_____________________________

Liviu Tamas et al 23
ORiGiNAL ARticLeS
abstract
Received for publication: Nov. 21, 2005. Revised: Feb. 15, 2006.
rEZUMat
1
Department Of Biochemistry, Molecular Biology Division,
2
Pediatric Clinic
No. 2, Victor Babes University of Medicine and Pharmacy, Timisoara,
3
National
Center of Cystic Fibrosis, Timisoara
Correspondence to:
Liviu Tamas, Department of Biochemistry, Victor Babes University of Medicine
and Pharmacy, 2 E. Murgu Sq., 300041 Timisoara, Tel: +40744767343
Email: tliviu33@yahoo.com
IntrodUctIon
Cystic fbrosis is the most common autosomal
recessive disease in Caucasian populations, having a
GEnEtIc analysIs of cftr MUtatIons
In cystIc fIbrosIs patIEnts froM roManIa
Liviu tamas
1
, ioan Popa
2
, Liviu Pop
2
, Andrei Anghel
1
, Zagorca Popa
3
,
catalin Marian
1
Objectives: Cystic fibrosis is the most common autosomal recessive disease in Caucasian populations. The aim of this study was to improve the number of
cystic fibrosis mutations detected in patients from the National Center of Cystic Fibrosis from Timisoara and to establish a solid method of genetic analysis for
cystic fibrosis mutations in Timisoara. Material and methods: The study included a retrospective part, which consisted of analyzing the genetic tests results
for 79 patients from the National Center of Cystic Fibrosis from Timisoara, already investigated in collaboration with Royal Manchester Children's Hospital
- Genetic Unit (UK), and an original, prospective part, in which we selected 17 patients in evidence at the Center for genetic analysis, based on clinical findings
and sweat test. 29 mutations were investigated and the detection was performed using a Elucigene
TM
CF29 kit, which detects point mutations or small deletions
in deoxyribonucleic acid (DNA) using a method based on ARMS allele specific amplification technology. DNA was extracted from lymphocytes from peripheral
blood samples, genomic DNA was amplified by PCR and the PCR products were visualized on a UV transiluminator after electrophoresis on agarose gel and
staining with ethidium bromide. Results: We identified 18 mutated alleles from a total number of 34 alleles and three mutations: F508, G542X and I148T. We
combined the new, original data with data from the retrospective part and we obtained new estimates of the frequency for CF mutations in Romania. Conclusion:
The most frequent mutation in Western and Central Europe, F508 (70%) has a lower frequency in Romania (47,92%). There are still many mutations that
remain unidentified (34 %) by investigating only the usual mutations. The great number of mutations and polymorphisms identified up to date (25) reflects the
genetic heterogeneity of Romanian population.
Key Words: cystic fibrosis, children, F508 mutation
Obiective: Fibroza chistic\ reprezint\ cea mai frecvent\ boal\ autosomal recesiv\ la popula]iile caucaziene. Scopul acestui studiu a fost cre[terea num\rului de
muta]ii detectate la pacien]ii cu fibroz\ chistic\ din Centrul Na]ional de Fibroz\ Chistic\ din Timi[oara [i implementarea unei metode solide de analiz\ genetic\
pentru muta]iile CFTR n Timi[oara. Material [i metode: Studiul a fost compus dintr-o parte retrospectiv\ care a constat din analiza rezultatelor testelor
genetice pentru 79 de pacien]i de la Centrul Na]ional de Fibroz\ Chistic\ din Timi[oara, care au fost investiga]i prin colaborarea cu Royal Manchester Children's
Hospital - Genetic Unit (UK), [i o parte original\, prospectiv\ n care am selectat 17 pacien]i afla]i n eviden]a Centrului pentru efectuarea analizei genetice [i
identificarea muta]iilor, selec]ia realizndu-se pe baza simptomatologiei clinice [i a valorilor testului sudorii. Au fost investigate 29 de muta]ii, iar detec]ia
muta]iilor CFTR s-a realizat utiliznd kitul ElucigeneTM CF29, care detecteaz\ muta]ii punctiforme sau mici dele]ii n catena de ADN (acidul dezoxiribonucleic) [i
folose[te o metod\ bazat\ pe tehnologia de amplificare specific\ a alelelor, ARMS. Dup\ extrac]ia ADN din limfocitele din probe de snge periferic, am amplificat
ADN genomic prin PCR, iar produ[ii PCR rezulta]i (ampliconii) au fost vizualiza]i ntr-un transiluminator cu lumin\ UV dup\ electroforeza lor n gel de agaroz\
colorat cu bromur\ de etidium. Rezultate: Am identificat 18 alele mutante dintr-un num\r total de 34 de alele [i 3 muta]ii: F508, G542X [i I148T. Apoi, prin
combinarea datelor noi, originale, cu datele din partea retrospectiv\ a studiului, am ob]inut noi estim\ri ale frecven]ei muta]iilor din fibroza chistic\ n Romnia.
Concluzii: F508, cea mai frecvent\ muta]ie n Europa de vest [i central\ (70%) are o frecven]\ mai redus\ n Romania (47,92%). Multe muta]ii au r\mas
neidentificate (34%) n urma investiga]iei numai a muta]iilor uzuale. Num\rul mare de muta]ii [i polimorfisme identificate pn\ n prezent (25) demonstreaz\
heterogenitatea genetic\ a popula]iei Romniei.
Cuvinte cheie: fibroz\ chistic\, copii, muta]ie F508
frequency of 1 in 2000 - 2500 live births and a carrier
frequency of 1 in 25 - 30.
1,2
It is caused by mutations
in the cystic fbrosis transmembrane conductance
regulator (CFTR) gene on chromosome 7, which
codes a membrane-associated protein of 1480 amino
acid residues that forms a cAMP-regulated and ATP-
gated chloride channel.
1-7

