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Analysis of allele distribution at the HLA-DRB1*04 gene, as one of the frequent ones among Croatians, and their HLA-A-B-DRB1 haplotypes in the Croatian population was performed in this study. Using LABType® SSO and PCR-SSP method, 11 DRB1*04 subtypes were observed, of which DRB1*04:01 was the most frequent (28.0%) followed by DRB1*04:02 (26.3%), DRB1*04:03 (22.3%), and DRB1*04:04 (14.2%). The significant haplotypes (with highest P value) for given DRB1*04 allele were the following combinations: HLA-B*15:01-DRB1*04:01, HLA-B*38:01-DRB1*04:02, HLA-B*35:03-DRB1*04:03, HLA-B*35:03-DRB1*04:08, HLA-B*14:01-DRB1*04:04, and HLA-B*49-DRB1*04:05. Marked differences in the distribution of our most frequent haplotypes of HLA-B-DRB1*04 (HLA-B*38:01-DRB1*04:02 and HLA-B*15:01-DRB1*04:01) were found in comparison to other European populations investigated so far. Additionally, comparison of HLA-A-B-DRB1*04 haplotypes showed that although there are similarities in the haplotype structure between our and other populations, there are also noteworthy differences. In summary, the identification of conserved and unusual DRB1*04 haplotypes in the present study of Croats should have important clinical implications for donor-recipient matching in the hematopoietic stem cell transplantation program, help in the understanding of HLA polymorphisms in different European populations, and also prove to be very useful in the determination of possible susceptibility genes involved in HLA-DRB1*04-associated diseases.

The analysis of short tandem repeat (STR) markers using polymerase chain reaction (PCR) method has become a widely applied technique in forensic individual identification, rapid detection of chromosome aneuploidies in prenatal and postnatal diagnosis, as well as paternity testing (1-5). Until now, a large number of autosomal and Y-chromosomal markers has been forensically evaluated and used for various purposes. Although X-chromosomal markers have been increasingly applied in both forensic and medical field, their role has not been as extensively investigated as that of autosomal and Y-chromosomal markers. Several investigations have documented the accuracy of fluorescent PCR using STR loci for the rapid prenatal diagnoses of numerical disorders affecting the chromosomes 21, 18, and 13 (6-8). However, the low polymorphism of the most chromosome X and Y markers has hampered the use of the PCR-STR approach for the detection of numerical disorders of sex chromosomes, such as the Turner (45, X) or Klinefelter (47, XXY) syndromes (1,7). A few years ago, a group of authors reported on the application of PCR-STR method in the detection of X-chromosome abnormalities (9,10). One of the biggest challenges is the Turner syndrome, in which a sufficient number of STR loci has to be included to be sure that the individual has only a single X chromosome. The aims of the present study were to investigate the diagnostic informativeness of 5 X-linked STR markers: DXS9895 (Xpter-Xp22.1), GATA172D05 (Xq26.1), DXS6810 (Xq12-Xq21.33), DXS6803 (Xq24-Xq27), and HPRTB (Xq27.3) in the Croatian population and to evaluate the diagnostic value of these 5 loci.

The aim of the present study was to compare haplotypes of the most frequent B*27 alleles among Croatians (B*2702 and *2705) and the rare B*2730 allele. For this purpose, 37 families with members carrying human leukocyte antigen (HLA)-B27 were selected. All individuals were analysed for eight microsatellites (Msats): D6S2927, short tandem repeat - MHC class I-related gene (STR_MICA), D6S2793, D6S2811, tumor necrosis factor a (TNFa), tumor necrosis factor d (TNFd), D6S273 and D6S1014, while individuals carrying the HLA-B27 specificity were subtyped. Of 39 analysed haplotypes, 20 individuals had B*2702, 15 subjects were positive for the B*2705 allele, the B*2730 allele was found in three haplotypes from different families, while one individual carried the B*2703 allele. HLA-A3 and -DRB1*16 were shared by all three B*2730 haplotypes. The DRB1*16 allele was also observed in the majority of B*2702 haplotypes (76.5%), while HLA-A3 was, after HLA-A2, the second most frequent HLA-A specificity in B*2702 haplotypes. No such correlation was found for the B*2705 haplotypes. Msat analysis showed that B*2730 haplotypes also share the same allele at all tested Msats. The D6S2927, D6S2793, MICA and TNFd Msats were not useful in distinguishing B*2702 and B*2705 alleles because D6S2927-213bp, STR_MICA-179bp, D6S2793-206bp, D6S2811-83bp and TNFd-130bp were detected in almost all cases. Conversely, for the TNFa, D6S273 and D6S1014 loci, haplotypes carrying B*2702 and B*2730 shared a single Msat allele in the majority of cases (TNFa-113bp, D6S1014-134bp and D6S273-134bp), which was not observed for B*2705 haplotypes. In conclusion, the similarity between B*2702 and B*2730 DNA sequences as well as their sharing of the same haplotypic combinations corroborates the proposed mechanism of B*2730 evolution from B*2702 by interallelic recombination.

