We analysed frequencies of two single‐nucleotide polymorphisms (SNP) in the interferon‐γ (IFN‐γ) receptor‐1 (IFNGR1) gene promoter (G‐611A, T‐56C) in tuberculosis patients (n = 244) and compared them with controls (n = 521). These frequencies were not significantly different, whether analysed independently or as haplotypes. Because these SNP affect transcription, the results suggest that the expression of the IFNGR1 gene does not confer susceptibility to disease in patients from Croatia. Further analysis revealed a significant association between the protective (CA)n polymorphism (22 repeats, 192 FA1), located in the fifth intron of the IFNGR1 gene (+16682), and GT promoter haplotype (−611; −56) that showed the strongest expression capacity. In addition to this cis relationship, the (CA)22 allele was correlated in trans with an IFN‐γ SNP (IFNG G + 2109A), which might affect the transcription of the IFNG gene. These results suggest that a particular combination of IFNG and IFNGR1 SNP might offer a better protection against tuberculosis in this population.

Monoamine oxidase (MAO), a mitochondrial flavine containing enzyme, exists in two isoenzymes, MAO-A and MAO-B. Platelets contain MAOB subtype, proposed to be a biomarker for different personality characteristics and vulnerability for substance abuse. The most common polymorphism of MAO-B gene, a single base change (A or G) occurs in intron 13. It has been proposed to be a functional polymorphism, controlling the activity of MAO-B in platelets. The aim of the study was to determine the association between platelet MAO-B activity and MAO-B intron 13 polymorphism in 225 racially and ethnically uniform healthy Caucasian men of the Croatian origin. Our results showed that platelet MAO-B activity did not differ between subjects subdivided into those with «A allele» or «G allele». This polymorphism of the MAO-B gene did not control the activity of the MAO-B in platelets. Platelet MAO-B activity was associated only with the smoking status, and it was significantly decreased in smokers when compared to nonsmokers. No significant association was found between MAO-B polymorphism and smoking status. In healthy individuals of the Croatian origin, the studied MAO-B polymorphism showed a lack of functional importance in regulating MAO-B activity in platelets. Since different populations may vary in the association between functional polymorphism and the MAO-B activity, and the genotype of transcription factor AP-2β was reported to be associated with altered platelet MAO-B activity, and with specific personality traits, further studies on different populations should be conducted to elucidate the molecular mechanism/s regulating platelet MAO-B activity.

The aim of this study was to investigate the allelic frequency of 35delG mutation in patients with recessive, nonsyndromic hearing loss (NSHL) compared to normal hearing individuals in the Croatian population. For this purpose, we analyzed 27 unrelated individuals with nonsyndromic hearing loss and 342 healthy individuals. The method we used is based on the principle of polymerase chain reaction (PCR)-mediated, site-directed mutagenesis, followed by a BsiYI digestion. Among patients with NSHL, the 35delG mutation was found on 51.85% alleles. Carrier frequency among healthy control individuals was 1 in 68.4 (1.5%). The patients, found to be wild-type, either in heterozygous or homozygous form, were further tested by direct sequencing. Among them, two different mutations were observed, W24X and 313del14. Relatively high prevalence of 35delG mutation among patients with NSHL indicate that it is an important cause of NSHL in Croatia. Early diagnosis by identification of the 35delG mutation would greatly improve genetic counseling, as well as treatment and management of deafness in Croatia.

Cystic fibrosis is one of the most common recessive disorders in Caucasian affecting approximately 1 in 3000 individuals. More than 1000 mutations have been identified in cystic fibrosis transmembrane conductance regulator gene (CFTR). The most frequent mutation, accounted for about 67% CF chromosomes, is Δ F508. Only four others (G542X, N1303K, G551D and W1282X) have frequencies higher than 1% ; most other are rare and specific for some population subgroups. The southern-east region of Europe is highly heterogenic and CF mutation analysis can be facilitated by association studies between intragenic polymorphic haplotypes. The aim of this study was to reveal the frequency of 29 CF mutations along with the distribution of three polymorphic loci (IVS 1 CA, IVS 8 CA and IVS 17b CA) and associated hapoltypes in diseased population. A total of 41 unrelated CF patients from Croatia were included in this study which revealed 6 different mutations accounted for 68, 29% diseased alleles. The most frequent mutation was Δ F508 (58, 74%), followed by G542X (3.66%) and N1303K (2.44%). Polymorphic loci analysis revealed high level of heterogeneity, 15 different haplotypes were found. The most frequent associated with CF was 21-23-13 (23, 2%), followed by 21-17-13 being the most frequent one (31, 3%). According to our results it can be concluded that genetic background of cystic fibrosis in Croatian population is more complex than it was expected. Much more work is needed to get inside the exact population screening with available kits and association studies.