The CFTR protein, which is a low conductance
chloride channel localized at the apical membrane of
epithelial cells, belongs to the large superfamily of
ATP-binding cassette (ABC) transporters.
8-10

It is composed of two symmetrical halves, each
possessing a membrane-spanning domain (MSD)
and a nucleotide-binding domain (NBD, or the ABC
_____________________________
24 TMJ 2006, Vol. 56, No. 1
domain).
1,11
The regulatory (R) domain is the link
between the two halves and the phosphorylation of
the regulatory domain regulates channel activity.
10,11
Patients with cystic fbrosis have abnormal chloride
conduction across the apical membrane of epithelial
cells, causing inspissated secretions in the airways,
pancreas, intestines, and vas deferens.
5
The main clinical symptomatology of the disease
consists of:
a. lung disease with the following pulmonary
symptomatology: bronchiectasis, atelectasis, hyper-
infation, airway obstruction, recurrent/chronic
pneumonia, Pseudomonas sp. chronic infections.
b. gastrointestinal and nutritional abnormalities:
pancreatic insuffciency, chronic diarrhea, failure to
thrive, meconium ileus and rectal prolapse.
c. High values for the sweat test (high chloride
concentration in sweat).
1,5,12-16
The aim of this study was to improve the number
of detected CFTR mutations in cystic fbrosis patients
from Romania, to calculate a new frequency for CFTR
mutations and to establish a solid method of genetic
analysis for cystic fbrosis mutations in Timisoara.

MatErIals and MEthods
The patients were selected over a period of two
years based on typical clinical fndings and sweat test
values. The selection was made from patients who are
in evidence at the National Center of Cystic Fibrosis
from Timisoara.
We analyzed 34 chromosomes from 17 patients
which presented clinical manifestations of the disease
and high values for the sweat test. We also investigated
patients who presented normal or borderline
values for the sweat test, but had suggestive clinical
manifestations.
1,12
Genetic analysis was performed on samples of
whole blood (EDTA). The samples of blood were
either immediately analyzed or frozen at 200
0
C for
later analysis.
17

