De novo mutations in idiopathic male infertility-A pilot study.

STUDY QUESTION: Are de novo mutations in the human genome associated with male infertility? SUMMARY ANSWER: We identified de novo mutations in five candidate genes: SEMA5A, NEURL4, BRD2, CD1D, and CD63. WHAT IS KNOWN ALREADY: Epidemiological and genetic studies have consistently indicated contribution of genetic factors to the etiology of male infertility, suggesting that more than 1500 genes are involved in spermatogenesis. STUDY DESIGN, SIZE, DURATION: First, we searched for de novo mutations in patients with idiopathic azoospermia with whole-exome sequencing (WES). To evaluate the potential functional impact of de novo identified mutations, we analyzed their expression differences on independent testis samples with normal and impaired spermatogenesis. In the next step, we tested additional group of azoospermic patients for mutations in identified genes with de novo mutations. In addition to the analysis of de novo mutations in patients with idiopathic azoospermia, we considered other models of inheritance and searched for candidate genes harboring rare maternally inherited variants and biallelic autosomal and X-chromosome hemizygous variants. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed WES in 13 infertile males with idiopathic azoospermia and their parents. Potential functional impact of de novo identified mutations was evaluated by global gene expression profiling on 20 independent testis samples. To replicate the results, we performed WES in further 16 independent azoospermic males, which were screened for the variants in the same genes. Library preparation was performed with Nextera Coding Exome Capture Kit (Illumina), with subsequent sequencing on Illumina HiSeq 2500 platform. MAIN RESULTS AND THE ROLE OF CHANCE: We identified 11 de novo mutations in 10 genes of which 5 were considered potentially associated with azoospermia: SEMA5A, NEURL4, BRD2, CD1D, and CD63. All candidate genes showed significant differential expression in testis samples composed of patients with severely impaired and normal spermatogenesis. Additionally, we identified rare, potentially pathogenic mutations in the genes previously implicated in male infertility-a maternally inherited heterozygous frameshift variant in FKBPL gene and inframe deletion in UPF2 gene, homozygous frameshift variant in CLCA4 gene, and a heterozygous missense variant NR0B1 gene, which represent promising candidates for further clinical implication. LIMITATIONS OF THE STUDY, REASONS FOR CAUTION: We provided limited functional support for involvement of de novo identified genes in pathogenesis of male infertility, based on expression analysis. Additionally, the sample size was limited. WIDER IMPLICATIONS OF THE FINDINGS: We provide support that de novo mutations might contribute to male infertility and propose five genes as potentially implicated in its pathogenesis.

Polymorphism RAD51 172G > T in Serbian patients with colorectal cancer.

PURPOSE: The RAD51 gene plays an important role in homologous strand exchange in DNA repair. Two common single nucleotide polymorphisms in this gene, 135G>C and 172G>T, were associated with altered gene transcription. While 135G>C was already linked to breast and colorectal cancers in certain populations, 172G>T is far less investigated, although sporadic studies showed it could be a prognostic factor for some cancer lesions. The purpose of this study was to investigate RAD51 172G>T polymorphism in Serbian population, its association with colorectal carcinoma, as well as correlation with disease characteristics and response to neoadjuvant chemoradiotherapy. METHODS: The 172G>T polymorphism was evaluated by PCR-RFLP method in blood samples of 209 colorectal cancer patients and 43 healthy subjects who served as controls. The distribution of genotypes was also analyzed in respect to several tumor characteristics in cases where histopathological data were available. RESULTS: A significant association between the RAD51 172G>T polymoprhism and desmoplastic reaction of colorectal cancer was demonstrated. The 172G allele was found to be significantly more frequent in patients with more intensive desmoplastic response of the tumor tissue. CONCLUSIONS: The results of our study suggest that the 172T allele of RAD51 may be a favorable prognostic factor in Serbian patients with colorectal cancer, although larger prospective studies are required to confirm this finding.

Immunohistochemical expression of proliferative markers in renal cell carcinoma.

