Clinical Chemistry 45: 957-962, 1999;
(Clinical Chemistry. 1999;45:957-962.)
© 1999 American Association for Clinical Chemistry, Inc.
Detection of Five Rare Cystic Fibrosis Mutations Peculiar to Southern Italy: Implications in Screening for the Disease and Phenotype Characterization for Patients with Homozygote Mutations
Giuseppe Castaldo1,2,
Antonella Fuccio1,
Cécile Cazeneuve3,
Luigi Picci4,
Donatello Salvatore5,
Valeria Raia6,
Maurizio Scarpa4,
Michel Goossens3 and
Francesco Salvatore1,a
1
Centro di Ingegneria Genetica scarl and Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli "Federico II", 80131 Naples, Italy.
2
Facoltà di Scienze, Università del Molise,
I-86170 Isernia, Italy.
3
Laboratoire de Biochimie et de Génétique
Moléculaire, INSERM U468, Hôpital Henri-Mondor, F-94010
Créteil, France.
4
Dipartimento di Pediatria, Università di Padova,
I-35100 Padua, Italy.
5
Divisione di Pediatria, Ospedale Civile, I-85100
Potenza, Italy.
6
Dipartimento di Pediatria, Università di Napoli
"Federico II", 80131 Naples, Italy.
a Address correspondence to this author at: Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli "Federico II", via S. Pansini 5, 80131 Napoli, Italy. Fax 39 081 746 3650; e-mail salvator{at}unina.it
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Abstract
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Background: The search for the eight most frequent mutations
(i.e., 
F508, G542X, W1282X, N1303K, 1717-1G
A, R553X, 2183AAG,
and I148T) by allele-specific oligonucleotide dot-blot analysis
revealed 78% of 396 cystic fibrosis alleles in Southern Italy. The
observation of frequent haplotypes on the unidentified cystic fibrosis
alleles suggested that a few mutations could account for a large number
of unidentified alleles.
Methods: We screened most of the coding sequence of the cystic
fibrosis transmembrane regulator gene by denaturing gradient gel
electrophoresis to determine the spectrum of these mutations in 68
unrelated cystic fibrosis patients bearing one or both unidentified
mutations.
Results: The screening revealed five mutations, R1158X,
711+1GT, 4016insT, L1065P, and G1244E, each of which had a frequency
of 1.3–1.8% (7% collectively). The 7% increase in the detection
rate (85% vs 78%) reduces by >50% the residual risk of being cystic
fibrosis carriers for couples who had tested negative by molecular
analysis. We therefore designed a second allele-specific
oligonucleotide set to analyze the five mutations. Among the patients
analyzed, one patient homozygous for the L1065P mutation expressed a
mild pulmonary and intestinal form of the disease with pancreatic
insufficiency. Two other patients, homozygous for mutations R1158X and
4016insT, both expressed a severe cystic fibrosis phenotype.
Conclusions: Five cystic fibrosis mutations are peculiar to
patients from Southern Italy. The method described for their analysis
is efficient, inexpensive, and can be semi-automated by use of a
robotic workstation. The results obtained in patients from Southern
Italy may have an impact on laboratories in other countries, given the
large migrations of populations from Southern Italy to other countries
in the last two centuries.© 1999 American Association for
Clinical Chemistry
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Introduction
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Cystic fibrosis
(CF)1
is the most frequent lethal inherited disease among
Caucasians, having a prevalence of ~1 in 2500 newborns. Since the
cloning of the cystic fibrosis transmembrane regulator
(CFTR) gene in 1989 (1)(2)(3), >800
mutations have been detected. A few mutations (i.e., F508, N1303K,
G542X, and R553X) are frequent worldwide; the other mutations are
regional or "private" mutations. Several mutations are peculiar to
specific ethnic groups, i.e., W1282X is frequent among Ashkenazi
(4), T338I is typical among Sardinians
(5), and 2183AAG and R1162X are frequent in Northeastern
Italy (5).
Consequently, to be able to provide the molecular analysis of CF for
diagnostic purposes, laboratories must know the most frequent mutations
in an individual's ethnic group. Mutation mapping is fundamental for
"cascade" screenings of CF families (6), because
screening programs on general populations are limited by the genetic
heterogeneity of the disease.
