1. health and fitness
2. disease

Hum Genet (2001) 108 : 290–298
DOI 10.1007/s004390100490
O R I G I N A L I N V E S T I G AT I O N
C. Le Maréchal · M. P. Audrézet · I. Quéré ·
O. Raguénès · S. Langonné · C. Férec
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
Received: 8 January 2001 / Accepted: 12 February 2001 / Published online: 4 April 2001
© Springer-Verlag 2001
Abstract More than 900 mutations and more than 200 different polymorphisms have now been reported in the cystic fibrosis transmembrane conductance regulator (CFTR)
gene. Ten years after the cloning of the CFTR gene, the
complete scanning of the 27 exons to identify known and
novel mutations remains challenging. Rapid accurate
identification of mutated alleles is important for prenatal
diagnosis, for cascade screening in families at risk of cystic fibrosis (CF) and for understanding the correlation between genotype and phenotype. In this study, we report
the successful use of denaturing ion-pair reverse-phase
high performance liquid chromatography (D-HPLC) to
analyse rapidly the complete coding sequence of the
CFTR gene. With 27 pairs of polymerase chain reaction
primers, we optimised the temperature conditions required for the analysis of each amplicon and validated
thetest conditions on samples from a panel of 1552 CF patients who came from France and other European countries and who had mutations and polymorphisms located
in the various melting domains of the gene. D-HPLC
identified 415 mutated alleles previously characterised by
denaturing gradient gel electrophoresis and DNA sequencing, plus 74 novel mutations reported here.This new
technique for screening DNA for sequence variation was
extremely accurate (it identified 100% of the CFTR alleles tested so far) and rapid (the complete CFTR gene
could be analysed in less than a week). Our approach
should reduce the number of untyped CF alleles in populations and thus decrease the residual risk in couples at
risk of CF. This technique may be important not only for
CF,but also for many other genes with a high frequency of
point mutations at a variety of sites.
C. Le Maréchal · C. Férec (✉)
EFS-Bretagne, EPI 01-15, Site de Brest,
CHU, Brest; France
e-mail: [email protected],
Tel.: +33-2-98445064, Fax: +33-2-98430555
M. P. Audrézet · I. Quéré · O. Raguénès · S. Langonné · C. Férec
Laboratoire de Génétique Moléculaire, EPI 01-15,
CHU, 29200 Brest, France
Introduction
With an incidence of about 1 in 3000 live births in Caucasians of European extraction (Welsh et al. 1995), cystic
fibrosis (CF) is one of the most common lethal diseases in
childhood. CF can result from many different mutations in
the cystic fibrosis transmembrane conductance regulator
(CFTR) gene (ABCC7, MIM 602421). In addition to the
∆F508 deletion, which accounts for about 66% of all CF
alleles world-wide, more than 900 different mutations
have been reported throughout the 27 exons of the CFTR
gene by the Cystic Fibrosis Genetic Analysis Consortium
(http://www.genet.sickkids.on.ca).
CF mutations have also been found in other clinical
diseases such as congenital bilateral absence of the vas
deferens (CBAVD; Chillon et al. 1995; de Braekeleer and
Ferec 1996; Dumur et al. 1990; Mercier et al. 1995), disseminated bronchiectasis (Girodon et al. 1997; Pignatti et
al. 1995) and chronic pancreatitis (Cohn et al. 1998;
Sharer et al. 1998). The particularly large number of different alleles, combined with marked variation in their
distribution and frequency according to geographic and
ethnic origin (Estivill et al. 1997; Tsui 1992) makes clinical testing for mutated alleles particularly difficult. It is
only in a few populations, such as those in Brittany (France)
and in Quebec (Saguenay Lac St Jean) that 98%–100% of
the mutated alleles have been identified so far (de Braekeleer et al. 1998; Férec et al. 1992).
During the last ten years, the development of technology for mutation screening has been extremely productive
with the appearance of powerful techniques such as polymerase chain reaction (PCR), single strand conformational polymorphism (SSCP) (Orita et al. 1989), chemical
and enzymatic cleavages (Cotton et al. 1988; Babon et al.
1999), denaturing gradient gel electrophoresis (DGGE)
(Lerman and Silverstein 1987) and, more recently, denaturing ion-pair reverse-phase high-performance liquid
chromatography (D-HPLC) (Oefner and Underhill 1998).
D-HPLC is an automated technology for mutation screening based on the separation of heteroduplexes from ho-
291
moduplexes on a stationary phase under partially denaturing conditions. This technique was originally used to
identify single nucleotide polymorphisms (SNPs) on the
Y chromosome (Underhill et al. 1996).
