intensity) and one
duplication (1.5-fold increase) were detected in four patients
who already carried a mild mutation (Tables II and III).
No rearrangement was found in the patients with no identified
point mutation. Patient #1, heterozygous for (TG)12(T)5,
exhibited a deletion of exons 17a to 18; by the use of specific
primers, the presence of the known 3120 þ 1.6 kbdel8.6 kb
No. of
patients
No. of
unidentified
alleles
No. of
rearrangements
No CFTR mutation [?] þ [?]
14
One CFTR mutation
29
[F508del] þ [?]
9
[711 þ 1G . T] þ [?]
1
[(TG)13(T)5] þ [?]
1
[(TG)12(T)5] þ [?]
5
[(TG)11(T)5] þ [?]
1
[(TG)11(T)5;V562I] þ [ ?]
2
[D443Y;G576A;R668C] þ [?]
1
[G576A;R668C] þ [?]
1
[R668C] þ [?]
1
[Q1352H(G . C)] þ [?]
2
[R170H] þ [?]
1
[V938G] þ [?]
1
[A959V] þ [?]
1
[G1069R] þ [?]
1
[V1153E] þ [?]
1
Two CFTR mutations
15
[R117H] þ [(TG)13(T)5]
1
[R117H] þ [(TG)12(T)5]
1
[R117H] þ [(TG)11(T)5]
1
[R117H] þ [M952I]
1
[D1152H] þ [(TG)12(T)5]
2
[D1152H] þ [Y1032C]
1
[(TG)11(T)5;V562I] þ [L997F]
1
[(TG)11(T)5;V562I] þ [S977F]
1
1
[E1473X] þ [(TG)13(T)5]
[V232D] þ [(TG)12(T)5]
1
[R334W] þ [(TG)12(T)5]
1
[G622D] þ [(TG)12(T)5]
1
[3272-26A . G] þ [(TG)11(T)5] 1
[Q1352H(G . C)] þ [I556V]
1
Apparent homozygosity
3
[R117H] þ [R117H]
1
[D110H] þ [D110H]
1
[R74W;D1270 N] þ
1
[R74W;D1270 N]
28
29
0
3
Total
61
2
1
0– 15
0
0– 3
1
1
57–75
4
(or CFTRdele17a_18) (Lerer et al., 1999) was confirmed.
Patient #2, heterozygous for the mild V938G missense
mutation, had a deletion of exons 22 and 23, for which sequencing analysis showed the same breakpoints as those previously
described (Audrézet et al., 2004). Patient #3 had already been
referred to in Niel et al. (2004): he was apparently homozygous
for R117H with IVS8(T)7 in cis and was found to carry a
complete deletion of the CFTR gene, inherited from his
mother. Patient #4, heterozygous for (TG)12(T)5, exhibited a
duplication pattern involving exons 11 to 13. The presence of
the duplication was confirmed upon four independent
QFM-PCR experiments. The use of different sets of primers
targeted on normal and duplicated exons showed that the
duplication may not affect the whole exon 13 (Figure 1).
Rearrangements, found in 6.9% of 58 unknown alleles
(57 alleles from the first two groups of patients and one
allele from the patient who was apparently homozygous for
R117H), thus accounted for 0.9% of CBAVD alleles and 1%
of the identified alleles. The QFM-PCR assay was sensitive
enough to detect a number of microdeletions/insertions
within exons, which modified fragment size, such as
1287
I.Ratbi et al.
