Preconceptional identification of cystic fibrosis carriers in the Sardinian population: A pilot screening program.
ABSTRACT In Sardinia the mutational spectrum of CFTR gene is well defined. A mutation detection rate of 94% can be achieved by screening for 15 CFTR mutations with a frequency higher than 0.5%. The efficiency of this molecular test suggests that Sardinians may represent a suitable population for a preconceptional screening.
Five hundred couples of Sardinia descent were screened for 38 mutations using a semi-automated reverse-dot blot and PCR-gel electrophoresis assays. This mutation panel included the 15 most frequent CF alleles in Sardinia.
We identified 38 CF carriers, revealing an overall frequency of 1/25 (4%). The most common CF allele was the p.Thr338Ile (T338I) (65%), followed by the p.Phe508del (F508del) (22.5%). We also identified one couple at risk and an asymptomatic female homozygote for the p.Thr338Ile allele.
In spite of the low number of the couples tested, the results herein reported demonstrate the efficacy and efficiency of the preconceptional screening program and the high participation rate of the Sardinian population (99%).
- SourceAvailable from: Iris Lavon[show abstract] [hide abstract]
ABSTRACT: To determine the distribution and frequency of cystic fibrosis (CF) mutations in the Israeli population, we have screened 96 patients for 11 relatively common mutations. Five mutations--delta F508, G542X, W1282X, N1303K, and 3849 + 10kb C-->T--were found to account for 97% of the CF alleles in the Ashkenazi Jews. In contrast, of the 11 mutations tested, only delta F508 was detected in Jewish patients of Sephardic or Oriental origin, accounting for 43% of the CF alleles. Four mutations--delta F508, G542X, W1282X, and N1303K--accounted for 55% of the CF alleles in Arab patients. In a pilot screening study, a random sample of 424 Ashkenazi individuals was analyzed for three mutations--delta F508, W1282X, and G542X. Thirteen individuals were detected as heterozygotes (six for delta F508 and seven for W1282X), predicting a heterozygote frequency of 1:29. This is similar to the frequency of carriers in the Caucasian population of northern European ancestry. On the basis of these data, the Ashkenazi population is considered to be a candidate for CF heterozygote screening.The American Journal of Human Genetics 11/1992; 51(5):951-6. · 11.20 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: We have analyzed 640 Spanish cystic fibrosis (CF) families for mutations in the CFTR gene by direct mutation analysis, microsatellite haplotypes, denaturing gradient gel electrophoresis, single-strand conformation analysis and direct sequencing. Seventy-five mutations account for 90.2% of CF chromosomes. Among these we have detected seven novel CFTR mutations, including four missense (G85V, T582R, R851L and F1074L), two nonsense (E692X and Q1281X) and one splice site mutation (711+3A-->T). Three variants, two in intronic regions (406-112A/T and 3850-129T/C) and one in the coding region (741C/T) were also identified. Mutations G85V, T582R, R851L, E692X and Q1281X are severe, with lung and pancreatic involvement; 711+3A-->T could be responsible for a pancreatic sufficiency/insufficiency variable phenotype; and F1074L was associated with a mild phenotype. These data demonstrate the highest molecular heterogeneity reported so far in CF, indicating that a wide mutation screening is necessary to characterize 90% of the Spanish CF alleles.Human Genetics 12/1997; 101(3):365-70. · 4.63 Impact Factor
Preconceptional identification of cystic fibrosis carriers in the Sardinian
population: A pilot screening program
Alessandra Coianaa,1, Valeria Faa'b,1, Daniela Cartaa, Rosalba Pudduc,
Antonio Caob, Maria Cristina Rosatellia,⁎
aDipartimento di Scienze Biomediche e Biotecnologie, Università degli Studi di Cagliari, Italy. Via Jenner 09121 Cagliari, Italy
bIstituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Cittadella Universitaria di Monserrato, SS 554 Km 4500,
09042 Monserrato, Cagliari, Italy
cServizio di Screening e Consulenza Genetica, Ospedale Regionale per le Microcitemie, Cagliari Italy. Via Jenner 09121 Cagliari, Italy
Received 12 January 2011; received in revised form 23 February 2011; accepted 24 February 2011
Background: In Sardinia the mutational spectrum of CFTR gene is well defined. A mutation detection rate of 94% can be achieved by screening
for 15 CFTR mutations with a frequency higher than 0.5%. The efficiency of this molecular test suggests that Sardinians may represent a suitable
population for a preconceptional screening.
