The future for genetic studies in reproduction

Genetic factors contribute to risk of many common diseases affecting reproduction and fertility. In recent years, methods for genome-wide association studies(GWAS) have revolutionized gene discovery forcommontraits and diseases. Results of GWAS are documented in the Catalog of Published Genome-Wide Association Studies at the National Human Genome Research Institute and report over 70 publications for 32 traits and diseases associated with reproduction. These include endometriosis, uterine fibroids, age at menarche and age at menopause. Results that pass appropriate stringent levels of significance are generally well replicated in independent studies. Examples of genetic variation affecting twinning rate, infertility, endometriosis and age at menarche demonstrate that the spectrum of disease-related variants for reproductive traits is similar to most other common diseases.GWAS 'hits' provide novel insights into biological pathways and the translational value of these studies lies in discovery of novel gene targets for biomarkers, drug development and greater understanding of environmental factors contributing to disease risk. Results also show that genetic data can help define sub-types of disease and co-morbidity with other traits and diseases. To date, many studies on reproductive traits have used relatively small samples. Future genetic marker studies in large samples with detailed phenotypic and clinical information will yield new insights into disease risk, disease classification and co-morbidity for many diseases associated with reproduction and infertility.

Genetic variants underlying risk of endometriosis: Insights from meta-analysis of eight genome-wide association and replication datasets

BACKGROUND Endometriosis is a heritable common gynaecological condition influenced by multiple genetic and environmental factors. Genome-wide association studies (GWASs) have proved successful in identifying common genetic variants of moderate effects for various complex diseases. To date, eight GWAS and replication studies from multiple populations have been published on endometriosis. In this review, we investigate the consistency and heterogeneity of the results across all the studies and their implications for an improved understanding of the aetiology of the condition. METHODS Meta-analyses were conducted on four GWASs and four replication studies including a total of 11 506 cases and 32 678 controls, and on the subset of studies that investigated associations for revised American Fertility Society (rAFS) Stage III/IV including 2859 cases. The datasets included 9039 cases and 27 343 controls of European (Australia, Belgium, Italy, UK, USA) and 2467 cases and 5335 controls of Japanese ancestry. Fixed and Han and Elkin random-effects models, and heterogeneity statistics (Cochran's Q test), were used to investigate the evidence of the nine reported genome-wide significant loci across datasets and populations. RESULTS Meta-analysis showed that seven out of nine loci had consistent directions of effect across studies and populations, and six out of nine remained genome-wide significant (P < 5 × 10(-8)), including rs12700667 on 7p15.2 (P = 1.6 × 10(-9)), rs7521902 near WNT4 (P = 1.8 × 10(-15)), rs10859871 near VEZT (P = 4.7 × 10(-15)), rs1537377 near CDKN2B-AS1 (P = 1.5 × 10(-8)), rs7739264 near ID4 (P = 6.2 × 10(-10)) and rs13394619 in GREB1 (P = 4.5 × 10(-8)). In addition to the six loci, two showed borderline genome-wide significant associations with Stage III/IV endometriosis, including rs1250248 in FN1 (P = 8 × 10(-8)) and rs4141819 on 2p14 (P = 9.2 × 10(-8)). Two independent inter-genic loci, rs4141819 and rs6734792 on chromosome 2, showed significant evidence of heterogeneity across datasets (P < 0.005). Eight of the nine loci had stronger effect sizes among Stage III/IV cases, implying that they are likely to be implicated in the development of moderate to severe, or ovarian, disease. While three out of nine loci were inter-genic, the remaining were in or near genes with known functions of biological relevance to endometriosis, varying from roles in developmental pathways to cellular growth/carcinogenesis. CONCLUSIONS Our meta-analysis shows remarkable consistency in endometriosis GWAS results across studies, with little evidence of population-based heterogeneity. They also show that the phenotypic classifications used in GWAS to date have been limited. Stronger associations with Stage III/IV disease observed for most loci emphasize the importance for future studies to include detailed sub-phenotype information. Functional studies in relevant tissues are needed to understand the effect of the variants on downstream biological pathways.

Genome-wide mapping of cystitis due to Streptococcus agalactiae and Escherichia coli in mice identifies a unique bladder transcriptome that signifies pathogen-specific antimicrobial defense against urinary tract infection

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli; however, Gram-positive organisms including Streptococcus agalactiae, or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35 year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; qRT-PCR was used to analyze selected gene responses identified in array datasets. A surprisingly small significant gene list of 172 genes was identified at 24h; this compared to 2507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2h. Bioinformatics analyses including integrative systems-level network mapping revealed multiple activated biological pathways in the GBS cystitis transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens.

