Transcriptional repression of peroxisome proliferator-activated receptor beta/delta in murine keratinocytes by CCAAT/enhancer-binding proteins.

The roles of peroxisome proliferator-activated receptors (PPARs) and CCAAT/enhancer-binding proteins (C/EBPs) in keratinocyte and sebocyte differentiation suggest that both families of transcription factors closely interact in the skin. Initial characterization of the mouse PPARbeta promoter revealed an AP-1 site that is crucial for the regulation of PPARbeta expression in response to inflammatory cytokines in the skin. We now present evidence for a novel regulatory mechanism of the expression of the PPARbeta gene by which two members of the C/EBP family of transcription factors inhibit its basal promoter activity in mouse keratinocytes. We first demonstrate that C/EBPalpha and C/EBPbeta, but not C/EBPdelta, inhibit the expression of PPARbeta through the recruitment of a transcriptional repressor complex containing HDAC-1 to a specific C/EBP binding site on the PPARbeta promoter. Consistent with this repression, the expression patterns of PPARbeta and C/EBPs are mutually exclusive in keratinocytes of the interfollicular epidermis and hair follicles in mouse developing skin. This work reveals the importance of the regulatory interplay between PPARbeta and C/EBP transcription factors in the control of proliferation and differentiation in this organ. Such insights are crucial for the understanding of the molecular control regulating the balance between proliferation and differentiation in many cell types including keratinocytes.

Systems Genetics of Metabolism: The Use of the BXD Murine Reference Panel for Multiscalar Integration of Traits.

Metabolic homeostasis is achieved by complex molecular and cellular networks that differ significantly among individuals and are difficult to model with genetically engineered lines of mice optimized to study single gene function. Here, we systematically acquired metabolic phenotypes by using the EUMODIC EMPReSS protocols across a large panel of isogenic but diverse strains of mice (BXD type) to study the genetic control of metabolism. We generated and analyzed 140 classical phenotypes and deposited these in an open-access web service for systems genetics (www.genenetwork.org). Heritability, influence of sex, and genetic modifiers of traits were examined singly and jointly by using quantitative-trait locus (QTL) and expression QTL-mapping methods. Traits and networks were linked to loci encompassing both known variants and novel candidate genes, including alkaline phosphatase (ALPL), here linked to hypophosphatasia. The assembled and curated phenotypes provide key resources and exemplars that can be used to dissect complex metabolic traits and disorders.

Mutation in the human homeobox gene NKX5-3 causes an oculo-auricular syndrome.

Several dysmorphic syndromes affect the development of both the eye and the ear, but only a few are restricted to the eye and the external ear. We describe a developmental defect affecting the eye and the external ear in three members of a consanguineous family. This syndrome is characterized by ophthalmic anomalies (microcornea, microphthalmia, anterior-segment dysgenesis, cataract, coloboma of various parts of the eye, abnormalities of the retinal pigment epithelium, and rod-cone dystrophy) and a particular cleft ear lobule. Linkage analysis and mutation screening revealed in the first exon of the NKX5-3 gene a homozygous 26 nucleotide deletion, generating a truncating protein that lacked the complete homeodomain. Morpholino knockdown expression of the zebrafish nkx5-3 induced microphthalmia and disorganization of the developing retina, thus confirming that this gene represents an additional member implicated in axial patterning of the retina.

Inhibition and enhancement of in vitro helper activity of apocytochrome c-specific murine T cell populations and clones by anti-apocytochrome c-specific monoclonal antibodies.

A murine monoclonal antibody (mAb) specific for apocytochrome c was found to be able to either inhibit or enhance the helper activity of mouse apocytochrome c-specific T cell clones and populations in a hapten (trinitrophenyl)-carrier (apocytochrome c) system of T-B cell cooperation. This effect of the mAb was carrier specific, could not be ascribed simply to a shift in the kinetics of the antibody response and was observed using apocytochrome c T helper cells of different mouse haplotypes. In addition, the anti-apocytochrome c mAb was able to inhibit specific T helper cell activity even when the T cells were triggered with antigen-presenting cells pulsed with antigen. Taken together, these results suggested that the mAb was inhibiting helper activity due to its ability to modify the interaction between T cells and antigen-presenting cells.

Revealing a subclinical salt-losing phenotype in heterozygous carriers of the novel S562P mutation in the alpha subunit of the epithelial sodium channel.