The population from Croatia consisted of 150 Caucasian individuals. All individuals were tested for HLA-A, -B alleles using PCR-RLS method.

Population studies of two short tandem repeats (STR) loci (D15S642 and D15S659) were carried out in a sample of 130 unrelated healthy individuals. After PCR amplification, samples were run on 6% polyacrylamide gel in automated sequencer (ALFexpress). Twelve different alleles were identified at D15S642 locus and 11 alleles at D15S659 locus. The most frequent alleles at D15S642 were allele 2 (16.7%), allele 8 (16.3%) and allele 9 (14.0%), while the most frequent genotype was 2-2. Among 11 different alleles at D15659, the most frequent were allele 9 (22.1%), allele 3 (19.1%) and allele 8 (18.4%). Genotype 9-8 showed the highest frequency (9.6%) at D15S659 locus. The observed heterozygosities for these two loci were 0.81818 for D15S642 and 0.83088 for D15S659. The PIC was calculated as follows: 0.88 for D15S642 and 0.83 for D15S659. No significant deviations from Hardy–Weinberg equilibrium could be observed for these systems. The results indicate that these two loci are useful genetic markers for paternity testing as well as for prenatal or postnatal diagnosis.

In order to increase the number of STR loci for forensic DNA analysis as well as for prenatal diagnosis of Down syndrom in the Croatian population, we have studied three tetranucleotide STR loci on chromosome 21 (D21S1411, D21S1414, and D21S1435). One hundred and fifty-six unrelated healthy individuals were tested in this study. The results clearly demonstrated that the most polymorphic locus (20 different alleles) was D21S1414 with PIC value of 0.9279, while at D21S1435 locus seven different alleles were observed with PIC value of 0.7706. The third locus, D21S1411, also showed great polymorphism with 15 distinct alleles, PIC value 0.8920.

During the 3-year period, 111 cases of paternity disputes were processed in our laboratory. The analysis was performed on nine STR (TH01, VWA31, FES/FPS, F13A01, SE33, D1S1656, D12S391, D18S535, and D22S683) loci and one VNTR (D1S80) locus. Out of 111 cases, 22 were exclusions. In all cases, exclusion was confirmed on at least five loci. STR loci that were informative in majority of cases were SE33 and D12S391. The least informative loci were FES/FPS and F13A01. In 21 cases exclusions were also confirmed by HLA class I, while only in 17 cases HLA class II alleles excluded the alleged father.

We have analysed polymorphism at 4 STR loci in our population.All tested loci showed high level of informativness and heterozygosity.

The KIR locus with genes involved in immune processes is among the most polymorphic and structurally diverse human loci. KIR genes encode activating and inhibitory receptors that differ in specificity for HLA class I ligands and signaling potential. These receptors are expressed principally by natural killer (NK) cells and subpopulations of T cells. This study represents the first report of the distribution of KIR genes, KIR genotypes and KIR/HLA pairs in 121 unrelated healthy Croatian individuals. Twenty-three different genotypes were observed in the Croatian population and all 16 KIR genes known to date were found. The most frequent KIR genotype was the AA genotype. All individuals had at least one inhibitory KIR/HLA pair with the majority of individuals with three inhibitory KIR/HLA pairs. The most frequent KIR/HLA pair was the KIR2DL3/C1 group. Our results demonstrated the similarity of the Croatian population's KIR repertoire with other Caucasian populations reported so far.

The triple A syndrome (Allgrove syndrome, OMIM #231550) is caused by autosomal recessively inherited mutations in the AAAS gene on chromosome 12q13 encoding the nuclear pore protein ALADIN. This multisystemic disease is characterised by achalasia, alacrima, adrenal insufficiency and neurological impairment. We analyse long-term clinical follow-up and results of sequencing of the AAAS gene in eight patients with triple A syndrome aged from 2 to 35 years. At the time of diagnosis, all patients presented with alacrima, neurological dysfunction, dermatological abnormalities, seven of them with adrenal insufficiency and five of them with achalasia. Sequencing of the AAAS gene identified the p.S263P mutation in five of eight patients, supporting the hypothesis that this mutation is a founder mutation in Slavic population. One of the patients is homozygous for the p.S263P mutation, two are compound heterozygous for the p.S263P and the p.G14fs mutation, two are compound heterozygous for the p.S263Pro mutation and p.S296Y mutation, two are compound heterozygous for the p.G14fs and the p.Q387X mutations and one is homozygous for the p.Q387X mutation. In the course of the follow-up time of 4–29 years, progression of existing and appearance of new symptoms developed. Although severe, many of these symptoms presented in all six young adult patients are often overlooked or neglected: postural hypotension with blurred vision and syncope, hyposalivation resulting with complete edentulosis, talocrular contractures with permanent walking difficulties and erectile dysfunction in male patients. Triple A syndrome is a progressive debilitating disorder which may seriously affect quality of life and even be life-threatening in patients with severe neurological impairment. Conclusion: Long-term follow-up of patients with triple A syndrome revealed a variety of the clinical features involving many systems. Progressive natural course of the disease may seriously affect quality of life and even be life-threatening in patients with severe neurological impairment.