No genetic support for a contribution of prostaglandins to the aetiology of androgenetic alopecia

MADAM, Androgenetic alopecia (AGA) is a common age-dependent trait, characterized by a progressive loss of hair from the scalp. The hair loss may commence during puberty and up to 80% of white men experience some degree of AGA during their lifetime.1 Research has established that two essential aetiological factors for AGA are a genetic predisposition and the presence of androgens (male sex hormones).1,2 A recent meta-analysis of genome-wide association studies (GWAS) has increased the number of identified loci associated with this trait at the molecular level to a total of eight.3 However, despite these successes, a large fraction of the genetic contribution remains to be identified. One way to identify further genetic loci is to combine the resource of GWAS datasets with knowledge about specific biological factors likely to be involved in the development of disease. The focused evaluation of a limited number of candidate genes in GWAS datasets avoids the necessity for extensive correction for multiple testing, which typically limits the power for detecting genetic loci at a genome-wide level.4 Because the presence of genetic association suggests that candidate genes are likely to operate early in the causative chain of events leading to the phenotype, this approach may also function to favour biological pathways for their importance in the development of AGA.

Androgenetic alopecia: Identification of four genetic risk loci and evidence for the contribution of WNT signaling to its etiology

The pathogenesis of androgenetic alopecia (AGA, male-pattern baldness) is driven by androgens, and genetic predisposition is the major prerequisite. Candidate gene and genome-wide association studies have reported that single-nucleotide polymorphisms (SNPs) at eight different genomic loci are associated with AGA development. However, a significant fraction of the overall heritable risk still awaits identification. Furthermore, the understanding of the pathophysiology of AGA is incomplete, and each newly associated locus may provide novel insights into contributing biological pathways. The aim of this study was to identify unknown AGA risk loci by replicating SNPs at the 12 genomic loci that showed suggestive association (5 x 10(-8)

Multistage genome-wide association meta-analyses identified two new loci for bone mineral density

Aiming to identify novel genetic variants and to confirm previously identified genetic variants associated with bone mineral density (BMD), we conducted a three-stage genome-wide association (GWA) meta-analysis in 27 061 study subjects. Stage 1 meta-analyzed seven GWA samples and 11 140 subjects for BMDs at the lumbar spine, hip and femoral neck, followed by a Stage 2 in silico replication of 33 SNPs in 9258 subjects, and by a Stage 3 de novo validation of three SNPs in 6663 subjects. Combining evidence from all the stages, we have identified two novel loci that have not been reported previously at the genome-wide significance (GWS; 5.0 × 10-8) level: 14q24.2 (rs227425, P-value 3.98 × 10-13, SMOC1) in the combined sample of males and females and 21q22.13 (rs170183, P-value 4.15 × 10-9, CLDN14) in the female-specific sample. The two newly identified SNPs were also significant in the GEnetic Factors for OSteoporosis consortium (GEFOS, n 5 32 960) summary results. We have also independently confirmed 13 previously reported loci at the GWS level: 1p36.12 (ZBTB40), 1p31.3 (GPR177), 4p16.3 (FGFRL1), 4q22.1 (MEPE), 5q14.3 (MEF2C), 6q25.1 (C6orf97, ESR1), 7q21.3 (FLJ42280, SHFM1), 7q31.31 (FAM3C, WNT16), 8q24.12 (TNFRSF11B), 11p15.3 (SOX6), 11q13.4 (LRP5), 13q14.11 (AKAP11) and 16q24 (FOXL1). Gene expression analysis in osteogenic cells implied potential functional association of the two candidate genes (SMOC1 and CLDN14) in bone metabolism. Our findings independently confirm previously identified biological pathways underlying bone metabolism and contribute to the discovery of novel pathways, thus providing valuable insights into the intervention and treatment of osteoporosis. © The Author 2013. Published by Oxford University Press.