PURPOSE: The purpose of this study was to investigate into the expression of cyclin A and telomerase in renal cell carcinoma (RCC) and to analyze the relationship between expression and the clinicopathological characteristics of the tumor and their impact on survival. METHODS: The overall material included 74 samples of RCC and 4 of normal renal tissue. Primary cyclin A antibody from Santa Cruz Biotechnology and TERT MA5-16034 antibody from Thermo Fisher Scientific Inc were used. Staining was performed by streptavidin-biotin technique using DAKO LSAB+ kit. Statistical analyses were performed using of SPSS 23 Statistics software from IBM. RESULTS: No differences in cyclin A and telomerase expression among gender and age groups were found, nor did the tumor dimensions have any significant impact on expression. Also, tumor grades and stages did not differ. However, histological types differed in favor of the papillary type. A significant positive correlation between both markers, as well as between the expression and tumor stage and grade was noticed. Only the tumor stage had negative impact on survival. CONCLUSIONS: Although not affecting survival, the expression of cyclin A and telomerase increased with tumor stage and grade, suggesting that cyclin A and telomerase could be potential proliferative immunohistochemical markers of RCC.

Association between angiotensin-converting enzyme gene insertion/deletion polymorphism and susceptibility to preterm birth: A case-control study and meta-analysis.

BACKGROUND: Preterm birth is the largest contributor to newborn mortality, morbidity, and hospitalization in the first year of life worldwide. Previous studies have suggested the importance of genetic variation in the angiotensin-converting enzyme gene, including the angiotensin-converting enzyme gene insertion/deletion polymorphism, in association with preterm birth. The angiotensin-converting enzyme is a key component of the renin-angiotensin system that is involved in blood pressure homeostasis during pregnancy and also affects risk factors of preterm birth, including the regulation of fibrinolytic system, uteroplacental circulation, vascularization of the placenta, and inflammation. OBJECTIVE: The results of previous studies investigating the association between the insertion/deletion polymorphism and susceptibility to preterm birth have been inconsistent, therefore, we have performed a case-control study and conducted a meta-analysis of related studies to clarify this association. STUDY DESIGN: In a case-control genetic association study, performed on 217 women with a history of preterm birth and 158 women who experienced full-term pregnancy, the significances of associations between allelic and genotype frequencies and preterm birth were determined using Chi-square tests. Following the case-control study, PubMed, Scopus, Google Scholar, and HugeNavigator databases were systematically searched to identify relevant studies. Altogether, four eligible studies involving 369 cases and 559 controls were included in the meta-analysis. The strength of the association between the angiotensin-converting enzyme gene insertion/deletion polymorphism for preterm birth was estimated by odds ratios (ORs) and corresponding 95% confidence intervals (95% CIs), using a fixed-effects model (Mantel-Haenszel method). RESULTS: In our case-control study we did not detect a significant association of angiotensin-converting enzyme insertion/deletion alleles and genotypes with preterm birth. The results of the meta-analysis showed a significant association between the angiotensin-converting enzyme gene insertion/deletion and the risk of preterm birth under allelic, dominant, and recessive comparison genetic models (D vs. I: OR = 1.35, 95% CI = 1.11-1.65, p = 0.0033; DD + ID vs. II: OR = 1.52, 95% CI = 1.08-2.15, p = 0.0161; DD vs. ID + II: OR = 1.48, 95% CI = 1.07-2.04, p = 0.0184). CONCLUSIONS: The present meta-analysis suggests that the insertion/deletion polymorphism of the angiotensin-converting enzyme gene in mothers might be associated with preterm birth, however, further well-designed large replication studies involving various ethnicities are needed to confirm this association.

Genetic variation in the CLOCK gene is associated with idiopathic recurrent spontaneous abortion.

Physiological studies in animals and human support an important role of circadian system in reproduction. The aim of this study was to investigate the potential association of CLOCK gene polymorphisms with idiopathic recurrent spontaneous abortion (IRSA). We performed a case-control study. The study group consisted of 268 women with a history of three or more idiopathic recurrent spontaneous abortions and 284 women with at least two live births and no history of pathologic pregnancies all from Slovenia and Serbia. Two SNPs in the CLOCK gene were chosen and genotyped. The results showed a statistically significant difference in genotype distribution between the two groups in the CLOCK gene for rs6850524 and rs11932595. Our analysis showed that G allele under dominant model (GG+GC/CC) for rs6850524 (p = 2∙10-4, OR = 2.28, 95%CI = 1.46-3.56) as well as G allele under dominant model (GA+AA/AA) for rs11932595 (p = 0.04, OR = 1.47, 95%CI = 1.01-2.04) might be risk factors against IRSA. Our data suggest that genetic variability in the CLOCK gene is associated with IRSA warranting further confirmation and mechanistic investigations.