AIM: To investigate the influence of tumor necrosis factor (TNF) microsatellite polymorphisms on patient survival following hematopoietic stem cell transplantation. ----- METHODS: We analyzed TNFa, TNFb, and TNFd microsatellites among 100 patients who underwent allogeneic hematopoietic stem cell transplantation from a human leukocyte antigen (HLA)-identical sibling donor at the Internal Clinic of the University Hospital Center Zagreb in the period 2001-2009. The analysis was performed using polymerase chain reaction amplification and electrophoresis on a polyacrylamide gel in an automated sequencer. ----- RESULTS: There was no significant difference in patient survival with respect to the allele length at a given microsatellite. However, a significantly lower survival rate was noticed among patients who were positive for TNFa8 allele (P

Population study was carried out on the sample of 167 unrelated donors from the wider area of the Croatia's capital, Zagreb, using the short tandem reapet (STR) loci: TH01, VWFA31, FES/FPS, F13A01, D1S1656, D12S391, D18S535 and D22S683.

Istraživana je raznovrsnost četiri mikrosatelita HLA (D6S248, D6S2674, D6S2811 i D6S273) u skupini bolesnika s psorijatičnim artritisom (PsA) (N=22) i zdravim osobama (K; N=94) pozitivnima za gen HLA-B*27, te povezanost haplotipskih veza gena HLA-B*27 s PsA. Svi ispitanici bili su prethodno tipizirani za gene HLA-A i -B metodom PCR-SSP i bili su HLA-B*27 pozitivni. Mikrosateliti HLA su analizirani metodom PCR-STR i elektroforezom u ALFexpress sekvenceru. Analiza raspodjele alela mikrosatelita HLA pokazala je statistički značajne razlike za alele D6S273-3 (PsA-21,1 % naspram K-4,9 %; P=0,0013) i D6S273-4 (PsA-15,8 % naspram K-32,1 %; P=0,0180), dok je analiza haplotipskih veza pokazala da je kombinacija HLA-B*27/D6S273-4 bila statistički značajno smanjena u skupini bolesnika s PsA u odnosu na kontrolnu skupinu (10,5 % i 40,2 %; P=0,0164). Dobiveni rezultati navode na pretpostavku da je u blizini mikrosatelita D6S273 smješten gen koji igra ulogu u etiopatogenezi PsA, no za konačnu potvrdu nužno je povećati skupinu bolesnika., The polymorphism at HLA microsatellites (D6S248, D6S2674, D6S2811 and D6S273) among patients with psoriatic arthritis (PsA) (N=22) and healthy control subjects (K; N=94), as well as haplotypic associations between tested loci were analysed in this study. All subjects were previously typed for HLA-A and -B by PCR-SSP method and were HLA-B*27 positive. HLA microsatellites were analysed using PCR-STR method and electrophoresis in an ALFexpress sequencer. The results demonstrated statistically significant P value for following alleles: D6S273-3 (PsA-21.1 % vs. K-4.9 %; P=0.0013) and D6S273-4 (PsA-15.8 % vs. K-32.1 %; P=0.0180). Analysis of haplotypic associations showed the only statistically significant difference for combination HLA-B*27/D6S273-4 (PsA-10.5 % vs. K-40.2 %; P=0.0164). The results presented in this study lead to the assumption that some other gene/s involved in ethiology of PsA are located in the proximity of D6S273 microsatellite, but in order to reach a final conclusion, an increase in the number of patients is necessary.