Table III. Phenotype and genotype data of patients carrying CFTR rearrangements
Patient Current age (years) Origin
1
2
3
4
45
33
47
34
Syria
France/Southern Italy
France
Morocco
Allele 1
(TG)12(T)5
V938G
R117H
(TG)12(T)5
Allele2
Reference
Simple name
Extent
CFTRdele17a_18
CFTRdele22_23
CFTRdele1_24
CFTRdup11_13
8.6 kb deletion
1.5 kb deletion
3 Mb deletion
Unknown (4.9– 35.2 kb duplication)
Lerer et al. (1999)
Audrézet et al. (2004)
This study and Niel et al. (2004)
This study
Figure 1. Electropherogram from a semi-quantitative fluorescent multiplex (QFM) PCR experiment in patient #4 (blue), carrying a duplication
involving exon 12 and part of exon 13 at the heterozygous state (indicated by arrows), superimposed with that of a normal control (red). The
duplication is evidenced by a 1.5-fold increase in fluorescence intensity in the patient as compared to the normal control. The 416 bp 13(1)
fragment involving the first half of exon 13 is duplicated whereas the 294 bp 13(2) fragment corresponding to the end of exon 13 is not. This
indicates that the 30 breakpoint of the duplication is located next to the 30 end of exon 13. The duplication also involves exon 11 which is analysed
in another QFM-PCR experiment (not shown). The x-axis represents the computed length of the PCR products in base pairs as determined using an
internal lane standard (indicated in green). The y-axis shows fluorescent intensities in arbitrary units. Normalization of the profiles was performed
using F9 (chromosome X) or DSCR1 (chromosome 21) reference genes (Niel et al., 2004).
F508del, 2221dupA, as well as variants at the IVS8(TG)m(T)n
polymorphic site. Overall, the complete screening has enabled
the identification of 87.8% of CBAVD alleles.
Discussion
Screening for large CFTR rearrangements enriches the
mutational spectrum in CBAVD
Four large CFTR gene rearrangements were identified in four
of 61 CBAVD patients investigated (Tables II and III). We
have included all the patients with one or no detected mutation
after complete screening for point mutations (57 unidentified
alleles), as well as patients with two putative allelic mutations,
because their effect may not be deleterious enough to account
for the disease or because homozygous status could not be confirmed by family analysis (0 – 18 unidentified alleles). The four
rearrangements accounted for nearly 7% of the previously unidentified alleles. This proportion is lower than that found in
populations of CF patients, where 14– 23% of unidentified
alleles turned out to be large rearrangements (Audrézet et al.,
2004; Niel et al., 2004; Bombieri et al., 2005; Ferec et al.,
2006; Hantash et al., 2006b). This can be partly explained by
the fact that patients with isolated CBAVD usually carry
mutations of a wide range of effects but not two severe
mutations. Therefore, CFTR rearrangements, that had been
considered null mutations, are expected to be present at most
in one allele in CBAVD patients, in trans with a mild mutation,
1288
as was observed in the four cases of the present study. In other
respects, the proportion of 7% is much higher than that found
by Hantash in a recent study of 48 CBAVD patients
(Hantash et al., 2006a): a deletion, removing exons 22 to 24,
was found in one patient, which accounted for 1.4% of
unidentified alleles. This discrepancy could be attributed to a
difference in patients’ origin or to less stringent inclusion
criteria.
Among the four rearrangements identified in our study, two
had already been described: CFTRdele17a_18 (Lerer et al.,
1999) and CFTRdele22_23 (Audrézet et al., 2004). CFTRdele17a_18 is one of the first reported CFTR gene deletions
and was found to account for 13% of CF chromosomes in
Israeli Arab patients (Laufer-Cahana et al., 1999). It was
subsequently identified in other groups of CF patients (Niel
et al., 2004; Hantash et al., 2006b). In keeping with these
data, we found it in a patient from Syria. CFTRdele22_23,
initially reported in a French patient (Audrézet et al., 2004),
was also further identified in other CF patients originating
from Southern Europe (Audrézet M.P., personal communication). We found it in a patient of Italian descent. The availability of multiplex semi-quantitative PCR assays applied to
the CFTR gene (Audrézet et al., 2004; Niel et al., 2004;
Chevalier-Porst et al., 2005; Hantash et al., 2006b) has resulted
in an increasing description of large CFTR rearrangements
(www.genet.sickkids.on.ca/cftr). Like point mutations, some
appear quite frequent, especially in particular populations, as
CFTR gene rearrangements in CBAVD
is the case for CFTRdele2,3(21 kb) in Slavic patients (Dörk
et al., 2000), CFTRdele17a_18 and CFTRdele3_10,14b_16 in
Arab patients (Lerer et al., 1999; Niel et al., 2006) or CFTRdele17a_17b in Eastern France (Niel et al., 2004). This observation further underlines the need to take into account
patients’ origin as regards genetic counselling.