Genetic Mechanisms Underlying Autism Spectrum Disorders

Autism is a childhood-onset developmental disorder characterized by deficits in reciprocal social interaction, verbal and non-verbal communication, and dependence on routines and rituals. It belongs to a spectrum of disorders (autism spectrum disorders, ASDs) which share core symptoms but show considerable variation in severity. The whole spectrum affects 0.6-0.7% of children worldwide, inducing a substantial public health burden and causing suffering to the affected families. Despite having a very high heritability, ASDs have shown exceptional genetic heterogeneity, which has complicated the identification of risk variants and left the etiology largely unknown. However, recent studies suggest that rare, family-specific factors contribute significantly to the genetic basis of ASDs. In this study, we investigated the role of DISC1 (Disrupted-in-schizophrenia-1) in ASDs, and identified association with markers and haplotypes previously associated with psychiatric phenotypes. We identified four polymorphic micro-RNA target sites in the 3 UTR of DISC1, and showed that hsa-miR-559 regulates DISC1 expression in vitro in an allele-specific manner. We also analyzed an extended autism pedigree with genealogical roots in Central Finland reaching back to the 17th century. To take advantage of the beneficial characteristics of population isolates to gene mapping and reduced genetic heterogeneity observed in distantly related individuals, we performed a microsatellite-based genome-wide screen for linkage and linkage disequilibrium in this pedigree. We identified a putative autism susceptibility locus on chromosome 19p13.3 and obtained further support for previously reported loci at 1q23 and 15q11-q13. To follow-up these findings, we extended our study sample from the same sub-isolate and initiated a genome-wide analysis of homozygosity and allelic sharing using high-density SNP markers. We identified a small number of haplotypes shared by different subsets of the genealogically connected cases, along with convergent biological pathways from SNP and gene expression data, which highlighted axon guidance molecules in the pathogenesis of ASDs. In conclusion, the results obtained in this thesis show that multiple distinct genetic variants are responsible for the ASD phenotype even within single pedigrees from an isolated population. We suggest that targeted resequencing of the shared haplotypes, linkage regions, and other susceptibility loci is essential to identify the causal variants. We also report a possible micro-RNA mediated regulatory mechanism, which might partially explain the wide-range neurobiological effects of the DISC1 gene.

Gene Expression Profiling of Preovulatory Follicle in the Buffalo Cow: Effects of Increased IGF-I Concentration on Periovulatory Events

The preovulatory follicle in response to gonadotropin surge undergoes dramatic biochemical, and morphological changes orchestrated by expression changes in hundreds of genes. Employing well characterized bovine preovulatory follicle model, granulosa cells (GCs) and follicle wall were collected from the preovulatory follicle before, 1, 10 and 22 h post peak LH surge. Microarray analysis performed on GCs revealed that 450 and 111 genes were differentially expressed at 1 and 22 h post peak LH surge, respectively. For validation, qPCR and immunocytochemistry analyses were carried out for some of the differentially expressed genes. Expression analysis of many of these genes showed distinct expression patterns in GCs and the follicle wall. To study molecular functions and genetic networks, microarray data was analyzed using Ingenuity Pathway Analysis which revealed majority of the differentially expressed genes to cluster within processes like steroidogenesis, cell survival and cell differentiation. In the ovarian follicle, IGF-I is established to be an important regulator of the above mentioned molecular functions. Thus, further experiments were conducted to verify the effects of increased intrafollicular IGF-I levels on the expression of genes associated with the above mentioned processes. For this purpose, buffalo cows were administered with exogenous bGH to transiently increase circulating and intrafollicular concentrations of IGF-I. The results indicated that increased intrafollicular concentrations of IGF-I caused changes in expression of genes associated with steroidogenesis (StAR, SRF) and apoptosis (BCL-2, FKHR, PAWR). These results taken together suggest that onset of gonadotropin surge triggers activation of various biological pathways and that the effects of growth factors and peptides on gonadotropin actions could be examined during preovulatory follicle development.

Metal Complexes of Curcumin for Cellular Imaging, Targeting, and Photoinduced Anticancer Activity