The ideal technique for mutation detection has to be
sensitive, specific and robust (Cotton 1997). Although it
is unfortunately time-consuming and technically difficult
to implement, DGGEis probably one of the most powerful
techniques available for the detection of point mutations.
This is particularly true for large genes with many different mutations, such as CFTR. These limitations have led
us to search for a new technique and to focus on D-HPLC.
We now report the complete scanning of the CFTR gene
by D-HPLC. All the previously characterised mutated alleles tested 415 different nucleotide changes including
mutations (334), polymorphisms (50) and 31 complex
haplotypes, have been correctly identified, and we report
the identification of 74 novel mutations. We propose that
this technique should become standard for quick and efficient scanning of the CFTR gene as it allows rapid genotyping and has greatly improved our Bayesian calculations for couples at risk of CF.
Materials and methods
Sample collection
This study is the result of 10 years of CFTR analysis carried out
within our genetic laboratory. We analysed by DGGE the 27 exons
of the CFTR gene(ABCC7) in clinical situations involving CFTR
abnormalities (CF, CBAVD, bronchiectasis, chronic pancreatitis)
and also in a control population. This analysis revealed 359 mutated samples and SNPs distributed all over the coding sequence
from our cohort of1552 French CF patients and 253 CBAVD patients. Collaborations with several centres from various countries
and with members of the Cystic Fibrosis Genetic Analysis Consortium increased the diversity of our collection of mutated samples (56 different mutated samples). A list of the mutations and
SNPs tested is available upon request.
DNA amplification
DNA was isolated from blood cells by the salt precipitation
method. Primers were designed by using Primer Express software
(Applied Biosystems). PCR was performed in 50 µl containing
0,5 µM of each primer, 1–2.5 mM MgCl2, 1×PCR buffer II (Applied Biosystems), 200 µM each dNTP (Amersham-Pharmacia
Biotech), 0.2 U AmpliTaq DNA polymerase (Applied Biosystems), and 50 ng DNA. We chose a touchdown PCR protocol as
previously described (Don et al. 1991). This enabled us to minimise experimentation and reducethe number of thermocycler programs: indeed, two protocols were enough to amplify all 27 exons
of the CFTR gene. Cycling conditions were as follows: a denaturation step at 94°C for 3 min, 14 touchdown cycles with annealing
temperature decreasing 0.5°C per cycle (denaturation 94°C for 20 s,
annealing for 40 s, primer extension 72°C for 45 s), 25 cycles at
the final touchdown annealing temperature and a final elongation
step at 72°C for 7 min. Heteroduplexes were formed by denaturing
at 95°C and cooling by 1°C per minute to 65°C. Amplicons were
stored at 4°C until D-HPLC analysis. All reactions were carried
out using the GeneAmp PCR system 9700 (Applied Biosystems).
Specific sizes and quantities of amplicons were checked by
agarose gel electrophoresis. D-HPLC analysis of a wild-type sample amplified with 1, 1.5, 2 and 2.5 mM MgCl2 was performed to
find the magnesium concentration that gave no spurious products.
Table 1 summarises PCR conditions for the 27 exons of CFTR
gene.
D-HPLC analysis
Denaturing high performance liquid chromatography (D-HPLC)
analysis is a reverse-phase ion-pair high-performance liquid chromatography that allows the identification of heteroduplex molecules (Oefner and Underhill 1998). It was performed using the
Transgenomic WAVE system as described by Kuklin et al. (1998).
Aliquots of 3–5 µl crude PCR samples were loaded onto a preheated C18 reverse-phase column based on non-porous poly
(styrene-divinyl benzene) particles (DNASep column Transgenomic). DNA (homoduplexes with or without heteroduplexes)
was eluted from the column by a linear acetonitrile gradient in 0.1
mM triethylamine acetate buffer (TEAA; Transgenomic), pH 7, at
a constant flow rate of 0.9 ml/min. The gradient was formed by
mixing buffer A (0.1 mM TEAA) and buffer B (0.1 mM TEAA,
25% v/v acetonitrile). The temperature of the oven for optimal heteroduplex separationwith partial DNA denaturation was deduced
from the melting profile of the DNA sequence. Wavemaker 3.4.4
software (Transgenomic) was first used to compute melting curves
and to estimate the temperature for analysis. This was tested experimentally by injecting a wild-type sample onto the column at
approximately the calculatedtemperature (±2°C) by 1°C steps; we
chose the temperature just below that at which denaturation occurred (1 min retention time shift compared with non-denaturing
conditions of 50°C in most cases). If the sequence possessed several melting domains with more than 4°C difference, the melting
curve indicated the various temperatures that had to be investigated.
