Improved Staging of Ciliary Body and Choroidal Melanomas Based on Estimation of Tumor Volume and Competing Risk Analyses.

PURPOSE: The current, 8th edition of the American Joint Committee on Cancer (AJCC) anatomic classification and staging model for uveal melanoma does not fully separate survival estimates for patients with advanced stages of the disease (e.g., IIIB and IIIC). Furthermore, some tumors in higher size categories have a smaller volume than tumors in lower categories. Therefore, we developed a novel model for prognostication of metastatic mortality based on estimations of tumor volume. DESIGN: Retrospective, multicenter case series of patients with uveal melanoma involving the choroid, ciliary body, or both. PARTICIPANTS: Six thousand five hundred twenty-eight consecutively registered patients treated at 3 tertiary ocular oncology centers on 2 continents between 1981 and 2022. METHODS: Data on survival, tumor size, and extent were collected for all 6528 patients. Tumor volume was estimated using a simple equation based on largest basal diameter and thickness. Volume-based size categories and stages were developed and validated in independent patient cohorts using competing risk analyses, and correlations with cytogenetic and cytomorphologic features were examined. MAIN OUTCOME MEASURE: Cumulative incidence of metastatic death. RESULTS: The 6528 patients were distributed over 7 stages based on estimated tumor volume and anatomic extent (V stages IA, IB, IIA, IIB, IIIA, IIIB, and IIIC), with a 15-year incidence of metastatic death ranging from 7% to 77%. A new category, V1min, and corresponding stage IA, were introduced, indicating an excellent prognosis. Metastatic mortality in V stage IIIC was significantly higher than that in V stage IIIB (P = 0.03), whereas incidence curves crossed for patients in AJCC stages IIIC vs. IIIB (P = 0.53). Univariable and multivariable competing risk regressions demonstrated higher Wald statistics for V stages compared with AJCC stages (1152 vs. 1038 and 71 vs. 17, respectively). The frequency of monosomy 3, gain of chromosome 8q, and epithelioid cytomorphologic features increased with tumor volume (R2 = 0.70, R2 = 0.50, and R2 = 0.71, respectively; P < 0.001) and showed similar correlations with both AJCC and V stages. CONCLUSIONS: Anatomic classification and staging of ciliary body and choroidal melanomas based on estimation of tumor volume improves prognostication of metastatic mortality. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Genetic Analysis of Uveal Melanoma in 658 Patients Using the Cancer Genome Atlas Classification of Uveal Melanoma as A, B, C, and D.

PURPOSE: The Cancer Genome Atlas (TCGA) classification has been validated for uveal melanoma (UM) prognostication. We applied TCGA classification to UM biopsied using fine-needle aspiration biopsy (FNAB) to determine the predictability for metastasis and death. DESIGN: Retrospective cohort study. PARTICIPANTS: Patients with UM treated with plaque radiotherapy at Wills Eye Hospital, Philadelphia, Pennsylvania, from October 1, 2008, through December 31, 2018, who completed genetic analysis of chromosomes 3 and 8 after FNAB. METHODS: Tumors were classified as A, B, C, or D and were compared using the chi-square test, Fisher exact test, analysis of variance, and Kaplan-Meier analysis. MAIN OUTCOME MEASURES: Metastasis and death. RESULTS: Six hundred fifty-eight UM patients were categorized accordingly as TCGA class A (n = 342 [52%]), B (n = 91 [14%]), C (n = 118 [18%]), and D (n = 107 [16%]). More advanced tumor classification revealed older mean patient age (56 vs. 53 vs. 60 vs. 63 years, respectively; P < 0.001), worse presenting visual acuity (20/20-20/50: 81% vs. 67% vs. 71% vs. 66%, respectively; P < 0.001), greater distance from the optic disc (3.5 vs. 4.9 vs. 5.7 vs. 5.3 mm, respectively; P < 0.001), larger tumor basal diameter (10.3 vs. 12.9 vs. 13.9 vs. 15.3 mm, respectively; P < 0.001), and greater tumor thickness (4.3 vs. 6.1 vs. 6.6 vs. 7.5 mm, respectively; P < 0.001). After mean follow-up (47.6 vs. 47.6 vs. 42.9 vs. 28.7 months, respectively; P < 0.001), more advanced TCGA class was associated with increased risk of metastasis (3% vs. 10% vs. 25% vs. 41%, respectively; P < 0.001) and death (1% vs. 0% vs. 3% vs. 9%, respectively; P < 0.001). Compared with class A, the 5-year hazard ratio for metastasis increased at 4.1 (B vs. A; P = 0.01), 10.1 (C vs. A; P < 0.001), and 30.0 (D vs. A; P < 0.001). The 5-year hazard ratio for death increased at 3.1 (C vs. A; P = 0.11) and 13.7 (D vs. A; P < 0.001) with no deaths in class B. CONCLUSIONS: Grouping of UM using TCGA classification predicts the risk of melanoma-related metastasis and death.