Cell-free DNA as biomarker and source for mutation detection in primary colorectal cancer.

PURPOSE: To analyze if cell-free (cf)DNA levels and the presence of KRAS and BRAF mutations in serum could be used as diagnostic biomarkers in patients with primary colorectal cancer (CRC). METHODS: This study included 92 individuals who were operated due to primary CRC (N=52;study group) and to hemorrhoids (N=40;control group). Serum cfDNA levels were measured with real-time PCR (RT-PCR) using PicoGreen dsDNA quantitation reagent. Colorectal tissue and related blood and serum samples taken at the time of surgery were subjected to DNA extraction and analysis of KRAS and BRAF mutations based on multiplex SNaPshot assay and DNA sequencing. RESULTS: The average cfDNA concentration was lower in patients of the study group (20±7 ng/µL) in comparison to controls (34±9 ng/µL) and this difference was statistically significant (p<0.001). The SNaPshot analysis detected KRAS c35 mutations in colorectal tumor tissue in 14 cases, but the presence of the mutation was not confirmed in cfDNA extracted from blood samples of these patients. CONCLUSIONS: The level of serum cfDNA in CRC is decreased in comparison to patients with hemorrhoids, which questions the usefulness of cfDNA as cancer biomarker. Also, cfDNA does not appear to be suitable as a source for mutation detection in this disease.

SMAD4 gene promoter mutations in patients with thyroid tumors.

As a key component of the transforming growth factor beta (TGFB) pathway, which regulates the expression of thyroid-specific genes, tumor suppressor SMAD4 is crucial for thyroid development and function. Aberrant expression of SMAD4 in thyroid tumor tissue was reported and mutations affecting the coding region have been detected, but a potential role of mutations in SMAD4 gene regulatory regions remains unexplored. The aim of this study was to analyze SMAD4 gene promoters in thyroid tumors. A total of 76 thyroidectomy specimens were studied, including 42 malignant and 34 benign tumors. The presence of mutations in four SMAD4 gene promoters was analyzed in thyroid tumor tissue and peripheral blood by PCR and DNA sequencing. The expression and intracellular localization of endogenous SMAD4 protein in selected tumor samples was studied by immunostaining and confocal microscopy. Of three novel variants detected, two were within promoter A (-204T/C and -5C/T) and one in promoter D (-180delA). Unlike somatic mutations previously detected in the nearby region, germline mutation -180delA in promoter D doesn't appear to affect SMAD4 expression in the thyroid tumor tissue. However, all newly detected SMAD4 promoter variants affect predicted binding sites of transcription factors involved in cell cycle regulation and should be further characterized functionally. Although not directly involved in carcinogenesis, detected variants may alter SMAD4 transcriptional regulation to some extent. Considering that dosage dependence is of great importance for the role of SMAD4 protein as a tumor suppressor, potential clinical significance of SMAD4 gene promoter mutations is worth further investigation.

Genetic variation in circadian rhythm genes CLOCK and ARNTL as risk factor for male infertility.

BACKGROUND: The circadian system has a major role in maintaining homeostasis and proper body functions including reproductive capacity. The aim of this study was to examine whether there is an association between genetic variability in the primary clock genes CLOCK and ARNTL and male infertility in humans. METHODOLOGY/PRINCIPAL FINDINGS: We performed a case-control study, where we searched for an association between polymorphisms of CLOCK and ARNTL genes and male infertility in 961 Slovenian and Serbian Caucasian men. The study group consisted of 517 patients with idiopathic infertility and a control group of 444 fertile men. A statistically significant difference was found in genotype distribution between the two groups in the CLOCK gene: rs11932595 (p = 6·10(-5), q = 4·10(-4), OR equaled 1.9 with 95% CI 1.4-2.7), rs6811520 (p = 2·10(-3), q = 8·10(-3), OR = 1.7 with 95% CI 1.2-2.2) and rs6850524 (p = 0.01, q = 0.02, OR = 1.4 with 95% CI 1.1-1.9). Further analyses of haplotypes were consistent with genotyping results. CONCLUSIONS/SIGNIFICANCE: We provide evidence that genetic variability in the CLOCK gene might be associated with male infertility warranting further confirmation and mechanistic investigations.