Methods: Five hundred couples of Sardinia descent were screened for 38 mutations using a semi-automated reverse-dot blot and PCR-gel
electrophoresis assays. This mutation panel included the 15 most frequent CF alleles in Sardinia.
Results: We identified 38 CF carriers, revealing an overall frequency of 1/25 (4%). The most common CF allele was the p.Thr338Ile (T338I) (65%),
followed by the p.Phe508del (F508del) (22.5%). We also identified one couple at risk and an asymptomatic female homozygote for the p.Thr338Ile allele.
Conclusions: In spite of the low number of the couples tested, the results herein reported demonstrate the efficacy and efficiency of the
preconceptional screening program and the high participation rate of the Sardinian population (99%).
© 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.
Keywords: Cystic fibrosis; Preconceptional screening; Genetic counseling; Genetic testing
The prerequisites suggested for a prospective approach for
control of recessive genetic disease through heterozygote
screening include the following: high severity of the disease,
high prevalence in a defined population, the availability of
simple and accurate methods for carrier identification, as well as
of genetic counseling services to inform individuals and couples
at risk about the disease and the reproductive choices.
Differences in disease frequency can play a role in decisions
on screening for specific disorders, and countries with similar
disease prevalence often reach different decisions about the
appropriateness of it.
In the early 1990s, most genetic screening programs were at
the pilot stage, with a few notable exceptions. For example,
screening programs had been established for β-thalassemias 
and Tay–Sachs disease  in certain sub-populations. Since
then policy developments, advances in scientific understanding
and technological developments have led to the introduction of
screening tests for several other pathologies. Currently, cystic
fibrosis (CF, MIM 219700) represents one of the most frequent
diseases worldwide object of screening.
More than 1700 sequence variations have been identified in
the CFTR gene (MIM 602421), causing both cystic fibrosis and
⁎Corresponding author at: Dipartimento di Scienze Biomediche e Biotecnologie,
Laboratorio di Genetica Molecolare, via Jenner, 09121 Cagliari, Italy. Tel.: +39
0706095653; fax: +39 0706095654.
E-mail address: firstname.lastname@example.org (M.C. Rosatelli).
1These two authors contributed equally to this work.
1569-1993/$ - see front matter © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.
Journal of Cystic Fibrosis 10 (2011) 207–211
other associated phenotypes, which are collectively referred to
as CFTR-related disorders (CFTR-RD) (http://www.genet.
sickkids.on.ca/cftr/). These mutations often have geographic
or population variations in frequency. Because CF carriers are
asymptomatic, they can only be identified by the detection of
mutations in the CFTR gene.
The US National Institutes of Health, the American College
of Obstetricians and Gynecologists, and the American College
of Medical Genetics have recommended widespread CF genetic
screening of couples that are either planning a pregnancy or in
the early stages of pregnancy [3–5]. The different mutational
spectra associated with each specific populations cause
problems with the identification of CF carriers that complicate
the screening process as well as pre- and post-test genetic
counseling. For example, only 6 or 7 mutations account for over
95% of all CF chromosomes in Ashkenazi Jews , and 15–32
mutations are responsible for over 85% of all CF chromosomes
in most northern Europeans. In southern European populations,
however, up to 90 or more mutations need to be screened to
obtain a 90% mutation detection rate in CF patients [7,8].
