Bombieri C. and Pignatti P.F.
Genetic Testing (2001) 5:229-233.
CF mutation testing in Italy
Cristina Bombieri and Pier Franco Pignatti
Section of Biology and Genetics, DMIBG, University of Verona
Running title: CF mutation testing in Italy
Corresponding author:
Prof. Pier Franco Pignatti
Sezione di Biologia e Genetica; DMIBG; Università di Verona,
Strada Le Grazie, 8; I-37134 Verona; ITALY.
Phone: +39-045-584602; Fax: +39-045-8027180
E-mail: PierFranco.Pignatti@univr.it
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Bombieri C. and Pignatti P.F.
Abstract
In Italy, CF mutation frequency differences have been observed in different
regions. In the North Eastern Veneto and Trentino Alto Adige regions, a
complete CFTR gene screening in CF patients detected through a newborn
screening program has identified about 90% of the mutations. In these two
regions, current detection rate of a CF screening panel containing 16 among
the most common mutations is 86.6%. CF mutations in some other Italian
regions have not been so thoroughly analysed. Available data indicate that a
more general national screening panel comprising 31 mutations may detect
about 75% of CF mutations in Italy.
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Bombieri C. and Pignatti P.F.
Introduction
A law of 1993 dictates dispositions for the prevention and care of CF in Italy.
This includes primary prevention provisions, and the institution of CF Centers
for each of the 20 Italian regions, or for groups of the smaller regions. CF gene
mutation analysis has been performed to different degrees in different parts of
the country. No national neonatal or population screening program is
operative. Some regions have been enacting a CF neonatal screening program
for a long time, some for a more limited period. Neonatal screening is based
on immunoreactive trypsinogen test followed by genetic analysis (Castellani et
al., 1997). No region has yet launched a generalised population screening
program. Some regions have implemented a cascade screening program in at
risk families (Borgo et al., 1999). A few research groups have proposed
general population screening pilot programs, but published data are not yet
available. In this variegated, and rapidly evolving, picture we will report some
data on 1) a long running mutation testing program in two North Eastern
Italian regions, and 2) a national mutation analysis summary.
CF mutations in Veneto and Trentino-Alto Adige regions.
The analysis of the complete coding sequence of the CFTR gene was
performed in a cohort of CF patients from North-Eastern Italy (Bonizzato et
al., 1995).
The study cohort included all CF patients born from 10.1.1973 to 12.31.1981
in two regions: Veneto and Trentino Alto Adige. A total of 113 CF patients
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Bombieri C. and Pignatti P.F.
was screened. Methods used for mutation analysis were: RNA SSCP for the
first 12 gene exons, DNA sequencing for exon 13, and Denaturing Gradient
Gel Electrophoresis (DGGE) for exons 14-24. When two mutations were
identified in a patient, CF alleles were not further analysed. Twenty two
different mutations were identified in 203 alleles out of a total of 225 screened
alleles (90.2% detection rate), as detailed in Table 1.
CF mutation panel for the Veneto and Trentino-Alto Adige regions
The currently used panel of mutations includes all the mutations shown in
Table 1, with a frequency above 1%, and a few other mutations as described
below. Two of the four mutations detected twice in 225 alleles (0.9%) were
included. Mutation 3132 del TG was excluded as it was difficult to detect
unambiguously with a simple test, and mutation 2790-2 AG was excluded
as it was found in homozygosity in one patient only. Three of the 7 mutations
found only once in the 225 alleles tested were included, as they were present
in the mutation detection kits available: R347P, G541D, 3849+10 Kb CT.
The final mutation panel used therefore includes 16 mutations, as indicated in
Table 2. The cumulative frequency of these mutations is approximately
86.67%.
Italian CF mutations
The relative frequency of 60 CF mutations was investigated in several Italian
regions, and the geographical distribution of the 12 most frequent CF
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Bombieri C. and Pignatti P.F.
mutations screened in 3492 CF alleles was determined (Rendine et al., 1997).
In this study, the geographic origin of the alleles was established by assessing
the birthplace of the four grandparents, and assigning each allele, or fraction of
it if the grandparents were born in different places, to one of the Italian
regions. Several differences in mutation type and frequency were observed,
due to limited population migration within Italy. The Northern regions were
well differentiated from the Central-Southern regions. Within Northern
regions the Western and Eastern mutation frequency distribution was
remarkably distinct, the Western part being characterised by mutation 1717-1
GA, and the Eastern part by mutations R1162X, 2183 AAG, and 711+5
GA. Moreover, the island of Sardinia showed the presence of mutation
T338I, which seemed absent from other European CF alleles. Some regions
had a low number of chromosomes tested. The list of the 31 mutations, from
the collaborative Italian study cited above, with a frequency of at least 0.1% is
reported in Table 3. The cumulative frequency of these mutations is
approximately 75.69%. As immigration into Italy is increasing in recent years,
it is possible that the Italian CF mutation spectrum might change in the near
future.