The analytic gradient was 3.5 min long and buffer B increased
at 2% per minute. For each fragment, the initial and final concentrations of buffer B were adjusted to obtain a retention time between 3 and 5 min; the conditions are listed in Table 1. The column was then cleaned with 100% buffer B for 30 s and equilibrated at the start conditions for 2 min before the next injection.
Elution of DNA was detected by 260 nm UV absorbance. HSM
software regulated every parameter of the Wave system during
analysis and stored the data.
Data analysis
Two researchers interpreted the results independently. Four wildtypesamples were always used as negative controls to ensure that a
normal homoduplex profile was reproducibly obtained (retention
time and peak profile). Chromatograms were overlaid with one
from a wild-type. Samples with extra peaks (one, two or three
more)or with a difference in peak appearance were scored as positive.
Direct sequencing
Samples showing abnormal D-HPLC profiles were re-amplified
from genomic DNA. PCR products were purified on microcon 100
columns (Millipore). Direct sequencing was performed with the
BigDye Terminator cycle sequencing kit from Applied Biosystems, with 25 ng template, 3 pmol selected primer and RRmix as
supplied by the manufacturer. Cycle sequencing (25 cycles at 95°C
for 5 s, 60°C for 10 s and 72°C for 4 min) was performed with a
GeneAmp PCR system 9700 (Applied Biosystems). Centrifugation through a Centrisep spin column removed excess dye terminator. An aliquot of 10 µl Template Suppression Reagent (TSR)
was added to 10 µl purified reaction product, after which the products were denatured at 94°C for 2 min and kept on ice before being analysed by capillary electrophoresis on an ABIprism 310 (Applied Biosystems).
292
Table 1 PCR (primers sequences, final touchdown annealing temperature) and DHPLC (oven temperature, Acetonitril gradient) analysis conditions for 27 exons of the CFTR gene (ABCC7)
Exon
1
2
3
4
5
6a
6b
7
8
9
10
11
12
13
14A
14b
15
16
17a
17b
18
19
20
21
22
23
24
Primer
Sequences 5’→3’
Amplicon
length (bp)
MgCl2
(mM)
final
annealing
temp (°C)
Oven
temp
% B buffer
start/end
h1i5
h1i3
2i5b
2i3”
3i5
3i3
h4i5
4i3
5i5
5i3
h6ai5
h6ai3
6bi5B
6bi3
h7i5
7i3b
8i5b
8i3b
9i5C
9i3D
h10i5
C16D*
h11i5
11i3ter
H12i5
h12i3
13i5
TTgAgCggCAggCACC
gCACgTgTCTTTCCgAAgCT
CAAATCTgTATggAgACC
CAACTAAACAATgTACATgAAC
GAAATAggACAACTAAAATA
ATTCACCAgATTTCgTAGTC
CACATATggTATgACCCTCT
ATCCATCACTCgACCATgTT
GTTgAAATTATCTAACTTTC
AACTCCgCCTTTCCAgTTgT
TCCTTTTACTTgCTTTCTTTCA
TATgCATAgAgCAgTCCTggTT
GATTTACAgAgATCAgAg
gAggTggAAgTCTACCATgA
TgCTCAgATCTTCCATTCCAAg
AACTgATCTATTgACTgAT
AATgCATTAATgCTATTCTgATTC
AgTTAggTgTTTAgAgCAAACAA
TggggAATTATTTgAgAAAg
CTTCCAgCACTACAAACTAgAAA
TgATAATgACCTAATAATgAT
CATTCACAgTAgCTTACCCA
TgCCTTTCAAATTCAgATTgAgC
ACAgCAAATgCTTgCTAgACC
gAA TCg ATg Tgg TgA CCA TAT TgT
CCA gTA ggg CAg ATC AgA TTT gA
TgCTAAAATACgAgACATATTgC
181
1.5
56
63°C
52/59
194
1
50
58°C
51/58
259
2
50
437
2
56
56°C
58°C
59°C
52/59
51/58
54/61
192
2.5
50
55°C
51/58
344
2
50
221
2
56
56°C
61°C
56°C
58/65
53/60
52/59
390
1.5
56
190
2.5
56
55°C
61°C
54°C
57/64
51/58
55/62
258
1
56
56/63
386
1.5
56
197
1.5
56
55°C
58°C
50°C
56°C
57°C
58/65
56/63
54/61
366
2
50
55°C
57/64