Gene expression and genetic variation in response to endoplasmic reticulum stress in human cells.

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) results in the condition called "ER stress," which induces the unfolded protein response (UPR), a complex cellular process that includes changes in expression of many genes. Failure to restore homeostasis in the ER is associated with human diseases. To identify the underlying changes in gene expression in response to ER stress, we induced ER stress in human B cells and then measured gene expression at ten time points. We followed up those results by studying cells from 60 unrelated people. We rediscovered genes that were known to play a role in the ER-stress response and uncovered several thousand genes that are not known to be involved. Two of these are VLDLR and INHBE, which showed significant increase in expression after ER stress in B cells and in primary fibroblasts. To study the links between UPR and disease susceptibility, we identified ER-stress-responsive genes that are associated with human diseases and assessed individual differences in the ER-stress response. Many of the UPR genes are associated with Mendelian disorders, such as Wolfram syndrome, and complex diseases, including amyotrophic lateral sclerosis and diabetes. Data from two independent samples showed extensive individual variability in ER-stress response. Additional analyses with monozygotic twins revealed significant correlations within twin pairs in their responses to ER stress, thus showing evidence for heritable variation among individuals. These results have implications for basic understanding of ER function and its role in disease susceptibility.

A prognostic classification system for uveal melanoma based on a combination of patient age and sex, the American Joint Committee on Cancer and the Cancer Genome Atlas models.

PURPOSE: To revisit the independent importance of ciliary body involvement (CBI), monosomy 3 (M3), tumour size, histological and clinical factors in uveal melanoma (UM) and to devise a new prognostic classification based on a combination of the American Joint Committee on Cancer (AJCC) and the Cancer Genome Atlas (TCGA) models. METHODS: Two cohorts with a total of 1796 patients were included. Clinicopathological factors were compared between patients with and without CBI and M3. Development of the prognostic classification was performed in a training cohort and was then tested in two independent validation cohorts. RESULTS: Tumours with CBI were more common in women, had greater apical thickness, greater basal tumour diameter, greater rates of vasculogenic mimicry and greater rates of M3, were more often asymptomatic at diagnosis and had poorer 5- and 10-year globe conservation rates (p < 0.023). In multivariate logistic regression, patient age at diagnosis, tumour diameter and CBI were independent predictors of M3 (p < 0.001). In multivariate Cox regression, male sex, age at diagnosis, tumour diameter, M3 and CBI were independent predictors of metastasis. The proposed prognostic classification combined patient age, sex, CBI, extraocular extension, M3, 8q (optional) and tumour size, and demonstrated greater prognostic acumen than both AJCC 4 T categories and TCGA groups A to D in validation cohorts. CONCLUSIONS: Tumour size does not confound the prognostic implication of CBI, M3, male sex and age at diagnosis in UM. These factors were included in a new prognostic classification that outperforms AJCC T category and TCGA groups.

Mapping determinants of human gene expression by regional and genome-wide association.

