Human Androgen Receptor Assay Research Articles

Investigation of the role of X chromosome inactivation and androgen receptor CAG repeat polymorphisms in patients with recurrent pregnancy loss: a prospective case–control study

BackgroundPrevious research has revealed that skewed X chromosome inactivation (SXCI) and androgen receptor (AR) CAG polymorphisms are associated with increased risk of recurrent pregnancy loss (RPL); however, the results are conflicting, and the underlying mechanisms remain unclear. This study investigated the role of SXCI and AR CAG polymorphisms in patients with RPL and explored whether the underlying mechanisms were related to the ovarian reserve and preimplantation embryo aneuploidy.MethodsThis was a prospective case-control study carried out in a tertiary hospital-based reproductive medicine center. An external validation RPL cohort was recruited during the study period. Data on baseline and cycle characteristics were collected. X-chromosome inactivation (XCI) was measured using a human AR assay. AR polymorphisms were assessed using quantitative fluorescent polymerase chain reactions and direct sequencing. Blastocysts of the patients with RPL were tested by single nucleotide polymorphism microarray based preimplantation genetic testing for aneuploidy.ResultsIn total, 131 patients with idiopathic RPL and 126 controls were included for the case-control study. Patients with RPL exhibited a significantly more skewed XCI distribution pattern (67.71 ± 10.50 vs. 64.22 ± 10.62, p = 0.011), as well as significantly shorter bi-allelic mean (18.56 ± 1.97 vs. 19.34 ± 2.38, p = 0.005) and X-weighted bi-allelic mean (18.46 ± 2.02 vs. 19.38 ± 2.53, p = 0.001) of AR CAG repeats. Multivariate logistic regression models indicated that CAG repeat < 20, SXCI, and duration of stimulation were independently associated with the risk of RPL. However, SXCI and AR CAG polymorphisms were not associated with ovarian reserve or preimplantation embryo aneuploidy in the RPL group, and the same results were attained in a separate validation cohort of 363 patients with RPL.ConclusionSXCI and AR CAG polymorphisms are related to RPL; however, these two factors do not lead to RPL by affecting the ovarian reserve or increasing embryo aneuploidy. The roles of SXCI and AR CAG in RPL may involve other mechanisms that require further investigation.Trial registration:NCT02504281, https://www.clinicaltrials.gov (Date of registration, 21/07/2015; date of enrolment of the first subject, 30/07/2015).

ODP279 A Case of Nephrogenic Diabetes Insipidus Diagnosed at an Advanced Age in a Female Patient with an AVPR2 Gene Mutation and Skewed×Inactivation

Abstract Background Arginine vasopressin receptor 2 (AVPR2) gene mutationsare the most common cause of congenital nephrogenic diabetes insipidus (CNDI), and they are inherited in an X-linked recessive manner. Women are generally asymptomatic or mildly affected, but atypically severe cases have been reported. Skewed X inactivation, in which the X chromosome is unevenly active, is a cause of this atypical severe case.×chromosome inactivation is a developmental compensation mechanism that results in equal doses of X-linked genes between XX females and XY males and has been reported to be accentuated by aging. Clinical Case A 69-year-old woman had no family history of diabetes insipidus. Immediately after undergoing a craniotomy for subarachnoid hemorrhage in July, she showed marked polyuria of approximately 8000 mL/day and hypernatremia of 161 mEq/L. The use of desmopressin (15mcg/kg/day) did not improve herpolyuria. She was referred to our department in September for further investigation and treatment. Her urine osmolality remained hypotonic at approximately 60–80 mOsm/kg/H 2 O (50–1300 mOsm/kg/H 2 O) according to a water restriction test. Her urine osmolality increased slightly to 106 mOsm/kg/H 2 O in a desmopressin test. These results indicated that desmopressin was ineffective, and the patient was diagnosed with nephrogenic diabetes. Her urine output decreased to approximately 2500 mL/day with trichlormethiazide treatment. There was no significant anterior pituitary hypofunction. Genetic examination revealed a heterozygous mutation of the AVPR2 gene (c.656T&amp;gt;G, p. Leu219Arg) in her X chromosome, and her X chromosome activity ratio was 79: 21 in a human androgen receptor assay, indicating skewed X inactivation. Conclusion We observed a case of CNDI in an elderly woman with no family history of the disease. In this case, a combination of the skewed X inactivation, presumably accentuated by aging, and the onset of subarachnoid hemorrhage, reduced her drinking water and led to the diagnosis of CNDI.

