A Risk of Gonadoblastoma in Familial Swyer Syndrome-A Case Report and Literature Review.

A complete gonadal dysgenesis (CGD) with 46,XY karyotype is known as the Swyer syndrome and belongs to the group of 46,XY differences of sex development (DSD). The main problem in patients with Swyer syndrome is the delayed puberty and primary amenorrhea. Moreover, intrabdominal dysgenetic gonads in the patient with genetic material of a Y chromosome may conduce to the development of gonadal tumors, such as gonadoblastoma or germinoma. The management of such patients is based on preventive excision of dysgenetic gonads and long-term hormonal replacement therapy. Sporadic cases are considered more common than familial cases. This paper presents two siblings with Swyer syndrome in whom gonadoblastoma was found. A thorough review of familial CGD with 46,XY DSD in the literature from the last 15 years suggests that the risk of gonadal tumors could be increased in familial compared to sporadic cases (66.6% vs. 15-45%, respectively).

Circular RNAs Could Encode Unique Proteins and Affect Cancer Pathways.

circRNAs constitute a novel class of RNA, generally considered as non-coding RNAs; nonetheless, their coding potential has been under scrutiny. In this work, we systematically explored the predicted proteins of more than 160,000 circRNAs detected by exome capture RNA-sequencing and collected in the MiOncoCirc pan-cancer compendium, including normal and cancer samples from different types of tissues. For the functional evaluation, we compared their primary structure and domain composition with those derived from the same linear mRNAs. Among the 4362 circRNAs potentially encoding proteins with a unique primary structure and 1179 encoding proteins with a novel domain composition, 183 were differentially expressed in cancer. In particular, eight were associated with prognosis in acute myeloid leukemia. The functional classification of the dysregulated circRNA-encoded polypeptides showed an enrichment in the heme and cancer signaling, DNA-binding, and phosphorylation processes, and disclosed the roles of some circRNA-based effectors in cancer.

Association Between Parafibromin Expression and Presence of Brown Tumors and Jaw Tumors in Patients with Primary Hyperparathyroidism: Series of Cases with Review of the Literature.

BACKGROUND Brown and jaw tumors are rare entities of poorly understood etiology that are regarded as end-stage of bone remodeling in patients with long-lasting and chronic hyperparathyroidism. Jaw tumors are mainly diagnosed in jaw tumors syndrome (HPT-JT syndrome) and are caused by mutation in the CDC73 gene, encoding parafibromin, a tumor suppressing protein. The aim of this work is to present 4 cases of patients in whom the genetic mutation of the CDC73 gene and clinical presentation coexist in an unusual setting that has not yet been described. CASE REPORT We present cases of 4 patients with primary hyperparathyroidism. Three were diagnosed with brown tumors (located in long bones, ribs, iliac, shoulders) and 1 with brown and jaw tumors. Expression of parafibromin in affected parathyroid tissues were analyzed. In patients without positive parafibromin staining, we searched for CDC73 mutation using next-generation sequencing. Parafibromin staining was positive in 1 patient with brown tumors and was negative in 2 individuals with brown tumors and 1 with brown and jaw tumors. CDC73 mutation was detected in two-thirds of patients (60%) with negative staining for parafibromin and brown tumors. MEN1 mutation was found in the patient with brown tumor and positive staining for parafibromin. CONCLUSIONS Patients with hyperparathyroidism and coexistence of brown tumors or jaw tumors might have decreased expression of parafibromin in parathyroid adenoma tissue, which might be caused by CDC73 mutation and suggest a genetic predisposition. Further research on much larger study groups is needed.

Compound Haplotype Variants in CFH and CD46 Genes Determine Clinical Outcome of Atypical Hemolytic Uremic Syndrome (aHUS)-A Series of Cases from a Single Family.