Genetic alterations in SMAD4 and K-ras in Serbian patients with endometrial carcinoma.

OBJECTIVE: This study was aimed at analyzing alterations in K-ras gene and SMAD4 gene promoter in endometrial carcinoma tissue in Serbian patients. METHODS/MATERIALS: The study has encompassed 36 patients whose endometrial cancer tissue samples and peripheral blood samples were analyzed for the presence of alterations in the K-ras gene and the SMAD4 gene promoter. The detection of K-ras codon 12 mutation was performed by polymerase chain reaction restriction fragment length polymorphism technique. Analysis of mononucleotide repeat variants at -462T(15) and -4T(12) of the SMAD4 gene promoter was performed by capillary electrophoresis analysis of DNA fragments fluorescently labeled by polymerase chain reaction. RESULTS: Mutation in codon 12 of the K-ras gene was detected with relatively high frequency of 75.0% (27 of 36 cases). Analysis of 2 mononucleotide repeats in the SMAD4 gene promoter showed that in most cases, haplotypes -462T(15)/-4T(12) and -462T(16)/-4T(12) were present; whereas in one case, a novel haplotype -462T(15)/-4T(10) was detected. CONCLUSIONS: Findings on the role and potential significance of the K-ras codon 12 mutation and SMAD4 gene promoter variants in patients with endometrial carcinoma remain controversial, and their occurrence in this type of cancer should be further investigated.

Structural and functional analysis of SMAD4 gene promoter in malignant pancreatic and colorectal tissues: detection of two novel polymorphic nucleotide repeats.

BACKGROUND: The tumor suppressor gene SMAD4 (DPC4) encodes for the common intracellular mediator of the TGF-ß superfamily pathway, which regulates numerous cellular processes, such as cell proliferation, cell differentiation, apoptosis, cell fate and migration. This study was aimed to investigate the presence of genetic variants in SMAD4 gene promoter in malignant pancreatic and colorectal tissue and to analyze their functional consequences. METHODS: The study was performed on genomic DNA isolated from malignant tissue samples obtained on surgery from 50 patients with pancreatic carcinoma and 50 patients with colorectal cancer. Screening for mutations within an 800bp-long fragment of the SMAD4 gene promoter was performed by DNA sequencing and two mononucleotide repeats, at positions -462 and -4, were found to be polymorphic in malignant tissue. The exact number of thymidines in the tracts -462T(15) and -4T(12) was determined by PCR with fluorescently labeled primers followed by capillary electrophoresis. Functional analysis of -462T(15)/-4T(12) haplotypes was performed by luciferase reporter assays. RESULTS: Haplotype -462T(14)/-4T(10) was found in 85% of pancreatic cancer tissues, but it was not present in any of colorectal cancer tissues. Statistically significant reduction (p<0.001) in activity was observed in the haplotype -462T(14)/-4T(10) in comparison with the haplotypes -462T(15)/-4T(12) and -462T(14)/-4T(11). CONCLUSION: Results of this study indicate that novel genetic variant -4T(10) in the SMAD4 gene promoter affects its activity and that element -4T(12) may play a role in transcriptional regulation of SMAD4 gene expression. Obtained results, though preliminary, also indicate that SMAD4 gene promoter haplotype -462T(14)/-4T(10) may represent a genetic marker of potential relevance for pancreatic and colorectal cancer. The findings of this study should be confirmed by further investigation in these two and other tumors, on larger number of patients and with different tumor stages. Translational research aimed at investigating potential application of mononucleotide repeats -462T(15) and -4T(12) in SMAD4 gene promoter as molecular markers in cancer may also prove useful.