To study the genetic basis of natural variation in gene expression, we previously carried out genome-wide linkage analysis and mapped the determinants of approximately 1,000 expression phenotypes. In the present study, we carried out association analysis with dense sets of single-nucleotide polymorphism (SNP) markers from the International HapMap Project. For 374 phenotypes, the association study was performed with markers only from regions with strong linkage evidence; these regions all mapped close to the expressed gene. For a subset of 27 phenotypes, analysis of genome-wide association was performed with >770,000 markers. The association analysis with markers under the linkage peaks confirmed the linkage results and narrowed the candidate regulatory regions for many phenotypes with strong linkage evidence. The genome-wide association analysis yielded highly significant results that point to the same locations as the genome scans for about 50% of the phenotypes. For one candidate determinant, we carried out functional analyses and confirmed the variation in cis-acting regulatory activity. Our findings suggest that association studies with dense SNP maps will identify susceptibility loci or other determinants for some complex traits or diseases.

Genetic analysis of genome-wide variation in human gene expression.

Natural variation in gene expression is extensive in humans and other organisms, and variation in the baseline expression level of many genes has a heritable component. To localize the genetic determinants of these quantitative traits (expression phenotypes) in humans, we used microarrays to measure gene expression levels and performed genome-wide linkage analysis for expression levels of 3,554 genes in 14 large families. For approximately 1,000 expression phenotypes, there was significant evidence of linkage to specific chromosomal regions. Both cis- and trans-acting loci regulate variation in the expression levels of genes, although most act in trans. Many gene expression phenotypes are influenced by several genetic determinants. Furthermore, we found hotspots of transcriptional regulation where significant evidence of linkage for several expression phenotypes (up to 31) coincides, and expression levels of many genes that share the same regulatory region are significantly correlated. The combination of microarray techniques for phenotyping and linkage analysis for quantitative traits allows the genetic mapping of determinants that contribute to variation in human gene expression.

Accuracy of The Cancer Genome Atlas Classification vs American Joint Committee on Cancer Classification for Prediction of Metastasis in Patients With Uveal Melanoma.

Importance: The Cancer Genome Atlas (TCGA) classification is a newly emerging method for prediction of uveal melanoma (UM)-related metastasis and death. Limited information is available regarding the accuracy of the TCGA classification for prediction of metastasis in patients with UM. Objective: To investigate the accuracy of the TCGA classification for predicting UM-related metastasis compared with the American Joint Committee on Cancer (AJCC) classification. Design, Setting, and Participants: In this retrospective cohort study, patients with UM treated with plaque radiotherapy at a tertiary referral center from October 1, 2008, to December 31, 2018, were evaluated. All patients with tumors classified according to the American Joint Committee on Cancer Staging Manual, 8th Edition, and who completed pretreatment fine-needle aspiration biopsy sampling for genetic analysis of chromosomes 3 and 8 for TCGA classification were included. Tumors were classified into 4 categories, 17 subcategories, and 4 stages using AJCC classification and further grouped into 4 classes using TCGA classification. Main Outcomes and Measures: Value of TCGA classification vs AJCC classification for predicting UM-related metastasis. Results: Of 642 included patients, 331 (51.6%) were women, and the mean (SD) age was 58.0 (13.8) years. There were 642 tumors from 642 patients classified according to both AJCC and TCGA classifications. The mean (range) follow-up time for the entire cohort was 43.7 (1.4-159.2) months. At 5 years, TCGA classification showed higher value for prediction of metastasis (4 TCGA classes: Wald statistic, 94.8; hazard ratio [HR], 2.8; 95% CI, 2.3-3.5; P < .001; 4 AJCC categories: Wald statistic, 67.5; HR, 2.6; 95% CI, 2.1-3.2; P < .001; 17 AJCC subcategories: Wald statistic, 74.3; HR, 1.3; 95% CI, 1.2-1.3; P < .001; 4 AJCC stages: Wald statistic, 67.0; HR, not applicable; P < .001). After entering TCGA and AJCC classifications into a multivariate model, TCGA classification still showed higher value for prediction of metastasis (TCGA classification: Wald statistic, 61.5; HR, 2.4; 95% CI, 1.9-2.9; P < .001; AJCC classification: Wald statistic, 35.5; HR, 1.9; 95% CI, 1.5-2.4; P < .001). Conclusions and Relevance: These results suggest that TCGA classification provides accuracy that is superior to AJCC categories, subcategories, and stages for predicting metastasis from UM. When genetic testing is available, TCGA classification can provide a more accurate way to identify patients at high risk of metastasis who might benefit from adjuvant therapy.