This extensive heterogeneity in the distribution of CFTR
gene mutations in European populations makes the goal of a
mutation detection rate of 92–93% very hard to achieve. In
general, such screens should be designed to have high
sensitivity and the ability to detect all CF-causing mutations
with a frequency greater than 1% in the local population.
In Italy, the mean detection rate of the Italian CF alleles is
about 86% [9–11].
We recently reported the molecular characterization of the
large majority of Sardinian CF alleles (175/176) . A
mutation detection rate of 94% can be achieved by screening for
15 mutations with a frequency higher than 0.5% in the Sardinian
population. The high diagnostic efficiency of the molecular
CFTR test suggests that Sardinians may represent a suitable
population for preconceptional CF screening.
In this study we report the results of a pilot preconceptional
carrier screening of CF in Sardinian couples, with no family
history of CF. The aim was to establish the a priori risk of CF
carrier status in the Sardinian population and to offer a reliable
screening test to couples planning a pregnancy allowing them to
make informed decisions. Furthermore, Sardinian population,
intensively involved and sensitized in the carrier screening of
β-thalassemia, carried out successfully since the late 1970s, has
facilitated the organization of this survey.
2. Patients and methods
A total of 505 couples were enrolled from a group of people
who had voluntarily requested hematological screening for
β-thalassemia at the Genetic Screening and Counseling Service,
Thalassemic Regional Hospital, Cagliari.
All couples were of Sardinian descent, either planning a
pregnancy or in the early stage of pregnancy (3–10 weeks),
without family history of CF or CFTR-related disorders. The
individuals ranged in age from 18 to 54 years.
Five hundred couples, for a total of 1000 individuals, agreed
to participate in the molecular screening.
Prior to the collection of blood samples, the couples were
informed about the availability of the pilot study and then asked
to fill out an informed consent. The informed consent, approved
by the Local Ethic Committee of the Hospital, provided
information about the clinical aspects of CF, carrier screening
tests, the risk of having an affected child if both partners were
found to be carriers, and the consequences of a negative test
Participants were invited to fill in a self-administered
questionnaire in order to assess impact and understanding of
the CF screening among them. The questionnaire included
multiple choice questions on their knowledge of CF and their
reproductive intentions within the next 2 years.
DNA was extracted from 400 μl of peripheral blood using
Biomek NX (Beckman Coulter, California, USA) according to
The samples were screened for the 15 most common
Sardinian CF mutations , allowing an allele detection rate
of 94%. The Cystic Fibrosis Nuclear Laser kit (Nuclear Laser
Medicine, Italy), which screens by a reverse dot-blot assay 36
CF Italian mutations at once, included 13 of the 15 common
Sardinian mutations. Hybridization was performed using
ProfiBlot T48 (Tecan, Switzerland) according to the manufac-
The remainingtwoSardinian mutations,the c.54-5811_164+
2186del8108ins182 (exon 2 deletion) and the p.Asn287-
LysfsX19 (991del5) were detected by PAGE analysis .
Four patients with anomalous hybridization signal required
an extensive molecular analysis at both DNA and RNA levels.
Sequencing analysis of exon 10 and its flanking regions was
performed using the Big Dye Terminator cycle sequencing kit
(Applied Biosystems, California, USA) and the sequencing
reactions were performed on an ABI PRISM 3130XL (Applied
RNA was extracted from nasal epithelial cells collected
using cyto-brush from patients and a non-CF control subject.
cDNA synthesis was performed using the High Capacity cDNA
Archive kit (Applied Biosystems) according to the manufac-
turer's instructions. The cDNA spanning exons 8–12 was
amplified, and the resulting PCR products were resolved on 2%
agarose gel, and sequenced as described above.
If one of the partners in a couple was diagnosed as a carrier,
the other, testing negative for the 94% of mutations identified in
this screen, was further analyzed by a multiple ligation probe
amplification assay (MRC-Holland, the Netherlands), to
exclude the possibility of large CFTR gene rearrangements.