Atypical hemolytic uremic syndrome (aHUS) is a rare disease triggered by dysregulation of the alternative complement pathway, consisting of a characteristic triad of nonimmune hemolytic anemia, thrombocytopenia, and renal failure. The risk of aHUS onset, recurrence, and allograft loss depends on the genetic background of a patient. We show a series of cases from a single family whose five members were affected by aHUS and presented distinct clinical outcomes. Next-generation sequencing revealed combined mutations in both complement factor H and membrane cofactor protein CD46. Out of eight siblings, aHUS affected three adult brothers, and, subsequently, affected two children of an unaffected sister. The first patient died due to aHUS, and two other brothers underwent successful kidney transplantation with no aHUS recurrence. The younger, 10-month-old child presented with a severe course of the disease with cardiac involvement and persistent hemolytic anemia limited by eculizumab, while the 2-year-old recovered completely on eculizumab. The study shows a highly variable disease penetrance.

BMPR1B gene in brachydactyly type 2-A family with de novo R486W mutation and a disease phenotype.

BACKGROUND: Brachydactylies are a group of inherited conditions, characterized mainly by the presence of shortened fingers and toes. Based on the patients' phenotypes, brachydactylies have been subdivided into 10 subtypes. In this study, we have identified a family with two members affected by brachydactyly type A2 (BDA2). BDA2 is caused by mutations in three genes: BMPR1B, BMP2 or GDF5. So far only two studies have reported the BDA2 cases caused by mutations in the BMPR1B gene. METHODS: We employed next-generation sequencing to identify mutations in culpable genes. RESULTS AND CONCLUSION: In this paper, we report a case of BDA2 resulting from the presence of a heterozygous c.1456C>T, p.Arg486Trp variant in BMPR1B, which was previously associated with BDA2. The next generation sequencing analysis of the patients' family revealed that the mutation occurred de novo in the proband and was transmitted to his 26-month-old son. Although the same variant was confirmed in both patients, their phenotypes were different with more severe manifestation of the disease in the adult.

Bezafibrate Upregulates Mitochondrial Biogenesis and Influence Neural Differentiation of Human-Induced Pluripotent Stem Cells.

Bezafibrate (BZ) regulates mitochondrial biogenesis by activation of PPAR's receptors and enhancing the level of PGC-1α coactivator. In this report, we investigated the effect of BZ on the expression of genes (1) that are linked to different pathways involved in mitochondrial biogenesis, e.g., regulated by PPAR's receptors or PGC-1α coactivator, and (2) involved in neuronal or astroglial fate, during neural differentiation of hiPSC. The tested cell populations included hiPSC-derived neural stem cells (NSC), early neural progenitors (eNP), and neural progenitors (NP). RNA-seq analysis showed the expression of PPARA, PPARD receptors and excluded PPARG in all tested populations. The expression of PPARGC1A encoding PGC-1α was dependent on the stage of differentiation: NSC, eNP, and NP differed significantly as compared to hiPSC. In addition, BZ-evoked upregulation of PPARGC1A, GFAP, S100B, and DCX genes coexist with downregulation of MAP2 gene only at the eNP stage of differentiation. In the second task, we investigated the cell sensitivity and mitochondrial biogenesis upon BZ treatment. BZ influenced the cell viability, ROS level, mitochondrial membrane potential, and total cell number in concentration- and stage of differentiation-dependent manner. Induction of mitochondrial biogenesis evoked by BZ determined by the changes in the level of SDHA and COX-1 protein, and mtDNA copy number, as well as the expression of NRF1, PPARGC1A, and TFAM genes, was detected only at NP stage for all tested markers. Thus, developmental stage-specific sensitivity to BZ of neurally differentiating hiPSC can be linked to mitochondrial biogenesis, while fate commitment decisions to PGC-1α (encoded by PPARGC1A) pathway.

MicroRNA Analysis Using the Quantitative Real-Time PCR Reaction.

The analysis of microRNA expression patterns provides new insights into numerous cellular processes and their aberrances in diseases. Despite its potential pitfalls, the quantitative real-time polymerase chain reaction (qPCR) is the most commonly used tool for microRNA profiling. The method requires extraction and quality analysis of RNA, which is further reverse transcribed using specific primers and used as a template in a qPCR reaction. All these elements have been addressed in this chapter.

MicroRNA Analysis Using Next-Generation Sequencing.