CONSPECTUS: Curcumin is a polyphenolic species. As an active ingredient of turmeric, it is well-known for its traditional medicinal properties. The therapeutic values include antioxidant, anti-inflammatory, antiseptic, and anticancer activity with the last being primarily due to inhibition of the transcription factor NF-kappa B besides affecting several biological pathways to arrest tumor growth and its progression. Curcumin with all these positive qualities has only remained a potential candidate for cancer treatment over the years without seeing any proper usage because of its hydrolytic instability involving the diketo moiety in a cellular medium and its poor bioavailability. The situation has changed considerably in recent years with the observation that curcumin in monoanionic form could be stabilized on binding to a metal ion. The reports from our group and other groups have shown that curcumin in the metal-bound form retains its therapeutic potential. This has opened up new avenues to develop curcumin-based metal complexes as anticancer agents. Zinc(II) complexes of curcumin are shown to be stable in a cellular medium. They display moderate cytotoxicity against prostate cancer and neuroblastoma cell lines. A similar stabilization and cytotoxic effect is reported for (arene)ruthenium(II) complexes of curcumin against a variety of cell lines. The half-sandwich 1,3,5-triaza-7-phosphatricyclo-3.3.1.1]decane (RAPTA)-type ruthenium(II) complexes of curcumin are shown to be promising cytotoxic agents with low micromolar concentrations for a series of cancer cell lines. In a different approach, cobalt(III) complexes of curcumin are used for its cellular delivery in hypoxic tumor cells using intracellular agents that reduce the metal and release curcumin as a cytotoxin. Utilizing the photophysical and photochemical properties of the curcumin dye, we have designed and synthesized photoactive curcumin metal complexes that are used for cellular imaging by fluorescence microscopy and damaging the cancer cells on photoactivation in visible light while being minimally toxic in darkness. In this Account, we have made an attempt to review the current status of the chemistry of metal curcumin complexes and present results from our recent studies on curcumin complexes showing remarkable in vitro photocytotoxicity. The undesirable dark toxicity of the complexes can be reduced with suitable choice of the metal and the ancillary ligands in a ternary structure. The complexes can be directed to specific subcellular organelles. Selectivity by targeting cancer cells over normal cells can be achieved with suitable ligand design. We expect that this methodology is likely to provide an impetus toward developing curcumin-based photochemotherapeutics for anticancer treatment and cure.

Relationship between prion propensity and the rates of individual molecular steps of fibril assembly.

Peptides and proteins possess an inherent propensity to self-assemble into generic fibrillar nanostructures known as amyloid fibrils, some of which are involved in medical conditions such as Alzheimer disease. In certain cases, such structures can self-propagate in living systems as prions and transmit characteristic traits to the host organism. The mechanisms that allow certain amyloid species but not others to function as prions are not fully understood. Much progress in understanding the prion phenomenon has been achieved through the study of prions in yeast as this system has proved to be experimentally highly tractable; but quantitative understanding of the biophysics and kinetics of the assembly process has remained challenging. Here, we explore the assembly of two closely related homologues of the Ure2p protein from Saccharomyces cerevisiae and Saccharomyces paradoxus, and by using a combination of kinetic theory with solution and biosensor assays, we are able to compare the rates of the individual microscopic steps of prion fibril assembly. We find that for these proteins the fragmentation rate is encoded in the structure of the seed fibrils, whereas the elongation rate is principally determined by the nature of the soluble precursor protein. Our results further reveal that fibrils that elongate faster but fracture less frequently can lose their ability to propagate as prions. These findings illuminate the connections between the in vitro aggregation of proteins and the in vivo proliferation of prions, and provide a framework for the quantitative understanding of the parameters governing the behavior of amyloid fibrils in normal and aberrant biological pathways.

Probing protein aggregation with quartz crystal microbalances.

The supra-molecular self-assembly of peptides and proteins is a process which underlies a range of normal and aberrant biological pathways in nature, but one which remains challenging to monitor in a quantitative way. We discuss the experimental details of an approach to this problem which involves the direct measurement in vitro of mass changes of the aggregates as new molecules attach to them. The required mass sensitivity can be achieved by the use of a quartz crystal transducer-based microbalance. The technique should be broadly applicable to the study of protein aggregation, as well as to the identification and characterisation of inhibitors and modulators of this process.

Estresse ocupacional segundo o modelo demanda-controle e suas repercussões na saúde do trabalhador de enfermagem: análise das variações de cortisol salivar