Hearing loss is one of the most common congenital disorders affecting 1 to 3 of 1000 newborns. NSHI (nonsyndromic hearing impairment) is observed in about 70% of cases with a genetic background and is mostly caused by autosomal recessive mutations. Mutations in GJB2 gene, encoding gap junction beta 2 protein (connexin 26) are found in 50% Caucasians with profound NSHI. 35delG is the most frequent GJB2 mutation leading to NSHI in European populations, especially of Mediterranean descent, with carrier frequency up to 1/30. The aim of this study was to determine the allelic frequency of 35delG mutation in patients with NSHI and in normal hearing individuals in the Croatian population. The method we used is based on the principle of PCR-mediated site-directed mutagenesis, followed by a BsiYI digestion. PCR products were subsequently size-separated by electrophoresis on a 4% agarose gel and analyzed. Results: we analyzed 29 unrelated individuals with nonsyndromic sensory deafness and 342 normal hearing individuals. Among the patients with NSHI the 35delG mutation was found on 48.27% alleles. The carrier frequency among the healthy control individuals was 5 in 342 (1.46%). Conclusion: The relatively high prevalence of 35delG mutation in GJB2 gene among Croatian patients with NSHI shows that this mutation is one of the leading causes of NSHI in Croatia. Hence, early screening for 35delG mutation would greatly improve the genetic counselling, prevention and treatment strategies in our patients with NSHI.

Uza sve brži razvoj genetike, tehnike analize gena i racunalne tehnologije, pred lijecnicima-prakticarima razlicitih profila pojavljuje se sve veci broj razlicitih genetickih pretraga. Neke od njih su sastavni dio svakodnevne dijagnosticke prakse odnosno dijagnostickih algoritama, dok je korist drugih dvojbena.

The frequency of cystic fibrosis (CF) mutations varies among different ethnic groups. Our aim was to determine the frequency of common mutations of CF patients in Croatia, Southern European and Mediterranean country, in order to develop a practical CF mutation panel that can provide maximal information for our population. PATIENTS AND METHODS: A total of 47 CF patients were diagnosed and surveyed at the Children's Hospital Zagreb, Croatia. Most of the patients were screened for 16 common mutations while some of them were screened with INNO LiPa DNA probe assay for 29 CF mutations and Tn polymorphism. RESULTS: Among 47 CF patients were identified 7 different CFTR mutations: Δ F508, F117H, G542X, N1303K, 1717-1G-A, R1162X and 621+1G>T. Δ F508 mutation was present in 66%, which was consistent with Northwest-Southeast gradient within the European population. The G542X and R117H were found each in 3.2% of Croatian CF patients. The N1303K was present in 2.1% while R1162X, 1717-1G-A and 621+1g>T were found each in 1.1% of CF chromosomes. There were 19 Δ F508 homozygotes and 10 compound Δ F508 heterozygotes with severe (3 G542X, 2 N1303K, 1 R1162X, 1 621+1G>T, 1 1717-1G-A) or mild mutation (2 R117H) on other allele. Fifteen CF patients had one mutation unknown (14 N/Δ F508 and 1 N/R117H) while 3 patients have both mutations still unknown. CONCLUSIONS: A total of 79% of CF chromosomes in Croatian CF patients carry 7 different CFTR mutations:Δ F508, R117H, G542X, N1303K, 1717-1G-A, R116X and 621+1G>T. Further studies are necessary to identify other mutations in our population in order to enable more accurate genetic counseling of Croatian CF families and to provide meaningful carrier screening and testing for rare mutations in affected individuals.