906
1.5
50
13i3
TACACCTTATCCTAATCCTAT
h14ai5
14i3
H14bi5
14bi3b
H15i5
h15i3
h16i5
h16i3
h17ai5
h17ai3
h17Bi5
17Bi3B
18I5
H18I3
19i5
19i3B
h20i5
h20i3
21i5A
21i3
h22i5
h22i3
H23i5mod
h23i3
24i5C
24i3C
CACAATggTggCATgAAACT
gTATACATCCCCAAACTATCT
ggg Agg AAT TAg gTg AAg AT
TAC ATA CAA ACA TAg Tgg ATT
TgTATTggAAATTCAgTAAgTAACTTTgg
AgCCAgCACTgCCATTAgAAA
CTgAATgCgTCTACTgTgATCCA
TgTgggATTgCCTCAggTTT
AATCACTgACACACTTTgTCCACTT
TCAAATAgCTCTTATAgCTTTTTTACAAgATg
AAT gAC ATT TgT gAT ATg AT
CTTAAATgCTTAgCTAAAgT
AgTCgTTCACAgAAgAgAgA
AAT gAC AgA TAC ACA gTg ACC CTC A
GTgAAATTgTCTgCCATTCT
ACTCCATATAATAAAACATgTgTg
ATCTTCCACTggTgACAggA
AAAgACAgCAATgCATAACAA
AATgTTCACAAgggACTCCA
CAAAAgTACCTgTTgCTCCA
ATCAATTCAAATggTggCAggT
AATgATTCTgTTCCCACTgTgCT
CggCAAggTAAATACAgATCAT
GCA ggA ACT ATC ACA TgT gA
TCCCTgCTCTggTCTgACCTgC
CATgAggTgACTgTCCCACgAg
256
1.5
56
54°C
57°C
59°C
62°C
56°C
62/69
60/67
57/64
52/59
55/62
174
2
50
57°C
52/59
401
1.5
56
59°C
56/63
401
2
56
55°C
56/63
281
1.5
56
58°C
55/62
380
1.5
50
311
1.5
50
56°C
59°C
56°C
56/63
53/60
55/62
449
2.5
50
59°C
55/62
401
2
50
477
1.5
56
370
1
56
54°C
58°C
59°C
58°C
60
59/66
54/61
52/59
48/55
53/60
250
1.5
56
57°C
53/60
329
1
56
62°C
55/62
293
Results
D-HPLC allows the separation of homoduplexes and heteroduplexes in double-stranded DNA molecules up to 1 kb
in length. Taking advantage of our knowledge of the
CFTR gene and its mutations, which we have studied for
more than 10 years, we defined the primer and temperature conditions for each of the 27 exons of the CFTR
gene. The melting profiles were studied by using Wavemaker software. As an example, Fig. 1A illustrates the
melting profile obtained for exon 3 of the CFTR gene.
The coding sequence, localised between the two dotted
lines, has two melting domains. The 5’ domain has a melting temperature at 58°C, whereas the 3’ domain melts at
55°C. Two positive samples, with mutations localised in
the two different domains, were selected (E60X and
G85E). Analysis of the fragment was performed at 57°C
and 58°C and the experiment revealed that a 1°C shift
radically changed the studied domain. At 57°C, only the
low melting domain represented by the mutation G85E in
this example is analysed. At 58°C, only the 5’ part of the
sequence (highlighted; mutation E60X) is correctly analysed
(Fig. 1B).
Fig. 1 A Melting profile of
exon 3 of the CFTR gene: the
coding sequence is located between the two dotted lines and
the two mutations chosen are
positioned on the sequence.
B Chromatograms show the
shift of the studied domains
with only a 1°C change. At
57°C, the 3’ part is studied
(low melting) represented by
mutation G85E (green),
whereas at 58°C, the 5’ part
with mutation E60X (pink) is
analysed
For each of the 27 exons, all the melting domains were
validated with positive samples. This allowed us to be
sure that the domain was well studied at the exact temperature. For the majority ofthe fragments (22 exons), one
temperature was enough. Two temperatures were necessary for exons 3, 7, 17b and 21. Exon 13, which encodes
the R domain of the protein (725 bp), required four different analysis conditions (54°C, 57°C, 59°C and 62°C)
from the same amplicon.
We chose one additional condition for exons 6a and 20.