PDE8A genetic variation, polycystic ovary syndrome and androgen levels in women.

Polycystic ovary syndrome (PCOS) is characterized by excessive theca cell androgen secretion, dependent upon LH, which acts through the intermediacy of 3',5'-cyclic adenosine monophosphate (cAMP). cAMP signaling pathways are controlled through regulation of its synthesis by adenylyl cyclases, and cAMP degradation by phosphodiesterases (PDEs). PDE8A, a high-affinity cAMP-specific PDE is expressed in the ovary and testis. Leydig cells from mice with a targeted mutation in the Pde8a gene are sensitized to the action of LH in terms of testosterone production. These observations led us to evaluate the human PDE8A gene as a PCOS candidate gene, and the hypothesis that reduced PDE8A activity or expression would contribute to excessive ovarian androgen production. We identified a rare variant (R136Q; NM_002605.2 c.407G > A) and studied another known single nucleotide polymorphism (SNP) (rs62019510, N401S) in the PDE8A coding sequence causing non-synonymous amino acid substitutions, and a new SNP in the promoter region (NT_010274.16:g.490155G > A). Although PDE8A kinetics were consistent with reduced activity in theca cell lysates, study of the expressed variants did not confirm reduced activity in cell-free assays. Sub-cellular localization of the enzyme was also not different among the coding sequence variants. The PDE8A promoter SNP and a previously described promoter SNP did not affect promoter activity in in vitro assays. The more common coding sequence SNP (N401S), and the promoter SNPs were not associated with PCOS in our transmission/disequilibrium test-based analysis, nor where they associated with total testosterone or dehydroepiandrosterone sulfate levels. These findings exclude a significant role for PDE8A as a PCOS candidate gene, and as a Las major determinant of androgen levels in women.

Ovulatory response to treatment of polycystic ovary syndrome is associated with a polymorphism in the STK11 gene.

CONTEXT: Clomiphene and insulin sensitizers such as metformin are used to induce ovulation in polycystic ovary syndrome (PCOS), but the ovulatory response is variable, and the causes of this variation are poorly understood. OBJECTIVE: Our objective was to identify predictive genetic polymorphisms and other determinants of ovulatory response. DESIGN: This was a substudy of a multicenter randomized clinical trial. SETTING: This study was performed at academic medical centers and their affiliates. PARTICIPANTS: A total of 312 women with PCOS were included in the study. MAIN OUTCOME MEASURES: Historical, biometric, biochemical, and genetic parameters were performed. RESULTS: We found that the C allele of a single nucleotide polymorphism in the STK11 gene (expressed in liver; also known as LKB1) was associated with a significantly decreased chance of ovulation in PCOS women treated with metformin. In an analysis of ovulation per cycle, the adjusted odds ratio (OR) comparing the C/C genotype to the G/G genotype was 0.30 [95% confidence interval (CI) 0.14, 0.66], and the OR for the C/G genotype vs. the G/G genotype was also 0.30 (95% CI 0.14, 0.66). In an analysis of metformin-treated subjects, we found that the percentage of women who ovulated increased with the number of G alleles present: 48% (10 of 21) of C/C women, 67% (32 of 48) of C/G women, and 79% (15 of 19) of G/G women ovulated. We also found that increased frequency of ovulation was associated with lower body mass index (BMI) [adjusted OR of 2.36 (95% CI 1.65, 3.36) and 2.05 (95% CI 1.46, 2.88), respectively, for comparisons of BMI less than 30 vs. BMI equal to or more than 35, BMI 30-34 vs. BMI equal to or more than 35, in the analysis of ovulation per cycle], a lower free androgen index (FAI) [adjusted OR of 1.59 (95% CI 1.17, 2.18) for FAI<10 vs. FAI>or=10], and a shorter duration of attempting conception [adjusted OR of 1.63 (95% CI 1.20, 2.21) for<1.5 vs.>or=1.5 yr]. CONCLUSIONS: We have demonstrated that a polymorphism in STK11, a kinase gene expressed in liver and implicated in metformin action, is associated with ovulatory response to treatment with metformin alone in a prospective randomized trial. The interaction with the effects of changes in modifiable factors (e.g. BMI or FAI) requires further study.

