2006 Number 1 Articol CFTR

8/3/2019 2006 Number 1 Articol CFTR

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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: [email protected]

IntrodUctIon

Cystic brosis is the most common autosomal

recessive disease in Caucasian populations, having a

GEnEtIc analysIs of cftr MUtatIons

In cystIc fIbrosIs patIEnts froM roManIa

Lvu tamas1, ioan Popa2, Lvu Pop2, Andr Anghl1, Zagora Popa3,caaln Maran1

Objectives: Cystic fibrosis is the most common autosomal recessive disease in Caucasian populations. The aim of this study was to improve the number ofcystic 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. Wecombined 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 demuta]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 testelorgenetice 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, princombinarea 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\masneidentificate (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 brosis 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


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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 brosis 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-

ination, airway obstruction, recurrent/chronic

pneumonia, Pseudomonas sp. chronic infections.

b. gastrointestinal and nutritional abnormalities:

pancreatic insufciency, 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 brosis patients

from Romania, to calculate a new frequency for CFTR

mutations and to establish a solid method of genetic

analysis for cystic brosis mutations in Timisoara.

MatErIals and MEthods

The patients were selected over a period of two

years based on typical clinical ndings and sweat test

values. The selection was made from patients who arein 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 2000 C forlater analysis.17

For CFTR mutation detection we used the

ElucigeneTM 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 ELUCIGENETM CF29 kit uses

ARMS (Amplication Refractory Mutation System)allele specic amplication 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 specied conditions. Selection of appropriate

oligonucleotides allows specic mutant or normal

DNA sequences to be amplied and detected.

18-20

Genomic DNA was extracted from lymphocytes

from peripheral blood samples following the steps

presented in the ElucigeneTM CF29 kit protocol

(extraction with NH4Cl, NaCl, EDTA, NaOH and

Tris-base/HCl) and with two alternative methods:

using the Wizard Genomic DNA Purication 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 veried byelectrophoresis 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

amplication.

For one sample, genomic DNA was amplied

by PCR according with the ElucigeneTM 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 orTD), 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 amplication was carried out in a Touch Gene

Gradient Thermocycler (Techne, Massachusetts, USA)

using the amplication program from ElucigeneTM

CF29 kit protocol (activation of the AmpliTaq Gold

at 940C for 20 minutes linked to an amplication

cycling program of 30 seconds at 940C - denaturation,

2 minutes at 580C - annealing and 1 minute at 720C -

extension, for 35 cycles. This was linked to a 20-minutetime-delay le at 720C - extension on the nal 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 amolecular weight marker.


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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 andphotographed 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%) allelesremained unidentied. (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 identied 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 identied bythe Elucigene kit (G542X, I148T). Of the 17 patients

tested 6 had both mutations detected, 6 only one

mutation identied, 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 identied 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 theworld), 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 mutationsand the following mutations and polymorphisms

Case 1Ladder

Case 2 Case 3 NegativeControl

F508 NF508 M

F508 NF508 NF508 N

F508 M

Ladder Ladder Ladder

150 bp

200 bp

100 bp

250 bp

F508 MF508 M

F508 NF508 N F508 N

F508 M

F508 N

G542X

Ladder Ladder Ladder Ladder

Case 1 Case 2 Case 3 NegativeControl

Case 5

150 bp

200 bp

100 bp

250 bp

Ladder

150 bp

200 bp

100 bp

250 bpF508 N I148T


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26 TMJ 2006, Vol. 56, No. 1

were identied: 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 identication 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 identied as F508(48.73%), non - F508 30 alleles (18.98%) and 51

remained unidentied (unknown) - 32.27%. (Table 3)

Table 3. Frequencies of CFTR mutations observed in cystic fibrosis patientsfrom 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 fibrosispatients 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 fibrosispatients from Romania (total number of alleles = 192).

Table 6. New frequencies of the identified CFTR genotypes in cystic fibrosispatients 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 thefrequency of F508 remains low (47.92%), the non -

F508 alleles account for 17.19 % and the unidentied

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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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 signicantly

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 trafcking center

as the Mediterranean can still be observed today. When

one studies CF chromosomes in those populations of

the New World that were heavily inuenced 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 thathas 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 amplied 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 havethe 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 identied mutations

are each represented by a single allele.

There are also two mutations which have been

identied for the rst time in Romania: R735K and

1717-2 (A>G). 24 That is why the use of commercial

kits, like ElucigeneTM CF29 is not enough for detecting

a signicant percent (> 90%) of the mutations thatappear in patients with cystic brosis. 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 brosis 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

identied up to date reects the genetic heterogeneity

of Romanian population.25

2. The great number of unidentied 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

brosis patients.

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