The role of the prokineticin 2 pathway in human reproduction: evidence from the study of human and murine gene mutations.

A widely dispersed network of hypothalamic GnRH neurons controls the reproductive axis in mammals. Genetic investigation of the human disease model of isolated GnRH deficiency has revealed several key genes crucial for GnRH neuronal ontogeny and GnRH secretion. Among these genes, prokineticin 2 (PROK2), and PROK2 receptor (PROKR2) have recently emerged as critical regulators of reproduction in both mice and humans. Both prok2- and prokr2-deficient mice recapitulate the human Kallmann syndrome phenotype. Additionally, PROK2 and PROKR2 mutations are seen in humans with Kallmann syndrome, thus implicating this pathway in GnRH neuronal migration. However, PROK2/PROKR2 mutations are also seen in normosmic GnRH deficiency, suggesting a role for the prokineticin signaling system in GnRH biology that is beyond neuronal migration. This observation is particularly surprising because mature GnRH neurons do not express PROKR2. Moreover, mutations in both PROK2 and PROKR2 are predominantly detected in the heterozygous state with incomplete penetrance or variable expressivity frequently seen within and across pedigrees. In some of these pedigrees, a "second hit" or oligogenicity has been documented. Besides reproduction, a pleiotropic physiological role for PROK2 is now recognized, including regulation of pain perception, circadian rhythms, hematopoiesis, and immune response. Therefore, further detailed clinical studies of patients with PROK2/PROKR2 mutations will help to map the broader biological role of the PROK2/PROKR2 pathway and identify other interacting genes/proteins that mediate its molecular effects in humans.

New loci associated with birth weight identify genetic links between intrauterine growth and adult height and metabolism.

Birth weight within the normal range is associated with a variety of adult-onset diseases, but the mechanisms behind these associations are poorly understood. Previous genome-wide association studies of birth weight identified a variant in the ADCY5 gene associated both with birth weight and type 2 diabetes and a second variant, near CCNL1, with no obvious link to adult traits. In an expanded genome-wide association meta-analysis and follow-up study of birth weight (of up to 69,308 individuals of European descent from 43 studies), we have now extended the number of loci associated at genome-wide significance to 7, accounting for a similar proportion of variance as maternal smoking. Five of the loci are known to be associated with other phenotypes: ADCY5 and CDKAL1 with type 2 diabetes, ADRB1 with adult blood pressure and HMGA2 and LCORL with adult height. Our findings highlight genetic links between fetal growth and postnatal growth and metabolism.

Cooperation between the transcription factors p63 and IRF6 is essential to prevent cleft palate in mice.

Cleft palate is a common congenital disorder that affects up to 1 in 2,500 live human births and results in considerable morbidity to affected individuals and their families. The etiology of cleft palate is complex, with both genetic and environmental factors implicated. Mutations in the transcription factor-encoding genes p63 and interferon regulatory factor 6 (IRF6) have individually been identified as causes of cleft palate; however, a relationship between the key transcription factors p63 and IRF6 has not been determined. Here, we used both mouse models and human primary keratinocytes from patients with cleft palate to demonstrate that IRF6 and p63 interact epistatically during development of the secondary palate. Mice simultaneously carrying a heterozygous deletion of p63 and the Irf6 knockin mutation R84C, which causes cleft palate in humans, displayed ectodermal abnormalities that led to cleft palate. Furthermore, we showed that p63 transactivated IRF6 by binding to an upstream enhancer element; genetic variation within this enhancer element is associated with increased susceptibility to cleft lip. Our findings therefore identify p63 as a key regulatory molecule during palate development and provide a mechanism for the cooperative role of p63 and IRF6 in orofacial development in mice and humans.

Analysis of potential transcriptomic biomarkers for Huntington's disease in peripheral blood.

Highly quantitative biomarkers of neurodegenerative disease remain an important need in the urgent quest for disease-modifying therapies. For Huntington's disease (HD), a genetic test is available (trait marker), but necessary state markers are still in development. In this report, we describe a large battery of transcriptomic tests explored as state biomarker candidates. In an attempt to exploit the known neuroinflammatory and transcriptional perturbations of disease, we measured relevant mRNAs in peripheral blood cells. The performance of these potential markers was weak overall, with only one mRNA, immediate early response 3 (IER3), showing a modest but significant increase of 32% in HD samples compared with controls. No statistically significant differences were found for any other mRNAs tested, including a panel of 12 RNA biomarkers identified in a previous report [Borovecki F, Lovrecic L, Zhou J, Jeong H, Then F, Rosas HD, Hersch SM, Hogarth P, Bouzou B, Jensen RV, et al. (2005) Proc Natl Acad Sci USA 102:11023-11028]. The present results may nonetheless inform the future design and testing of HD biomarker strategies.