2.3. Mutation nomenclature
The gene variants at the protein level were named as
recommended in the Human Genome Variation Society
(HGVS) web page (http://www.hgvs.org/mutnomen/). For
208A. Coiana et al. / Journal of Cystic Fibrosis 10 (2011) 207–211
variation described at the nucleotide level, the A of the ATG
translation start codon was numbered as +133 in accordance
with the current CFTR gene numbering based on cDNA
sequence (GenBank NM_000492.3) and on the CF mutation
database. These variations were also given in parentheses
following the approved nomenclature format (A of the ATG
translation start codon as +1).
2.4. Statistical analysis
Data are reported as counts and percentages. Comparison
between qualitative variables was performed by using Chi-
This study involved 500 couples of Sardinia descent, who
had no family history of CF and were either planning a
pregnancy or in the early stages of pregnancy (3–10 weeks of
gestation). They were enrolled from a group of patients
requesting voluntary screening for β-thalassemia. The majority
were from the south of Sardinia (77.8%), with the remainder
from the middle (20.1%), and north (2.1%) part of the island.
The questionnaire was returned by 700 out of 1000
participants (70%). Among this group, 42% had not previously
heard about CF. The remaining individuals who knew about the
disease received their information from advertising promotions/
newspapers/magazines (60.9%), general practitioner/gynecolo-
gists (8.6%), and other sources (30.5%) (Table 1).
The main reason given by couples for participating in the
screening program was that they perceived as serious the risk
associated with being a carrier of cystic fibrosis and with having
a child with CF.
Seventy per cent of couples (350/500) completed the
questionnaire, demonstrating a positive impact to the screening
program. Among these, 200 (57.1%) were planning to have a
child in the next 2 years.
All the couples were screened for 38 mutations (Table 2),
using a reverse dot-blot assay for 36 and a PCR-gel
electrophoresis assay for the other 2 (c.54-5811_164+
2187del8108ins182 and the p.Asn287LysfsX19).This mutation
panel included the 15 most frequent CF alleles in Sardinia .
The mutations detected and their frequencies are listed in
Table 2. The most common CF allele was the p.Thr338Ile
(T338I) (65%), followed by the p.Phe508del (F508del)
(22.5%). The p.Asn1303Lys, c.2051_2052delAAinsG, c.489+
1GNT, c.54-5811_164+2187del8108ins182, and p.Arg347Pro
mutations were found once, with a frequency of 2.5% each. The
p.Arg347Pro mutation, never detected in Sardinian patients
until now, was reported to have an overall frequency of 0.8% in
the Italian population (WHO, ).
Moreover, one couple at risk (p.Phe508del/c.489+1GNT)
planning a pregnancy was identified. The couple was previously
diagnosed to be at risk for β-thalassemia. Due to the higher risk
to conceive an affected child (43.75%), they decided to undergo
prenatal diagnosis for both diseases.
Knowledge questionnaire: overall results.
Knowledge questionnaire completed
Knowledge questionnaire not completed
Knowledge about CF
By mass media
By general practitioner/gynecologists
By occupation (high school teachers, physicians, nurses, etc.)
By friends/relatives previously screened
By acquaintance with a CF patient
List and frequency (%) of CF mutations included in the screening test.
exon 2 del (c.54-5811_164+2187del8108ins182)
The 3849+10kbCNT (c.3717+12191CNT), G85E (p.Gly85Glu), 2789+5GNA
(c.579+5GNA), 711+1GNT (c.579+1GNA), 4016insT (p.Ser1297PhefsX5),
G542X (p.Gly542X), 1717-1GNA (c.1585-1GNA), R553X (p.Arg553X),
Q552X (p.Gln552X), G551D (p.Gly551Asp), S549R (ANC) (p.Ser549Arg),
I507del (p.Ile507del), F508C (p.Phe508Cys), I502T (p.Ile502Thr), 1706del17
(p.Gln525LeufsX37), 1677delTA (p.Tyr515X), R117H (p.Arg117His),
D1152H (p.Asp1152His), L1065P (p.Leu1065Pro), R1066H (p.Arg1066His),
L1077P (p.Leu1077Pro), 4382delA (p.Glu1418ArgfsX14), R1162X (p.Arg1162X),
R1158X (p.Arg1158X), 1259 insA (p.Gln378AlafsX4), 852del22 (p.Gly241-
GlufsX13), S912X (p.Ser912X), and 991del5bp (p.Asn287LysfsX19) mutations
included in the CF panel were not detected in the population tested. Sardinian
mutations leading a detection rate of 94% are in bold.