A complete CFTR gene deletion was found in one patient who
had previously been referred to in Niel et al. (2004) and who was
apparently homozygous for R117H. The patient was further
investigated for the presence of mild CF symptoms but none
was found. Characterization of the breakpoints has shown that
the deletion actually extends over 3 Mb of chromosome 7 and
removes other genes (Niel et al., manuscript in preparation).
This finding highlights the importance of looking for rearrangements in apparently homozygous patients, especially when the
genotype may not explain the disease phenotype.
The duplication involving exons 11 to 13 was found in a
patient from Morocco, a country where very few data are
available on CF and related diseases. Although duplications
are more difficult to detect than deletions, because they result
in 1.5-fold increase in fluorescence intensity, when compared
with 2-fold reduction in case of a deletion, the duplication
pattern was clear from the different experiments performed at
separate times. The use of distinct pairs of primers for the
large exon 13 allowed to show that the 30 breakpoint of the
duplication is actually located next to the end of exon 13.
Although the position and orientation of the duplication have
not been determined yet, we postulate that the rearrangement
is located inside the CFTR gene and interferes with the transcription or translation process, thus resulting in a null mutation.
Comprehensive mutation and genotype spectrum in CBAVD
Overall, with comprehensive CFTR gene study, 390 out of 444
alleles were identified, which corresponds to a mutation detection rate of 87.8%. This rate is somewhat higher than those
described elsewhere for large CBAVD groups from various
European origins (Chillon et al., 1995a; Dörk et al., 1997;
Casals et al., 2000; Claustres et al., 2000), probably because
only CBAVD patients with documented semen biochemical
markers and transrectal but no renal ultrasound anomaly
were selected in our series. This is also in keeping with the
low proportion of patients who carry no CFTR mutation. Of
the 222 patients, 82.0% had two CFTR mutations, 11.7% had
one identified CFTR mutation and only 6.3% had no detected
mutation.
A total of 99 different molecular anomalies were found,
which have been described in patients with a broad range of
phenotypes, from classical CF to CBAVD. Most of them
were found only once. Among the most frequent defects, 10
were observed at a frequency above 1% and their cumulated
frequency reached 74.6% among the identified mutations
(Table I). This mutation spectrum is similar to those already
described in other European populations (Chillon et al.,
1995a; Dörk et al., 1997; Claustres et al., 2000).
Beside F508del, the IVS8(T)5 variant was the second most
frequent mutation encountered in the present study. It is considered as a mild CFTR defect whose disease penetrance
increases with the adjacent (TG)m length (Cuppens et al.,
1998; Groman et al., 2004). (TG)11(T)5 penetrance reaching
only 11% (Groman et al., 2004), we systematically investigated the whole CFTR gene to look for other cis mutations in
the 14 patients carrying this variant and we found V562I in
five of them. Three of the five carried a mutation on the
other chromosome: L997F, S977F and W1282X. Although
V562I has been considered a severe CF mutation, a series of
arguments question its severe deleterious effect: its presence
in trans of the severe W1282X mutation, the case of a V562I
homozygous CF patient who carried in cis the frameshift
2347delG (Girodon et al., unpublished data), and the fact
that residue V562 is not conserved in other proteins containing
the ATP-binding cassette motif. We suggest reconsidering
V562I as a mild and CBAVD-associated mutation.