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A Reply to the Comment by Jing-Dong Bao and Yan Zhou.

Flow-microfluorometric monitoring of oligoclonal CD8+ T cell responses to an immunodominant Moloney leukemia virus-encoded epitope in vivo.

The TCR repertoire of CD8+ T cells specific for Moloney murine leukemia virus (M-MuLV)-associated Ags has been investigated in vitro and in vivo. Analysis of a large panel of established CD8+ CTL clones specific for M-MuLV indicated an overwhelming bias for V beta4 in BALB/c mice and for V beta5.2 in C57BL/6 mice. These V beta biases were already detectable in mixed lymphocyte:tumor cell cultures established from virus-immune spleen cells. Furthermore, direct ex vivo analysis of PBL from BALB/c or C57BL/6 mice immunized with syngeneic M-MuLV-infected tumor cells revealed a dramatic increase in CD8+ cells expressing V beta4 or V beta5.2, respectively. M-MuLV-specific CD8+ cells with an activated (CD62L-) phenotype persisted in blood of immunized mice for at least 2 mo, and exhibited decreased TCR and CD8 levels compared with their naive counterparts. In C57BL/6 mice, most M-MuLV-specific CD8+ CTL clones and immune PBL coexpressed V alpha3.2 in association with V beta5.2. Moreover, these V beta5.2+ V alpha3.2+ cells were shown to recognize the recently described H-2Db-restricted epitope (CCLCLTVFL) encoded in the leader sequence of the M-MuLV gag polyprotein. Collectively, our data demonstrate a highly restricted TCR repertoire in the CD8+ T cell response to M-MuLV-associated Ags in vivo, and suggest the potential utility of flow-microfluorometric analysis of V beta and V alpha expression in the diagnosis and monitoring of viral infections.

A global analysis of Y-chromosomal haplotype diversity for 23 STR loci.

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent.

The limits of brain determinacy.

The genes do not control everything that happens in a cell or an organism, because thermally induced molecular movements and conformation changes are beyond genetic control. The importance of uncontrolled events has been argued from the differences between isogenic organisms reared in virtually identical environments, but these might alternatively be attributed to subtle, undetected differences in the environment. The present review focuses on the uncontrolled events themselves in the context of the developing brain. These are considered at cellular and circuit levels because even if cellular physiology was perfectly controlled by the genes (which it is not), the interactions between different cells might still be uncoordinated. A further complication is that the brain contains mechanisms that buffer noise and others that amplify it. The final resultant of the battle between these contrary mechanisms is that developmental stochasticity is sufficiently low to make neurobehavioural defects uncommon, but a chance component of neural development remains. Thus, our brains and behaviour are not entirely determined by a combination of genes-plus-environment.

The puzzles of the prokineticin 2 pathway in human reproduction.

Prokineticin, 1 (PROK1) and prokineticin 2 (PROK2), are two closely related proteins that were identified as the mammalian homologs of their two amphibian homologs, mamba intestinal toxin (MIT-1) and Bv8. MIT-1 was initially identified as a non-toxic constituent in the venom of the black mamba snake (Dendroaspis polylepis) (Joubert and Strydom, 1980) while Bv8 was identified in the skin secretion of the toad, Bombina variegate (Mollay et al., 1999). All three homologs stimulate gastrointestinal motility thus accounting for their family name "prokineticins" (Schweitz et al., 1990, 1999). However, since its initial description, both PROK1 and PROK2 have been found to regulate a dazzling array of biological functions throughout the body. In particular, PROK1 acts as a potent angiogenic mitogen on endocrine vascular epithelium, thus earning its other name, Endocrine gland-vascular endothelial factor (EG-VEGF) (LeCouter et al., 2002). In contrast, the PROK2 signaling pathway is a critical regulator of olfactory bulb morphogenesis and sexual maturation in mammals and this function is the focus of this review.

ER stress activates the NLRP3 inflammasome via an UPR-independent pathway.