CF related risk after mutation analysis.
Given the detection rates described above of 86.67% for the Veneto and
Trentino Alto Adige regions, or of 75.69% for the Italian population as a
whole, respectively, the residual risk of carrying a mutation or of having an
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Bombieri C. and Pignatti P.F.
affected child after a negative mutation test, can be calculated as indicated in
Table 4.
The calculations are based on a CF incidence of 1/2705, derived from the most
recent neonatal screening program data for the Veneto and Trentino Alto
Adige regions from 1.1.1993 to 12.31.2000: 146 cases were detected out of
394,961 screened (Castellani et al., 2001). CF allele frequency is therefore
0.019.
Potential for the detection of couples at risk by mutation analysis.
Given the detection rates above mentioned for the Veneto and Trentino Alto
Adige regions or for Italy as a whole, respectively, the potential for the
detection of couples at risk of having an affected child was calculated, and it is
given in Table 5. Also, the detection rate of 95% is added, a level
recommended for population-based screening by a National Institutes of
Health workshop on population screening for the CF gene (Workshop on
population screening for the cystic fibrosis gene, 1990).
Discussion.
The frequency of F508 is less in Italy than in other North and Central
European regions, and is similar to other Mediterranean area regions (Estivill
et al, 1997). Within Italy, notable differences in mutation type and frequency
are observed. The data here reported indicate the present procedure for CF
mutation analysis in two North-Eastern Italian regions, and a more general list
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Bombieri C. and Pignatti P.F.
of Italian population mutations which may be suggested for screening, when
no regional data are available. No commercial kit is available based on this
list. It would be desirable to have a more detailed knowledge of Italian CF
mutations from a new national study involving a complete gene screening in a
large number of CF patients from all Italian regions, and from immigrant
groups. This would allow all regions to screen most efficiently. A
collaborative project may be initiated soon in this regard. The analysis of other
mutations should be considered, depending on population origin of the
individual to be tested.
The availability of commercial kits makes CF mutation analysis often more
convenient. But the sheer number of mutations present in the kits is not a good
parameter to evaluate their efficiency. Sometimes these kits, having been
developed for other populations, may miss frequent mutations to be tested in
the Italian population: in this case it is necessary to add population specific
mutation tests. It is therefore recommended to refer to mutation frequency in
the population to be tested, and include the most common mutations in the
population.
Caution should be used when quoting test results because of the possibility of
errors, as highlighted in the annual CF quality control tests run by the
European Union CF Concerted Action (Dequeker and Cassiman 2000).
Following these quality control results, recommendations for quality
improvement in genetic testing for CF have been proposed (Dequeker et al.,
2000).
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Bombieri C. and Pignatti P.F.
Finally, it should be remembered that genetic testing for CF mutations should
always be done in collaboration with an expert clinical geneticist for providing
informations on risk assessment, therapeutic and reproductive options, and the
comparative consideration of CF related diseases in CFTR mutation carriers
(Report of a joint WHO/CF(M)A/ECFTN meeting, 2001).
Acknowledgements
The Verona CF Center (G. Mastella, G. Borgo, C. Castellani) and the
European
Union
CF
Thematic
Network,
web
site
http://www.med.kuleuven.ac.be/cme/cf/cfnetwork.htm (J.J. Cassiman and E.
Dequeker) for inspiration and stimulation; Roche Molecular Systems (R.
Saiki) for making available CF testing reverse blot strips for research
purposes; the Italian Ministry of Health, CF Project, law 548/93, and the
Italian Ministry of University and Research for funding research on CF
mutation classification and on genotype-phenotype correlations, respectively.
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Bombieri C. and Pignatti P.F.
References
Bonizzato A, Bisceglia L, Marigo C, Nicolis E, Bombieri C, Castellani C,
Borgo G,et al (1995) Analysis of the complete coding region of the CFTR
gene in a cohort of CF patients from North-Easter Italy: identification of
90% of the mutations. Hum Genet 95: 397-402.
Borgo G, Castellani C, Bonizzato A, rolfini R, Altieri S, Zanolla L, Mastella
G. (1999) Carrier testing program in a high-risk cystic fibrosis population
from North Eastern italy . Community Genet .2:82-90 .