For CFTR mutation detection we used the
Elucigene
TM
CF29 kit which can identify 29 mutations:
1717-1G>A, G542X, W1282X, N1303K, F508,
3849+10kbC>T, 621+1G>T, R553X, G551D, R117H,
R1162X, R334W, A455E, 2183AA>G, 3659delC,
1078delT, I507, R347P, S1251N, E60X, D1152H,
3120+1G>A, 2789+5G>A, 1898+1G>A, 711+1G>T,
G85E, 2184delA, I148T and R560T. The method
employed by the ELUCIGENE
TM
CF29 kit uses
ARMS (Amplifcation Refractory Mutation System)
allele specifc amplifcation technology, which detects
point mutations or small deletions in deoxyribonucleic
acid (DNA). The principle of ARMS is that
oligonucleotides with a 3 mismatched residue will not
function as Polymerase Chain Reaction (PCR) primers
under specifed conditions. Selection of appropriate
oligonucleotides allows specifc mutant or normal
DNA sequences to be amplifed and detected.
18-20
Genomic DNA was extracted from lymphocytes
from peripheral blood samples following the steps
presented in the Elucigene
TM
CF29 kit protocol
(extraction with NH4Cl, NaCl, EDTA, NaOH and
Tris-base/HCl) and with two alternative methods:
using the Wizard Genomic DNA Purifcation Kit
(Promega, Madisson, USA)and Qiagen QIAamp
DNA Blood Mini Kit (250).
17
The quantity and quality
of DNA extracted by the three methods was similar
and further analysis of extracted DNA produced
optimal results. DNA concentration was verifed by
electrophoresis on agarose gel (0.4%) with a DNA
weight marker (10 ng/l), obtaining results in the
interval 10-30 ng/l, optimal concentrations for PCR
amplifcation.
For one sample, genomic DNA was amplifed
by PCR according with the Elucigene
TM
CF29 kit
protocol using a 25 l reaction mixture for each of
the four primers mix: 5 l DNA and 20 l of reaction
mixture - 5,5 l enzyme(AmpliTaq Gold) dilution
(with sterile deionised water, dilution buffer and
loading dye) + 16,5 l primers mix(TA, TB, TC or
TD), from which we used 20 l for mixing with DNA
in 4 thin-walled PCR vials. A negative DNA control
was included in each PCR run.
18
PCR amplifcation was carried out in a Touch Gene
Gradient Thermocycler (Techne, Massachusetts, USA)
using the amplifcation program from Elucigene
TM

CF29 kit protocol (activation of the AmpliTaq Gold
at 94
0
C for 20 minutes linked to an amplifcation
cycling program of 30 seconds at 94
0
C - denaturation,
2 minutes at 58
0
C - annealing and 1 minute at 72
0
C -
extension, for 35 cycles. This was linked to a 20-minute
time-delay fle at 72
0
C - extension on the fnal cycle).
18
The next step was the electrophoresis of the
PCR products (25 l) on a 3% agarose (NuSieve
3:1, Cambrex BioScience) gel using tris-borate with
ethidium bromide (TBE/EtBr) as running buffer in
a Sub-Cell GT DNA Electrophoresis system (Bio-
Rad). A 50 Base-Pair Ladder (Amersham-Pharmacia
Biotech) at 1.5 mg/15 ml was prepared in the Loading
Dye supplied (80 ml distilled water/10 ml Loading Dye
/10 ml 50 Base-Pair Ladder). 25 ml of this dilution
was loaded on the gel and run adjacent to samples as a
molecular weight marker.
_____________________________
Liviu Tamas et al 25
Electrophoresis was carried out at 5 V/cm between
electrodes (for a gel 15 x 25 cm and a distance of 30
cm between electrodes we used 150 V) until the dye
front had migrated 5 10 cm from the loading wells
towards the anode (1.5 to 2 hours).
After electrophoresis the gels were placed on a
UV transilluminator at 260 nm, then visualized and
photographed with a Polaroid