Renal X-inactivation in female individuals with X-linked Alport syndrome primarily determined by age

X-linked Alport syndrome (AS) caused by hemizygous disease-causing variants in COL4A5 primarily affects males. Females with a heterozygous state show a diverse phenotypic spectrum ranging from microscopic hematuria to end-stage kidney disease (ESKD) and extrarenal manifestations. In other X-linked diseases, skewed X-inactivation leads to preferential silencing of one X-chromosome and thus can determine the phenotype in females. We aimed to show a correlation between X-inactivation in blood and urine-derived renal cells and clinical phenotype of females with a heterozygous disease-causing variant in COL4A5 compared to healthy controls. A total of 56 females with a heterozygous disease-causing COL4A5 variant and a mean age of 31.6 ± 18.3 SD years were included in this study. A total of 94% had hematuria, 62% proteinuria >200 mg/day, yet only 7% had decreased eGFR. Using human androgen receptor assay X-inactivation was examined in blood cells of all 56 individuals, in urine-derived cells of 27 of these individuals and in all healthy controls. X-inactivation did not correlate with age of first manifestation, proteinuria or eGFR neither in blood, nor in urine. The degree of X-inactivation showed a moderate association with age, especially in urine-derived cells of the patient cohort (rho = 0.403, p = 0.037). Determination of X-inactivation allelity revealed a shift of X-inactivation toward the COL4A5 variant bearing allele. This is the first study examining X-inactivation of urine-derived cells from female individuals with AS. A correlation between phenotype and X-inactivation could not be observed suspecting other genetic modifiers shaping the phenotype in female individuals with AS. The association of X-inactivation with age in urine-derived cells suggests an escape-mechanism inactivating the COL4A5 variant carrying allele in female individuals with AS.

Preferential X Chromosome Inactivation as a Mechanism to Explain Female Preponderance in Myasthenia Gravis.

Myasthenia gravis (MG) is a neuromuscular autoimmune disease characterized by prevalence in young women (3:1). Several mechanisms proposed as explanations for gender bias, including skewed X chromosome inactivation (XCI) and dosage or sex hormones, are often involved in the development of autoimmunity. The skewed XCI pattern can lead to an unbalanced expression of some X-linked genes, as observed in several autoimmune disorders characterized by female predominance. No data are yet available regarding XCI and MG. We hypothesize that the preferential XCI pattern may contribute to the female bias observed in the onset of MG, especially among younger women. XCI analysis was performed on blood samples of 284 women between the ages of 20 and 82. XCI was tested using the Human Androgen Receptor Assay (HUMARA). XCI patterns were classified as random (XCI < 75%) and preferential (XCI ≥ 75%). In 121 informative patients, the frequency of skewed XCI patterns was 47%, significantly higher than in healthy controls (17%; p ≤ 0.00001). Interestingly, the phenomenon was observed mainly in younger patients (<45 years; p ≤ 0.00001). Furthermore, considering the XCI pattern and the other clinical characteristics of patients, no significant differences were found. In conclusion, we observed preferential XCI in MG female patients, suggesting its potential role in the aetiology of MG, as observed in other autoimmune diseases in women.

Use of the FMR1 Gene Methylation Status to Assess the X-Chromosome Inactivation Pattern: A Stepwise Analysis.

X-chromosome inactivation (XCI) is a developmental process to compensate the imbalance in the dosage of X-chromosomal genes in females. A skewing of the XCI pattern may suggest a carrier status for an X-linked disease or explain the presence of a severe phenotype. In these cases, it is important to determine the XCI pattern, conventionally using the gold standard Human Androgen-Receptor Assay (HUMARA), based on the analysis of the methylation status at a polymorphic CAG region in the first exon of the human androgen receptor gene (AR). The aim of this study was to evaluate whether the methylation status of the fragile mental retardation protein translational regulator gene (FMR1) can provide an XCI pattern similar to that obtained by HUMARA. A set of 48 female carriers of FMR1 gene normal-sized alleles was examined using two assays: HUMARA and a FMR1 methylation PCR (mPCR). Ranges were defined to establish the XCI pattern using the methylation pattern of the FMR1 gene by mPCR. Overall, a 77% concordance of the XCI patterns was obtained between the two assays, which led us to propose a set of key points and a stepwise analysis towards obtaining an accurate result for the XCI pattern and to minimize the underlying pitfalls.