This permitted us to explore the intronic sequence amplified with the primers, containing respectively the 875+40
A→G and 4005+33 A→G polymorphisms.
Exon 10 of the CFTR gene contains the most frequent
mutation, ∆F508, which corresponds to a 3-bp deletion
and accounts for about 66% of CF chromosomes worldwide. Analysis of this fragment under non-denaturing
condition (at 50°C) permits us to search specifically for
this mutation and to distinguish it from the frequent variant M470 V that appears identical under the classical
analysis condition (56°C) for exon 10 (Fig. 2).
Our series of 415 mutated alleles included 198 transitions, 124 transversions and 63 insertion/deletions as representatives of the abnormalities observed in the CFTR
294
Fig. 2 Exon 10: analysis under
non-denaturing conditions
(50°C) allows the identification
of ∆F508. At 56°C, the profiles
of ∆F508 and M470V are identical
gene. This collection of samples corresponds to the 415 previously known variants plus 74 novel nucleotide changes,
three of which were identified for the first time by
D-HPLC (Table 2). All 415 of the 415 positive sequences
tested were identified under the conditions specified for
each exon. Thus, the sensitivity of D-HPLC for the CFTR
gene is 100% in this collection of mutated samples.
As soon as the conditions of analysis were established
for each exon, we studied the unidentified chromosomes
from our cohort of 1552 French CF patients. At the end of
this study, only 61 chromosomes remained unidentified,
corresponding to a detection rate of more than 98% of the
mutated alleles.
Discussion
Since the CFTR gene was cloned in 1989, more than 900
mutations have been reported spread throughout the gene.
Mutations have also been reported in many related diseases in adults, such CBAVD (Chillon et al. 1995; Dumur
et al. 1990; Mercier et al. 1995), disseminated bronchiectasis (Pignatti et al. 1995) and pancreatitis (Sharer et al.
1998).
Apart from ∆F508 and a few mutations that are found
all over the world, most of these mutated alleles are private mutations (http://www.genet.sickkids.on.ca). The ideal
screening technique should identify known mutations and
novel private ones that could lie anywhere in the gene.
Since the arrival of the PCR technique, many methods
have been used to screen for specific mutations. Heteroduplex analysis and restriction enzyme analysis are the
most common methods (Dequeker and Cassiman 1998).
Other techniques, such as dot-blot or reverse dot-blot
(Cuppens et al. 1992), or allele specific mutation detection systems (Ferrie et al. 1992) are also commonly used.
More recently, Applied Biosystems has developed commercial kits based on the oligonucleotide ligation assay
and allowing the detection of 31 mutations (Brinson et al.
1997). This test is robust but persistently fails to identify
certain mutations that are common in particular ethnic
groups. Scanning methods are thus essential, as they can
identify known mutations and novel ones. The most frequently used are SSCP (Orita et al. 1989) and the DGGE
method initially proposed by Lerman and Silverstein
(1987). To date, DGGE is the most popular and sensitive
technique for CFTR gene analysis. Nevertheless, these
methods still have limitations, basically with regard to the
time that they require and to their sensitivity.
In order to develop a powerful, rapid and robust
method to identify mutant CF alleles, we have used the DHPLC method initially described by Oefner and Underhill
(1995). D-HPLC allows the separationof homoduplexes
and heteroduplexes in double-stranded DNA molecules
up to 1 kb, based on a difference in denaturation characteristics. We have designed pairs of PCR primers for each
of the 27 exons and exon/intron boundaries and have determined the temperature conditions required to obtain the
best resolution. During the last 10 years, we have collected a large number of CF alleles from various European
countries (e.g. France, Belgium, Italy, Ireland, Slovenia,
Russia, Bulgaria; Mercier et al. 1993; Audrézet et al.
1993, 1994; Verlingue et al. 1995) and from native American people (Mercier et al. 1994). Being comprised of 415
characterised alleles and 74 undescribed mutations previously identified by DGGE, this collection has provided a
good test of the ability of D-HPLC to identify mutations.