Instant Camera (Cole-

Parmer) or a digital camera.
rEsUlts
Genetic analysis led to the detection of 3 different
mutations: F508, G542X and I148T.
The most prevalent mutation, F508, was found
in 15/34 CF chromosomes (Figures 1, 2), with the
second most common allele being the G542X (2/34)
(Fig. 2) and I148T (1/34). (Fig. 3) 16 (47.06%) alleles
remained unidentifed. (Table 1)
Figure 1. Identification of two heterozygote genotypes (F508/X).
Figure 2. Identification of one compose heterozygote genotype (F508/
G542X).
Figure 3. Identification of one heterozygote genotype (I148T/X).
Table 1. Frequencies of CFTR mutations observed in cystic fibrosis
analyzed patients (total number of alleles = 34).
In Table 2 we show the different CFTR genotypes.
The most frequent genotype was F508/F508
(5/17). Other genotypes identifed were: F508/
unknown observed in 4 cases, F508/non-F508
found in 1 patient, non-F508/unknown in 2 and
unknown/unknown, in 5. We named as unknown the
alleles with mutations that couldnt be detected by
the Elucigene kit, due to its limitations (it can detect
only 29 mutations) and non-F508 the alleles with
mutations, other than F508, that were identifed by
the Elucigene kit (G542X, I148T). Of the 17 patients
tested 6 had both mutations detected, 6 only one
mutation identifed, and in 5 both mutations remained
unknown.
Table 2. Frequencies of the identified CFTR genotypes (total number = 17).
dIscUssIons
To date more than 1400 mutations and
polymorphisms have been identifed in the CFTR
gene.
21,22
The most frequent mutation, F508, accounts
for approximately 70% of the CF chromosomes in
northern and central European countries (and in the
world), but the frequencies and types of mutations in
different populations vary considerably depending on
the ethnic and geographical origin of the population
tested.
23
Wide variations in disease manifestations are
observed among affected CF patients and a multitude
of disease-causing mutations have been found in the
CFTR gene.
In Romania, previous studies on patients from the
National Center of Cystic Fibrosis from Timisoara,
consisted of genetic analysis for CFTR mutations
and the following mutations and polymorphisms
Case 1
Ladder
Case 2 Case 3
Negative
Control
F508 N
F508 M
F508 N
F508 N
F508 N
F508 M
Ladder Ladder Ladder
150 bp
200 bp
100 bp
250 bp
F508 M
F508 M
F508 N
F508 N F508 N
F508 M
F508 N
G542X
Ladder Ladder
Ladder Ladder
Case 1 Case 2 Case 3 Negative
Control
Case 5
150 bp
200 bp
100 bp
250 bp
Ladder
150 bp
200 bp
100 bp
250 bp F508 N
I148T
M0tatIea 80her ef aIIeIes % 000IatIve [%)
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_____________________________
26 TMJ 2006, Vol. 56, No. 1
were identifed: F508, G542X, N1303K, W1282X,
CFTR del exon 2,3 (21 kb), 2183 AA >G, 621 + 1
G >T, R735K, 1717-2 (A>G), G817V, 2694 T/G
(polymorphism), 3272-26 A>G, 457 TAT > G, 3849
+ 10 kb (C>T), R1162X, S1235R, 3849 G>A, 2694
T/C (polymorphism), 1898 (G>A), I148T, G576X,
R533X, R117H, IVS8-5T variant and IVS8-7T variant
(polymorphisms).
24-28
The molecular diagnostic for identifcation of
CFTR gene mutations at patients from Timisoara
started to be performed since 1990, right after the
discovery of CFTR gene in 1989 by Lap-Che Tsui
and J.R.R Riordan. In 15 years, by collaboration with
Royal Manchester Childrens Hospital Genetic Unit
(UK), blood samples from 79 patients (158 alleles)
were analyzed and 25 mutations and polymorphisms
were detected, including two new mutations - R735K
and 1717-2 (A>G). 77 alleles were identifed as F508
(48.73%), non - F508 30 alleles (18.98%) and 51
remained unidentifed (unknown) - 32.27%. (Table 3)
Table 3. Frequencies of CFTR mutations observed in cystic fibrosis patients
from Romania (total number of alleles = 158).
The 79 genotypes analyzed were distributed as
follows: 26 genotypes were F508/F508, 8 - F508/
X, 16 - F508/non-F508, 4-non-F508/non-
F508, 5-non-F508/X and 20 unknown (X/X).
24-29