No evidence for preferential X-chromosome inactivation as the main cause of divergent phenotypes in sisters with X-linked hypohidrotic ectodermal dysplasia

BackgroundX-linked hypohidrotic ectodermal dysplasia (XLHED), a rare genetic disorder, affects the normal development of ectodermal derivatives, such as hair, skin, teeth, and sweat glands. It is caused by pathogenic variants of the gene EDA and defined by a triad of hypotrichosis, hypo- or anodontia, and hypo- or anhidrosis which may lead to life-threatening hyperthermia. Although female carriers are less severely affected than male patients, they display symptoms, too, with high phenotypic variability. This study aimed to elucidate whether phenotypic differences in female XLHED patients with identical EDA genotypes might be explained by deviating X-chromosome inactivation (XI) patterns.MethodsSix families, each consisting of two sisters with the same EDA variant and their parents (with either mother or father being carrier of the variant), participated in this study. XLHED-related data like sweating ability, dental status, facial dysmorphism, and skin issues were assessed. We determined the women`s individual XI patterns in peripheral blood leukocytes by the human androgen receptor assay and collated the results with phenotypic features.ResultsThe surprisingly large inter- and intrafamilial variability of symptoms in affected females was not explicable by the pathogenic variants. Our cohort showed no higher rate of nonrandom XI in peripheral blood leukocytes than the general female population. Furthermore, skewed XI patterns in favour of the mutated alleles were not associated with more severe phenotypes.ConclusionsWe found no evidence for preferential XI in female XLHED patients and no distinct correlation between XLHED-related phenotypic features and XI patterns. Phenotypic variability seems to be evoked by other genetic or epigenetic factors.

Molecular Basis and Enzymatic Properties of Glucose 6-Phosphate Dehydrogenase Volendam, Leading to Chronic Nonspherocytic Anemia, Granulocyte Dysfunction, and Increased Susceptibility to Infections

AbstractWe have investigated the blood cells from a woman with a low degree of chronic nonspherocytic hemolytic anemia and frequent bacterial infections accompanied by icterus and anemia. The activity of glucose 6-phosphate dehydrogenase (G6PD) in her red blood cells (RBCs) was below detection level, and in her leukocytes less than 3% of normal. In cultured skin fibroblasts, G6PD activity was approximately 15% of normal, with 4- to 5-fold increased Michaelis constant (Km) for NADP and for glucose 6-phosphate. Activated neutrophils showed a decreased respiratory burst. Family studies showed normal G6PD activity in the RBCs from all family members, including both parents and the 2 daughters of the patient. Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA showed a novel, heterozygous 514C→T mutation, predicting a Pro172→Ser replacement. Analysis of G6PD RNA from the patient’s leukocytes and fibroblasts showed only transcripts with the 514C→T mutation. This was explained by the pattern of X-chromosome inactivation, studied by means of the human androgen receptor (HUMARA) assay, which proved to be skewed in the patient, her mother, and one of the patient’s daughters. Thus, the patient has inherited a de novo mutation in G6PD from her father and an X-chromosome inactivation determinant from her mother, causing exclusive expression of the mutated G6PD allele. Purified mutant protein from an Escherichia coli expression system showed strongly decreased specific activity, increased Km for NADP and for glucose 6-phosphate, and increased heat lability, which indicates that the defective phenotype is due to 2 synergistic molecular dysfunctions: decreased catalytic efficiency and protein instability.