By using the molecular tools presented here, we have
shown that the sensitivity of the technique is 100%; furthermore, we report the identification of 74 novel mutations. The technique is both inexpensive and quick: for
the scanning of the whole coding sequence, we assess the
cost at US $50 (excluding labour costs) and the complete
coding sequence of the gene can now be studied in less
than a week. The sensitivity and specificity of D-HPLC
have also been analysed by Ellis and co-workers (2000),
295
Table 2 Novel nucleotide changes identified in the CFTR gene and detected by D-HPLC
Exon/
intron
Mutant name
Nucleic acid change
Amino acid change
1
2
2
2
2
3
3
3
3
4
4
4
5
5
6a
6a
6a
6a
6a
6b
7
7
7
7
7
8
8
8
9
10
10
10
10
185+1 G to T
186 – 13 C to G
211 Del G
237 Ins A
296+2 T to C
W 57 X2
306 InsA
306 Ins C
W 79 X
A 96 E
L 127 X
541 Del CTCC
L 165 S
R 170 C
L 206 F
A 209 S
A 209 A
C 225 X
G 241 R
905 Del G
A 309 A
V 322 M
R 334 Q
Q 353 H
1248+1 G to C
L 383 L
W 401 X
E 403 D
1367 Del C
1525 – 2 A to G
G 480 G
1576 Ins T
H 484 R
G to T at 185+1
C to G at 186–13
Deletion of G at 211
Insertion A at 237
296+2 T to C
G to A at 303
Insertion of A at 306
Insertion of C at 306
G to A at 368
C to A at 419
T to G at 512
Deletion of CTCC at 541
T to C at 626
C to T at 640
G to T at 750
G to T at 757
A to G at 759
T to A at 807
G to A at 852
Deletion of Gat 905
C to G at 1059
G to A at 1096
G to A at 1133
A to C at 1191
G to C at 1248+1
G to A at 1281
G to A at 1334
G to C at 1341
Deletion of C at 1367
T to C at 1572
Insertion of T at 1576
A to G at 1583
Gly to Gly at 480 (GGT to GGC)
10
11
I506 V
1717 – 19 T to C
A to G at 1648
T to C at 1717–19
Ileto Val at 506 (ATC to GTC)
Silent
Splicing ?
11
11
12
12
13
14a
14a
14a
14b
14b
14b
15
15
15
G 544 G
1802 Del C
Y 569 X
1898+5 G to A
2335 Del A
E 831 X
C 866 Y
V 868 V
2752 – 1 G to T
2752 – 97 C to T
W 882 X
S 895 T
F 932 S
3040+23 T to C
T to G at 1764
Deletion of C at 1802
T to A at 1839
G to A at 1898+5
Deletion of A at 2335
G to T at 2623
G to A at 2729
G to A at 2736
G to T at 2752–1
C to T at 2752–97
G to A at 2777
G to C at 2816
T to C at 2927
T to C at 3040 +23
Gly to Gly at 544 (GGT to GGG)
Silent
Frameshift
Nonsense
Splicing
Frameshift
Nonsense
Missense
Silent
Splicing
Silent
Nonsense
Missense
Missense
Silent
Trp to Stop at 57 (TGG to TGA)
Trp to Stop at 79 (TGG to TAG)
Ala to Glu at 96 (GCA to GAA)
Leu to Stop at 127 (TTA to TGA)
Leu to Ser at 165 (TTA to TCA)
Arg to Cys at 170 (CGT to TGT)
Leu to Phe at 206 (TTG to TTT)
Ala to Ser at 209 (GCA toTCA)
Ala to Ala at 209 (GCA to GCG)
Cys to Stop at 225 (TGT to TGA)
Gly to Arg at 241 (GGG to AGG)
Ala to Ala at 309 (GCC to GCG)
Val to Met at 322 (GTG to ATG)
Arg to Gln at 334 (CGG toCAG)
Gln to His at 353 (CAA to CAC)
Leu to Leu at 383 (TTG to TTA)
Trp to Stop at 401 (TGG to TAG)
Glu to Asp at 403 (GAG to CAG)
His to Arg at 484 (CAC to CGC)
Tyr to Stop at 569 (TAT to TAA)
Glu to Stop at 831 (GAG to TAG)
Cys to Tyr at 866 (TGC to TAC)
Val to Val at 868 (GTA to GTG)
Trp to Stop at 882 (TGG to TAG)
Ser to Thr at 895 (AGT to ACT)
Phe to Ser at 932 (TTC to TCC)
Effect on amino
acid sequence
Patient
Splicing
Silent
Frameshift
Frameshift
Splicing
Nonsense
Frameshift
Frameshift
Nonsense
Missense
Nonsense
Frameshift
Missense
Missense
Missense
Missense
Silent
Nonsense
Missense
Frameshift
Silent
Silent
Missense
Missense
Splicing
Silent
Nonsense
Missense
Frameshift
Splicing
Silent
Frameshift
Missense
CF patient
CF patient
CF patient
CF patient
CF patient
CF patient