[Y chromosome microdeletions in infertile male candidates for microfertilization].

INTRODUCTION: Y chromosome microdeletions are the second most frequent genetic cause of male infertility after Klinefelter's syndrome. OBJECTIVE: The aim of the study was to determine the frequency ofY chromosome microdeletions in a group of infertile men with an idiophatic cause of infertility, candidates for microfertilization (Intra-cytoplasmic Sperm Injection--ICSI) in Serbia and to correlate genotype-phenotype in patients with Y chromosome microdeletions. METHOD: One hundred and sixty patients with low sperm count (less than 5 x 10(6) spermatozoa/ml) were enrolled in the study. Forty patients were excluded from the study: ten because they were diagnosed with cytogenetic abnormality and thirty patients were diagnosed with other known causes of infertility. The control group consisted of 150 men who fathered at least one child in the last two years. Genomic DNA was extracted from peripheral blood samples and two multiplex polymerase chain reactions (PCR) analyses were performed using specific primers to confirm the presence or absence of Y chromosome microdeletions. RESULTS: Microdeletions were detected in 12 of 120 (10%) cases, while no deletions were detected in the control group. Of total number of 12 deletions, nine were detected in AZFc region (75%), one in AZFa (8%), and two in AZFbc (17%). CONCLUSION: Testing for Y chromosome microdeletions should be considered as an important element in diagnosis and genetic counselling of infertile couples in Serbia. Decisions regarding the assisted reproduction should be made based on the detailed clinical, endocrinological and cytogenetic examinations, spermogram, presence or absence and type of AZF microdeletions and CFTR gene mutations.

Analysis of Y chromosome microdeletions and CFTR gene mutations as genetic markers of infertility in Serbian men.

BACKGROUND/AIM: Impaired fertility of a male partner is the main cause of infertility in up to one half of all infertile couples. At the genetic level, male infertility can be caused by chromosome aberrations or gene mutations. The presence and types of Y chromosome microdeletions and cystic fybrosis transmembrane conductance regulator (CFTR) gene mutations as genetic cause of male infertility was tested in Serbian men. The aim of this study was to analyze CFTR gene mutations and Y chromosome microdelations as potential causes of male infertility in Serbian patients, as well as to test the hypothesis that CFTR mutations in infertile men are predominantly located in the several last exons of the gene. METHODS: This study has encompassed 33 men with oligo- or azoospermia. The screening for Y chromosome microdeletions in the azoospermia factor (AZF) region was performed by multiplex PCR analysis. The screening of the CFTR gene was performed by denaturing gradient gel electrophoresis (DGGE) method. RESULTS: Deletions on Y chromosome were detected in four patients, predominantly in AZFc region (four of total six deletions). Mutations in the CFTR gene were detected on eight out of 66 analyzed chromosomes of infertile men. The most common mutation was F508del (six of total eight mutations). CONCLUSION: This study confirmed that both Y chromosome microdeletions and CFTR gene mutations played important role in etiology of male infertility in Serbian infertile men. Genetic testing for Y chromosome microdeletions and CFTR gene mutations has been introduced in routine daignostics and offered to couples undergoing assisted reproduction techniques. Considering that both the type of Y chromosome microdeletion and the type of CFTR mutation have a prognostic value, it is recomended that AZF and CFTR genotyping should not only be performed in patients with reduced sperm quality before undergoing assisted reproduction, but also for the purpose of preimplantation and prenatal diagnostics in couples in which in vitro fertilization has been performed successfully.

[Recommendations for treatment of individuals at risk for hereditary breast and ovarian carcinoma]. / Preporuke za lecenje osoba sa rizikom za nasledni karcinom dojke i jajnika.

Germline mutations in BRCA 1 and BRCA 2 gene have been recognized as hereditary predisposition for breast and ovarian cancer for many years. The optimal clinical management of individuals at risk for hereditary breast and ovarian cancer are not completely defined. Current surveillance options are restricted in their effectiveness as well as limitations of the techniques. Surgical prevention interventions are effective, but may be complicated by physical and psychological morbidity. Genetic testing, which plays a role in defining risk, requires careful pre- and post-test counseling to discuss the limitations of testing itself and available management strategies.