Similarly, given the migration and genetic mixing that started at the
beginning of this century, awareness of CF mutations "peculiar" to
each ethnic group is necessary to increase the "detection rate" of
CF alleles and to evaluate correctly the residual risk of being a CF
carrier after molecular analysis. Finally, analysis of the clinical
expression of CF patients homozygous for rare mutations can improve our
knowledge of the genotype-phenotype correlation of the disease
(7)(8).
In a "pilot" study, we identified a panel of the most frequent
mutations in CF patients from Southern Italy (9). Eight
mutations led to the identification of ~80% of CF chromosomes. In
the same study, we reported that a few CF haplotypes, determined by the
study of three polymorphisms, were frequently observed on CF
chromosomes carrying an unknown mutation. This observation prompted us
to: (a) search for these unidentified mutations by screening
most of the coding regions and intronic boundaries of the
CFTR gene, using denaturing gradient gel electrophoresis
(DGGE); and to (b) set up a rapid detection method to
analyze the five most frequent mutations identified through the
screening.
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Patients and Methods
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patients
We studied 198 unrelated patients bearing CF, from the Campania
(6 x 106 inhabitants) and Basilicata
(1 x 106 inhabitants) regions of Southern
Italy at least up to the second generation. These patients represent
the whole known CF population of the Basilicata region and 85% of the
known CF population of Campania. The diagnosis, based on clinical data,
was always confirmed by the sweat analysis of chloride (cutoff, 60
mEq/L). All patients were first analyzed for eight CF mutations, i.e.,
F508, N1303K, G542X, W1282X, 1717-1GA, R553X, 2183AAG, and
I148T (9). The 68 patients bearing one or both unknown
mutations (88 CF alleles remained uncharacterized) were screened by
DGGE for most of the coding regions and intronic boundaries of the
CFTR gene (excluding the following exons: 1, 2, 10, 16, and
22). The same patients were also analyzed for four intragenic (i.e.,
IVS8CA, IVS17bTA, IVS17bCA, and M470V) and two extragenic (i.e., XV2c
and KM19) polymorphisms (10)(11)(12)(13)(14)(15).
methods
The eight CF mutations (i.e., F508, N1303K, G542X, W1282X,
1717-1GA, R553X, 2183AAG, and I148T) were identified with a
semi-automated procedure based on a single multiplex PCR amplification
followed by the allele-specific oligonucleotide (ASO) identification we
described previously (9). XV2c and KM19 polymorphisms were
analyzed by PCR amplification followed by TaqI and
PstI digestion, respectively
(10)(11). Polymorphisms IVS8CA (12),
IVS17bTA, and IVS17bCA (13) were identified by PCR
amplification followed by polyacrylamide gel electrophoresis. The DGGE
screening of the CFTR exons was performed as described
previously (14)(15). The M470V polymorphism was
also analyzed by DGGE of exon 10 (14)(15). The
CFTR exons displaying a shift in the DGGE pattern were
sequenced using the Sanger protocol (16) with an automated
procedure in which the four terminator reactions are marked with
fluorescent dideoxynucleotides. The fragments were analyzed with the
373A apparatus of Applied Biosystems (Perkin-Elmer).
aso dot-blot procedure for the analysis of the five "rare" cf
mutations
To analyze routinely the five mutations (i.e., L1065P, 711+1GT,
R1158X, 4016insT, and G1244E) we set up a procedure based on a single
multiplex PCR amplification followed by ASO dot-blot hybridization. The
amplification was performed using the primers shown in Table 1
, in a mixture (50 µL) containing the following: 50 mmol/L
KCl, 10 mmol/L Tris-HCl (pH 8.5), 2.5 nmol of each of the four
deoxyribonucleoside triphosphates, 20 pmol of each of the two primers,
and 1 U of Taq DNA polymerase (Perkin-Elmer Cetus). The PCR conditions
were as follows: 10 PCR cycles of 30 s at 94 °C, 30 s from
65 °C to 56 °C, and 30 s at 72 °C; and 25 PCR cycles of
30 s at 94 °C, 30 s at 55 °C, and 30 s at
72 °C. After the multiplex PCR amplification, the five mutations
were analyzed using ASO dot-blot analysis, using the pair of
oligonucleotides shown in Table 1
for each hybridization.