OBJECTIVE: Pseudohypoaldosteronism type I (PHA1) is a rare inborn disease causing severe salt loss. Mutations in the three coding genes of the epithelial sodium channel (ENaC) are responsible for the systemic autosomal recessive form. So far, no phenotype has been reported in heterozygous carriers. PATIENTS: A consanguineous family from Somalia giving birth to a neonate suffering from PHA1 was studied including clinical and hormonal characteristics of the family, mutational analysis of the SCNN1A, SCNN1B, SCNN1G and CFTR genes and in vitro analysis of the functional consequences of a mutant ENaC channel. RESULTS: CFTR mutations have been excluded. SCNN1A gene analysis revealed a novel homozygous c.1684T > C mutation resulting in a S562P substitution in the alphaENaC protein of the patient. Functional analysis showed a significantly reduced S562P channel function compared to ENaC wild type. Protein synthesis and channel subunit assembly were not altered by the S562P mutation. Co-expression of mutant and wild-type channels revealed a dominant negative effect. In heterozygote carriers, sweat sodium and chloride concentrations were increased without additional hormonal or clinical phenotypes. CONCLUSION: Hence, the novel mutation S562P is causing systemic PHA1 in the homozygous state. A thorough clinical investigation of the heterozygote SCNN1A mutation carriers revealed increased sweat sodium and chloride levels consistent with a dominant effect of the mutant S562P allele. Whether this subclinical phenotype is of any consequence for the otherwise asymptomatic heterozygous carriers has to be elucidated.

A murine genital-challenge model is a sensitive measure of protective antibodies against human papillomavirus infection.

The available virus-like particle (VLP)-based prophylactic vaccines against specific human papillomavirus (HPV) types afford close to 100% protection against the type-associated lesions and disease. Based on papillomavirus animal models, it is likely that protection against genital lesions in humans is mediated by HPV type-restricted neutralizing antibodies that transudate or exudate at the sites of genital infection. However, a correlate of protection was not established in the clinical trials because few disease cases occurred, and true incident infection could not be reliably distinguished from the emergence or reactivation of prevalent infection. In addition, the current assays for measuring vaccine-induced antibodies, even the gold standard HPV pseudovirion (PsV) in vitro neutralization assay, may not be sensitive enough to measure the minimum level of antibodies needed for protection. Here, we characterize the recently developed model of genital challenge with HPV PsV and determine the minimal amounts of VLP-induced neutralizing antibodies that can afford protection from genital infection in vivo after transfer into recipient mice. Our data show that serum antibody levels >100-fold lower than those detectable by in vitro PsV neutralization assays are sufficient to confer protection against an HPV PsV genital infection in this model. The results clearly demonstrate that, remarkably, the in vivo assay is substantially more sensitive than in vitro PsV neutralization and thus may be better suited for studies to establish correlates of protection.

Study of chromosome rearrangements associated with the trpE26 mutation of Bacillus subtilis.

Chromosome rearrangements involved in the formation of merodiploid strains in the Bacillus subtilis 168-166 system were explained by postulating the existence of intrachromosomal homology regions. This working hypothesis was tested by analysing sequences and restriction patterns of the, as yet uncharacterized, junctions between chromosome segments undergoing rearrangements in parent, 168 trpC2 and 166 trpE26, as well as in derived merodiploid strains. Identification, at the Ia/Ib chromosome junction of both parent strains, of a 1.3 kb segment nearly identical to a segment of prophage SPbeta established the existence of one of the postulated homology sequences. Inspection of relevant junctions revealed that a set of different homology regions, derived from prophage SPbeta, plays a key role in the formation of so-called trpE30, trpE30+, as well as of new class I merodiploids. Analysis of junctions involved in the transfer of the trpE26 mutation, i.e. simultaneous translocation of chromosome segment C and rotation of the terminal relative to the origin moiety of the chromosome, did not confirm the presence of any sequence suitable for homologous recombination. We propose a model involving simultaneous introduction of four donor DNA molecules, each comprising a different relevant junction, and their pairing with the junction regions of the recipient chromosome. The resolution of this structure, resting on homologous recombination, would confer the donor chromosome structure to the recipient, achieving some kind of 'transstamping'. In addition, a rather regular pattern of inverse and direct short sequence repeats in regions flanking the breaking points could be correlated with the initial, X-ray-induced, rearrangement.