(Table 4)
Table 4. Frequencies of the identified CFTR genotypes in cystic fibrosis
patients from Romania (total number = 79).
The actual prospective part of the study consisted
of the genetic analysis on 17 new patients (the
analyses were realized by the Division of Molecular
Biology, Biochemistry Department from Victor Babes
University of Medicine and Pharmacy, Timisoara).
The new, original results (17 patients, 34 alleles)
were combined with the previous results from the
retrospective part of the study (79 patients, 158 alleles)
and we obtained the following distributions, presented
in Tables 5 and 6 (96 patients, 192 alleles).
Table 5. New frequencies of CFTR mutations observed in cystic fibrosis
patients from Romania (total number of alleles = 192).
Table 6. New frequencies of the identified CFTR genotypes in cystic fibrosis
patients from Romania (total number = 96).
Also, it is noteworthy that the number of alleles
with F508 mutation (15) is similar with the number of
alleles with unknown mutations (16), which supports the
idea that the Romanian population is an heterogeneous
one regarding CFTR mutations. (Table 1)
In northern and central Europe the frequency
of F508 is approximately 70%, but in Romania the
frequency of F508 remains low (47.92%), the non -
F508 alleles account for 17.19 % and the unidentifed
mutations still have a high frequency (34.89%). The
genetic heterogeneity of Romanian population for
the CFTR gene (25 mutations detected) and the low
frequency of F508 are similar with the situation from
other eastern or southern European countries (Hungary
F508 - 54.9%, Greece - F508 - 52.9%, Bulgaria
- F508 - 63.6%, Macedonia - F508 - 54.3%). It is
interesting to observe that a low frequency of F508
appears also in other latin European countries like Italy
- 50.9%, Portugal - 44.7% and Spain - 52.7%.
Also like Romania, Spain, Bulgaria, Greece, and
Turkey have some of the most diverse mutational arrays
in Europe, on average approximately 25 mutations.
This is most likely due to their geographic situation,
serving as historic gateways into Europe from the
Middle East, Africa, and associated waterways.
Countries from central, northern, western,
and northeastern Europe show a large degree of
homogeneity among CFTR mutations (Austria,
Belarus, Belgium, Denmark, Estonia, Finland, France,
Germany, Lithuania, the Netherlands, Norway,
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_____________________________
Liviu Tamas et al 27
Poland, Russia, Sweden, Switzerland, and the Ukraine)
and a reduced mutational arrays (approximately 10
mutations).
The second most frequent mutation in Romania
is G542X (4 alleles/192 alleles), which is also second
in Europe and in the world. G542X is most common
in the Mediterranean regions of Europe and Africa.
G542X has been implicated as a mutation that was
introduced into the Mediterranean region by the
migration of Phoenicians.
28

Across Europe, G542X is found in signifcantly
higher proportions in countries, and in regions of
countries, which border the Mediterranean Sea.
G542X has its highest rates of prevalence in the north
of Africa and in the south of Spain (South Spain,
14.4%; Tunisia, 8.9%). Interestingly enough, these two
regions correspond to two major ancient Phoenician
cities, Carthagne and Carthage, respectively.
28

The genetic results of the introduction of this
mutation into such a busy trade and traffcking center
as the Mediterranean can still be observed today. When
one studies CF chromosomes in those populations of
the New World that were heavily infuenced by such
countries as Spain and Italy (countries from South and
Central America), the G542X mutation frequencies
are virtually identical.
28
The mutation N1303K (which has also a higher
frequency comparing with the others mutations with
the exception of F508) is another CFTR allele that
has a similar distribution patterns as G542X, and
may also have been introduced via the Mediterranean
route.
W1282X is a mutation of single origin that has
historically been associated with the Ashkenazi Jews.
This mutations frequency has been amplifed to almost
double that of the F508 mutation in this distinct
population.
28-48
In Romania W128X, N1303K are more frequent
then the others mutations (2 alleles each from 192
alleles) and there also several mutations which have
the same frequency: CFTR dele 2,3 (21kb)37, 2183 AA
>G, 621 + 1 G >T and I148T. The I148T mutation has
a higher frequency in the French-Canadian population
(9,1%), while in other populations it is a less frequent
mutation (< 1%).
21
The rest of the identifed mutations
are each represented by a single allele.
There are also two mutations which have been
identifed for the frst time in Romania: R735K and
1717-2 (A>G). 24 That is why the use of commercial
kits, like Elucigene
TM
CF29 is not enough for detecting
a signifcant percent (> 90%) of the mutations that
appear in patients with cystic fbrosis. Therefore, for
further improvement of genetic diagnostic we need to
apply different methods like DNA direct sequencing or
DGGE (Denaturing Gradient Gel Elecrophoresis).
48-56
The actual study shows the possibility of molecular
diagnosis for the majority of cystic fbrosis patients
and can offer for some cases the option of prenatal
diagnosis.
conclUsIons
1. The low frequency of F508 (48%) in Romania
and the great number of mutations and polymorphisms
identifed up to date refects the genetic heterogeneity
of Romanian population.
25
2. The great number of unidentifed mutations
(34%) imposes the continuation of the study using
multiple methods of mutation detection in order to
detect them.
3. We succeeded to establish locally, in Timisoara,
a solid method of molecular diagnostic for cystic
fbrosis patients.

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