Molekularno-citogenetičko proučavanje heterocromatina u nekih kornjaša

Heterochromatin characteristics have been studied in Leptinotarsa decemlineata belonging to the leaf beetles (Chrysomelidae) and compared to the thoroughly analysed heterochromatin of the mealworm beetle Tenebrio molitor, representing darkling beetles (Tenebrionidae). C-banding reveals heterochromatin in pericentromeric regions of all chromosomes of both species; however heterochromatic blocks of L. decemlineata are significantly smaller in size. Digestion of L. decemlineata genomic DNA with 12 restriction enzymes reveals presence of bands characteristic for repetitive DNA, but again in a significantly lower amount than those found in genomic DNA of T. molitor. While T. molitor exhibits complete correspondence of restriction enzyme effects on naked DNA and mitotic chromosomes, L. decemlineata mitotic chromosomes are resistant to the digestion with restriction enzymes. The results show that two species have similar organization of heterochromatin on chromosomes but differ significantly in its amount. In addition, difference in accessibility to in situ restriction enzyme digestion indicates specificity of heterochromatin structure in the two species.Proučavan je heterokromatin u krumpirove zlatice Leptinotarsa decemlineata kao predstavnice zlatnica (Chrysomelidae), te je uspoređen s detaljno opisanim heterokromatinom brašnara Tenebrio molitor, kao predstavnika crnokrilaca (Tenebrionidae). Metoda C-pruganja je pokazala prisutnost heterokromatina u pericentromernim područjima svih kromosoma obiju vrsta, iako je njegov udio znatno manji u krumpirove zlatice. Razgradnja genomske DNA krumpirove zlatice sa 12 restrikcijskih enzima otkrila je prisutnost ponovljene DNA, ali opet u znatno manjoj količini u odnosu na brašnara. Iako kod brašnara T. molitor postoji podudarnost u razgradnji DNA i mitotskih kromosoma restrikcijskim enzimima, mitotski kromosomi krumpirove zlatice su otporni na razgradnju restrikcijskim enzimima. Rezultati pokazuju da obje vrste imaju podjednaku organizaciju heterokromatina na kromosomima unatoč velikoj razlici u količini. Pored toga, razlika u razgradnji mitotskih kromosoma restrikcijskim enzimima ukazuje na specifičnost heterokromatinske strukture u ovih vrsta

Pojavnost genskog polimorfizma receptora 2039A>G folikularno stimulirajućeg hormona i rizik muške neplodnosti u albanskoj populaciji