Concomitant Occurrence of Polyclonal Hematopoiesis and Cell-Autonomous Megakaryopoiesis in Triple-Negative Essential Thrombocythemia

Somatic mutations in JAK2, MPL, and CALR are found in approximately 80% of patients with essential thrombocythemia (ET), whereas the remaining patients are negative for disease-defining mutations and are defined as triple-negative (TN). Studies have shown that some patients with TN-ET harbor non-canonical mutations in JAK2 and MPL; however, the failure to identify recurrent mutations in most patients has made the pathogenesis of TN-ET ambiguous (Milosevic Feenstra et al. Blood 2016, Cabagnols et al. Blood 2016). In this study, we screened 483 patients suspected as having ET in a single center, performed mutation analysis for JAK2 V617F, CALR exon 9, and MPL exon 10, and centrally reviewed bone marrow specimens. We identified 23 patients with TN-ET based on the WHO 2016 criteria. Sequencing analysis of these patients revealed non-canonical mutations in JAK2 and MPL in 4 cases. Whole exome-sequencing analysis of genomic DNA from peripheral blood and CD3-positive cells from 9 patients revealed that 2 patients harbored somatic mutations in other genes; 7 patients showed no detectable somatic mutation. A STAT5 reporter assay revealed that unlike JAK2 V617F and MPL W515L, all non-canonical mutants of JAK2 or MPL activated STAT5 similar to wild-type proteins, suggesting that these mutations did not drive the disease. Statistical analysis of clinical records revealed that patients with TN-ET were mostly young (median age of 36.0 years), female (18/23, 78.3%), and had neither a history of thrombosis nor progression to secondary myelofibrosis and leukemia, demonstrating the unique characteristics of TN-ET. The presence of clonal hematopoiesis, analyzed using genomic DNA purified from granulocytes of peripheral blood from female patients in a human androgen receptor assay, revealed that only 1 out of 15 patients was clonal. Hypothesizing that TN-ET was reactive thrombocytosis, the concentrations of cytokines promoting platelet production such as thrombopoietin (TPO) and interleukin-6 (IL-6) in the serum were analyzed. However, no significant differences in concentrations were observed among ET with driver mutations, TN-ET, and healthy individuals. We next examined the capacity of hematopoietic stem cells from patients with TN-ET to form megakaryocytic colonies. CD34-positive cells purified from cryopreserved bone marrow cells were cultured in the absence or presence of TPO. CD34-positive cells derived from patients with TN-ET exhibited an equivalent capacity to form megakaryocytic colonies compared to those from patients with ET harboring a driver mutation, even in the absence of TPO (Figure 1). Thus, in TN-ET, megakaryopoiesis may have been induced in a cell-autonomous manner. In 10 patients with TN-ET with available blood count data, no sign of thrombocytosis was observed before ET development, indicating that thrombocytosis was not hereditary but rather occurred via an alternate mechanism, such as aberrations in epigenomic regulation that induced cellular transformation (Ohnishi et al, Cell 2015). Taken together, TN-ET is a distinctive disease entity associated with polyclonal hematopoiesis and paradoxically caused by hematopoietic stem cells harboring a capacity for cell-autonomous megakaryopoiesis. Figure 1 Disclosures Komatsu: Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; PPMX: Consultancy, Research Funding; Meiji Seika Pharma Co., Ltd.: Patents &amp; Royalties: PCT/JP2020/008434, Research Funding; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding; Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy.

A female patient with retinoblastoma and severe intellectual disability carrying an X;13 balanced translocation without rearrangement in the RB1 gene: a case report

BackgroundFemale carriers of a balanced X; autosome translocation generally undergo selective inactivation of the normal X chromosome. This is because inactivation of critical genes within the autosomal region of the derivative translocation chromosome would compromise cellular function. We here report a female patient with bilateral retinoblastoma and a severe intellectual disability who carries a reciprocal X-autosomal translocation.Case presentationCytogenetic and molecular analyses, a HUMARA (Human androgen receptor) assay, and methylation specific PCR (MSP) and bisulfite sequencing were performed using peripheral blood samples from the patient. The patient’s karyotype was 46,X,t(X;13)(q28;q14.1) by G-banding analysis. Further cytogenetic analysis located the entire RB1 gene and its regulatory region on der(X) with no translocation disruption. The X-inactivation pattern in the peripheral blood was highly skewed but not completely selected. MSP and deep sequencing of bisulfite-treated DNA revealed that an extensive 13q region, including the RB1 promoter, was unusually methylated in a subset of cells.ConclusionsThe der(X) region harboring the RB1 gene was inactivated in a subset of somatic cells, including the retinal cells, in the patient subject which acted as the first hit in the development of her retinoblastoma. In addition, the patient’s intellectual disability may be attributable to the inactivation of the der(X), leading to a 13q deletion syndrome-like phenotype, or to an active X-linked gene on der (13) leading to Xq28 functional disomy.