Statistical properties of population differentiation estimators under stepwise mutation in a finite island model.

Microsatellite loci mutate at an extremely high rate and are generally thought to evolve through a stepwise mutation model. Several differentiation statistics taking into account the particular mutation scheme of the microsatellite have been proposed. The most commonly used is R(ST) which is independent of the mutation rate under a generalized stepwise mutation model. F(ST) and R(ST) are commonly reported in the literature, but often differ widely. Here we compare their statistical performances using individual-based simulations of a finite island model. The simulations were run under different levels of gene flow, mutation rates, population number and sizes. In addition to the per locus statistical properties, we compare two ways of combining R(ST) over loci. Our simulations show that even under a strict stepwise mutation model, no statistic is best overall. All estimators suffer to different extents from large bias and variance. While R(ST) better reflects population differentiation in populations characterized by very low gene-exchange, F(ST) gives better estimates in cases of high levels of gene flow. The number of loci sampled (12, 24, or 96) has only a minor effect on the relative performance of the estimators under study. For all estimators there is a striking effect of the number of samples, with the differentiation estimates showing very odd distributions for two samples.

Sex-chromosome turnovers induced by deleterious mutation load.

In sharp contrast with mammals and birds, many cold-blooded vertebrates present homomorphic sex chromosomes. Empirical evidence supports a role for frequent turnovers, which replace nonrecombining sex chromosomes before they have time to decay. Three main mechanisms have been proposed for such turnovers, relying either on neutral processes, sex-ratio selection, or intrinsic benefits of the new sex-determining genes (due, e.g., to linkage with sexually antagonistic mutations). Here, we suggest an additional mechanism, arising from the load of deleterious mutations that accumulate on nonrecombining sex chromosomes. In the absence of dosage compensation, this load should progressively lower survival rate in the heterogametic sex. Turnovers should occur when this cost outweighs the benefits gained from any sexually antagonistic genes carried by the nonrecombining sex chromosome. We use individual-based simulations of a Muller's ratchet process to test this prediction, and investigate how the relevant parameters (effective population size, strength and dominance of deleterious mutations, size of nonrecombining segment, and strength of sexually antagonistic selection) are expected to affect the rate of turnovers.

Novel FGF8 mutations associated with recessive holoprosencephaly, craniofacial defects, and hypothalamo-pituitary dysfunction.

Context: Fibroblast growth factor (FGF) 8 is important for GnRH neuronal development with human mutations resulting in Kallmann syndrome. Murine data suggest a role for Fgf8 in hypothalamo-pituitary development; however, its role in the etiology of wider hypothalamo-pituitary dysfunction in humans is unknown.Objective: The objective of this study was to screen for FGF8 mutations in patients with septo-optic dysplasia (n = 374) or holoprosencephaly (HPE)/midline clefts (n = 47).Methods: FGF8 was analyzed by PCR and direct sequencing. Ethnically matched controls were then screened for mutated alleles (n = 480-686). Localization of Fgf8/FGF8 expression was analyzed by in situ hybridization in developing murine and human embryos. Finally, Fgf8 hypomorphic mice (Fgf8(loxPNeo/-)) were analyzed for the presence of forebrain and hypothalamo-pituitary defects.Results: A homozygous p.R189H mutation was identified in a female patient of consanguineous parentage with semilobar HPE, diabetes insipidus, and TSH and ACTH insufficiency. Second, a heterozygous p.Q216E mutation was identified in a female patient with an absent corpus callosum, hypoplastic optic nerves, and Moebius syndrome. FGF8 was expressed in the ventral diencephalon and anterior commissural plate but not in Rathke's pouch, strongly suggesting early onset hypothalamic and corpus callosal defects in these patients. This was consolidated by significantly reduced vasopressin and oxytocin staining neurons in the hypothalamus of Fgf8 hypomorphic mice compared with controls along with variable hypothalamo-pituitary defects and HPE.Conclusion: We implicate FGF8 in the etiology of recessive HPE and potentially septo-optic dysplasia/Moebius syndrome for the first time to our knowledge. Furthermore, FGF8 is important for the development of the ventral diencephalon, hypothalamus, and pituitary. (J Clin Endocrinol Metab 96: E1709-E1718, 2011)

The T309G Murine Double Minute 2 Gene Polymorphism Is an Independent Prognostic Factor for Patients with Renal Cell Carcinoma.