CBAVD genotypes were varied, the most frequent combined
F508del either with the IVS8(T)5 variant (28.0% of 222) or
with R117H (6.3%). They were also heterogeneous in their
expected effect, as previously documented (Lissens et al.,
1996; Claustres et al., 2000; Stuhrmann and Dörk, 2000). No
patient carried two severe CF mutations. Some genotypes
had already been described in patients with moderate or late
CF, such as those combining F508del with L206W, D1152H,
3272-26A.G or 2789 þ 5G.A. These patients might
develop other symptoms related to a mild form of CF, and
thus need long-term follow-up. On the other hand, these
mild, possibly variable CF mutations were found in our
CBAVD population in combination with milder, non
CF-causing defects, such as the IVS8(T)5 variant. Moreover,
genotypes combining two mild alleles were found, such as
[R117H] þ [(TG)13(T)5], [(TG)11(T)5;V562I] þ [L997F] or
homozygosity for [R74W;D1270N]. Patients carrying such
genotypes were thus screened for rearrangements but all
tested negative. In these cases, however, as well as in those
with only one or no detectable CFTR mutation, we cannot
rule out the presence of mutations undetected with the
methods used or because they lie inside introns or regulatory
regions. In other respects, the proportion of cases without a
detectable mutation after extensive gene screening by classical
techniques and QFM-PCR (14 of 222) is higher than expected
using calculation of genotype frequencies from allele frequencies according to Hardy – Weinberg equilibrium, and further
strengthens the hypothesis that a number of these cases
are not related to CFTR mutations (Mercier et al., 1995; RaveHarel et al., 1995). Other factors might be responsible for
CBAVD in these 14 patients and in others with one CFTR
mutation as well or, even, in patients with two mild mutations,
potentially in a multifactorial context.
Revised CFTR molecular strategy and implications
for genetic counselling
The results presented here highlight the importance of carrying
out a complete CFTR gene analysis, including screening for
rearrangements in CBAVD patients with strict diagnosis criteria, as regards diagnostic and genetic counselling matters.
Given the varied CBAVD genotype combinations and severities, molecular characterization of both CFTR alleles is of
great concern for couples who request assisted reproduction.
Strategy for identification of CFTR defects would be a
1289
I.Ratbi et al.
Table IV. Strategy for identification of CFTR mutations in our group of 222 CBAVD patients
Strategy
No. of alleles
Cumulative detection
rate among 444
alleles (%)
Cumulative detection
rate among 390
identified alleles (%)
Search for 30 frequent CF mutations and the IVS8(T)5 variant
Search for 6 other frequent defects: [R668C;G576A;D443Y],
L206W, D1152H, V562I, [R74W;D1270 N], Q1352Ha
Extensive analysis of the 27 exons to search for other point
mutations
Screening for rearrangements
Total
280
30
63.1
69.8
71.8
79.5
76
86.9
99.0
4
390
87.8
100
a
Q1352H was found in Asian patients.
multistep process, implying collaboration networks between
routine and specialized laboratories where scanning tools and
screening for rearrangements are available. The strategy
would combine (Table IV): (i) the search for frequent
mutations using commercial kits; (ii) complementary screening
for the other six frequent CBAVD defects; (iii) extensive
analysis of the 27 exons to search for other point mutations
and (iv) screening for rearrangements by QFM-PCR. The
first two steps, which may be available in routine CF laboratories, would have enabled detection of almost 80% of the
390 identified alleles in our CBAVD population. In other
respects, screening for large deletions that are frequent in particular populations could also be performed in routine laboratories using specific and simple tools, as for the Slavic
CFTRdele2,3 (Dörk et al., 2000) or CFTRdele17a_18 (Lerer
et al., 1999).
Confirmation of compound heterozygous status in CBAVD
patients by studying the parents could be crucial and the
parents should therefore be referred for genetic counselling.
Moreover, whenever assisted reproduction is requested,
CBAVD patients’ partners should be screened for at least frequent CF mutations, and their geographical or ethnic origin
taken into account. In cases where a CF mutation is identified
in the partner, the couples have a high risk of having a CF child,
depending on the patient’s genotype, and may request prenatal
or preimplantation diagnosis. As an illustration, patient #1’s
partner, a Bosnian Muslim woman, was found to carry
2184insA, a CF mutation that is not included in commercial
kit panels. The couple therefore ran a 25% risk of having a
CF child but only obtained this information 10 years after the
first referral because the large duplication was identified
recently and only carriage for IVS8(T)5 was known for the
patient. CBAVD patients should also be informed that their
relatives may be CF carriers and could benefit from genetic
counselling. Again, knowledge of the actual mutation status
greatly facilitates cascade screening in relatives.
