Genomic Instability of the APC Gene Found in Glioblastoma

The etiology and pathogenesis of tumors of the central nervous system are still inadequately explained. This study analyses tumor suppressor gene— adenomatous polyposis coli (APC) in 28 patients with glioblastoma, the most aggressive form of glial tumors. APC protein has a structural role in adherens junctions, but also plays a signaling role as a negative regulator of the wnt pathway. Our interest in the APC gene stemmed principally from the findings that the wild-type APC protein is highly expressed in the central nervous system, and upon the finding that it is critically involved in particular syndromes, among which brain tumors play a significant role. Glioblastoma samples were tested for gene instability by PCR/loss of heterozygosity using RFLP method. Two polymorphic markers were used: an Rsa I polymorphic site in exon 11, and an Msp I polymorphic site in exon 15. The results of our analysis for both markers showed allelic loss of the APC gene in 40% of our sample out of 25 heterozygous patients (informativeness 89%). Another 20% of samples demonstrated allelic imbalance of the APC allele in tumor tissue. Altogether, there were 15 samples (60%) demonstrating instability of this tumor suppressor gene. Despite increasing knowledge on glioma biology and genetics, the prognostic tools for glioblastoma still need improvement. Our findings on genomic instability of the APC gene may contribute to better understanding of glioblastoma genetic profile and could be used as a prognostic marker of disease evolution and progression

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

Clinical and pathohistological characteristics of Alport spectrum disorder caused by COL4A4 mutation c.193-2A>C: a case series

Aim To present the pathohistological and clinical charac - teristics of five Croatian families with Alport spectrum dis - orders caused by splice acceptor pathogenic variant c.193- 2A>C in COL4A4 at the genomic position chr2:227985866. Methods The study enrolled five probands with kidney bi - opsy analysis and five family members. Mutation screening was performed with Illumina MiSeq platform. The patho - genic variant was confirmed with standard dye-terminator sequencing. Results The only homozygous patient, aged two, had pro - teinuria and hematuria with preserved kidney function and no extrarenal manifestations. This patient had chang - es characteristic for Alport syndrome observed on elec - tron microscopy of the kidney biopsy. In the heterozygous group, six patients had hematuria, four biopsied probands had proteinuria, and only one had moderately reduced kidney function. Heterozygous probands had variable kid - ney biopsy findings. Three patients had thin glomerular basement membrane nephropathy visible on electron mi - croscopy and focal segmental glomerulosclerosis on light microscopy, two of them with focal lamellation on elec - tron microscopy. One heterozygous patient had changes characteristic for Alport syndrome on electron microscopy without focal segmental glomerulosclerosis. Conclusion The homozygous patient had hematuria and proteinuria with preserved kidney function. The heterozy - gous patients presented with reasonably mild clinical phe - notype and variable pathohistological findings

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

Izraženost beta-katenina u malignom melanomu

Beta-catenin is bound to E-cadherin in adherens junction formation, but also functions as a signaling molecule in the WNT pathway. We investigated beta-catenin expression in 41 superficial spreading melanomas. Our melanoma sample was analyzed by immunohistochemistry and evaluated by image analysis as staining density, i.e. light permeability (LP). In normal skin beta-catenin showed homogenous membranous staining of the epidermis. Comparison of relative LP for beta-catenin in tumor tissue and adjacent skin did not show any differences (157.8 compared to 156.6; t = . 1.087, P = 0.283). However, the cellular location of beta-catenin changed considerably in melanoma. The protein was observed in the cytoplasm in 32% of patients, in 29% in the cell membrane, in 24% in both the cytoplasm and membrane, in 5% in the cytoplasm and nucleus, while in 9.8% of patients beta-catenin could not be observed. There was a marked difference in beta-catenin distribution correlated to the Clark stages of melanoma progression. Patients with Clark 4 and 5 had significantly less beta-catenin than patients with Clark 2 and 3 (χ2 = 11.3; P=0.01). Our results suggest that changes of beta-catenin levels have roles in melanoma development mechanisms and could be used as markers of disease progression.Beta-katenin vezan je za molekulu E-kadherina u zonulama adherens, ali također funkcionira i kao signalna molekula u putu prijenosa signala WNT. U ovom radu istraživali smo ekspresiju ovoga proteina u uzorku od 41 superficijelno širećeg malignog melanoma. Metoda imunohistokemije korištena je za analizu beta-kateninske izraženosti, a razina ekspresije kvantificirana je image analizom kao gustoća obojenja, odnosno, propusnost svjetla. U zdravoj koži beta-katenin pokazivao je homogeno membransko obojenje u epidermisu. Usporedba relativne propusnosti svjetla za izraženost beta-katenina u tumorskom tkivu i pripadajućoj koži nije pokazala razlike u propusnosti svjetla (157.8 prema 156.6; t = .1.087, P = 0.283). Međutim, stanični smještaj ispitivanog proteina uvelike se promijenio. Opazili smo citoplazmatski smještaj beta-katenina u 32% bolesnika, membranski smještaj u 29%, citoplazmatski i membranski u 24%, a u samo 5% citoplazmatski i smještaj u jezgri tumorskih stanica. U 10% ispitanih bolesnika nismo detektirali beta-kateninsku izraženost. Znakovita razlika primijećena je u razdiobi beta-katenina po stupnjevima melanomske progresije (Clark). Bolesnici s Clarkom 4 i 5 imali su znakovito manje beta-katenina od bolesnika s Clarkom 2 i 3 (χ2 = 11.3; P=0.01). Naši rezultati pokazuju da promjene u izraženosti beta-katenina imaju ulogu u mehanizmima nastanka melanoma i da postoji mogućnost korištenja beta-katenina kao biljega progresije bolesti

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

E-cadherin and beta-catenin expression patterns in malignant melanoma assessed by image analysis

BACKGROUND: We investigated the expression of E-cadherin and beta-catenin in melanoma. Both proteins are components of adherens junctions but also play signalling roles in the wnt signal transduction pathway. ----- MATERIALS AND METHODS: Seventy malignant melanomas were analysed by immunohistochemistry and evaluated by image analysis as staining density, i.e. light permeability (LP). ----- RESULTS: Comparison of mean values of relative LP for E-cadherin and beta-catenin in tumor tissue shows that levels of E-cadherin protein are significantly lower (259.67-116.23; t=22.7; p=0.000). The comparison of mean values of the relative LP of E-cadherin in melanoma to the LP in the adjacent normal skin also shows that the expression of E-cadherin in tumor is significantly lower (256.06-169.87; t=11.55, p=0.000). beta-catenin was observed in the cytoplasm in 30.6% of patients, in 24.2% in the cell membrane, in 21% in both the cytoplasm and membrane, in 1.6% in the membrane and nucleus and in 4.8% in the cytoplasm and nucleus, whereas in 17.7% of patients beta-catenin could not be observed. Patients with Clark 4 and 5 had significantly less beta-catenin than patients with Clark 2 and 3 (chi2=12.854; p=0.005). ----- CONCLUSIONS: Changes in E-cadherin and beta-catenin levels have important roles in melanoma and could be used as molecular markers of disease progression