View this table:
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Table 1. Primers for a multiplex PCR amplification and internal
oligonucleotides for the ASO dot-blot analysis of the five CF mutations
newly identified in Southern
Italy.
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Results
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The DGGE screening allowed us to identify 20 different mutations;
five of these, i.e., R1158X, G1244E, 4016insT, 711+1GT, and L1065P,
were observed with a frequency >1.0% among 396 CF alleles from
Southern Italy.
An example of the multiplex DGGE analysis is shown in Fig. 1
. The homozygote (Fig. 1A
) and heterozygote (Fig. 1B
) patterns
of exon 17b are clearly altered; sequence analysis revealed the L1065P
mutation. The mutation creates a restriction site for both
MnlI and BslI. The incidence of the L1065P
mutation in our series of 396 CF chromosomes was 1.3%. One patient
homozygous for L1065P showed a mild pulmonary and gastrointestinal form
of CF, with a moderate pancreatic insufficiency and mild liver
involvement. The patient (present age, 18 years), born without meconium
ileus, was diagnosed at the age of 1 year; the sweat chloride result
was 93 mEq/L.
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Figure 1. Multiplex DGGE analysis of CFTR exons 11,
17b, and 14b.
(A), an example of an altered homozygote pattern of exon
17b (lane 1) compared with a wild-type pattern
(lane N). (B), two examples of an altered
heterozygote DGGE pattern of exon 17b (lanes 1 and
2) compared with a wild-type pattern (lane
N). The sequence analysis revealed the L1065P mutation.
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Fig. 2
shows an example of the DNA sequences in a homozygote and a
heterozygote patient for the 4016insT mutation (exon 21), compared with
the wild-type sequence. In the heterozygote patient, the
mutation produced an overlapping of the sequence of the mutated allele
with the wild-type allele after nucleotide 4016. The mutation neither
creates nor suppresses restriction sites. In the series of 396 CF
chromosomes, the 4016insT mutation was observed in seven chromosomes
(1.8%), among which was one homozygote patient. The latter, born
without meconium ileus, was diagnosed at the age of 2 years; the sweat
chloride result was 70 mEq/L. The patient expressed a severe
respiratory phenotype with pancreatic insufficiency and cholestasis.
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Figure 2. Sequence analysis of the 4016insT (exon 21) mutation in a
homozygote (bottom) and in an heterozygote
(top) patient.
Wild-type sequence is shown in the middle.
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The R1158X mutation was identified through the altered DGGE pattern of
exon 19, followed by sequence analysis. The mutation suppresses a
restriction site for SfaNI. The incidence of R1158X among CF
chromosomes from our regions was 1.3%. The mutation was identified in
homozygosity in a patient expressing a severe respiratory and
gastrointestinal form of the disease with a moderate pancreatic
insufficiency. The patient, born without meconium ileus, was diagnosed
at the age of 3 months on the basis of pulmonary distress and failure
to thrive. The sweat chloride result was 98 mEq/L. After the diagnosis,
the patient showed a very severe pulmonary expression. At the age of 19
years, he experienced lung transplantation; 1 year later he died. Table 2
shows the main phenotypic features of the three homozygous
patients described.
Mutation 711+1GT (exon 5) was identified in five CF patients
(1.3%), always in compound heterozygosity with other CF mutations; the
mutation cannot be analyzed by restriction enzymes. Finally, the
G1244EGA mutation was identified through the DGGE screening of exon
20 followed by DNA sequence analysis in five chromosomes from our
series (1.3%). The mutation suppresses a restriction site for
MboII. Table 3
shows the haplotype associated with the five mutations for six
polymorphisms.
An example of the improved ASO dot-blot procedure used to analyze the
five mutations is shown in Fig. 3
for R1158X (Fig. 3A
), L1065P (Fig. 3B
), 4016insT (Fig. 3C
),
711+1GT (Fig. 3D
), and G1244EGA (Fig. 3E
). The ASO dot-blot
clearly distinguishes homozygous, heterozygous, and wild-type subjects.