Acknowledgements
The authors are indebted to the clinicians who referred the patients:
Dr M. Albert, Poissy; Dr E. Amar, Neuilly; Dr F. Fellmann, Besançon;
Dr J. Flori, Strasbourg; Dr E. Gautier, Neuilly; Dr E. Ginglinger,
Mulhouse; Dr F. Girard-Lemaire, Strasbourg; Dr V. Izard, Le
Kremlin-Bicêtre; Pr P. Jonveaux, Nancy; Dr J. Lespinasse, Chambéry;
Dr M.O. Peter, Mulhouse; Dr B. Simon-Bouy, Versailles; Dr J.C.
Soufir, Le Kremlin-Bicêtre; Pr A. Toutain, Tours. Annick Lefloch,
1290
Brigitte Boissier, Rachel Medina and Fabienne Rossi are thanked
for valuable technical assistance. David Kerridge is acknowledged
for re-reading the manuscript.
References
Anguiano A, Oates RD, Amos JA, Dean M, Gerrard B, Stewart C, Maher TA,
White MB and Milunsky A (1992) Congenital bilateral absence of the vas
deferens—a primarily genital form of cystic fibrosis. JAMA 267,1794–
1797.
Audrézet MP, Chen JM, Raguenes O, Chuzhanova N, Giteau K, Le Marechal
C, Quere I, Cooper DN and Ferec C (2004) Genomic rearrangements in the
CFTR gene: extensive allelic heterogeneity and diverse mutational
mechanisms. Hum Mutat 23,343–357.
Bienvenu T, Adjiman M, Thiounn N, Jeanpierre M, Hubert D, Lepercq J,
Francoual C, Wolf J-P, Izard V, Jouannet P, et al. (1997) Molecular
diagnosis of congenital bilateral absence of the vas deferens: analyses of
the CFTR gene in 64 french patients. Ann Genet 40,5–9.
Bobadilla JL, Macek M, Jr, Fine JP and Farrell PM (2002) Cystic fibrosis: a
worldwide analysis of CFTR mutations—correlation with incidence data
and application to screening. Hum Mutat 19,575–606.
Bombieri C, Bonizzato A, Castellani C, Assael BM and Pignatti PF (2005)
Frequency of large CFTR gene rearrangements in Italian CF patients.
Eur J Hum Genet 13,687– 689.
Bombieri C, Giorgi S, Carles S, de Cid R, Belpinati F, Tandoi C, Pallares-Ruiz
N, Lazaro C, Ciminelli BM, Romey MC, et al. (2000) A new approach for
identifying non-pathogenic mutations. An analysis of the cystic fibrosis
transmembrane regulator gene in normal individuals. Hum Genet
106,172–178.
Casals T, Bassas L, Egozcue S, Ramos MD, Gimenez J, Segura A, Garcia F,
Carrera M, Larriba S, Sarquella J, et al. (2000) Heterogeneity for
mutations in the CFTR gene and clinical correlations in patients with
congenital absence of the vas deferens. Hum Reprod 15,1476– 1483.
Chevalier-Porst F, Souche G and Bozon D (2005) Identification and
characterization of three large deletions and a deletion/polymorphism in
the CFTR gene. Hum Mutat 25,504.
Chillon M, Casals T, Mercier B, Brassas L, Lissens W, Silber S, Romey M,
Ruiz-Romero J, Verlingue C, Claustres M, et al. (1995a) Mutations in the
cystic fibrosis gene in patients with congenital absence of the vas deferens.
N Engl J Med 332(22),1475–1480.
Chillon M, Dörk T, Casals T, Gimenez J, Fonknechten N, Will K, Ramos D,
Nunes V and Estivill X (1995b) A novel donor splice site in intron 11 of
the CFTR gene created by mutation 1811 þ 1.6kbA-.G produces a new
exon: high frequency in Spanish cystic fibrosis chromosomes and
association with severe phenotype. Am J Hum Genet 56,623–629.
Claustres M, Guittard C, Bozon D, Chevallier F, Verlingue C, Férec C, Girodon
E, Cazeneuve C, Bienvenu T, Lalau G, et al. (2000) Spectrum of CFTR
mutations in cystic fibrosis and in congenital absence of the vas deferens
in France. Hum Mutat 16,143– 156.