PRiMeUM: A Model for Predicting Risk of Metastasis in Uveal Melanoma.

Purpose: To create an interactive web-based tool for the Prediction of Risk of Metastasis in Uveal Melanoma (PRiMeUM) that can provide a personalized risk estimate of developing metastases within 48 months of primary uveal melanoma (UM) treatment. The model utilizes routinely collected clinical and tumor characteristics on 1227 UM, with the option of including chromosome information when available. Methods: Using a cohort of 1227 UM cases, Cox proportional hazard modeling was used to assess significant predictors of metastasis including clinical and chromosomal characteristics. A multivariate model to predict risk of metastasis was evaluated using machine learning methods including logistic regression, decision trees, survival random forest, and survival-based regression models. Based on cross-validation results, a logistic regression classifier was developed to compute an individualized risk of metastasis based on clinical and chromosomal information. Results: The PRiMeUM model provides prognostic information for personalized risk of metastasis in UM. The accuracy of the risk prediction ranged between 80% (using chromosomal features only), 83% using clinical features only (age, sex, tumor location, and size), and 85% (clinical and chromosomal information). Kaplan-Meier analysis showed these risk scores to be highly predictive of metastasis (P < 0.0001). Conclusions: PRiMeUM provides a tool for predicting an individual's personal risk of metastasis based on their individual and tumor characteristics. It will aid physicians with decisions concerning frequency of systemic surveillance and can be used as a criterion for entering clinical trials for adjuvant therapies.

Phosphorylation of pRb: mechanism for RB pathway inactivation in MYCN-amplified retinoblastoma.

A small, but unique subgroup of retinoblastoma has been identified with no detectable mutation in the retinoblastoma gene (RB1) and with high levels of MYCN gene amplification. This manuscript investigated alternate pathways of inactivating pRb, the encoded protein in these tumors. We analyzed the mutation status of the RB1 gene and MYCN copy number in a series of 245 unilateral retinoblastomas, and the phosphorylation status of pRb in a subset of five tumors using immunohistochemistry. There were 203 tumors with two mutations in RB1 (RB1-/- , 83%), 29 with one (RB1+/- , 12%) and 13 with no detectable mutations (RB1+/+ , 5%). Eighteen tumors carried MYCN amplification between 29 and 110 copies: 12 had two (RB1-/- ) or one RB1 (RB1+/- ) mutations, while six had no mutations (RB1+/+ ). Immunohistochemical staining of tumor sections with antibodies against pRb and phosphorylated Rb (ppRb) displayed high levels of pRb and ppRb in both RB1+/+ and RB1+/- tumors with MYCN amplification compared to no expression of these proteins in a classic RB1-/- , MYCN-low tumor. These results establish that high MYCN amplification can be present in retinoblastoma with or without coding sequence mutations in the RB1 gene. The functional state of pRb is inferred to be inactive due to phosphorylation of pRb in the MYCN-amplified retinoblastoma without coding sequence mutations. This makes inactivation of RB1 by gene mutation or its protein product, pRb, by protein phosphorylation, a necessary condition for initiating retinoblastoma tumorigenesis, independent of MYCN amplification.

Linkage and association with type 1 diabetes on chromosome 1q42.

