Replication of TCF4 through Association and Linkage Studies in Late-Onset Fuchs Endothelial Corneal Dystrophy

Fuchs endothelial corneal dystrophy (FECD) is a common, late-onset disorder of the corneal endothelium. Although progress has been made in understanding the genetic basis of FECD by studying large families in which the phenotype is transmitted in an autosomal dominant fashion, a recently reported genome-wide association study identified common alleles at a locus on chromosome 18 near TCF4 which confer susceptibility to FECD. Here, we report the findings of our independent validation study for TCF4 using the largest FECD dataset to date (450 FECD cases and 340 normal controls). Logistic regression with sex as a covariate was performed for three genetic models: dominant (DOM), additive (ADD), and recessive (REC). We found significant association with rs613872, the target marker reported by Baratz et al.(2010), for all three genetic models (DOM: P = 9.33×10−35; ADD: P = 7.48×10−30; REC: P = 5.27×10−6). To strengthen the association study, we also conducted a genome-wide linkage scan on 64 multiplex families, composed primarily of affected sibling pairs (ASPs), using both parametric and non-parametric two-point and multipoint analyses. The most significant linkage region localizes to chromosome 18 from 69.94cM to 85.29cM, with a peak multipoint HLOD = 2.5 at rs1145315 (75.58cM) under the DOM model, mapping 1.5 Mb proximal to rs613872. In summary, our study presents evidence to support the role of the intronic TCF4 single nucleotide polymorphism rs613872 in late-onset FECD through both association and linkage studies

Photoperiodic Modulation of Circadian Clock and Reproductive Axis Gene Expression in the Pre-Pubertal European Sea Bass Brain

The acquisition of reproductive competence requires the activation of the brain-pituitary-gonad (BPG) axis, which in most vertebrates, including fishes, is initiated by changes in photoperiod. In the European sea bass long-term exposure to continuous light (LL) alters the rhythm of reproductive hormones, delays spermatogenesis and reduces the incidence of precocious males. In contrast, an early shift from long to short photoperiod (AP) accelerates spermatogenesis. However, how photoperiod affects key genes in the brain to trigger the onset of puberty is still largely unknown. Here, we investigated if the integration of the light stimulus by clock proteins is sufficient to activate key genes that trigger the BPG axis in the European sea bass. We found that the clock genes clock, npas2, bmal1 and the BPG genes gnrh, kiss and kissr share conserved transcription factor frameworks in their promoters, suggesting co-regulation. Other gene promoters of the BGP axis were also predicted to be co-regulated by the same frameworks. Co-regulation was confirmed through gene expression analysis of brains from males exposed to LL or AP photoperiod compared to natural conditions: LL fish had suppressed gnrh1, kiss2, galr1b and esr1, while AP fish had stimulated npas2, gnrh1, gnrh2, kiss2, kiss1rb and galr1b compared to NP. It is concluded that fish exposed to different photoperiods present significant expression differences in some clock and reproductive axis related genes well before the first detectable endocrine and morphological responses of the BPG axis.European Community [222719 - LIFECYCLE]; Foundation for Science and Technology of Portugal (FCT) [SFRH/BPD/66742/2009, PEst-C/MAR/LA0015/2011]; Valencian Regional Goverment [Prometeo II/2014/051]; Spanish Ministry of Science and Innovation (MICINN) [CSD 2007-0002]info:eu-repo/semantics/publishedVersio

Reproductive Hormone-Dependent and -Independent Contributions to Developmental Changes in Kisspeptin in GnRH-Deficient Hypogonadal Mice

Kisspeptin is a potent activator of GnRH-induced gonadotropin secretion and is a proposed central regulator of pubertal onset. In mice, there is a neuroanatomical separation of two discrete kisspeptin neuronal populations, which are sexually dimorphic and are believed to make distinct contributions to reproductive physiology. Within these kisspeptin neuron populations, Kiss1 expression is directly regulated by sex hormones, thereby confounding the roles of sex differences and early activational events that drive the establishment of kisspeptin neurons. In order to better understand sex steroid hormone-dependent and -independent effects on the maturation of kisspeptin neurons, hypogonadal (hpg) mice deficient in GnRH and its downstream effectors were used to determine changes in the developmental kisspeptin expression. In hpg mice, sex differences in Kiss1 mRNA levels and kisspeptin immunoreactivity, typically present at 30 days of age, were absent in the anteroventral periventricular nucleus (AVPV). Although immunoreactive kisspeptin increased from 10 to 30 days of age to levels intermediate between wild type (WT) females and males, corresponding increases in Kiss1 mRNA were not detected. In contrast, the hpg arcuate nucleus (ARC) demonstrated a 10-fold increase in Kiss1 mRNA between 10 and 30 days in both females and males, suggesting that the ARC is a significant center for sex steroid-independent pubertal kisspeptin expression. Interestingly, the normal positive feedback response of AVPV kisspeptin neurons to estrogen observed in WT mice was lost in hpg females, suggesting that exposure to reproductive hormones during development may contribute to the establishment of the ovulatory gonadotropin surge mechanism. Overall, these studies suggest that the onset of pubertal kisspeptin expression is not dependent on reproductive hormones, but that gonadal sex steroids critically shape the hypothalamic kisspeptin neuronal subpopulations to make distinct contributions to the activation and control of the reproductive hormone cascade at the time of puberty

Kisspeptin signaling is required for the luteinizing hormone response in anestrous ewes following the introduction of males

The introduction of a novel male stimulates the hypothalamic-pituitary-gonadal axis of female sheep during seasonal anestrus, leading to the resumption of follicle maturation and ovulation. How this pheromone cue activates pulsatile secretion of gonadotropin releasing hormone (GnRH)/luteinizing hormone (LH) is unknown. We hypothesised that pheromones activate kisspeptin neurons, the product of which is critical for the stimulation of GnRH neurons and fertility. During the non-breeding season, female sheep were exposed to novel males and blood samples collected for analysis of plasma LH profiles. Females without exposure to males served as controls. In addition, one hour before male exposure, a kisspeptin antagonist (P-271) or vehicle was infused into the lateral ventricle and continued for the entire period of male exposure. Introduction of a male led to elevated mean LH levels, due to increased LH pulse amplitude and pulse frequency in females, when compared to females not exposed to a male. Infusion of P-271 abolished this effect of male exposure. Brains were collected after the male effect stimulus and we observed an increase in the percentage of kisspeptin neurons co-expressing Fos, by immunohistochemistry. In addition, the per-cell expression of Kiss1 mRNA was increased in the rostral and mid (but not the caudal) arcuate nucleus (ARC) after male exposure in both aCSF and P-271 treated ewes, but the per-cell content of neurokinin B mRNA was decreased. There was also a generalized increase in Fos positive cells in the rostral and mid ARC as well as the ventromedial hypothalamus of females exposed to males. We conclude that introduction of male sheep to seasonally anestrous female sheep activates kisspeptin neurons and other cells in the hypothalamus, leading to increased GnRH/LH secretion