Next-generation sequencing (NGS) enables the analysis of both microRNA expression and sequence, allowing for elucidation of a comprehensive landscape of miRNAs in a given tissue and sample type. NGS analysis requires high-quality RNA extraction and preparation of microRNA libraries. In this chapter, we describe the methods used for RNA extraction from tissue specimens, serum, cytological slides, and formalin-fixed paraffin-embedded samples. Although the described library preparation and sequencing approaches are based on Illumina NextSeq 500 sequencing technology, the presented principles shall be compatible with other commercially available sequencing platforms.

The rs2910164 Genetic Variant of miR-146a-3p Is Associated with Increased Overall Mortality in Patients with Follicular Variant Papillary Thyroid Carcinoma.

Aberrant expression of the sodium-iodide symporter (NIS) and the resistance to post-operative radioactive iodide treatment is a crucial cause of higher mortality of some thyroid cancer patients. In this study, we analyzed the impact of miR-146a on the expression and function of NIS and on the overall survival of thyroid cancer patients. The study included 2441 patients (2163 women; 278 men); including 359 cases with follicular variant of papillary thyroid carcinoma (fvPTC). miR:NIS interactions were analyzed in cell lines using in vivo binding and inhibition assays and radioactive iodine uptake assays. Tumor/blood DNA was used for rs2910164 genotyping. Overall survival was assessed retrospectively. In the results, we showed that miR-146a-3p directly binds to and inhibits NIS. Inhibition of miR-146a-3p restores the expression and function of NIS, increasing radioactive iodine uptake. Rs2910164 functional variant within miR-146a-3p is associated with increased overall mortality among fvPTC female patients. The deaths per 1000 person-years were 29.7 in CC carriers vs. 5.08 in GG/GC-carriers (HR = 6.21, p = 0.006). Higher mortality of CC vs. GG/GC carriers was also observed in patients with lower clinical stage (HR = 22.72, p < 0.001), smaller tumor size (pT1/pT2) (HR = 25.05, p < 0.001), lack of extrathyroidal invasion (HR = 9.03, p = 0.02), lack of nodular invasion (HR = 7.84, p = 0.002), lack of metastases (HR = 6.5, p = 0.005) and older (age at diagnosis >50 years) (HR = 7.8, p = 0.002). MiR-146a-3p underwent somatic mutations in 16.1% of analyzed specimens, mainly towards the deleterious C allele. In this report we propose a novel molecular marker of the clinical outcome of fvPTC patients. Rs2910164 increases the overall mortality with inhibition of NIS and disruption of radioiodine uptake as a possible mechanism.

Guidelines of Polish National Societies Diagnostics and Treatment of Thyroid Carcinoma. 2018 Update.

Significant advances have been made in thyroid can-cer research in recent years, therefore relevant clinical guidelines need to be updated. The current Polish guidelines "Diagnostics and Treatment of Thyroid Carcinoma" have been formulated at the "Thyroid Cancer and Other Malignancies of Endocrine Glands" conference held in Wisla in November 2015 [1].

Functional analysis of a novel, thyroglobulin-embedded microRNA gene deregulated in papillary thyroid carcinoma.

MicroRNAs, non-coding regulators of gene expression, are known culprits of thyroid cancer. Using next-generation sequencing, we identified a novel microRNA gene, encoded within an important thyroid regulator - thyroglobulin, and analyzed its functionality in the thyroid gland. In vitro and in silico analyses proved that the novel miR-TG is processed from the precursor, and co-expressed with thyroglobulin. Both genes are specific for thyroid tissue and downregulated in papillary thyroid carcinoma by 44% (p = 0.04) and 48% (p = 0.001), respectively. Putative target genes for miR-TG were identified using in silico tools, which pinpointed MAP4K4, an oncogene upregulated in thyroid cancer. Analysis of transcriptome by RNA-seq revealed that overexpression of miR-TG in PTC-derived cell line led to downregulation of several genes, including MAP4K4 (fold change 0,82; p = 0.036). The finding was confirmed by SQ-PCR (fold change 071; p = 0.004). Direct interaction between miR-TG and MAP4K4 was confirmed in the luciferase assay (p = 0.0006). Functional studies showed increase proliferation in K1 cell line transfected with miR-TG. We propose that in normal thyroid miR-TG plays a fine-tuning effect on the maintenance of MAPK pathway, inhibiting the expression of miR's target MAP4K4. This regulation is disturbed in cancer due to downregulation of the novel, thyroglobulin-embedded microRNA, characterized in this study.