CF patient
CF patient
CF patient
CF patient
CF patient
CF patient
CF patient
Control
CF patient
CF patient
CF patient
CF patient
CF patient
CF patient
Control
CF patient
Control
CF patient
CF patient
Control
CF patient
CF patient
CF patient
CF patient
CF patient
CF patient
Neonatal
hypertrypsinaemia
Control
Neonatal
hypertrypsinaemia
Control
CF patient
CF patient
CF patient
CF patient
CF patient
CF patient
CF patient
CF patient
Control
CF patient
Control
Control
Control
296
Table 2 (continued)
Exon/
intron
Mutant name
Nucleic acid change
Amino acid change
Effect on amino
acid sequence
Patient
16
17a
17a
17b
17b
17b
17b
17b
18
18
S 977 F
G 1003 X
Q 1042 X
L 1059 L
R 1066 S
T 1115 T
3499+6 A to G
3499+7 T to G
Delta M 1140
M 1140 K
C to Tat 3062
G to T at 3139
C to T at 3256
A to G at 3309
C to A at 3328
C to A at 3477
A to G at 3499
T to G at 3499+7
Deletion of 3 pb
T to A at 3551
Ser to Phe at 977 (TCC to TTC)
Gly to Stop at 1003 (GGA to TGA)
Gln to Stop at 1042 (CAA to TAA)
Leu to Leu at 1059 (TTA to TTG)
Arg to Ser at 1066 (CGT to AGT)
Thr to Thr at 1115 (ACC to ACA)
Missense
Nonsense
Nonsense
Silent
Missense
Silent
Splicing
Splicing
Frameshift
Missense
19
19
19
20
20
S 1159 F
S 1161 R
S 1206 X
F 1257 L
4005+33 A to G
C to T at 3608
C to G at 3615
C to G at 3749
T to G at 3903
A to G at 4005 +33
Ser to Phe at 1159 (TCT to TTT)
Ser to Arg at 1161 (AGC to AGG)
Ser to Stop at 1206 (TCA to TGA)
Phe to Leu at 1257 (TTT to TTG)
Missense
Missense
Nonsense
Missense
Splicing
21
21
21
21
21
22
22
22
23
24
V1293I
4015 Del A
N 1303 I
P 1306 P
E 1308 X
4172 Del GC
R 1358 S
I 1366 T
4374+10 T to C
D 1477 D
G to A at 4009
Deletion of A at 4015
A to T at 4040
C to T at 4050
G to T at 4064
Deletion of GC at 4172
A to T at 4206
T to C at 4229
T to C at 4374+ 10
T to C at 4563
Val to Ile at 1293
Missense
Frameshift
Missense
Silent
Nonsense
Frameshift
Missense
Missense
Splicing
Silent
CF patient
CF patient
CF patient
Control
CF patient
Control
CF patient
Control
CF patient
Bronchiectasis
CF patient
CF patient
CF patient
CF patient
Bronchiectasis
Control
CF patient
CF patient
CF patient
CF patient
CF patient
Control
Control
CF patient
Control
Met to Lys at 1140 (ATG to AAG)
Asn to Ile at 1303 (AAC to ATC)
Pro to Pro at 1306 (CCC to CCT)
Glu to Stop at 1308 (GAA to TAA)
Arg to Ser at 1358 (AGA to AGT)
Ile to Thr at 1366 (ATC to ACC)
Asp to Asp at 1477 (GAT to GAC)
who have compared the sensitivity of fluorescent-SSCP
(F/SSCP) and D-HPLC from a collection of 67 different
mutations from different genes (ABCC7, MIM 602421,
VHL, MIM 193300; Gross et al. 1999). They report a specificity of 100% and a sensitivity of 95% for the ABCC7
gene, a result comparable to others reported in the literature (Liu et al. 1998; O’Donovan et al. 1998). The 100%
sensitivity that we have obtained is probably attributable
to our prior experience with DGGE. The positions of
primers and the choice of the appropriate temperature for
each fragment was partly based on our previously defined
DGGE conditions.
Our significant collection of various point mutations
distributed in all the different melting domains of the coding sequence of the gene has been particularly helpful in
optimising the experimental analysis conditions. However, homozygous mutations do not generally alter the
stability of DNA fragments. Analysis of DNA from an
obligate carrier (mother or father) or of a mixture of PCR
products from the patient and a normal control in order to
create heteroduplexes, overcomes this problem.