Cilj: Transplantacija od nesrodnog podudarnog davatelja napretkom metodologije određivanja HLA sustava (engl. Hystocompatibility Leukocie Antigen) posljednjih se godina primjenjuje jednako kao i transplantacija od srodnog davatelja. Cilj ovog rada je prikazati iskustva liječenja alogeničnom transplantacijom od HLA podudarnog nesrodnog darivatelja u Zavodu za hematologiju KBC-a Zagreb. Metode: U razdoblju od 1991. do 2010. u Zavodu za hematologiju KBC-a Zagreb alogenom transplantacijom od HLA podudarnog nesrodnog darivatelja liječen je 71 bolesnik prosječne dobi od 28 godina (raspon: 1 mjesec – 61 godinu). Razlozi liječenja bili su akutna leukemija (34 bolesnika), kronična mijeloična leukemija (16 bolesnika), kronična limfocitna leukemija (4 bolesnika), limfomi i mijelom (7 bolesnika), teška aplastična anemija ili druge bolesti (8 bolesnika). Nesrodni darivatelji bili su HLA podudarnosti 6/6 (niska razina razlučivanja), HLA podudarnost 8/8, 8/10, 9/10 ili 10/10 (visoka razina razlučivanja). U pripremi za transplantaciju primijenjeni su ili standardni mijeloablativni program (ozračenje cijelog tijela i ciklofosfamid ili busulfan i ciklofosfamid) ili programi smanjenog intenziteta s fludarabinom. Rezultati: Vjerojatnost ukupnog preživljenja je 24 %. Plato krivulje preživljenja postiže se nakon 18 mjeseci. Trend boljeg terapijskog odgovora s preživljenjem od 37 % postignut je u bolesnika HLA podudarnosti 10/10. Neposredni uzroci smrtnosti u ranoj fazi nakon transplantacije su infekcije, GvHD, ponovna pojava bolesti i multiorgansko zatajenje. Rasprava i zaključak: Transplantacija od nesrodnog darivatelja primjenom optimalnog podobnog davatelja u ranoj fazi bolesti standardni je terapijski postupak mnogih zloćudnih tumora krvotvornog sustava i teške aplastične anemije., Aim: Stem cell transplantation from HLA-matched unrelated donor (MUD) is a standard therapy similar to allografting from related donor. Here we present the treatment outcome of MUD transplantation in the University Hospital Center Zagreb. Methods: From 1991 to 2010 at the Department of Hematology, University Hospital Center Zagreb 71 patients (average of 28 years, range 1 month to 61 year) underwent MUD transplantation; the reason for transplantation was acute leukemia (34 patients), chronic myeloid leukemia (16 patients), chronic lymphocytic leukemia (4 patients) lymphoma and myeloma (8 patients) and severe aplastic anemia or other disorders (8 patients). According to HLA, the donors were compatible in 6 out of 6 antigens (with low resolution) or in 8 out of 8 antigens, and 8 out of 10, 9 out of 10 and 10 out ten 10 antigens (with high resolution). For conditioning, patients received either standard myeloablative regimen (total body irradiation with cyclophosphamide or busulfan and cyclophosphamide) or fludarabine based reduced intensity regimen. Results: The probability of overall survival was 24 %. The plateau was reached after follow-up of 18 month. Trend for better treatment outcome and survival of 37 % was documented for patients months received stem cells from donors compatible in 10 out of 10 antigens. The principal causes of death after allografting were infections, GvHD, relaps and multiorgan failure. Discussion and conclusion: MUD-transplantation especially with the donor compatible in 10 out of 10 antigens performed in early phase of disease is currently the standard treatment for many malignant hematopoietic tumors and severe aplastic anemia.

Objectives: To analyze the distribution of HLA alleles and HLA microsatellite alleles in Croatian patients with testicular carcinoma, compare it with that of healthy controls and investigate whether the polymorphism within the HLA region could be associated with the development of testicular cancer. Methods: Genomic DNA was isolated from the peripheral blood of 24 patients with testicular germ cell tumors (TGCT). Patients and controls were typed for HLA class I and class II polymorphism by the PCR-SSO method. Nine HLA microsatellites were analyzed by PCR and electrophoresis in an automated sequencer. Results: No significant deviation in the distribution of frequencies at HLA class I alleles was observed between patients and controls. Among HLA class II alleles, a statistically significant increase in the frequency of the HLA-DPB1*1701 allele was found among patients. The frequency of the HLA-DRB1*07-DQA1*0201-DQB1*0202 haplotype was increased in patients in comparison to the controls. Analysis of HLA microsatellites showed an increased frequency of D6S291-3 allele (pcorr = 0.0455, OR = 3.05) among patients. Conclusions: The observed association of the disease and the DPB1*1701 allele as well as with the D6S291-3 allele suggests that this part of the HLA region might be involved in the pathogenesis of TGCT. Our data provide a basis for further studies about the correlation between the HLA region and testicular cancer.