Next-generation sequencing reveals microRNA markers of adrenocortical tumors malignancy.

BACKGROUND: Adrenocortical carcinoma is a rare finding among common adrenocortical tumors, but it is highly aggressive and requires early detection and treatment. Still, the differential diagnosis between benign and malignant lesions is difficult even for experienced pathologists and there is a significant need for novel diagnostic methods. In this study we aimed to reveal a complete set of microRNAs expressed in the adrenal gland and to identify easily detectable, stable and objective biomarkers of adrenocortical malignancy. METHODS: We employed next-generation sequencing to analyze microRNA profiles in a unique set of 51 samples, assigned to either a learning dataset including 7 adrenocortical carcinomas (ACCs), 8 adrenocortical adenomas (AAs) and 8 control samples (NAs), or a validation dataset including 8 ACCs, 10 AAs and 10 NAs. The results were validated in real-time Q-PCR. RESULTS: We detected 411 miRNAs expressed in 1763 length isoforms in the examined samples. Fifteen miRNAs differentiate between malignant (ACC) and non-malignant (AA + NA) tissue in the test set of independent samples. Expression levels of 6 microRNAs, miR-503-5p, miR-483-3p, miR-450a-5p, miR-210, miR-483-5p, miR-421, predict sample status (malignancy/non-malignancy) with at least 95% accuracy in both datasets. The best single-gene malignancy marker, miR-483-3p, has been validated by real-time RT PCR. CONCLUSIONS: As a result of the study we propose clinically valid and easily detectable biomarkers of adrenocortical malignancy that may significantly facilitate morphological examination. Since microRNAs can be detected in blood, the study brings tools for development of non-invasive diagnostics of adrenocortical carcinomas.

Variants in microRNA genes in familial papillary thyroid carcinoma.

Papillary Thyroid Carcinoma (PTC) displays one of the highest familiality scores of all cancers as measured by case-control studies, yet only a handful of genes have been implicated until now. Variants in microRNAs have been associated with the risk of several cancers including PTC but the magnitude of this involvement is unclear. This study was designed to test to what extent genomic variants in microRNAs contribute to PTC risk. We used SOLiD technology to sequence 321 genomic regions encoding 427 miRNAs in one affected individual from each of 80 PTC families. After excluding variants with frequency ≥ 1% in 1000 Genomes Phase 1 (n = 1092) we detected 1978 variants. After further functional filtering steps 25 variants in pre-miRs remained. Co-segregation was observed for six out of 16 tested miRNA variants with PTC in the families, namely let-7e, miR-181b, miR-135a, miR-15b, miR-320, and miR-484. Expression of miR-135a and miR-181b was tested in normal thyroid and tumor tissue from patients that carry the variants and a decrease in expression was observed. In vitro assays were applied to measure the effect of the variants on microRNAs' maturation. Four out of six variants were tested. Only the let-7e and miR-181b variants showed an effect on processing leading to lower levels of mature miRNA. These two variants were not detected in 1170 sporadic PTC cases nor in 1404 controls. Taken together, our data show that high penetrance germline sequence variants of miRNAs potentially predispose to a fraction of all PTC but are not common.

Regulatory mechanisms in arterial hypertension: role of microRNA in pathophysiology and therapy.

Multiple factors underlie the pathophysiology of hypertension, involving endothelial dysregulation, vascular smooth muscle dysfunction, increased oxidative stress, sympathetic nervous system activation and altered renin -angiotensin -aldosterone regulatory activity. A class of non-coding RNA called microRNA, consisting of 17-25 nucleotides, exert regulatory function over these processes. This paper summarizes the currently available data from preclinical and clinical studies on miRNA in the development of hypertension as well as the impact of anti-hypertensive treatment on their plasma expression. We present microRNAs' characteristics, their biogenesis and role in the regulation of blood pressure together with their potential diagnostic and therapeutic application in clinical practice.

Family of microRNA-146 Regulates RARß in Papillary Thyroid Carcinoma.