Type 1 diabetes is a complex disorder with multiple genetic loci and environmental factors contributing to disease etiology. In the current study, a human type 1 diabetes candidate region on chromosome 1q42 was mapped at high marker density in a panel of 616 multiplex type 1 diabetic families. To facilitate the identification and evaluation of candidate genes, a physical map of the 7-cM region surrounding the maximum logarithm of odds (LOD) score (2.46, P = 0.0004) was constructed. Genes were identified in the 500-kb region surrounding the marker yielding the peak LOD score and evaluated for polymorphism by resequencing. Single-nucleotide polymorphisms (SNPs) identified in these genes as well as other anonymous markers were tested for allelic association with type 1 diabetes by both family-based and case-control methods. A haplotype formed by common alleles at three adjacent markers (D1S225, D1S2383, and D1S251) was preferentially transmitted to affected offspring in type 1 diabetic families (nominal P = 0.006). These findings extend the evidence supporting the existence of a type 1 diabetes susceptibility locus on chromosome 1q42 and identify a candidate region amenable to positional cloning efforts.

Molecular karyotyping for detection of prognostic markers in fine needle aspiration biopsy samples of uveal melanoma.

Uveal melanoma is the most common cancer of the eye in which approximately 50 % of cases develop metastases that are fatal within 2-15 years. Thus it is critical to identify prognostic markers to select high-risk patients into an adjuvant treatment. Chromosomal copy number alterations have been associated with poor prognosis. Historically the gold standard for identifying chromosomal aberrations had been fluorescent in situ hybridization. But in recent years other techniques have been developed that allow very rapid molecular analysis for estimation of chromosomal copy number with finer resolution. These include microsatellite analysis, multiple ligation-dependent probe amplification, and, most recently, genome-wide single-nucleotide polymorphism array analysis. These various procedures have identified loss of all or part of chromosome 3 (monosomy), losses of 1p, 6q, or 8p, or gains of 6p or 8q which, together with tumor location, morphology, and size, can be used to accurately predict the risk of metastasis.

Chromosome 3 status combined with BAP1 and EIF1AX mutation profiles are associated with metastasis in uveal melanoma.

PURPOSE: Somatic mutations in GNAQ, GNA11, SF3B1, EIF1AX, and BAP1 have been identified in uveal melanoma (UM). The aim of this study was to determine whether mutations in these genes in primary tumors were associated with metastases in individuals diagnosed with UM. METHODS: A total of 63 UM cases who developed a metastasis within 48 months of primary treatment and 53 UM controls who were metastasis-free over a similar time period were selected for the study. Primary UM cases were screened for mutations in GNAQ, GNA11, SF3B1, EIF1AX, and BAP1. The association of these mutations with tumor characteristics, chromosome 3 copy number, and metastatic status was analyzed by logistic regression to estimate the odds of developing metastasis within 48 months. RESULTS: As expected, tumor diameter, thickness, cilio-choroidal location, and chromosome 3 monosomy were all significantly (P < 0.02) associated with the presence of metastasis. In univariate analysis, GNA11 (odds ratio [OR] 2.5, 95% confidence interval [CI] 1.1-5.5) and BAP1 (OR 6.3, 95% CI 2.7-14.4) mutations were positively associated and EIF1AX mutation (OR 0.13, 95% CI 0.034-0.47) was inversely associated with metastatic status at 48 months after UM treatment. After adjustment for covariates, a chromosome 3 monosomy/BAP1-mutation/EIF1AX-wild-type (WT) mutation profile was strongly associated (OR 37.5, 95% CI 4.3-414) with the presence of metastasis compared with a chromosome 3 disomy/BAP1-WT/EIF1AX mutation profile. CONCLUSIONS: The results suggest that knowledge of mutations in BAP1 and EIF1AX can enhance prognostication of UM beyond that determined by chromosome 3 and tumor characteristics. Tumors with chromosome 3 disomy/BAP1-WT/EIF1AX-WT have a 10-fold increased risk of metastasis at 48 months compared with disomy-3/BAP1-WT/EIF1AX mutant tumors.

Genomic profile of 320 uveal melanoma cases: chromosome 8p-loss and metastatic outcome.