Casals T., Bassas L., Egozcue S., Ramos M.D., Gimenez J., Segura A., Garcia
F., Carrera M., Larriba S., Sarquella J., Estivill X. (2000) Heterogeneity
for mutations in the CFTR gene and clinical correlations in patients with
congenital absence of the vas deferens. Hum. Repr.15:1476-83.
Castellani C, Bonizzato A, Cabrini G, Mastella G. (1997). Newborn screening
strategy for cystic fibrosis: a field study in an area with high allelic
heterogeneity. 86: 497-502.
Castellani C., (2001). Bollettino dello Screening Neonatale per Fibrosi cistica.
A cura del Servizio Clinico di Screening Neonatale per Fibrosi Cistica.
Centro Regionale Veneto di Ricerca Prevenzione e Cura per la Fibrosi
cistica, Azienda Ospedaliera di Verona. Num.5, Marzo 2001.
Dequeker E. and Cassiman J.J. (2000). Genetic testing and quality control in
diagnostic laboratories. Nat. Genet. 25:259-260.
Dequeker E., Cuppens H., Dodge J., Estivill X., Goossens M., Pignatti P.F.,
Scheffer H., Schwarz M., Tummler B., Cassiman J.J. (2000)
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Recommendations for quality improvement in genetic testing for cystic
fibrosis European Concerted Action on Cystic Fibrosis. Eur. J. Hum.
Genet. 8, suppl.2: S2-S24.
Dork T., Dworniczak B., Aulehla-Scholz C., Wieczorek D., Bohm I.,
Mayerova A., Seydewitz H. H., Nieschlag E., Meschede D., Horst J.,
Pander H. J., Sperling H., Ratjen F., Passarge E., Schmidtke J. and
Stuhrmanhn M. (1997). Distinct spectrum of CFTR gene mutations in
congenital absence of vas deferens. Hum. Genet. 100:365-377.
Estivill X., Bancells C., Ramos C., and the Biomed CF Mutation Analysis
Consortium (1997). Geographic distribution and regional origin of 272
Cystic Fibrosis mutations in European populations. Hum. Mut. 10:135154.
Garnerone S., Arduino C., Fontana D., Rolle L., Manenti M., Bombieri C.,
Benetazzo M.G., Pignatti P.F., Carbonara A.O. (1995) Analysis of a polyT variant intron 8 of the CFTR gene in Italian patients with congenital
Bilateral Absence of the Vas Deferens (CBAVD). Am. J. Hum. Genet. 57,
Suppl. 4: abs.1958, A337.
Grody W.W., Cutting G.R., Klinger K.W., Richards C.S., Watson M.S.,
Desnicl R.J. (Subcommittee on Cystic fibrosis Screening, Accreditation of
Genetic Services Committee, ACMG) (2001). Laboratory standards and
guidelines for population-based Cystic Fibrosis Carrier Screening.
Genetics in Medicine 3: 149-154.
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Lemna W.K. et al., Mutations analysis for heterozygote detection and the
prenatal diagnosis of Cystic Fibrosis. New Eng. J. Med. 322: 291-296;
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Mak V., Zielenski J., Tsui L.C., Durie P., Zini A., Martin S., Longley T.B.,
Jarvi K.A. (1999) Proportion of cystic fibrosis gene mutations not detected
by routine testing in men with obstructive azoospermia. JAMA
281.23:2217-2224.
Pradal U., Castellani C., Delmarco A., Mastella G. (1998) Nasal potential
Difference in congenital Bilateral Absence of Vas Deferens. Am. J. Respir.
Crit. Care Med. 158:896-901.
Rendine S, Calafell F, Capello N, Gagliardini R, Caramia G, Rigillo N,
Silvetti M, et al (1997) Genetic history of cystic fibrosis mutations in Italy.
I. Regional Distribution. Ann Hum Genet 61: 411-424.
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3, 2001. Classification of cystic fibrosis and related disorders. World
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from the national Institutes of Health workshop on population screening
for the Cystic Fibrosis gene. New Eng. J. Med. 323: 70-71; 1990.
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Bombieri C. and Pignatti P.F.