Immunohistochemical and genetic analysis of respiratory epithelial adenomatoid hamartomas and seromucinous hamartomas: are they precursor lesions to sinonasal low-grade tubulopapillary adenocarcinomas?

Respiratory epithelial adenomatoid hamartoma (REAH) and seromucinous hamartoma (SH) are rare tumor-like lesions of the nasal cavity, paranasal sinuses, and nasopharynx. The pathogenesis of REAH/SH is still unclear. Neoplastic proliferation, chronic mechanical irritation, inflammation, or possible embryological tissue misplacement are speculated as possible mechanisms of their development. Low-grade tubulopapillary adenocarcinoma (LGTA) is a rare variant of nonsalivary, nonintestinal type sinonasal adenocarcinoma. The aim of this study was to evaluate the immunohistochemical and genetic profiles of 10 cases of REAH/SH, with serous, mucinous, and respiratory components evaluated separately and to compare these findings with the features of 9 cases of LGTA. All cases of REAH/SH and LGTA were analyzed immunohistochemically with a cocktail of mucin antigens (MUC1, MUC2, MUC4, MUC5AC, MUC6) and with epithelial (CK7, CK20, CDX2, SATB2) and myoepithelial markers (S100 protein, p63, SOX10). The next-generation sequencing assay was performed using FusionPlex Solid Tumor Kit (ArcherDx) in 10 cases of REAH/SH, and the EGFR-ZNF267 gene fusion was detected in 1 of them. Two female REAH/SH cases were assessed for the presence of clonality. Using the human androgen receptor assay, 1 case was proved to be clonal. The serous component of REAH/SH was positive for CK7/MUC1 and SOX10 similarly to LGTA. Although REAH/SH and LGTA are histopathologically and clinically separate entities, the overlap in their morphological and immunohistochemical profiles suggests that REAH/SH might be a precursor lesion of LGTA.

Phenotypic profile of expanded NK cells in chronic lymphoproliferative disorders: a surrogate marker for NK-cell clonality.

Currently, the lack of a universal and specific marker of clonality hampers the diagnosis and classification of chronic expansions of natural killer (NK) cells. Here we investigated the utility of flow cytometric detection of aberrant/altered NK-cell phenotypes as a surrogate marker for clonality, in the diagnostic work-up of chronic lymphoproliferative disorders of NK cells (CLPD-NK). For this purpose, a large panel of markers was evaluated by multiparametric flow cytometry on peripheral blood (PB) CD56(low) NK cells from 60 patients, including 23 subjects with predefined clonal (n = 9) and polyclonal (n = 14) CD56(low) NK-cell expansions, and 37 with CLPD-NK of undetermined clonality; also, PB samples from 10 healthy adults were included. Clonality was established using the human androgen receptor (HUMARA) assay. Clonal NK cells were found to show decreased expression of CD7, CD11b and CD38, and higher CD2, CD94 and HLADR levels vs. normal NK cells, together with a restricted repertoire of expression of the CD158a, CD158b and CD161 killer-associated receptors. In turn, NK cells from both clonal and polyclonal CLPD-NK showed similar/overlapping phenotypic profiles, except for high and more homogeneous expression of CD94 and HLADR, which was restricted to clonal CLPD-NK. We conclude that the CD94(hi)/HLADR+ phenotypic profile proved to be a useful surrogate marker for NK-cell clonality.

Evaluation of clonal origin of malignant mesothelioma.