In humans, the region configurations DR1, DR8, DR51, DR52 and DR53 are known to display copy number as well as allelic variation, rendering high resolution typing of HLA-DRB haplotypes cumbersome. Advantage was taken of microsatellite D6S2878, present in all DRB genes/pseudogenes with an intact exon 2-intron 2 segment. This DRB-STR is highly polymorphic in composition and length. Recently, it was proven that all exon 2 sequences could be linked to a certain DRB-STR that segregates with the respective DRB allele. Because haplotypes show differential copy numbers and compositions of exon 2- positive DRB genes/pseudogenes, unique DRB-STR patterns could be described that appear to be specific for a particular DRB haplotype. The aim of this workshop project was to approve and to qualify this simple typing protocol in a larger panel covering different European populations. All participants succeeded in correctly defining the DRB-STR amplicons varying from 135 to 222 base pair (bp) lengths. The panel of 101 samples covered 50 DRB alleles distributed over 37 different haplotypes as defined by exon 2 sequence-based typing. These haplotypes could be refined into 105 haplotypes by DRB-STR typing. Thus, discrimination of exon 2-identical DRB alleles was feasible, as well as the exact description of three different crossing-over events that resulted in the generation of hybrid DR region configurations. This typing procedure appears to be a quick and highly robust technique that can easily be performed by different laboratories, even without experience in microsatellite typing ; thus, it is suitable for a variety of researchers in diverse research areas.

Congenital adrenal hyperplasia (CAH) due to steroid 11-beta hydroxylase deficiency (11beta- OHD) is a rare genetic disorder of steroidogenesis transmitted as an autosomal recessive trait. We describe a new case of 11beta-OHD CAH caused by compound heterozygosity for a novel mutation in intron 7 and previously described mutation in exon 8 of CYP 11B1 gene. A 2.5-year-old boy of Croatian descent presented with accelerated growth and bone age, borderline hypertension, and pseudoprecocious puberty. Hormonal studies established diagnosis of 11beta-OHD: elevated plasma levels of 11- deoxycortisol, 17-hydroxyprogesterone, androstenedione and testosterone, low levels of cortisol and aldosterone, and suppressed plasma renin activity. Sequencing of the CYP11B1 gene identified compound heterozygous mutation consisting of a novel splicing mutation in intron 7 (IVS 7DS+4A to G) and R448H mutation in exon 8 previously reported mostly in Moroccan Jews. This is the first patient with CAH due to 11beta- OHD in Croatia (and Slavic population in general) in whom molecular diagnosis of CYP11B1 gene was performed.

U ovom radu analizirani su produženi haplotipovi gena HLA-B*27 na uzorku od 42 obitelji iz Hrvatske. Svim ispitanicima uzeto je 2ml periferne krvi iz koje je izolirana DNA pomoću komercijalnog kita. Aleli gena HLA-B*27 određeni su lančanom reakcijom polimerazom s početnicama specifičnim za određeni alel (PCR-SSP), a aleli mikrosatelitskih lokusa D6S2927, STR_MICA i TNFa umnoženi su početnicama specifičnima za alele mikrosatelitskih lokusa (PCR-STR). Analizom produženih haplotipova HLA-A-B*27-D6S2927-STR_MICA-TNFa-DRB1 utvrđena je najjača neravnoteža udruživanja između alela gena HLA-B*27 i mikrosatelitskih alela D6S2927-1 i STR_MICA-A4. Analiza je pokazala da je rijetki alel B*2730 uvijek prisutan u produženom haplotipu: HLA-A3-B*2730-D6S2927-1-STR_MICA-A4-TNFa-9-DRB1*16. Rezultati ovog rada primijenit će se u daljnjim istraživanjima povezanosti mikrosatelitskih alela i spondiloartropatija te pomoći u boljem razumijevanju vezanja peptida na molekule HLA razreda I, kao i drugih procesa koji se javljaju tijekom imunološkog odgovora., In the present study the extended haplotypes of HLA-B*27 gene were analysed in the sample of 42 Croatian families. Peripheral blood (2ml) was collected from all the individuals and genomic DNA was extracted using a commercial kit. The HLA alleles were determined using the method PCR sequence specific primers (PCR-SSP) while the microsatellite alleles were amplified using the method PCR-STR. Analysis of HLA-A-B*27-D6S2927-STR_MICA-TNFa-DRB1 extended haplotypes demonstrated the strongest linkage disequilibrium between HLA-B*27 alleles and microsatellite alleles D6S2927-1 and STR_MICA-A4. Analysis indicated that the rare allele B*2730 is always present in the extended haplotype HLA-A3-B*2730-D6S2927-1-STR_MICA-A4-TNFa-9-DRB1*16. The results of the present study will be applied in future studies of association between microsatellite alleles and spondyloarthropathies and contribute to a better understanding of peptide binding to HLA class I molecules, as well as other aspects of immune response.