Table 1: CF mutations identified in two Italian regions (Veneto and
Trentino Alto Adige)
Mutation
F508
R1162X
2183 AAG
N1303K
G542X
711+5 GA
1717-1 GA
G85E
R553X
2789+5 GA
Q552X
621+1 GT
W1282X
R347P
G551D
3849+10 Kb CT
3132 del TG
2790-2 AG
457 TATG
1717-8 GA
R709X
1898+3 AG
Total
N. alleles Frequency Cumulative
with
%
Frequency %
mutation
107
47.6
47.56
22
9.8
57.33
21
9.3
66.67
9
4.0
70.67
6
2.7
73.33
6
2.7
76.00
5
2.2
78.22
3
1.3
79.56
3
1.3
80.89
3
1.3
82.22
3
1.3
83.56
2
0.9
84.44
2
0.9
85.33
1
0.4
85.77
1
0.4
86.21
1
0.4
86.67*
2
2
1
1
1
1
_______
203
0.9
0.9
0.4
0.4
0.4
0.4
_____
90.22
87.54
88.43
88.87
89.31
89.75
90.22
* 16 mutation screening panel.
Numbers refer to CFTR gene alleles carrying the specified mutation, over total
tested alleles (N = 225) from the affected subjects CF cohort, as indicated in
the text (From Bonizzato et al., 1995).
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Bombieri C. and Pignatti P.F.
Table 2: CF mutation panel (Veneto and Trentino Alto Adige) Italian
regions)


F508
R1162X
2183 AAG
N1303K
G542X
711+5 GA
1717-1 GA
G85E
R553X
2789+5 GA
Q552X
621+1 GT
W1282X
R347P
G551D
3849+10 Kb CT
Note: Contrary to what suggested for the US population (Grody et al., 2001),
R117H mutation (and its reflex IVS8-5T test) is not included in the panel as it
is not commonly found in the Italian CF population (Bonizzato et al., 1995;
Estivill et al., 1997; Rendine et al., 1997). R117H is searched only in male
infertility because of literature data in other populations (Dork et al, 1995;
Casals et al., 2000; Mak et al., 1999) or in the Italian population (Garnerone et
al., 1995; Pradal et al., 1998).
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Table 3 : CF gene mutations in Italy
Mutation
F508
N1303K
G542X
2183 AAG
R1162X
1717-1 GA
W1282X
R553X
T338I
R347P
711+5 GA
G85E
621+1 GT
R334W
R352Q
S549N
R347H
L1077P
R1158X
541del C
R1066H
E585X
Q552X
D1152H
2790-2 AG
3132 del TG
3667ins 4
I507
1898+3 AG
G1244E
1784 del G
N. alleles
screened
3442
3056
3082
2596
2580
2892
2600
2882
2306
2642
2454
1980
2594
2366
2112
2118
2184
1840
1878
1884
1918
1922
2172
1824
1862
1862
1876
1914
1920
1960
2052
Frequency Cumulative
%
Frequency %
51.07
51.07
4.84
55.91
4.83
60.75
2.66
63.41
2.42
65.83
2.11
67.94
1.23
69.17
1.15
70.31
0.69
71.01
0.61
71.61
0.57
72.18
0.40
72.59
0.39
72.97
0.30
73.27
0.24
73.50
0.24
73.74
0.18
73.92
0.16
74.09
0.16
74.25
0.16
74.40
0.16
74.56
0.16
74.72
0.14
74.86
0.11
74.97
0.11
75.07
0.11
75.18
0.11
75.29
0.10
75.39
0.10
75.50
0.10
75.60
0.10
75.69
(From Rendine et al., 1997)
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Table 4: CF-related risk after mutation analysis for carrier status in Italy
% of CF
mutations
detectable
Carrier risk for
persons with
negative test
Risk of CF in offspring
for couples tested
One parent
positive
Neither parent
positive
ccr
¼ ccr
¼ (ccr)2
0
1/26.5
n.a.
1/2705*
75.69
1/106
1/424
1/44,944
86.67
1/193
1/772
1/148,996
ccr = 2pq(1-d) / 2pq(1-d)+p2 (Lemna et al., 1990):
75.69% : Italian cumulative frequency (see table 3)
86.67%: Veneto and Trentino Alto Adige frequency (see table1)
n.a. = not applicable
* 1/2705: CF frequency in North-East Italy from carrier screening as described
in the text.
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Bombieri C. and Pignatti P.F.
Table 5: Potential for the detection of the couples at risk with the use of
population based screening for carriers in Italy
% of CF
mutations
detectable
Detection of carrier status
Both parents
One parent
Neither parent
d
d2
2d(1-d)
(1-d)2
75.69
57.3 %
36.8 %
5.9 %
86.67
75.1 %
23.1 %
1.8 %
95
90.3 %
9.5 %
0.3 %
75.69% : Italian cumulative frequency (see table 3)
86.67%: Veneto and Trentino Alto Adige frequency (see table 1)
16