The purpose of this study was to determine the prevalence of allele and genotype variants of the follicle-stimulating hormone receptor (FSHR) gene polymorphic region at position Asn680Ser in the Albanian male population and associate them with the clinical parameters of infertility. The study included 114 infertile men (mean age 35.04±5.85 years) stratified according to the level of spermatogenetic impairment (oligoasthenozoospermia, asthenozoospermia and normospermia) and 112 fertile men (mean age 36.44±7.05 years) with normal semen parameters. Genotyping of the FSHR gene at position 680 was performed by TaqMan genotyping assay. All the participants underwent semen analysis, and serum reproductive hormones (FSH, luteinizing hormone, prolactin and testosterone) were also measured. The FSHR Asn680Ser genotype frequencies were as follows: Asn/Ser 42%, Ser/Ser 33.9% and Asn/Asn 24.1% in the control group, and Asn/Ser 56.1%, Ser/Ser 22.8% and Asn/Asn 21.1% in the whole group of infertile men (χ2-test: P=0.08). There was no statistically significant correlation between serum hormone levels and semen characteristics or between fertility status and FSHR Asn680Ser gene variants in the control group and the group of infertile men. However, adjusted logistic regression analysis (age, body mass index, smoking and alcohol as covariates) revealed increased odds ratio for male infertility among heterozygous Asn/Ser genotype carriers associated with lower values of semen parameters (normal morphology, concentration, total sperm count and motility). In conclusion, our case-control study further confirmed previous reports on no significant association between the FSHR Asn680Ser polymorphisms and male infertility. Nevertheless, the data presented herein indicate that the Asn/Ser genotype may increase the risk of male infertility in Albanian population.Cilj ovoga istraživanja bio je odrediti pojavnost alela i varijante genotipa receptora folikularno stimulirajućeg hormona (FSHR) na poziciji Asn680Ser kod muškaraca albanske populacije u odnosu na kliničke parametre neplodnosti. Istraživanje je obuhvatilo 114 neplodnih muškaraca (srednja dob 35,04±5,85 godina) svrstanih prema razini oštećenja spermiograma (oligoastenozoospermija, astenozoospermija i normospermija) te 112 plodnih muškaraca (srednja dob 36,44±7,05 godina) s urednim nalazom spermiograma. Genotipizacija gena FSHR na poziciji 680 učinjena je primjenom TaqMan probe. Kod svih sudionika istraživanja učinjena je analiza sjemena i reprodukcijskih hormona uključujući FSH, luteinizirajući hormon, prolaktin i testosteron. U kontrolnoj skupini ispitanika kod FSHR Asn680Ser genotipa utvrđena pojavnost Asn/Ser bila je 42%, Ser/Ser 33,9% i Asn/Asn 24,1%, dok se u skupini neplodnih ispitanika incidencija kretala od 56,1% za Asn/Ser, 22,8% za Ser/Ser i 21,1% za Asn/Asn (χ2-test; p=0,08). Nije ustanovljena značajna statistička povezanost između razine hormona, karakteristika sjemena, stanja plodnosti u varijanti gena FSHR Asn680Ser u kontrolnoj skupini u odnosu na ispitanike u skupini neplodnih muškaraca. Ipak, primjenom prilagođene, logističke i regresijske analize (dob, indeks tjelesne mase, pušenje i alkohol kao kovarijable) utvrđeno je da postoji veća vjerojatnost javljanja muške neplodnosti kod nositelja heterozigota Asn/Ser koji su povezani sa sniženim vrijednostima parametara sjemena (morfologija, koncentracija, ukupan broj i pokretljivost). Zaključno možemo utvrditi da ovo istraživanje potvrđuje ranija izvješća kako ne postoji značajna povezanost između polimorfizma FSHR Asn680Ser i muške neplodnosti. Ipak, navedeni podaci upućuju na to da Asn/Ser genotip može povisiti rizik muške neplodnosti u albanskoj populaciji

Careful preoperative planning of aortic valve surgery – impact of echocardiography and CT parameters

Objective: We can observe an increase in incidence and prevalence of patients with aortic valve stenosis in the general population. The gold standard in aortic valve therapy is aortic valve replacement. Preoperative planning is essential for good outcomes, as the severity of stenosis and calcifications can sometimes be extremely progressive and even involve the aortic root and ascending aorta. There is not enough research on comparation of CT scan analysis of aortic valve stenosis and echocardiography which is the golden standard of disease diagnosis.1-3 Patients and Methods: We have analyzed 88 patients [age: 70.01±9.066 (mean±SD); female: n=45, 51.1%]. Among the patients, 12 had bicuspid aortic valve leaflet structure while the rest of the patients (n=76, 86.4%) exhibited TAV stenosis. Degree of aortic stenosis was assessed according to mean pressure gradient (MPG), peak pressure gradient (PPG), aortic valve area (AVA) indexed aortic valve area (AVAi) and maximum speed through aortic valve (Vmax). These were compared with calcium score (AVCS) calculated from CT scan. All of these patients were observed in the operating room during surgery and valves analyzed after explantation. All of the patients underwent aortic valve replacement. Results: Average AVCS values (median + IQ range) were 3306.3 (1995.4 – 4820.6) [female: 2215 (1463.35 – 3372.85); male: 4093.5 (3133.3 – 5274.4). Average AVCS values for BAV patients were 3063.5 (3323.125 – 4868.9) and 3106.55 (1965.375) – 4780.125) for TAV patients. There were significant correlations between AVCS and AVAi (Spearman’s ρ=−0.24, P=0.025), PPG (ρ= 0.38, P< 0.001), MPG (ρ= 0.36, P= 0.001) V max (ρ= 0.37, P < 0.001) and gender (ρ= 0.485, P < 0.001) while AVA values showed no significant correlation with AVCS (ρ= -0.066, P = 0.540). Overall survival was similar not depending of severity of calcifications and stenosis, however clamp time and surgery time were longer for patients with severely calcified valves which means calcium scoring as a parameter should also be taken in consideration during preoperative planning. Conclusion: Careful preoperative planning is essential for good outcome of surgery, here we have proven the connection between echocardiography parameters of aortic stenosis and calcium score calculated by CT scan