The ASO analysis confirmed the presence of the five mutations in all of
the patients identified by DGGE and gave negative results for a control
group of 50 CF patients bearing other mutations.
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Figure 3. ASO dot-blot analysis of five CF mutations.
Mutations were hybridized with the wild-type oligonucleotide
(left) and with the mutated oligonucleotide
(right). The numbers 1 to
4 refer to single individuals for each of the tested
mutations. For R1158X (A) and L1065P (B),
1 and 2 are heterozygotes for the
mutation, 3 is a homozygote for the mutation, and
4 is a healthy control. For 4016insT (C),
1 and 2 are healthy controls,
3 is a homozygote for the mutation, and 4
is a heterozygote for the mutation. For 711+1GT (D)
and G1244E (E), 1 and 2
are healthy controls, and 3 and 4 are
heterozygotes for the mutation.
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Discussion
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The DGGE screening of the CFTR coding regions revealed
five mutations frequent in Southern Italy. These mutations, although
detected sporadically in other population cohorts, seem to be
peculiar to Southern Italy. In fact, none of them was described
during the screening of the whole coding region of the CFTR
gene in a German population (17); only 711+1G was
reported in <1.0% of 600 French CF patients (18), and
4016insT was reported on a single CF allele in a Welsh subject
(19). Mutation R1158X has a frequency of 0.8% in Greece
(20); only one allele bearing the mutation has been
described in Spain (21), and two alleles have been described
in France (22).
Our data confirm some genetic differences in the CF mutations between
Southern and Northern Italy (5). Several mutations highly
frequent in Northern Italy (R1162X and 711+5GA) have not been
detected in Southern Italy, neither has T338I, which is peculiar to
Sardinia (5). On the contrary, none of the five mutations
described in the present study has been identified during screening of
the whole coding region of the CFTR gene in Northeastern
Italy (5)(23). Mutation L1065P has been reported
in ~3% of CF chromosomes (24) from Sicily (Southern
Italy).
The concordant haplotype obtained for all five mutations among our
patients indicates the recent origin of these mutations (25)
and excludes a recurrent origin. Different haplotypes have been
described only for R1158X, which suggests a recurrent origin
(19) or rather, several recombinant events. Mutation R1158X,
which has a frequency of 0.8% in the Greek population
(18)(20), could have been introduced into
Southern Italy by the ancient Greeks who colonized this geographic
area. More recently, the mutation could have spread from Southern to
Northern Italy and Europe. This hypothesis is reinforced by the
analysis of the polymorphism associated to the CFTR gene. We
observed the same haplotype in all five alleles bearing the R1158X
mutation, i.e., 1, 2, 6, 7, 17 (XV2c, KM19, IVS8CA, IVS17bTA, and
IVS17bCA). The chromosome described in Northern Italy has the same
haplotype 1,2 for XV2c and KM19 (26). Of the two chromosomes
bearing the R1158X mutation described in France, one showed haplotype
2, 2, 16, 7, 17, and the other showed haplotype 2, 2, 16, 45, 13,
suggesting the recurrent origin of the mutation or a recombinant event
(18)(22). The only difference between the first
French patient and the subjects in our study is the XV2c
dimorphic locus, i.e., the one most distant from the CFTR
gene. A recombinant event between the XV2c locus and the
CFTR gene is not inconceivable. The mutation detected in the
second French patient could derive from a second, more recent,
recombinant event within the CFTR gene, between exons 8 and
19.
The mutation detection rate of 85% of CF alleles with the analysis of
only 13 CF mutations is surprising considering the genetic
heterogeneity of the population of Southern Italy (5). In
Spain (21), >40 CF mutations identify ~78% of CF
alleles, whereas in France, 47 different mutations identify 86% of CF
chromosomes (18). The presence of several mutations peculiar
to Southern Italy could depend on the high rate of consanguinity among
CF carriers from our regions, as is suggested by the high incidence of
CF patients homozygous for rare CF mutations.