Etiologija slusnog ostecenja vrlo je raznolika. Vanjski cimbenici uzrokom su oko 40% slusnih ostecenja, dok je oko 60% ostecenja sluha uzrokovano genetickim cimbenicima. Oko 20-50% nasljedne nesindromske zamjedbene gluhoce uzrokovano je mutacijama u genima GJB2 i GJB6. Zastupljenost pojedinih mutacija pokazuje regionalne razlike. Cilj: 1) provesti prospektivno istraživanje etiologije slusnog ostecenja u grupi osoba trajnim ostecenjem sluha obrađenih u Ambulanti za klinicku genetiku 2) utvrditi ucestalost mutacija gena GJB2 i GJB6 u populaciji hrvatskih bolesnika sa NSHL 3) predložiti optimalan model organizacije infrastrukture i nacina obrade prirođenog slusnog ostecenja kod djece dijagnosticirane putem sveobuhvatnog novorođenackog probira u Hrvatskoj. Rezultati: tijekom 2003. godine obradili smo 148 djece s ostecenjem sluha, 36 s provodnom nagluhosti, 92 sa zamjedbenom, te 20 s mijesanom nagluhosti. U 14 (9, 6%) utvrđeni su vanjski cimbenici kao najvjerojatniji uzrok slusnog ostecenja. Od 134 djece s ostecenjem sluha za koje se pretpostavlja da su geneticki uvjetovani, 73 (54%) je bilo sindromske, a 61 (46%) nesindromske gluhoce. Preliminarni rezultati pokazuju da je trecina od dosad analiziranih bolesnika s nesindromskom perceptivnom gluhocom homozigotno ili heterozigotno za najcesce mutacije u GJB2 genu. Predložen je dijagnosticki postupnik obrade slusnog ostecenja. Ispitivanje etiologije gluhoce u Hrvatskoj omoguciti ce ucinkovitije planiranje dijagnostickih i preventivnih programa za zastitu zdravlja djece sa ostecenjem sluha.

Cisticna fibroza (CR) je autosomno recesivna bolest koja nastaje kao posljedica mutacije u samo jednom genu, genu CFTR (od eng. cystic fibrosis transmembrane conductance regulator gene). Produkt gena je protein CFTR koji djeluje kao prenosilac iona klora kroz membrane epitelnih stanica. U normalnim stanciama, posredstvom ciklickog adenozin monofosfata (c-AMP), protok klorida kroz kanal, u izvanstanicni mukus, tece nesmetano. Kao posljedica toga kloridni ioni, zajedno s ionima natrija i vodom, održavaju normalnu viskoznost mukusa. Međutim, u CF osoba koje u svojim stanicama nose mutirani protein, nece doci do c-AMP-om stimulirane aktivacije kanala. Takve stanice ne luce efikasno kloride i vodu i pokazuju povecanu apsorpciju natrijevih iona. Sve to dovodi do stvaranja dehidriranog mukusa cemu se mogu pripisati sve patoloske posljedice cisticne fibroze. Unutar gena CFTR do danas je opisano preko 1000 mutacija, od kojih su mnoge vrlo rijetke, a njihova raznolikost i razlicita zastupljenost unutar jedne populacije u mnogome otežavaju dijagnostiku cisticne fibroze. Najucestalija mutacija je delecija tri para baza u desetom eksonu, Δ F508, s prosjecnom ucestaloscu od 67%. U genu CFTR opisan je i velik broj polimorfizama (preko 200) koji se primjenjuju u standardnoj dijagnostici, pogotovo unutar obitelji s nepoznatom mutacijom. Do danas je objavljeno niz studija koje govore o važnosti primjene polimorfnih biljega u dijagnosticiranju i pracenju nasljeđivanja cisticne fibroze. Također, poznato je da su i pojedine mutacije znacajno cesce povezane s pojedinim polimorfnim biljezima. Rezultati studije, provedene u nasem laboratoriju, pokazali su, dosad nepoznatu, raspodjelu polimorfnih biljega unutar gena CFTR u stanovnika Hrvatske. Služit ce kao smjernica u dijagnosticiranju cisticne fibroze metodama molekularne dijagnostike, narocito unutar obitelji koje su opterecene cisticnom fibrozom, a gdje nisu pronađene mutacije gena