BackgroundThe hypothesis that most cancers are of monoclonal origin is often accepted as a fact in the scientific community. This dogma arose decades ago, primarily from the study of hematopoietic malignancies and sarcomas, which originate as monoclonal tumors. The possible clonal origin of malignant mesothelioma (MM) has not been investigated. Asbestos inhalation induces a chronic inflammatory response at sites of fiber deposition that may lead to malignant transformation after 30-50 years latency. As many mesothelial cells are simultaneously exposed to asbestos fibers and to asbestos-induced inflammation, it may be possible that more than one cell undergoes malignant transformation during the process that gives rise to MM, and result in a polyclonal malignancy.Methods and resultsTo investigate the clonality patterns of MM, we used the HUMARA (Human Androgen Receptor) assay to examine 16 biopsies from 14 women MM patients. Out of 16 samples, one was non-informative due to skewed Lyonization in its normal adjacent tissue. Fourteen out of the 15 informative samples revealed two electrophoretically distinct methylated HUMARA alleles, the Corrected Allele Ratio (CR) calculated on the allele peak areas indicating polyclonal origin MM.ConclusionsOur results show that MM originate as polyclonal tumors and suggest that the carcinogenic “field effect” of mineral fibers leads to several premalignant clones that give rise to these polyclonal malignancies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0301-3) contains supplementary material, which is available to authorized users.

Abstract 3188: Evaluation of clonal origin of malignant mesothelioma/polyclonal origin of malignant mesothelioma

Abstract Tumors are generally thought to be monoclonal in origin, although this paradigm stemmed decades ago mostly from the study of hematopoietic malignancies and sarcomas. The clonal origin of malignant mesothelioma, a deadly cancer resistant to the current therapies, has never been investigated before. We used the HUMARA (human androgen receptor) assay to examine the pattern of clonality in 14 sporadic and 2 familial Malignant Mesotheliomas (MM). Of 16 specimens studied, 14 were informative and nearly all (13/14) revealed two electrophoretically distinct methylated HUMARA alleles, indicating a polyclonal origin for these tumors. This discovery has important clinical implications, in that an accurate assessment of tumor clonality is key to the design of novel molecular strategies for the treatment of MM. Citation Format: Sabahattin Comertpay, Rosanna Mezzapelle, Mika Tanji, Oriana Strianese, Harvey I. Pass, Tracey Weigel, Joseph Friedberg, Paul Sugarbaker, Thomas Krausz, Ena Wang, Giovanni Gaudino, Haining Yang, Amy Powers, Barbara Parsons, Sandra Pastorino, Michele Carbone. Evaluation of clonal origin of malignant mesothelioma/polyclonal origin of malignant mesothelioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3188. doi:10.1158/1538-7445.AM2014-3188

Indolent T-lymphoblastic Proliferation With Disseminated Multinodal Involvement and Partial CD33 Expression

Although indolent T-lymphoblastic proliferations (iT-LBP) are rare, this diagnosis should be excluded in any patient with an extrathymic proliferation of immature TdT+T cells. Unlike T-lymphoblastic leukemia/lymphoma, patients with iT-LBP do not require chemotherapy. We report a case of iT-LBP with disseminated multinodal involvement in an otherwise healthy 49-year-old woman. Multiple lymph node biopsies were performed over the course of several months demonstrating persistent and anatomically diffuse involvement. Over 18 months, and without therapy, she has remained healthy, and her lymphadenopathy significantly improved. No bone marrow or peripheral blood involvement was ever identified. Atypical T cells showed an immunophenotypic spectrum of T-cell antigen expression with partial CD33 on a subset of T cells detected by both flow cytometry and immunohistochemistry. Both T-cell clonality and Human Androgen Receptor Assay (HUMARA) studies, performed on lymph node biopsy specimens, were negative. This case represents the first detailed clinical, morphologic, molecular, and immunophenotypic description of disseminated multinodal involvement by nonclonal iT-LBP with partial CD33 expression on T cells.

The leiomyomatous stroma in renal cell carcinomas is polyclonal and not part of the neoplastic process.

Some renal epithelial neoplasms, such as renal angiomyoadenomatous tumor, clear cell papillary renal cell carcinoma and renal cell carcinoma with smooth muscle stroma, contain a variably prominent smooth muscle stromal component. Whether or not this leiomyomatous stroma is part of the neoplastic proliferation has not been firmly established. We studied the clonality status of 14 renal cell carcinomas with a prominent smooth muscle stromal component (four renal angiomyoadenomatous tumors/clear cell papillary carcinomas, five clear cell carcinomas, two papillary carcinomas, and three renal cell carcinomas with smooth muscle rich stroma) using the human androgen receptor assay (HUMARA). We found the leiomyomatous stromal component in all analyzable (8/14) cases to be polyclonal and therefore reactive rather than neoplastic. Based on morphological observations, we propose that the non-neoplastic leiomyomatous stromal component is likely derived from smooth muscle cells of large caliber veins located at the peripheral capsular region or within the collagenous septae of the tumors.