Retinoic acid is a promising tool in adjuvant cancer therapies, including refractory thyroid cancer, and its biological role is mediated by the retinoic acid receptor beta (RARß). However, expression of RARß is lowered in papillary thyroid carcinoma (PTC), contributing to promotion of tumor growth and inefficiency of retinoic acid and radioactive iodine treatment. The causes of aberrant RARB expression are largely unknown. We hypothesized that the culpable mechanisms include the action of microRNAs from the miR-146 family, previously identified as significantly upregulated in PTC tumors. To test this hypothesis, we assessed the expression of RARB as well as miR-146a-5p and miR-146b-5p in 48 PTC tumor/normal tissue pairs by Taqman assay to reveal that the expression of RARB was 3.28-fold decreased, and miR-146b-5p was 28.9-fold increased in PTC tumors. Direct interaction between miRs and RARB was determined in the luciferase assay and further confirmed in cell lines, where overexpression of miR-146a-5p and miR-146b-5p caused a 31% and 33% decrease in endogenous RARB mRNA levels. Inhibition of miR-146a and miR-146b resulted in 62.5% and 45.4% increase of RARB, respectively, and a concomitant decrease in proliferation rates of thyroid cancer cell lines, analyzed in xCELLigence system.We showed that two microRNAs of the miR-146 family directly regulate RARB. Inhibition of miRs resulted in restoration of RARB expression and decreased rates of proliferation of thyroid cancer cells. By restoring RARB levels, microRNA inhibitors may become part of an adjuvant therapy in thyroid cancer patients.

Diagnostics and Treatment of Thyroid Carcinoma.

Revised Guidelines of Polish National Societies Prepared on the initiative of the Polish Group for Endocrine Tumours approved in their final version between November 16th and 28th, 2015 by the Scientific Committee of the V Conference "Thyroid Cancer and other malignancies of endocrine glands" organised between November 14th and 17th, 2015 in Wisla, Poland; called by the following Societies: Polish Endocrine Society, Polish Society of Oncology, Polish Thyroid Association, Polish Society of Pathologists, Society of Polish Surgeons, Polish Society of Surgical Oncology, Polish Society of Clinical Oncology, Polish Society of Radiation Oncology, Polish Society of Nuclear Medicine, Polish Society of Paediatric Endocrinology, Polish Society of Paediatric Surgeons, Polish Society of Ultrasonography Gliwice-Wisla, 2015 DECLARATION: These recommendations are created by the group of delegates of the National Societies, which declare their willingness to participate in the preparation of the revised version of the Polish Guidelines. The members of the Working Group have been chosen from the specialists involved in medical care of patients with thyroid carcinoma. Directly before the preparation of the Polish national recommendations the American Thyroid Association (ATA) published its own guidelines together with a wide comment fulfilling evidence-based medicine (EBM) criteria. ATA Guidelines are consistent with National Comprehensive Cancer Network (NCCN) Recommendation. According to the members of the Working Group, it is necessary to adapt them to both the specific Polish epidemiological situation as well as to the rules referring to the Polish health system. Therefore, the Polish recommendations constitute a consensus of the experts' group, based on ATA information. The experts analysed previous Polish Guidelines, published in 2010, and other available data, and after discussion summed up the results in the form of these guidelines. It should be added that Part II, which constitutes a pathological part, has been available at the website of the Polish Society of Pathologists for acceptance of the members of the Society, and no essential comments have been proposed. The Members of the Group decided that a subgroup elected from among them would update the Guidelines, according to EBM rules, every year. The Revised Guidelines should help physicians to make reasonable choices in their daily practice; however, the final decision concerning an individual patient should be made by the caring physician responsible for treatment, or optimally by a therapeutic tumour board together with the patient, and should take into consideration the patient's health condition. It should be emphasised that the recommendations may not constitute a strict standard of clinical management imposed on medical staff. The data from clinical trials concerning numerous clinical situations are scarce. In such moments the opinion of the management may differ from the recommendations after considering possible benefits and disadvantages for the patient.

Association between GWAS-Derived rs966423 Genetic Variant and Overall Mortality in Patients with Differentiated Thyroid Cancer.

