CYSTIC FIBROSIS MUTATION SPECTRUM
IN NORTH MACEDONIA:
A STEP TOWARD PERSONALIZED THERAPY Terzic M1, Jakimovska M1, Fustik S2, Jakovska T3, Sukarova-Stefanovska E1, Plaseska-Karanfilska D1,* *Corresponding Author: Professor Dijana Plaseska-Karanfilska, MD, PhD, Research Center for Genetic
Engineering and Biotechnology “Georgi D.Efremov,” Macedonian Academy of Sciences and Arts,
Av. Krste Misirkov 2, 1000 Skopje, Republic of North Macedonia. Tel: +389-23-235-400/264.
E-mail: dijana@manu. edu.mk page: 35
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MATERIALS AND METHODS
Materials. A total of 158 patients (ages between
newborn and 36 years) with positive (Cl– >60.0 mEqv/L)
or borderline(Cl– = 40.0-60.0 mEqv/L) sweat test result
and clinical features consistent with a diagnosis of CF:
respiratory (repeated episodes of bronchopneumonia,
chronic cough and sputum production, wheezing, sinus
disease with nasal polyps) and non respiratory, i.e., digestive
(meconium ileus, failure to thrive, malnutrition,
steatorrhea); dehydration with salt depletion; hepatobiliary
disease or recurrent pancreatitis were referred for molecular
characterization of CF, mainly from the Department
of Pediatrics and the Institute for Respiratory Diseases,
Skopje, Republic of North Macedonia. Of the 158 patients,
97 were of Macedonian ethnic background, 59 were of
the Albanian ethnic background. Of the remaining two
patients, one was of Turkish ethnicity and the other of
Bosnian ethnic background. Informed written consent was
obtained from all individuals included in the study. The
study was approved by the Ethics Committee of the Macedonian
Academy of Sciences and Arts, Skopje, Republic
of North Macedonia.
Methods. DNA was isolated from peripheral blood
using the standard phenol-chloroform extraction method.
Initially, all patients were screened for a panel of CFTR
mutations with SNaPshot analysis as published by Noveski
et al. [8] (mutations screened: F508del, G542X, N1303K,
621+1G>T, G551D, R553X, R1162X, W1282X, R117H,
2184insA and 1717-1G>A). The analysis of the amplified
fragments was performed on the ABI PRISM® 3130 Genetic
Analyzer (Thermo Fisher Scientific, Waltham, MA, USA).
For the patients whose genotypes were not determined with
the initial screening, several additional methods were applied.
These include: INNO LiPA CFTR 19 and INNO LiPA
CFTR+17 Tn Update detection kit for 36 mutations in the
CFTR gene (Fujirebio Diagnostics AB, Göteborg, Sweden);
multiplex ligation-dependent probe amplification (MLPA), for detection of deletions/duplications in the CFTR gene
(SALSA MLPA KIT P091 CFTR; MRC-Holland, Amsterdam,
The Netherlands); next generation sequencing (NGS),
with TruSight Inherited gene panel performed on MiSeq
Illumina Personal Sequencer and data analyses on the Illumina
Variant Studio (Illumina Inc., San Diego, CA, USA).
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