Cohn JA, Friedman KJ, Noone PG, Knowles MR, Silverman LM and Jowel PS
(1998) Relation between mutations of the cystic fibrosis gene and idiopathic
pancreatitis. N Engl J Med 339,653–658.
Costes B, Fanen P, Goossens M and Ghanem N (1993) A rapid, efficient and
sensitive assay for simultaneous analysis of multiple cystic fibrosis
mutations. Hum Mutat 2,185–191.
CFTR gene rearrangements in CBAVD
Costes B, Girodon E, Ghanem N, Flori E, Jardin A, Soufir JC and Goossens M
(1995) Frequent occurrence of the CFTR intron 8 (TG)n5T allele in men with
congenital bilateral absence of the vas deferens. Eur J Hum Genet 3,285–293.
Cuppens H, Lin W, Jaspers M, Costes B, Teng H, Vankeerberghen A, Jorissen
M, Droogmans G, Reynaert I, Goossens M, et al. (1998) Polyvariant mutant
cystic fibrosis trensmembrane conductance regulator genes. J Clin Invest
101,487–496.
Dörk T, Dworniczak B, Aulehla-Scholz C, Wieczorek D, Bohm I, Mayerova A,
Seydewitz HH, Nieschlag E, Meschede D, Horst J, et al. (1997) Distinct
spectrum of CFTR gene mutations in congenital absence of vas deferens.
Hum Genet 100,365–377.
Dörk T, Macek M, Jr, Mekus F, Tummler B, Tzountzouris J, Casals T,
Krebsova A, Koudova M, Sakmaryova I, Macek M, Sr, et al. (2000)
Characterisation of a novel 21-kb deletion, CFTRdele2,3(21 kb), in the
CFTR gene: a cystic fibrosis mutation of Slavic origin common in Central
and East Europe. Hum Genet 106,259– 268.
Dumur V, Gervais R, Rigot JM, Lafitte JJ, Manouvrier S, Biserte J, Mazeman
M and Roussel P (1990) Abnormal distribution of DF508 allele in
azoospermic men with congenital aplasia of the epidymis and vas
deferens. Lancet 336,512.
Estivill X, Bancells C, Ramos C, and Consortium TBCMA (1997) Geographic
distribution and regional origin of 272 cystic fibrosis mutations in European
populations. Hum Mutat 10(2),135 –154.
Fanen P, Ghanem N, Vidaud M, Besmond C, Martin J, Costes B, Plassa F and
Goossens M (1992) Molecular characterization of cystic fibrosis: 16 novel
mutations identified by analysis of the whole cystic fibrosis
transmembrane conductance regulator (CFTR) coding regions and splice
site junctions. Genomics 3,770– 776.
Ferec C, Casals T, Chuzhanova N, Macek MJ, Bienvenu T, Holubova A,
King C, McDevitt T, Castellani C, Farrell PM, et al. (2006) Gross
genomic rearrangements involving deletions in the CFTR gene:
characterization of six new events from a large cohort of hitherto
unidentified cystic fibrosis chromosomes and meta-analysis of the
underlying mechanisms. Eur J Hum Genet 14,567– 576.
Girodon E, Cazeneuve C, Lebargy F, Chinet T, Costes B, Ghanem N, Martin J,
Lemay S, Scheid P, Housset B, et al. (1997) CFTR gene mutations in adults
with disseminated bronchiectasis. Eur J Hum Genet 5,149–155.
Grangeia A, Niel F, Carvalho F, Fernandes S, Ardalan A, Girodon E, Silva J,
Ferras L, Sousa M and Barros A (2004) Characterization of cystic fibrosis
conductance transmembrane regulator gene mutations and IVS8 poly(T)
variants in Portuguese patients with congenital absence of the vas
deferens. Hum Reprod 19,2502–2508.
Groman JD, Hefferon TW, Casals T, Bassas L, Estivill X, Des Georges M,
Guittard C, Koudova M, Fallin MD, Nemeth K, et al. (2004) Variation in
a repeat sequence determines whether a common variant of the cystic
fibrosis transmembrane conductance regulator gene is pathogenic or
benign. Am J Hum Genet 74,176–179.