Association of polymorphic variants in serotonin re-uptake transporter gene with Crohn’s disease: a retrospective casecontrol study

Aim To analyze the distribution of SLC6A4 gene polymorphisms in Crohn’s disease (CD) patients and their association with the disease. Methods We evaluated the presence/absence of promoter (5-HTTLPR, rs25531) and intron 2 (STin2 VNTR) polymorphic variants of SLC6A4 gene in a retrospective case-control study including 192 CD patients and 157 healthy controls (HC). Genotyping was performed by polymerase chain reaction. The association of polymorphisms with CD and its clinical subtypes was analyzed using χ2 and Fisher exact test, binary logistic regression, and haplotype analysis.Results CD patients and healthy controls had similar sex (88 [45.8%] vs 84 [53.5%] women, respectively; P = 0.154) and age (41.3 ± 12.8 years vs 41.7 ± 8.8 years, respectively, P = 0.091) distribution. Significant differences were observed in the STin2 genotype and allele distribution between CD patients and healthy controls (P = 0.003 and P = 0.002, respectively) and between the corresponding female subgroups (P = 0.004 and P = 0.007, respectively), with a significant negative association of biallelic ss (STin2.9 and Stin2.10) STin2 genotype with CD (P = 0.013, age- and sexadjusted odds ratio [OR] 0.5, 95% confidence interval [CI] 0.29-0.86; women: P = 0.006, age-adjusted OR 0.32, 95% CI 0.14-0.72) and a significantly higher S-STin2.12 (5-HTTLPR/ rs25531: S-STin2: STin2.12) haplotype distribution in CD patients (P = 0.004, OR 1.62, 95% CI 1.16-2.26). There was no significant association between 5-HTTLRP and rs25531 genotype or allele frequencies and CD and between any SLC6A4 polymorphic loci with clinical CD subtypes. Conclusion STin2 VNTR polymorphism of SLC6A4 gene may contribute to CD pathogenesis

Clinical and histopathological characteristics of COL4A3 c.2881+1G>A variant causing Alport spectrum disorders in Croatian population

Alport syndrome (AS) and thin basement membrane nephropathy (TBMN) are part of the spectrum of kidney disorders caused by pathogenic variants in α3, α4, or α5 chains of the collagen type IV, the major structural component of the glomerular basement membrane (GBM). Using targeted next-generation sequencing (NGS), 34 AS/TBMN patients (58.8% male) from 12 unrelated families were found positive for heterozygous c.2881+1G>A variant of the COL4A3gene, that is considered disease-causing. All patients were from the continental or island part of Croatia. Clinical, laboratory, and histopathological data collected from the medical records were analyzed and compared to understand the clinical course and prognosis of the affected patients. At the time of biopsy or first clinical evaluation, the mean age was 31 years (median: 35 years; range: 1 – 72 years). Hematuria was present in 33 patients (97.1%) and 19 (55.9%) patients had proteinuria. There were 6 (17.6%) patients with hearing loss, 4 (11.8%) with ocular lesions, and 11 (32.4%) with hypertension. Twenty-three (67.6%) patients had proteinuria at follow-up, and 5 (14.7%) patients with the median age of 48 years (range: 27-55) progressed to kidney failure, started dialysis, or underwent kidney transplantation. Of the 13 patients who underwent kidney biopsy, 4 (30.8%) developed focal segmental glomerulosclerosis (FSGS), and 8 (66.7%) showed lamellation of the GBM, including all patients with FSGS. It is essential to conduct a detailed analysis of each collagen type IV genetic variant to optimize the prognosis and therapeutic approach for affected patients