Clonality analysis of Kaposi's sarcoma lesions by human androgen receptor assay

Objective To analyze the clonality in Kaposi's sarcoma (KS) lesions by evaluating Xchromosome inactivation pattern in the human androgen receptor (HUMARA) gene.Methods Twenty-five paraffinembedded tissue specimens were collected from female patients with KS (n =15) or cutaneous hemangioma (n =10).DNA was extracted from these specimens,and digested with the methylation-sensitive restriction endonuclease Hpa Ⅱ.PCR was performed to amplify the HUMARA gene,and the amplicons were separated on a 10％ denaturing polyacrylamied gel and stained with ethidium bromide (EB).The loss of heterozygosity of the HUMARA gene was defined as the presence of two DNA fragments before and one fragment after the endonuclease digestion.The clonality in KS lesions was assessed based on the above results.Results Among the 15 patients with KS,13 (86.7％) were heterozygous for the HUMARA gene,of which,92.31％ (12/13) showed loss of heterozygosity of the HUMARA gene on X-chromosome,suggesting a monoclonal origin.Of the 10 patients with hemangioma,9 were heterozygous for the HUMARA gene,and only one lost heterozygosity of the HUMARA gene.The heterozygosity rate for HUMARA gene was significantly different between the patients with KS and hemangioma (P ＜ 0.01).No statistical difference was observed in the clonality status of KS between patients of different nationality,at different stages,or between patients with and without complicated human immunodeficiency virus (HIV) infection (all P ＞ 0.05).Conclusion KS is monoclonal in origin. Key words: Sarcoma, Kaposi; X Chromosome inactivation; Androgen receptor gene; Clonality analysis

Assessment of combined antiandrogenic effects of binary parabens mixtures in a yeast-based reporter assay

To date, toxicological studies of endocrine disrupting chemicals (EDCs) have typically focused on single chemical exposures and associated effects. However, exposure to EDCs mixtures in the environment is common. Antiandrogens represent a group of EDCs, which draw increasing attention due to their resultant demasculinization and sexual disruption of aquatic organisms. Although there are a number of in vivo and in vitro studies investigating the combined effects of antiandrogen mixtures, these studies are mainly on selected model compounds such as flutamide, procymidone, and vinclozolin. The aim of the present study is to investigate the combined antiandrogenic effects of parabens, which are widely used antiandrogens in industrial and domestic commodities. A yeast-based human androgen receptor (hAR) assay (YAS) was applied to assess the antiandrogenic activities of n-propylparaben (nPrP), iso-propylparaben (iPrP), methylparaben (MeP), and 4-n-pentylphenol (PeP), as well as the binary mixtures of nPrP with each of the other three antiandrogens. All of the four compounds could exhibit antiandrogenic activity via the hAR. A linear interaction model was applied to quantitatively analyze the interaction between nPrP and each of the other three antiandrogens. The isoboles method was modified to show the variation of combined effects as the concentrations of mixed antiandrogens were changed. Graphs were constructed to show isoeffective curves of three binary mixtures based on the fitted linear interaction model and to evaluate the interaction of the mixed antiandrogens (synergism or antagonism). The combined effect of equimolar combinations of the three mixtures was also considered with the nonlinear isoboles method. The main effect parameters and interaction effect parameters in the linear interaction models of the three mixtures were different from zero. The results showed that any two antiandrogens in their binary mixtures tended to exert equal antiandrogenic activity in the linear concentration ranges. The antiandrogenicity of the binary mixture and the concentration of nPrP were fitted to a sigmoidal model if the concentrations of the other antiandrogens (iPrP, MeP, and PeP) in the mixture were lower than the AR saturation concentrations. Some concave isoboles above the additivity line appeared in all the three mixtures. There were some synergistic effects of the binary mixture of nPrP and MeP at low concentrations in the linear concentration ranges. Interesting, when the antiandrogens concentrations approached the saturation, the interaction between chemicals were antagonistic for all the three mixtures tested. When the toxicity of the three mixtures was assessed using nonlinear isoboles, only antagonism was observed for equimolar combinations of nPrP and iPrP as the concentrations were increased from the no-observed-effect-concentration (NOEC) to effective concentration of 80%. In addition, the interactions were changed from synergistic to antagonistic as effective concentrations were increased in the equimolar combinations of nPrP and MeP, as well as nPrP and PeP. The combined effects of three binary antiandrogens mixtures in the linear ranges were successfully evaluated by curve fitting and isoboles. The combined effects of specific binary mixtures varied depending on the concentrations of the chemicals in the mixtures. At low concentrations in the linear concentration ranges, there was synergistic interaction existing in the binary mixture of nPrP and MeP. The interaction tended to be antagonistic as the antiandrogens approached saturation concentrations in mixtures of nPrP with each of the other three antiandrogens. The synergistic interaction was also found in the equimolar combinations of nPrP and MeP, as well as nPrP and PeP, at low concentrations with another method of nonlinear isoboles. The mixture activities of binary antiandrogens had a tendency towards antagonism at high concentrations and synergism at low concentrations.