Uncontrolled endoplasmic reticulum (ER) stress responses are proposed to contribute to the pathology of chronic inflammatory diseases such as type 2 diabetes or atherosclerosis. However, the connection between ER stress and inflammation remains largely unexplored. Here, we show that ER stress causes activation of the NLRP3 inflammasome, with subsequent release of the pro-inflammatory cytokine interleukin-1β. This ER-triggered proinflammatory signal shares the same requirement for reactive oxygen species production and potassium efflux compared with other known NLRP3 inflammasome activators, but is independent of the classical unfolded protein response (UPR). We thus propose that the NLRP3 inflammasome senses and responds to ER stress downstream of a previously uncharacterized ER stress response signaling pathway distinct from the UPR, thus providing mechanistic insight to the link between ER stress and chronic inflammatory diseases.

Autoimmunity-Associated LYP-W620 Does Not Impair Thymic Negative Selection of Autoreactive T Cells.

A C1858T (R620W) variation in the PTPN22 gene encoding the tyrosine phosphatase LYP is a major risk factor for human autoimmunity. LYP is a known negative regulator of signaling through the T cell receptor (TCR), and murine Ptpn22 plays a role in thymic selection. However, the mechanism of action of the R620W variant in autoimmunity remains unclear. One model holds that LYP-W620 is a gain-of-function phosphatase that causes alterations in thymic negative selection and/or thymic output of regulatory T cells (Treg) through inhibition of thymic TCR signaling. To test this model, we generated mice in which the human LYP-W620 variant or its phosphatase-inactive mutant are expressed in developing thymocytes under control of the proximal Lck promoter. We found that LYP-W620 expression results in diminished thymocyte TCR signaling, thus modeling a "gain-of-function" of LYP at the signaling level. However, LYP-W620 transgenic mice display no alterations of thymic negative selection and no anomalies in thymic output of CD4(+)Foxp3(+) Treg were detected in these mice. Lck promoter-directed expression of the human transgene also causes no alteration in thymic repertoire or increase in disease severity in a model of rheumatoid arthritis, which depends on skewed thymic selection of CD4(+) T cells. Our data suggest that a gain-of-function of LYP is unlikely to increase risk of autoimmunity through alterations of thymic selection and that LYP likely acts in the periphery perhaps selectively in regulatory T cells or in another cell type to increase risk of autoimmunity.

International collaborative proficiency study of Human Papillomavirus type 16 serology.

We performed an international proficiency study of Human Papillomavirus (HPV) type 16 serology. A common methodology for serology based on virus-like particle (VLP) ELISA was used by 10 laboratories in 6 continents. The laboratories used the same VLP reference reagent, which was selected as the most stable, sensitive and specific VLP preparation out of VLPs donated from 5 different sources. A blinded proficiency panel consisting of 52 serum samples from women with PCR-verified HPV 16-infection, 11 control serum samples from virginal women and the WHO HPV 16 International Standard (IS) serum were distributed. The mean plus 3 standard deviations of the negative control serum samples was the most generally useful "cut-off" criterion for distinguishing positive and negative samples. Using sensitivity of at least 50% and a specificity of 100% as proficiency criteria, 6/10 laboratories were proficient. In conclusion, an international Standard Operating Procedure for HPV serology, an international reporting system in International Units (IU) and a common "cut-off" criterion have been evaluated in an international HPV serology proficiency study.

Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle.

The Caulobacter DNA methyltransferase CcrM is one of five master cell-cycle regulators. CcrM is transiently present near the end of DNA replication when it rapidly methylates the adenine in hemimethylated GANTC sequences. The timing of transcription of two master regulator genes and two cell division genes is controlled by the methylation state of GANTC sites in their promoters. To explore the global extent of this regulatory mechanism, we determined the methylation state of the entire chromosome at every base pair at five time points in the cell cycle using single-molecule, real-time sequencing. The methylation state of 4,515 GANTC sites, preferentially positioned in intergenic regions, changed progressively from full to hemimethylation as the replication forks advanced. However, 27 GANTC sites remained unmethylated throughout the cell cycle, suggesting that these protected sites could participate in epigenetic regulatory functions. An analysis of the time of activation of every cell-cycle regulatory transcription start site, coupled to both the position of a GANTC site in their promoter regions and the time in the cell cycle when the GANTC site transitions from full to hemimethylation, allowed the identification of 59 genes as candidates for epigenetic regulation. In addition, we identified two previously unidentified N(6)-methyladenine motifs and showed that they maintained a constant methylation state throughout the cell cycle. The cognate methyltransferase was identified for one of these motifs as well as for one of two 5-methylcytosine motifs.