PURPOSE: Uveal melanoma (UM) was a fatal malignancy in 40% to 50% of cases. The aim of this study is to evaluate the independent contributions of chromosome 1, 3, 6, and 8 abnormalities for prognostication of metastasis, and to define multichromosome copy number aberration (CNA) signatures that can be used to evaluate risk. METHODS: A series of 320 UM were analyzed for chromosome 1, 3, 6, and 8 abnormalities using whole genome single-nucleotide polymorphism arrays. Results for changes in six chromosomal regions were analyzed using univariate and multivariate Cox proportional hazard modeling to identify significant predictors of metastasis and CNA signatures. RESULTS: Univariate Cox analysis indicated that losses of chromosome 3, 1p, 6q, and 8p and gain of 8q, as well as sex, source of tumor tissue (fine-needle aspiration biopsy [FNAB] compared with tumor from an enucleated eye), tumor basal diameter and height, and ciliary body involvement were all significant predictors of poor metastatic outcome. In the multivariate analysis, loss of chromosome 3 and 8p remained significant after adjusting for the effects of all other variables, as did sex, tissue source, and basal diameter. Multivariate analysis of the joint effects of changes in the six chromosomal regions showed that six signatures, including chromosome 3-loss, 1p-loss, 8p-loss, and/or 8q-gain had hazard ratios (HR) ranging from 7.90 to 37.25. CONCLUSIONS: In UM, tumor size and location, tissue source, and sex were all significantly associated with increased metastasis. In addition, chromosome 3-loss and 8p-loss were found to be independent predictors of poor metastatic outcome and CNA signatures were identified that can add a specific HR value for classification of risk categories.

Catechol-O-methyltransferase (COMT) single nucleotide polymorphisms and haplotypes are not major risk factors for polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is an endocrine disorder that affects 5-8% of reproductive age women. The primary features of PCOS are hyperandrogenemia, chronic anovulation and infertility. It has been suggested that defects in ovarian steroid metabolism contribute to the follicular growth arrest and abnormal production of ovarian steroid hormones that are characteristic of PCOS. 2-Methoxyestradiol (2-ME) is formed by the action of catechol-O-methyltransferase (COMT) on 2-hydroxyestradiol. COMT expression is increased in the follicles and ovarian stroma of women with PCOS. Moreover, 2-ME decreases granulosa cell proliferation and steroidogenesis, raising the possibility that ovarian dysfunction associated with PCOS is due, in part, to increased synthesis of 2-ME resulting from increased COMT activity. Four single-nucleotide polymorphisms (SNPs) (rs6269, rs4633, rs4818, rs4680) in the COMT gene characterize haplotypes, which are associated with large variations in COMT enzymatic activity. The aim of this study was to determine whether individual COMT SNPs and the COMT haplotypes are associated with PCOS using a family-based test of association and linkage. Additionally, we examined the relationships between COMT SNPs and haplotypes with quantitative variables usually assessed in the evaluation of women with PCOS. There were no significant correlations between genotype and total testosterone, non-SHBG bound testosterone and BMI. However, we found that the prolactin level in women with PCOS varied significantly with COMT haplotype, and suggest that this association reflects a genetic factor influencing the stress response. Our findings suggest that common variants and haplotypes of the COMT gene are not major contributors to risk for PCOS, but that COMT genotype may influence prolactin levels.

Polymorphisms in the SHBG gene influence serum SHBG levels in women with polycystic ovary syndrome.