Cardiac myxoma the great imitators: comprehensive histopathological and molecular approach

Cardiac myxomas are rare benign and slowly proliferating neoplasms of uncertain histogenesis with heterogeneous histomorphology and variable and sometimes clinically quite malignant pathological manifestations. Majority of cardiac myxoma occur sporadically while a relatively small proportion of diagnosed cases develop as a part of Carney complex syndrome with established familial pattern of inheritance. Although histologically indistinguishable these two forms of cardiac myxoma exhibit distinct cytogenetic make-up and apparent pathological differences important for their clinical presentation and prognosis. Additional problem is presented with secondary lesions with more aggressive histology and significantly faster cell proliferation suggesting their successive malignant alteration. Surgical resection of cardiac myxoma is currently the only treatment of choice. However, to avoid potentially hazardous operating procedures and possible postoperative complications and to prevent recurrence of the neoplastic lesions it is necessary to develop alternative approaches and identify a possible drug targets for their successful pharmacological treatment. Due to the rarity of the disease, a small number of cases in one institution and lack of comprehensive experimental data particularly concerning the cases of metastatic dissemination and secondary lesions with malignant nature, a comprehensive multi-institutional approach is required for better understanding of their molecular pathology and illumination of key molecular, genetic as well as epigenetic markers and regulatory pathways responsible for their development. In this article we provide comprehensive pathohistological, molecular and cytogenetic overview of sporadic cardiac myxoma cases restating the major hypothesis concerning their histogenesis and emphasizing potential approaches for their further reexamination

Non-phosphorylated Tyr-1248 form of human epidermal growth factor receptor 2 (HER2) predicts resistance to trastuzumab therapy and poor disease-free survival of HER2- positive breast cancer patients

Aim To determine the predictive value of phosphorylat - ed human epidermal growth factor receptor 2 (pHER2Y1248) status in breast cancer (BC) patients undergoing trastu - zumab-based adjuvant therapy. Methods Immunohistochemical status of pHER2Y1248, EGFR/HER1, HER3, and HER4 was determined in 124 consec - utive HER2-positive BC patients (median age [range] =57 years [49.0-64.0]) treated at the University Hospital for Tu - mors, Zagreb, between 2008 and 2011. The median followup was 84 months (60.0-84.0). Prognostic factors of disease free survival (DFS) rate were evaluated with Kaplan-Meier/ log-rank test and Cox regression analysis. Results pHER2Y1248, HER1, HER3, and HER4 were expressed in 66.1%, 9.7%, 70.2%, and 71.0% of patients, respective - ly. Disease progression (DP) was observed in 17.1% of pHER2Y1248-positive and 47.6% of pHER2Y1248-negative BCs (P=0.001). Kaplan-Meier analysis showed a worse five-year DFS in pHER2Y1248-negative patients who were older than 60 years ( P <0.001) and had positive lymph node status ( P 2.0 cm ( P <0.001); higher histologi - cal grade ( P <0.001); HER2E intrinsic subtype ( P <0.001), negative hormone receptors ( P <0.001); negative HER1 status ( P <0.001), positive HER3 ( P =0.002); and/or positive HER4 ( P =0.002) status. The only negative prognostic factor for five-year DFS in multivariate Cox regression analysis was pHER2Y1248-negative (hazard ratio [HR] 3.6, 95% confidence interval [CI] 1.8-7.2, P <0.001) and lymph node-positive sta - tus (HR 3.6, 95% CI 1.3-9.8, P =0.014). Conclusion pHER2Y1248 predicts sensitivity to trastuzumab and a better five-year DFS regardless of any other prognos - tic parameter. In HER2-positive BC patients. Non-phospho - rylated HER2Y1248 is a strong predictor of trastuzumab resis - tance and a poor DFS

Non-phosphorylated Tyr-1248 form of human epidermal growth factor receptor 2 (HER2) predicts resistance to trastuzumab therapy and poor disease-free survival of HER2-positive breast cancer patients