aGene variants were named as indicated in the Cystic Fibrosis Nuclear Laser
kit. These variants are also given in parentheses as recommended by HGVS
209A. Coiana et al. / Journal of Cystic Fibrosis 10 (2011) 207–211
An asymptomatic 43 year old female homozygous for the p.
Thr338Ile (T338I) allele was also identified (Table 3) whose
parents, both T338I heterozygotes, were first cousins. The
genotype was confirmed by sequencing analysis of exon 7 of
CFTR gene. She was referred to the Regional Reference Centre
for Cystic Fibrosis for clinical evaluation. The sweat chloride
concentration was 93.7 mmol/L. Clinical lung and pancreatic
involvement was absent and the woman reported severe
dehydration in summer resulting from abnormal loss of sweat
electrolytes. During the genetic counseling session, she did not
show concern about her condition.
A total of 38 CF carriers were identified, resulting in an
overall frequency of 1/25 (4%).
A medical report and details of the genetic test was reported
to the participants during the post-test genetic counseling
session. The residual risk for having an affected child was
calculated for all the couples (Table 3). In addition, all subjects
who were diagnosed as carriers were invited to inform their
relatives about the opportunity to be tested for CF (cascade
3.1. Difficultytodetectthe p.Gln525LeufsX37 (1706del17) allele
An extensive molecular analysis either at DNA and RNA
level was performed on four patients because they completely
lacked a hybridization signal for the p.Gln525LeufsX37
mutation in exon 10, such that neither the wild type nor the
mutated alleles were detected. To determine the reason for the
assay failure, exon 10 and its flanking regions were sequenced.
These four individuals resulted homozygous for both the wild-
type p.Gln525LeufsX37 allele and the mutated c.1584GNA
(1716GNA) allele. The latter is a synonymous variation which
prevents the p.Gln525LeufsX37 probes from annealing to the
DNA. In heterozygous state, this variation has been reported to
cause exon 10 skipping and be associated either with idiopathic
chronic pancreatitis or CBAVD [13,14]. To define the effect of
this mutation in the homozygous state on the CFTR transcript,
RNA from two of these individuals was analyzed. PCR
spanning exons 8–12 of the cDNA resulted in two PCR
products of 650 and 458 bp respectively. Sequence analysis
showed that the 650 bp fragment contained five correctly
spliced exons, while the 458 bp fragment lacked exon 10
completely. A control individual not having this mutation
produced only the normal 650 bp fragment, as expected. To
date, this variation is considered to have no clinical con-
sequences . In our study, the c.1584GNA variant has a
frequency of 0.4%.
The success of a genetic screening program is mainly a
product of the educational support that fully informs the
population about the disease, thereby offering them the
opportunity of making an informed decision about reproduc-
tion. The thalassemia screening program that has been
performed in Sardinia since the late 1970s is an example of a
successful genetic screening program .
Following this model of success, we report a pilot
preconceptional carrier screening of CF in Sardinian couples,
with no family history of CF. The aim was to establish the a
priori risk of CF carrier status in the Sardinian population and to
offer a reliable screening test to couples planning a pregnancy
who wanted to know their risk of conceiving a CF child. This
CF molecular screening test was offered to 505 couples enrolled
from among the individuals voluntarily requesting screening for
β-thalassemia, and 500 (99%) accepted. Given that 38 CF
carriers were identified out of 1000 subjects with no previous
history of CF, the overall carrier frequency observed in the
general Sardinian population was 1/25 (4%).