Cilj: Idiopatska osteoporoza u muškaraca prvenstveno je posljedica niske vršne koštane mase koja je genetski uvjetovana. S obzirom na njegovu važnu ulogu u metabolizmu kosti, između brojnih gena kandidata u patogenezi osteoporoze, od posebnog je interesa gen za čimbenik rasta sličan inzulinu (IGF-1). Prethodna istraživanja pokazala su da je polimorfizam mikrosatelitne regije citozin-adenin (CA)n unutar gena za IGF-1 povezan sa serumskom koncentracijom IGF-I, stoga je primarni cilj ovog istraživanja bio analizirati utjecaj ovog polimorfizma na vršnu koštanu masu u muškaraca. Metode: U istraživanje su uključena 92 zdrava muškarca u dobi od 21 do 35 godina. Svakom je ispitaniku izmjerena mineralna gustoća kosti (BMD) u lumbalnoj kralježnici i proksimalnom dijelu bedrene kosti, koncentracije biljega koštane pregradnje, 25-OHD i spolnih hormona. Rezultati: Ispitanici u kojih je nađen alel (CA)18 (genotip 18+) imali su niži BMD u svim mjerenim područjima, no razlike između ispitivanih skupina nisu bile statistički značajne. Ipak, u homozigota (CA)18 nađen je značajno niži BMD u području vrata bedrene kosti (P=0,03), trohantera (P=0,01) i proksimalnog dijela bedrene kosti (P=0,04), dok razlike u koncentracijama biljega koštane pregradnje, 25-OHD, slobodnog testosterona i estradiola nisu bile statistički značajne. Rasprava: Rezultati ovog istraživanja ukazali su na mogući negativan utjecaj alela IGF-1 (CA)18 na vršnu koštanu masu u muškaraca. Ipak, za definitivne zaključke o ulozi tog polimorfizma u patogenezi osteoporoze potrebna su daljnja istraživanja na većem broju ispitanika i u različitim populacijama., Aim: Idiopathic osteoporosis in males is influenced predominantly by low peak bone mass as a feature under a strong genetic control. Among number of candidate genes, IGF-1 gene is of particular interest due to its important role in bone metabolism. It has been reported that IGF-1 citozin-adenin (CA)n polymorphism is related to serum IGF-1 concentration. Therefore, in the present study we examined the influence of certain IGF-1 (CA)n alleles on peak bone mass attainment in males. Methods: Study sample consisted of 92 unrelated healthy male volunteers, aged 21-35. In each subject, lumba spine and proximal femur bone mineral density (BMD), bone turnover markers, 25-OHD and sex hormones levels were measured. Results: IGF-1 (CA)18 allele (genotype 18+) were found to be �associated with low-lower BMD in all measured areas but the differences between analysed groups were not significant. However, homozygotes (CA)18 had significantly lower femoral neck (P=0,03), trochanter (P=0,01) and total hip (P=0,04) BMD whereas differences in bone turnover markers, 25-OHD, free testosterone and estradiol concentrations were not significant. Discussion: The results of the present study suggested possible negative effect of the IGF-1 (CA)18 allele on the peak bone mass attainment in males. However, for definitive conclusion about the role of this polymorphism in the pathogenesis of osteoporosis further studies in different populations and with larger number of participants are needed.

Polymorphism of TNFa, TNFb and TNFd microsatellites, linkage disequilibrium (LD) within TNF region as well as relationship of TNF microsatellites with alleles at HLA-A, -B and -DRB1 loci was investigated in this study. A sample of 160 Croatians, previously typed for HLA-A, -B and -DRB1 loci, was tested. Analysis was performed using electrophoresis of PCR products on a polyacrylamide gel in an ALFexpress sequencer. The results of the two-locus and three-locus haplotypic association study of TNF microsatellites were as follows: 1) the most frequent two-locus haplotypic associations were: TNFa10-TNFb4 (12.03% ; LD=2.32), TNFb4-TNFd4 (23.24% ; LD=2.54), and TNFa10-TNFd4 (14.06% ; LD=2.28) ; 2) the strongest LD was found for TNFa2-TNFb1 (LD=3.67 ; P