The possibility of identifying 85% of CF chromosomes through a rapid
molecular analysis allows us to estimate the residual risk of being
carrier or of having a CF-affected child in couples for whom molecular
analysis for CF was negative (see Table 4
). In fact, starting from an a priori risk of being a CF carrier
of 1:25 (4%), the negative molecular analysis for CF mutations reduces
the risk of being a carrier to 1:112.5 (0.88%) if the panel of
mutations has a detection rate of 78% (4% of an a priori risk x
22% risk of carrying an unidentified mutation). The risk becomes 1:167
(0.60%) if the panel of mutations has a detection rate of 85% (4% of
an a priori risk x 15% of risk of carrying an unidentified
mutation). Consequently, the risk of having a CF child for couples for
whom molecular analysis for the CF mutation was negative (see Table 4
)
is 1:50 625 if the detection rate of the mutations analyzed is 78%
(1/112.5 x 1/112.5 x 1/4) and becomes 1:111 556
(1/167 x 1/167 x 1/4) at a detection rate of 85%.
Similarly, if one member of the couple is a CF carrier and the other is
negative by molecular analysis, the risk of having a child affected by
CF is 1:450 (1/112.5 x 1/4) if the test has a detection rate of
78% and 1:668 (1/167 x 1/4) if the detection rate is 85%.
Consequently, on the basis of these findings, cascade screening can be
planned in the families of CF patients (6). The ASO dot-blot
procedure described is very easy and efficient because it is based on a
single multiplex amplification, which can be semi-automated with the
use of a robotic workstation (9), allowing the analysis of
large series of DNA samples. Using this procedure to analyze eight CF
mutations, we have made >1000 molecular CF diagnoses of homozygote or
heterozygote CF subjects over the last 4 years. Similarly, the ASO
analysis of the five new mutations gave unequivocal results in all CF
patients that had been characterized by DGGE and sequenced previously
and in all 100 control alleles. Furthermore, we now routinely screen CF
patients for the 13 mutations and have identified several other alleles
(data not shown) bearing the five new mutations in both CF patients and
subjects affected by congenital bilateral agenesis of the vasa
deferentes.
This study confirms that polymorphism analysis is a valid procedure
with which to asses the genetic heterogeneity of a population with
respect to a specific gene, and to evaluate the recurrent origin of a
mutation (22)(25)(27). Similarly, we
confirm the very high potential of DGGE as a tool for screening large
genes for unknown mutations (14)(15).
The phenotype analysis in the three homozygous patients confirmed, in
this case, the phenotype predicted by the molecular analysis. The
patient homozygous for R1158X (a nonsense mutation) and the patient
homozygous for 4016insT (a frameshift mutation) showed very severe
expression of CF (because the synthesis of the wild-type protein
was suppressed) compared with the patient homozygous for L1065P, a
missense mutation associated with the synthesis of a protein with a
single amino acid substitution.
In conclusion, the five so-called rare CF mutations described here
seem to be peculiar to Southern Italy. Their analysis, added to the
analysis of the eight most frequent CF mutations (9), allows
us to detect 85% of CF alleles from these regions and to calculate a
very low residual risk of having a child affected by CF for couples for
whom molecular analysis of the 13 mutations was negative. The use of an
additional multiplex PCR associated with a semi-automated ASO dot-blot
analysis represents a step forward in the strategy to eradicate the
disease from the regions studied. The identification of several rare
homozygotic mutations adds useful information to the genotype-phenotype
correlation for this disease. Lastly, the results obtained in CF
patients from Southern Italy may have an impact on laboratories in
other countries, given the large migrations of populations from
Southern Italy to other regions of Italy (i.e., Northern Italy) or to
countries (i.e., Switzerland, Germany, and North and South America) in
the last two centuries.
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Acknowledgments
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The work was funded in part by grants from the Ministero
dell'Università e Ricerca Scientifica e Tecnologica
(Grant PRIN 1997), Rome, Italy; Regione Campania (Grant LR 41/94),
Consiglio Nazionale delle Ricerche (P.F. Biotecnologie), and the
Ministero della Sanità (Rome).
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Footnotes
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1 Nonstandard abbreviations: CF, cystic fibrosis; CFTR, cystic fibrosis conductance regulator; DGGE, denaturing gradient gel electrophoresis; and ASO, allele-specific oligonucleotide. 
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Abstract
Introduction