O objeto de estudo foi o estresse no trabalho e os níveis de cortisol salivar. O objetivo geral foi avaliar a associação entre estresse no trabalho e variações de cortisol salivar de trabalhadores de Enfermagem inseridos na assistência hospitalar no Rio de Janeiro. A hipótese do estudo foi que existe associação entre trabalhadores expostos à alta exigência no trabalho e as variações de cortisol salivar. Trata-se de estudo epidemiológico observacional analítico seccional, realizado em hospital estadual localizado no Rio de Janeiro com amostra de 103 trabalhadores. Para avaliação dos aspectos psicossociais do trabalho, utilizou-se o questionário Job Content Questionnaire. O cortisol salivar foi medido através da coleta de 04 amostras de cada participante em um dia do plantão: ao acordar, 30 minutos depois, às 12h e 18 h. A coleta de dados foi realizada entre março e abril de 2012. Utilizou-se o programa SPSS 18.0 para análise dos dados. As dimensões demanda psicológica e controle e a subtração foram utilizadas sob a forma contínua nas análises de correlação com as covariáveis e desfecho. Os níveis de cortisol foram quantificados por meio de cinco índices: o cálculo da área sob a curva em relação ao zero ou base (AUCg), área sob a curva em relação ao aumento (AUCi), o aumento médio (MnInc), a excreção do cortisol no período pós acordar (AUCtrab) e a área sob a curva em relação ao zero ou base do ciclo diurno (AUCCD). Para avaliar a associação entre as covariáveis e exposição e desfecho utilizou-se os testes de Mann Whitney e Kruskall-Wallis. As covariáveis associadas à exposição ou ao desfecho com nível de significância de 20% (p≤0,20) foram testadas no modelo de regressão linear. Realizada análise de correlação utilizando-se o coeficiente de correlação de Spearmans. Como resultado encontrou-se que os trabalhadores de Enfermagem obtiveram médias para demanda psicológica e controle que tendem para o limite superior, bem como para a subtração, caracterizando alta demanda e alto controle, ou seja, trabalho ativo. O valor médio de cortisol observado ao acordar, 30 minutos após, 12h e 18h foi de 5,82 nmol/L) (4,86), 16,60 nmol/L ( 8,31), 7,49 nmol/L ( 6,97) e 3,93 nmol/L ( 3,15), respectivamente. O aumento do cortisol entre o acordar e 30 minutos após foi em média de 64%. Já para os índices de cortisol adotados observa-se o valor médio da MnInc, AUCg, AUCi, AUCtrab e AUCCD foi de 10,78 nmol/L (6,99), 5,61 nmol/L ( 2,92) 2,69 nmol/L ( 1,75), 32,51 nmol/L ( 21,99) e 107,99 nmol/L ( 61,63), respectivamente. Este estudo demonstrou que os níveis de cortisol salivar livre não estão associados à alta exigência no trabalho, mesmo quando ajustadas pelas possíveis variáveis de confusão ou modificadoras de efeito. A hipótese do estudo não foi confirmada. Os dados obtidos neste estudo revelaram aspectos importantes dos riscos psicossociais a que estão expostos os trabalhadores de Enfermagem durante o processo de trabalho, oferecendo subsídios para que sejam implementados programas de orientação e promoção à saúde do trabalhador e fornecem um contributo para entender os caminhos biológicos pelos quais o estresse no trabalho influencia a saúde.

金属离子与钙调蛋白相互作用的基质辅助激光解析电离飞行时间质谱研究

Calmodulin is a ubiquitous calcium-binding protein in eukaryote cells and engages in various important biological pathways. In the present study, binding interactions between several metal ions and calmodulin were nvestigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).The results revealed that the specific binding of metal ions with the protein could be detected using MALDI-TOF MS.

Common genetic variation and the control of HIV-1 in humans.

To extend the understanding of host genetic determinants of HIV-1 control, we performed a genome-wide association study in a cohort of 2,554 infected Caucasian subjects. The study was powered to detect common genetic variants explaining down to 1.3% of the variability in viral load at set point. We provide overwhelming confirmation of three associations previously reported in a genome-wide study and show further independent effects of both common and rare variants in the Major Histocompatibility Complex region (MHC). We also examined the polymorphisms reported in previous candidate gene studies and fail to support a role for any variant outside of the MHC or the chemokine receptor cluster on chromosome 3. In addition, we evaluated functional variants, copy-number polymorphisms, epistatic interactions, and biological pathways. This study thus represents a comprehensive assessment of common human genetic variation in HIV-1 control in Caucasians.

Molecular pathology of RUNX3 in human carcinogenesis

A major goal of molecular biology is to elucidate the mechanisms underlying cancer development and progression in order to achieve early detection, better diagnosis and staging and novel preventive and therapeutic strategies. We feel that an understanding of Runt-related transcription factor 3 (RUNX3)-regulated biological pathways will directly impact our knowledge of these areas of human carcinogenesis. The RUNX3 transcription factor is a downstream effector of the transforming growth factor-beta (TGF-beta) signaling pathway, and has a critical role in the regulation of cell proliferation and cell death by apoptosis, and in angiogenesis, cell adhesion and invasion. We previously identified RUNX3 as a major gastric tumor suppressor by establishing a causal relationship between loss of function and gastric carcinogenesis. More recently, we showed that RUNX3 functions as a bona fide initiator of colonic carcinogenesis by linking the Wnt oncogenic and TGF-beta tumor suppressive pathways. Apart from gastric and colorectal cancers. a multitude of epithelial cancers exhibit inactivation of RUNX3, thereby making it a putative tumor suppressor in human neoplasia. This review highlights our current understanding of the molecular mechanisms of RUNX3 inactivation in the context of cancer development and progression. (C) 2009 Elsevier B.V. All rights reserved.