Aims: To evaluate the incidence, gender, symptoms and age at diagnosis among patients with classical congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency in Croatia. Methods: Data were collected retrospectively for all classical CAH patients born or electively aborted following prenatal diagnosis between 01.01.1995 and 31.12.2006 and were compared with the data of the previously conducted study evaluating CAH patients discovered between 1964 and 1984. Results: During a 12-year period, 34 classical CAH patients were born. There were 20 salt-wasting (SW) (12 females/8 males) and 14 simple virilizing (SV) patients (7 females/7 males). If 3 female fetuses, electively aborted, were added, that would be a total of 37 CAH patients. With 532,942 live births and 34 CAH patients born over this period, incidence of classical CAH was estimated to 1:15,574 or 1:14,403 if 3 electively aborted fetuses were included. The lower incidence of SW boys compared to SW girls (8:12) and similar number of SW and SV boys (8:7) indicate that substantial proportion of SW boys die unrecognized. Owing to better health care, diagnosis was established significantly earlier in SW and SV girls compared to the period of 1964–1984 (p < 0.003). During 1995–2006, none of the patients died following the diagnosis of CAH, and there was no erroneous sex assignment. Conclusion: Despite of improvement in health care, diagnosis of CAH in Croatia is still delayed and some of the patients go unrecognized or die. Therefore, we think that the results of our study support the need for the introduction of newborn screening.

Idiopathic osteoporosis in males is influenced predominantly by low peak bone mass as a feature under a strong genetic control. Among a number of candidate genes, α-estrogen receptor (ERα) and CYP19 genes are of particular interest due to important role of estrogen in pathophysiology of osteoporosis. In the present study we examined the association of certain allelic combinations of ERα gene thymine–adenine (TA) polymorphism and aromatase gene TTTA polymorphism on bone mineral density (BMD) in young men. The study sample consisted of 92 unrelated healthy male volunteers, aged 21–35. In each subject, lumbar spine and proximal femur BMD, parameters of bone turnover and 25-OHD level were measured. Two ERα (TA) n alleles, allele 19 and allele 21, were found to be associated with lower BMD. The presence of allele 19 was associated with significantly lower lumbar spine (P = 0.006) and trochanter (P = 0.02) BMD while the subjects positive for allele 21 had significantly lower lumbar spine (P = 0.04), trochanter (P = 0.02) and total hip (P = 0.03) BMD. Men with CYP19 (TTTA)7-3/ERα (TA)19 allele combination had significantly lower lumbar spine BMD (P = 0.02) and those with CYP19 (TTTA)7-3/ERα (TA)21 allele combination had significantly lower BMD for all three measurements, i.e. lumbar spine (P = 0.02), femoral neck (P = 0.02) and total hip (P = 0.008). These particular combinations of high-risk alleles were associated with lower median lumbar spine, femoral neck and total hip BMD than either of the allele alone suggesting that negative effect of two risk alleles on peak bone mass add up.

The aim of the present study was to investigate polymorphism of D6S2927, STR_MICA, D6S2793, TNFa (D6S2792), TNFb and TNFd (D6S2789) microsatellites and linkage disequilibria between these loci and human leucocyte antigen (HLA)-B (previously tested) for better characterisation of extended HLA haplotypes. A total of 176 healthy unrelated Croatians were studied using polymerase chain reaction amplification and electrophoresis on6%polyacrylamide gel in ALFexpress sequencer. Eight HLA-B/D6S2927 haplotypic associations (B*07/D6S2927-4, B*08/D6S2927-3, B*18/D6S2927-3, B*27/D6S2927-1, B*35/ D6S2927-5, B*38/D6S2927-4, B*51/D6S2927-2 and B*61/D6S2927-1) showed strong association (P < 0.001, D > 0.5). Among 88 different HLA-B/STR_MICA haplotypic associations, seven combinations (B*07/STR_MICA-A5.1, B*08/ STR_MICA-A5.1, B*15/STR_MICA-A5, B*18/STR_MICA-A4, B*27/STR_ MICA-A4, B*38/STR_MICA-A9 and B*51/STR_MICA-A6) demonstrated high linkage (D 0.3) with significant P value (P

In this study we reported the results for the first time of applying Polymerase Chain Reaction-Short Tandem Repeats (PCR-STR) method in the field of detection of aneuploidies for chromosomes 21 and 18 in Croatians. The aims of the study were: (I) validation of the diagnostic informativeness of 6 STR loci (D18S51, D18S858, D18S535, D21S1435, D21S1411, and D21S1414) in sample of 205 unrelated healthy individuals ; (II) evaluation of diagnostic power of the PCR-STR method for those 6 microsatellites ; (III) establishment protocol for use STRs as routine method for rapid prenatal detection of trisomy 21 and 18. DNA samples were amplified by fluorescence-based PCR reaction, subjected to electrophoresis in automated laser fluorescence DNA sequencer (ALFexpress). Results of our study were: (I) all 6 tested loci are informative (68– 85% of heterozygous individuals) ; (II) comparison between PCR-STR method and conventional cytogenetics did not revealed any false positive or false negative results ; (III) in prenatal screening of 105 samples of uncultured amniotic fluid 6 (5.7%) samples with chromosomal abnormalities were identified.