PURPOSE: Five germline genetic variants (rs116909374, rs965513, rs944289, rs966423, and rs2439302) have been associated in genome-wide association studies (GWAS) with increased risk of differentiated thyroid cancer (DTC), but their role in mortality of patients has not been established. Also, no preoperative marker of the clinical outcome of thyroid cancer had yet been identified. The aim of the study was to investigate the relationship between the variants and overall mortality in patients with DTC. EXPERIMENTAL DESIGN: Retrospective study of 1,836 patients (1,643 women, 193 men) with median age at diagnosis of 49 years and overall median follow-up time of 8.7 years after initial treatment at a single comprehensive cancer center between 1990 and 2013. RESULTS: Among 5 variants, rs966423 was associated with increased mortality, which was 6.4% (33 of 518) versus 3.7% (47 of 1,259) in TT carriers versus CC/CT carriers (P = 0.017). The HR of TT versus TC/CC carriers was 1.6 [95% confidence interval (CI), 1.02-2.49; P = 0.038] after adjustment for age at diagnosis and sex. Importantly, the association of rs966423 with mortality remained valid when clinicopathologic risk factors were included in the model (HR, 1.89; 95% CI, 1.14-3.13; P = 0.014). Higher rs966423-associated patient mortality of TT versus CC/CT carriers was also observed in interaction with angioinvasion (adjusted HR, 3.48; 95% CI, 1.67-7.22; P < 0.001), lymph node metastasis (adjusted HR, 3.47; 95% CI, 1.16-10.4; P = 0.018), extrathyroidal invasion (adjusted HR, 2.07; 95% CI, 1.15-3.73; P = 0.013). CONCLUSIONS: The presence of the rs966423-TT genotype was associated with a significant increase in overall mortality of patients with DTC. Contrary to BRAF mutation and other somatic changes, the status of germline rs966423 is known before the treatment and might be used in the management of mortality risk by means of modification of therapy.

MECHANISMS IN ENDOCRINOLOGY: MicroRNA in diagnostics and therapy of thyroid cancer.

MicroRNAs, short non-coding regulators of the gene expression, are subjects of numerous investigations assessing their potential use in the diagnostics and management of human diseases. In this review, we focus on studies that analyze the utility of microRNAs as novel diagnostic and therapeutic tools in follicular cell-derived thyroid carcinomas. This very interesting and promising field brings new insight into future strategies for personalized medicine.

microRNA-339-5p modulates Na+/I- symporter-mediated radioiodide uptake.

Na(+)/I(-) symporter (NIS)-mediated radioiodide uptake (RAIU) serves as the basis for targeted ablation of thyroid cancer remnants. However, many patients with thyroid cancer have reduced NIS expression/function and hence do not benefit from radioiodine therapy. microRNA (miR) has emerged as a promising therapeutic target in many diseases; yet, the role of miRs in NIS-mediated RAIU has not been investigated. In silico analysis was used to identify miRs that may bind to the 3'UTR of human NIS (hNIS). The top candidate miR-339-5p directly bound to the 3'UTR of hNIS. miR-339-5p overexpression decreased NIS-mediated RAIU in HEK293 cells expressing exogenous hNIS, decreased the levels of NIS mRNA, and RAIU in transretinoic acid/hydrocortisone (tRA/H)-treated MCF-7 human breast cancer cells as well as thyrotropin-stimulated PCCl3 rat thyroid cells. Nanostring nCounter rat miR expression assay was conducted to identify miRs deregulated by TGFß, Akti-1/2, or 17-AAG known to modulate RAIU in PCCl3 cells. Among 38 miRs identified, 18 were conserved in humans. One of the 18 miRs, miR-195, was predicted to bind to the 3'UTR of hNIS and its overexpression decreased RAIU in tRA/H-treated MCF-7 cells. miR-339-5p was modestly increased in most papillary thyroid carcinomas (PTCs), yet miR-195 was significantly decreased in PTCs. Interestingly, the expression profiles of 18 miRs could be used to distinguish most PTCs from nonmalignant thyroid tissues. This is the first report, to our knowledge, demonstrating that hNIS-mediated RAIU can be modulated by miRs, and that the same miRs may also play roles in the development or maintenance of thyroid malignancy. Accordingly, miRs may serve as emerging targets to halt the progression of thyroid cancer and to enhance the efficacy of radioiodine therapy.