CONTEXT: Single-nucleotide polymorphisms (SNPs) in the SHBG gene are associated with type 2 diabetes mellitus. SHBG has also been proposed as a candidate gene for the polycystic ovary syndrome (PCOS). OBJECTIVE: The study aims were 1) to determine whether any of four SHBG SNPs (rs1779941, rs6297, rs6259, and rs727428) are associated with PCOS and 2) to determine whether SNP genotype influences SHBG levels in PCOS women. DESIGN: Using the transmission disequilibrium test, evidence of associations between SHBG SNPs and PCOS were analyzed. Additionally, correlations between SHBG levels and SNP genotype, body mass index, non-SHBG-bound testosterone, and insulin resistance estimated by the homeostasis model were determined. SETTING: The study was conducted at academic medical centers. PATIENTS OR OTHER PARTICIPANTS: A total of 430 families having a proband with PCOS were included in the family-based study. Associations between SNP genotypes, SHBG, and metabolic parameters were determined in 758 women with PCOS including probands from the family cohort. MAIN OUTCOME MEASURES: Primary outcome measures included transmission frequency of SNP alleles and correlation coefficients between SHBG and allele frequency/metabolic parameters. RESULTS: No evidence of association between SNPs of interest and PCOS was found. However, in multivariate analyses, SHBG levels varied significantly with rs1799941 and rs727428 genotype after controlling for body mass index, non-SHBG-bound testosterone, and homeostasis model for insulin resistance. CONCLUSIONS: Although SHBG SNPs associated with type 2 diabetes mellitus do not appear to be associated with PCOS status, rs1799941 and rs727428 genotypes are associated with SHBG levels independent of the effects of insulin resistance and obesity.

FTO and MC4R gene variants are associated with obesity in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility in women. It is also associated with metabolic disturbances that place women at increased risk for obesity and type 2 diabetes. There is strong evidence for familial clustering of PCOS and a genetic predisposition. However, the gene(s) responsible for the PCOS phenotypes have not been elucidated. This two-phase family-based and case-control genetic study was designed to address the question of whether SNPs identified as susceptibility loci for obesity in genome-wide association studies (GWAS) are also associated with PCOS and elevated BMI. Members of 439 families having at least one offspring with PCOS were genotyped for 15 SNPs previously shown to be associated with obesity. Linkage and association with PCOS was assessed using the transmission/disequilibrium test (TDT). These SNPs were also analyzed in an independent case-control study involving 395 women with PCOS and 176 healthy women with regular menstrual cycles. Only one of these 15 SNPs (rs2815752 in NEGR1) was found to have a nominally significant association with PCOS (χ(2) = 6.11, P = 0.013), but this association failed to replicate in the case-control study. While not associated with PCOS itself, five SNPs in FTO and two in MC4R were associated with BMI as assessed with a quantitative-TDT analysis, several of which replicated association with BMI in the case-control cohort. These findings demonstrate that certain SNPs associated with obesity contribute to elevated BMI in PCOS, but do not appear to play a major role in PCOS per se. These findings support the notion that PCOS phenotypes are a consequence of an oligogenic/polygenic mechanism.

Type 2 diabetes susceptibility single-nucleotide polymorphisms are not associated with polycystic ovary syndrome.

Two cohorts of women with polycystic ovary syndrome (PCOS), comprising 400 probands and affected sisters in 365 families and a case-control group including 395 women with PCOS and 171 healthy women with regular menstrual cycles, were studied to determine whether single-nucleotide polymorphisms (SNPs) identified as susceptibility loci in genomewide association studies of type 2 diabetes are also associated with PCOS. None of the 18 allelic variants in 10 genes previously shown to be associated with type 2 diabetes were found to be associated with PCOS, but some were associated with indices of beta cell function.

A variant in the fibrillin-3 gene is associated with TGF-ß and inhibin B levels in women with polycystic ovary syndrome.

In an attempt to evaluate the association between allele 8 (A8) of D19S884 in the fibrillin-3 gene and circulating transforming growth factor (TGF) ß and inhibin levels in women with polycystic ovary syndrome (PCOS), we studied 120 similarly aged women from families with PCOS and compared 40 women with PCOS who did not have A8 (A8- PCOS) with 40 women with PCOS who had A8 (A8+ PCOS) and 40 normally menstruating women who did not have either PCOS or A8 (A8- Non-PCOS). A8- PCOS is associated with higher levels of TGF-ß1 compared with A8+ PCOS or A8- Non-PCOS, similar levels of TGF-ß2 compared with A8+ PCOS but lower levels of TGF-ß2 compared with A8- Non-PCOS, and lower levels of inhibin B and aldosterone compared with A8+ PCOS.