Skewed X-inactivation patterns in ageing healthy and myelodysplastic haematopoiesis determined by a pyrosequencing based transcriptional clonality assay

BackgroundInvestigation of X-chromosome inactivation patterns (XCIP) by determination of differential CpG-methylation has been widely applied for investigation of female cell clonality. Using this approach the clonal origin of various tumours...

X chromosome inactivation pattern in BRCA gene mutation carriers

An association of preferential X chromosome inactivation (XCI) with BRCA gene status and breast/ovarian cancer risk has been reported. We evaluated XCI in a large group of BRCA mutation carriers compared to non-carriers and investigated associations between preferential XCI (⩾90:10) and age, mutated gene, cancer development and chemotherapy. XCI was analysed by human androgen receptor (HUMARA) assay and pyrosequencing in 437 BRCA1 or BRCA2 mutation carriers and 445 age-matched controls. The distribution of XCI patterns in the two groups was compared by logistic regression analysis. The association between preferential XCI and selected variables was investigated in both univariate and multivariate fashion. In univariate analyses preferential XCI was not significantly associated with the probability of being a BRCA mutation carrier, nor with cancer status, whereas chemotherapeutic regime and age both showed a significant association. In multivariate analysis only age maintained significance (odds ratio, 1.056; 95% confidence interval, 1.016–1.096). Our findings do not support the usefulness of XCI analysis for the identification of BRCA mutation carriers and cancer risk assessment. The increasing preferential XCI frequency with ageing and the association with chemotherapy justify extending the investigation to other categories of female cancer patients to identify possible X-linked loci implicated in cell survival.

Clinical significance of clonality assessment in JAK2V617F-negative essential thrombocythemia

JAK2V617F-negative essential thrombocythemia (ET) is a heterogeneous disease including clonal cases and others without evidence of clonality. However, it is unknown if the detection of myeloid clonality in JAK2V617F-negative ET patients confers a different clinical outcome than those in whom clonal hematopoiesis cannot be demonstrated. The objective of the present study was to evaluate the clinical significance of clonality assessment in patients with JAK2V617F-negative ET. Clonality investigation including mutational status of MPL, TET2, and ASXL1 genes and human androgen receptor (HUMARA) assay was performed in 73 JAK2V617F-negative cases out of 186 subjects consecutively diagnosed with ET in a single institution, at diagnosis or during follow-up. Mutations in MPL, TET2, and ASXL1 were observed in 7, 4, and 2 cases, respectively, whereas clonality by HUMARA assay was demonstrated in 21 out of 46 (46 %) female patients. With a median follow-up of 8 years, death, thrombosis, bleeding, and disease transformation were registered in 7, 10, 8, and 6 patients, respectively. No differences in thrombosis, bleeding or survival were observed according to clonality assessment. The probability of disease transformation at 10 years was higher in patients showing clonal hematopoiesis by presenting mutations in either MPL, TET2, or ASXL1 (64 versus 2 % in patients without mutations, p < 0.001) and in those with HUMARA clonality (35 versus 0 % in patients with polyclonal hematopoiesis, p < 0.004). In conclusion, disease transformation is associated with evidence of clonality in JAK2V617F-negative ET.