Our study showed that the most frequent mutation in
Sardinia is the p.Thr338Ile (T338I) (65%), a mild mutation
previously described by our group in Sardinian CF patients
either in homozygous state (11 patients) or in compound
heterozygous state with other severe mutations (18 patients)
[16,17]. All the patients are pancreatic sufficient with a sweat
In both groups age at diagnosis ranged from 2 months to
40 years. Patients homozygous for the T338I mutation are often
manifestation of patient compound heterozygotes for T338I and
other severe mutation are slightly more severe ranging from
isolated azoospermia, with or without CBAVD, to mild CF
phenotype characterized by a mild to moderate pulmonary
involvement. However in both homozygotes and compound
heterozygotes patients, the increased sweat electrolyte concen-
tration may lead, especially in warm-arid climatesand ininfancy,
to sporadic episodes of severe hypotonic dehydration associated
with hyponatremia, hypochloremia, metabolic alkalosis, that
often represent the moment in which the diagnosis is made.
Moreover seven patients belonging from both groups (3 patients
T338I/T338I, 2 patients T338I/F508del, 1 patient T338I/
2183AANG, 1 patient T338I/R1066H), affected by azoospermia
with no other CF symptoms, were occasionally identified when
they requested CF genetic test for assisted reproduction
technologies (ART) (Rosatelli, personal communication).
There is statistically significant difference between the
frequency of the p.Thr338Ile (T338I) allele (65%), highlighted
in the present study, and the findings of our previous study on
Sardinian CF patients (19.3%) (p=0.0001) . Conversely,
the difference between the frequency of the p.Phe508del
(F508del) mutation in CF Sardinian patients (50%) and in the
Overall results of preconceptional CF screening in general Sardinian population.
n (%)Risk to have
a CF childa
Couples at risk
One spouse CF carrier
One spouse homozygote for a CF mutation
Negative couples (sensitivity of 94%)
aPopulation risk: 1/2500.
cGenotype: p.Thr338Ile (T338I) homozygote.
210A. Coiana et al. / Journal of Cystic Fibrosis 10 (2011) 207–211
general Sardinian population (22.5%, this study) is not
statistically significant (p=0.375) (Fig. 1) . As alluded
above, this discrepancy is likely due to the mild phenotype of
patient p.Thr338Ile (T338I) homozygotes or compound hetero-
zygotes which can escape a diagnosis.
Due to the high frequency of a mild mutation resulting in mild
or asymptomatic phenotypes, careful management and genetic
counseling are of utmost importance. In the counseling session,
of acute episodes of metabolic alkalosis in infancy.
Our test, using an RDB assay comprising 36 different
mutations in combination with PAGE analysis, has been shown
tobea robustscreeningtestfor the evaluationof CF carrier status
affected by CF are born to parents who were not aware that they
could have a CF child; preconceptional carrier screening remain
thereby central to inform the couples about reproductive choices.
The results of this pilot screening are encouraging as
demonstrate the efficacy and efficiency of the preconceptional
screening program and the high participation rate (99%) of the
Conflict of interest statement
The authors declare no financial and personal relationships
with other people or organizations that could inappropriately
influence their work.
This work was supported by the Italian Cystic Fibrosis
Research Foundation (grant FFC# 5/2008) with the contribution
of “C.M.A.E. Club Milanese con Dompè Farmaceutici; Net
Center Padova; Sky Italia s.rl".
The authors would like to thank Dr Francesca Sessini, Dr
Angelo Ideo, and Prof Paolo Moi from the Genetic Screening and
Counseling Service, Thalassemic Regional Hospital, Cagliari for
genetic counseling; Antonella Tibbio, Francesca Culeddu, and
Gianna, from the Genetic Screening and Counseling Service,
and Maria Demurtas from the Molecular Genetic Laboratory,
Thalassemic Regional Hospital, Cagliari for technical support.
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211 A. Coiana et al. / Journal of Cystic Fibrosis 10 (2011) 207–211