Hantash FM, Milunsky A, Wang Z, Anderson B, Sun W, Anguiano A and
Strom CM (2006a) A large deletion in the CFTR gene in CBAVD. Genet
Med 8,93–95.
Hantash FM, Redman JB, Starn K, Anderson B, Buller A, McGinniss MJ, Quan
F, Peng M, Sun W and Strom CM (2006b) Novel and recurrent
rearrangements in the CFTR gene: clinical and laboratory implications for
cystic fibrosis screening. Hum Genet 17,1–11.
Laufer-Cahana A, Lerer I, Sagi M, Rachmilewitz-Minei T, Zamir C, Rivlin JR
and Abeliovich D (1999) Cystic fibrosis mutations in Israeli Arab patients.
Hum Mutat 14,543.
Le Marechal C, Audrezet MP, Quere I, Raguenes O, Langonne S and Ferec C
(2001) Complete and rapid scanning of the cystic fibrosis transmembrane
conductance regulator (CFTR) gene by denaturing high-performance
liquid chromatography (D-HPLC): major implications for genetic
counselling. Hum Genet 108,209– 298.
Lerer I, Laufer-Cahana A, Rivlin JR, Augarten A and Abeliovich D (1999)
A large deletion mutation in the CFTR gene (3120 þ 1kbdel8.6 kb): a
founder mutation in the Palestinian Arabs. Hum Mutat 13,337.
Lissens W, Mercier B, Tournaye H, Bonduelle M, Ferec C, Seneca S, Devroey
P, Silber S, Van Steirteghem A and Liebaers I (1996) Cystic fibrosis and
infertility caused by congenital bilateral absence of the vas deferens and
related clinical entities. Hum Reprod 11 (Suppl 4),55–78; discussion 79–80.
Mercier B, Verlingue C, Lissens W, Silber SJ, Novelli G, Bonduelle M,
Audrézet MP and Férec C (1995) Is congenital bilateral absence of vas
deferens a primary form of cystic fibrosis? Analyses of the CFTR Gene in
67 Patients. Am J Hum Genet 56,272– 277.
Niel F, Legendre M, Bienvenu T, Bieth E, Lalau G, Sermet I, Bondeux D,
Boukari R, Derelle J, Levy P, et al. (2006) A new large CFTR
rearrangement illustrates the importance of searching for complex alleles.
Hum Mutat 27,716–717.
Niel F, Martin J, Dastot-Le Moal F, Costes B, Boissier B, Delattre V, Goossens
M and Girodon E (2004) Rapid detection of CFTR gene rearrangements
impacts on genetic counselling in cystic fibrosis. J Med Genet 41(11),e118.
Pignatti PF, Bombieri C, Marigo C, Benetazzo M and Luisetti M (1995)
Increased incidence of cystic fibrosis gene mutations in adults with
disseminated bronchiectasis. Hum Mol Genet 4,635–639.
Rave-Harel N, Madgar R, Goshen R, Nissim-Rafinia M, Ziadni A, Rahat A,
Chiba O, Kalman YM, Brautbar C, Levinson D, et al. (1995) CFTR
haplotype analysis reveals genetics heterogeneity in the etiology of
congenital bilateral aplasia of the vas deferens. Am J Hum Genet
56,1359– 1366.
Sharer N, Schwarz M, Malone G, Howarth A, Painter J, Super M and Brazanga
J (1998) Mutations of the cystic fibrosis gene in patients with chronic
pancreatitis. N Engl J Med 339,645–652.
Stuhrmann M and Dörk T (2000) CFTR gene mutations and male infertility.
Andrologia 32,71–83.
Welsh MJ, Ramsey BW, Accurso F and Cutting GR (2001) Cystic fibrosis.
In Scriver CR, Beaudet AL, Sly WS and Valle D (eds) Metabolic and
Molecular Bases of Inherited Disease. McGraw-Hill, New York,
pp. 5121–5188.
Submitted on November 17, 2006; resubmitted on January 9, 2007; accepted on
January 16, 2007
1291