In this study we monitored mixed chimerism in 36 patients with various hematologic disorders. All of them underwent a classic conditioning regimen, 31 patients for related bone marrow transplantation (BMT) and 5 patients for unrelated BMT. DNA was isolated from peripheral blood, and samples were polymerase chain reaction (PCR) amplified for 5 short tandem repeat (STR) loci (TH01, VWA31, FES/FPS, F13A01, and SE33) and for one variable number of tandem repeats locus (D1S80). Samples were run on a 6% polyacrylamide gel in an automated ALFexpress sequencer. In all 36 donor-recipient pairs we found differences for at least two STR loci. In most cases the difference was observed for SE33 and D1S80 loci. Mixed chimerism (MC) was detected in 18 patients: 4 with unrelated BMT and 14 with related sibling donors. In 11 patients MC was detected in the early period after BMT, but was soon followed by full donor chimerism (FDC) in peripheral blood. In 5 cases patients MC appearing after FDC was established, and was predictive for the relapse. One patient showed alternating MC and FDC, but at the end showed only recipient cells and graft rejection. In conclusion, the PCR-STR analysis is a highly informative, fast, and simple screening method for monitoring chimerism in a BMT program.

Information about the chimeric status of patients is of great importance in comparison of different conditioning and prophylactic regimens as well as for the post-bone marrow transplantation (BMT) therapies. In some cases, mixed chimerism (MC) can also be predictive of relapse. Analysis of the short tandem repeats (STR) loci by polymerase chain reaction (PCR) is a choice method for this purpose. In this study, we monitored 15 patients after BMT. Twelve of them underwent classical-conditioning regimen while the remaining three patients were subjected to non-myeloablative conditioning (minitransplantation). Evaluation of chimerism was performed using five STR and one variable number of tandem repeats (VNTR) locus. Four additional loci were PCR-amplified in cases of minitransplantation. Samples were analyzed by electrophoresis in an ALFexpress sequencer. MC was detected in seven cases of which it was predictive of relapse for two patients, who suffered from acute lymphocytic leukemia (ALL). The PCR-STR method proved to be a fast and relatively simple method, while the tested STR loci showed a high level of informativeness.

Objective: To analyse long term follow up of neurological abnormalities (during 2 years to 23 years) in eight Croatian patients with triple A syndrome. Background The triple A syndrome is caused by autosomal recessively inherited mutation in AAAS gene on chromosome 12q13 encoding the nuclear pore protein ALADIN. This multisystem disease is characterized by achalasia, alacrimia, adrenal insufficiency and neurological impairment. Design/Methods: Clinical examination, electromyoneurography (EMNG) and molecular genetic analysis were performed in eight patients with triple A syndrome (five males and three females). Results: At the time of diagnosis all patients (aged 2 years to 8 years) presented with alacrimia, latent or manifest adrenal insufficiency, anisocoria in 2, optic atrophy in 4, motor and sensory polyneuropathy on the first exam in 2 siblings, muscle weakness and hypotrophy of distal muscle groups, cavus feet, hyperreflexia but absent triceps surae jerks, talocrural joints contractures, tremor and dysmetria in 5 patients. First EMG findings were normal at the age 6-11 years in 6/8 patients. The follow up EMG showed chronic partial denervation in all patients. Spontaneous activity (fibrillations) was registered in 3/8, compound muscle action potentials (CMAP) were polyphasic in 6/8 patients. Absent or low CMAP amplitude (0-0,5mV) was obtained in 6/8, decreased motor conduction velocity (29-40 m/s) in 8/8, absent F-wave potentials in 7/8, absent neural potentials in 5/8, and proximal conduction block in 5 patients. Mutation pSer263Pro was identified in 5 of 8 patients. One is homozygous and four are compound heterozygous for this mutation. Genotype/phenotype analysis confirmed lack of correlation in patients with triple A syndrome. Conclusions: Long term follow up neurography in patients with triple A syndrome showed progressive mixed motor and sensory polyneuropathy development with signs of pronounced demyelination and/or probably secondary axonal damage. Molecular results support the hypothesis that the pSer263Pro mutation is founder mutation in Slavic population. Disclosure: Dr. Barisic has nothing to disclose. Dr. Dumic has nothing to disclose. Dr. Kusec has nothing to disclose. Dr. Stingl has nothing to disclose. Dr. Lehman has nothing to disclose. Dr. Bunoza has nothing to disclose. Dr. Grdan has nothing to disclose. Dr. Koehler has nothing to disclose. Dr. Heubner has nothing to disclose.