Aim: To determine the predictive value of phosphorylated human epidermal growth factor receptor 2 (pHER2Y1248) status in breast cancer (BC) patients undergoing trastuzumab-based adjuvant therapy. ----- Methods: Immunohistochemical status of pHER2Y1248, EGFR/HER1, HER3, and HER4 was determined in 124 consecutive HER2-positive BC patients (median age [range]=57 years [49.0-64.0]) treated at the University Hospital for Tumors, Zagreb, between 2008 and 2011. The median follow-up was 84 months (60.0-84.0). Prognostic factors of disease free survival (DFS) rate were evaluated with Kaplan-Meier/log-rank test and Cox regression analysis. ----- Results: pHER2Y1248, HER1, HER3, and HER4 were expressed in 66.1%, 9.7%, 70.2%, and 71.0% of patients, respectively. Disease progression (DP) was observed in 17.1% of pHER2Y1248-positive and 47.6% of pHER2Y1248-negative BCs (P=0.001). Kaplan-Meier analysis showed a worse five-year DFS in pHER2Y1248-negative patients who were older than 60 years (P2.0 cm (P<0.001); higher histological grade (P<0.001); HER2E intrinsic subtype (P<0.001), negative hormone receptors (P<0.001); negative HER1 status (P<0.001), positive HER3 (P=0.002); and/or positive HER4 (P=0.002) status. The only negative prognostic factor for five-year DFS in multivariate Cox regression analysis was pHER2Y1248-negative (hazard ratio [HR] 3.6, 95% confidence interval [CI] 1.8-7.2, P<0.001) and lymph node-positive status (HR 3.6, 95% CI 1.3-9.8, P=0.014). ----- Conclusion: pHER2Y1248 predicts sensitivity to trastuzumab and a better five-year DFS regardless of any other prognostic parameter. In HER2-positive BC patients. Non-phosphorylated HER2Y1248 is a strong predictor of trastuzumab resistance and a poor DFS

Aberrant expression of SFRP1, SFRP3, DVL2 and DVL3 Wnt signaling pathway components in diffuse gastric carcinoma

Diffuse gastric carcinoma (DGC) is characterized by poorly cohesive cells, highly invasive growth patterns, poor prognosis and resistance to the majority of available systemic therapeutic strategies. It has been previously reported that the Wnt/β-catenin signaling pathway serves a prominent role in the tumorigenesis of gastric carcinoma. However, the mechanism underlying the dysregulation of this pathway in DGC has not been fully elucidated. Therefore, the present study aimed to investigate the expression profiles of Wnt antagonists, secreted frizzled-related protein 1 (SFRP1) and secreted frizzled-related protein 3 (SFRP3), and dishevelled protein family members, dishevelled segment polarity protein 2 (DVL2) and dishevelled segment polarity protein 3 (DVL3), in DGC tissues. The association between the expression levels of these factors and the clinicopathological parameters of the patients was determined. Protein and mRNA expression levels in 62 DGC tumor tissues and 62 normal gastric mucosal tissues obtained from patients with non-malignant disease were measured using immunohistochemical and reverse transcription-quantitative PCR (RT-qPCR) analysis. Significantly lower protein expression levels of SFRP1 (P<0.001) and SFRP3 (P<0.001), but significantly higher protein expression levels of DVL2 (P<0.001) and DVL3 (P<0.001) were observed in DGC tissues compared with in control tissues by immunohistochemistry. In addition, significantly lower expression levels of SFRP1 (P<0.05) and higher expression levels of DVL3 (P<0.05) were found in in DGC tissues compared with those in normal gastric mucosal tissues using RT-qPCR. According to correlation analysis between the SFRP1, SFRP3, DVL2 and DVL3 protein expression levels and the clinicopathological characteristics of patients with DGC, a statistically significant correlation was found between the SFRP3 volume density and T stage (r=0.304; P=0.017) and between the SFRP3 volume density and clinical stage (r=0.336; P=0.008). In conclusion, the findings of the present study suggested that the Wnt signaling pathway components SFRP1, SFRP3, DVL2 and DVL3 may be aberrantly expressed in DGC tissues, implicating their possible role in the development of this malignant disease. The present data also revealed a positive relationship between SFRP3 protein expression and the clinical and T stage of DGC