All PCR-based mutation screening approaches involving diploid DNA miss large deletions. For example, the
50-kb deletion described in a Spanish chromosome (Morral et al. 1993) or the 21-kb deletion (CFTRdelE2,3) re-
cently found in CF patients of Eastern and Western Slavic
descent (Dörk et al. 2000). They also miss mutations located deep inside introns, for example 3849+10 kb C→T
(Highsmith et al. 1994) or the 1811+1.6 kb A→G that is
common in Spain (Casals et al. 1997). Specific amplicons
can be proposed to search for these mutations. Other scanning methods, such as chemical cleavage, enzymatic
cleavage and the protein truncation test, have been used
for CFTR gene mutation analysis (Girodon-Boulandet et
al. 2000). All these are less sensitive and more time-consuming. Obviously, these scanning methods require direct
DNA sequencing to be performed to characterise the mutation.
Using D-HPLC and the technical protocols that we report in this paper, we have shown that, so far, all the previously known and new mutations can be identified. Despite the high level of heterogeneity of the CFTR mutation in Europe and United States, we think that a mutationdetection rate of 95% is achievable and that a detection rate of 98% can be reached in most of the countries of
European extraction. In our cohort of 1552 French CF patients, only 61 chromosomes remained unidentified, increasing the detection rate to more than 98%. Considering
that the tested population is highly heterogeneous, this is
a high level of point mutation detection.
297
This new tool thus greatly improves genetic counselling. For example, the residual risk of CF for a couple
(a partner of a heterozygote with 98% of mutations being
discarded) is now 1/5000 in our population with a carrier
rate of 1/25. Moreover, for the rare CF chromosomes, in
which the mutation remains unidentified, the characterisation of intragenic polymorphisms can provide us with information for prenatal diagnosis.
Ultrasound screening has become routine in pregnancy
and the discovery of a fetus with a hyperechogenic bowel
or ascities is suggestive of CF. In this emergency situation, we first screen for common known mutations. If one
parent is a carrier and the mutation is also present in the
fetus, we urgently have to scan the entire CFTR gene for
a second mutation. The residual risk for this fetus depends
upon the detection rate of the test used. For example, if
only the ∆F508 and the other most common mutations
(G551D, G542X, W1282X, 1717–1 G→A) are sought,
the detection rate is 70% and the residual risk is around
1/3. If D-HPLC is used, we can analyse, within a few
days, the whole gene and scan all the known and new private mutations with a detection rate of 98%. The residual
risk becomes 1/60 (V. Scottet et al. in preparation).
A thorough analysis of the CFTR gene is also much
needed in partners of CF patients. For these couples, the a
priori risk is 1/25 and a complete scanning of the gene allows one to reach a residual risk of 1/2500.
Although neonatal screening for CF is still a matter of
debate in the medical community, it has become a reality
in some countries. The main protocol includes a combined analysisofimmunoreactive trypsin (IRT) and mutation analysis. It is evident that the specific choice of the
exons analysed with respect to the distribution of mutations in the country will greatly improve the specificity of
the scanning protocol (Scottet et al.2000).
D-HPLC dramatically enhances our capacity to identify mutated alleles. However, we know from genotype/
phenotype correlation studies that the detection of a mutation alone is no longer sufficient to make a clinical diagnosis and assessment of CF. We have to determine the
precise sequence abnormality for at least two reasons.
First, genotypes can be assigned to one of five classes
based on the molecular outcome. Class I mutations cause
defective protein production; class II mutations are associated with defective protein processing; class III mutations cause defective regulation; class IV mutations correspond to mutations localised in the transmembrane domain of the protein and cause defective conductance; finally, class V mutations include those affecting the level
of normal mRNA transcript and, thus, of protein required
for normal function (Estivill 1996; Welsh and Smith
1993). The identification of class IV mutations, which affect channel conductance or channel gating, and class V
mutations, which reduce the level of normal CFTR protein by altering the promotor or splicing, is highly significant for genetic counselling. Mutations in these two
classes confer a milder phenotype with a longer median
life expectancy of about 50 years. Second, future therapeutic approaches will probably necessitate the design of
novel molecules directed at the underlying molecular defect of the CFTR protein. Precise genotype determination
will be critical in the choice of such therapy. The technology and experimental conditions described in this paper
should help to make this possible.
Acknowledgements This study was supported by the Agence
Bretonne d’Etude et de Recherche sur la Mucoviscidose (ABER-M)
and the Conseil Régional de Bretagne. We thank collegues in the
Cystic Fibrosis Genetic Analysis Consortium for providing us with
positive samples.
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