The purpose of this study was to evaluate the association of the T309G MDM2 gene polymorphism with renal cell carcinoma (RCC) risk, pathology, and cancer-specific survival (CSS). T309G MDM2 was genotyped in 449 Caucasians, including 240 with RCC and 209 cancer-free controls. The T309G MDM2 genotype was TT in 174 (38.8%), GT in 214 (47.7%), and GG in 61 (13.6%) subjects, without any significant differences between cases and controls on both univariable (p=0.58) and multivariable logistic regression (each p>0.25). Furthermore, T309G MDM2 was not linked with T stage (p=0.75), N stage (p=0.37), M stage (p=0.94), grade (p=0.21), and subtype (p=0.55). There was, however, a statistically significant association of T309G MDM2 with CSS (p=0.022): patients with TT had significantly worse survival than GG/GT (p=0.009), while those with GT and GG had similar outcomes (p=0.92). The 5-year survival rate for patients with TT, GT, and GG was 69.5%, 84.5%, and 89.7%, respectively. On the multivariable analysis, T309G was identified as an independent prognostic factor. The T309G MDM2 polymorphism is an independent prognostic factor for patients with RCC, with the TT genotype being associated with worse prognosis. In this study, there were no significant associations with RCC risk and pathology.

Macular dystrophy associated with the mitochondrial DNA A3243G mutation: pericentral pigment deposits or atrophy? Report of two cases and review of the literature.

BACKGROUND: The A3243G point mutation in mitochondrial DNA (mtDNA) is associated with MELAS (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) and MIDD syndromes (maternally inherited diabetes and deafness). Both MELAS and MIDD patients can present with visual symptoms due to a retinopathy, sometimes before the genetic diagnosis is made. CASE PRESENTATION: Patient 1: 46 year-old woman with diabetes mellitus and hearing loss was referred for an unspecified maculopathy detected during screening evaluation for diabetic retinopathy. Visual acuity was 20/20 in both eyes. Fundus examination showed bilateral macular and peripapillary hyperpigmented/depigmented areas.Patient 2: 45 year-old woman was referred for recent vision loss in her left eye. History was remarkable for chronic fatigue, migraine and diffuse muscular pain. Visual acuity was 20/20 in her right eye and 20/30 in her left eye. Fundus exhibited several nummular perifoveal islands of retinal pigment epithelium atrophy and adjacent pale deposits in both eyes.Retinal anatomy was investigated with autofluorescence, retinal angiography and optical coherence tomography. Retinal function was assessed with automated static perimetry, full-field and multifocal electroretinography and electro-oculography. Genetic testing of mtDNA identified a point mutation at the locus 3243. CONCLUSION: Observation of RPE abnormalities in the context of suggestive systemic findings should prompt mtDNA testing.

A novel homozygous R764H mutation in crumbs homolog 1 causes autosomal recessive retinitis pigmentosa.

PURPOSE: Retinitis pigmentosa (RP; MIM 268000) is a hereditary disease characterized by poor night vision and progressive loss of photoreceptors, eventually leading to blindness. This degenerative process primarily affects peripheral vision due to the loss of rods. Autosomal recessive RP (arRP) is clinically and genetically heterogeneous. It has been associated with mutations in different genes, including CRB1 (crumbs homolog 1). The aim of this study was to determine the causative gene in a Tunisian patient with arRP born to non-consanguineous parents. METHODS: Four accessible family members were included. They underwent full ophthalmic examination with best-corrected Snellen visual acuity, fundus photography and fluorescein angiography. Haplotype analysis was used to evaluate homozygosity in the family to 20 arRP loci. All exons and intron-exon junctions of candidate genes not excluded by haplotype analysis were PCR amplified and directly sequenced. RESULTS: The proband was a 43-year-old female patient. Best-corrected visual acuity was 20/63 (right eye) and 20/80 (left eye). Visual loss began during the third decade. Funduscopic examination and fluorescein angiography revealed typical advanced RP changes with bone spicule-like pigment deposits in the posterior pole and the midperiphery along with retinal atrophy, narrowing of the vessels, and waxy optic discs. Haplotype analysis revealed homozygosity with microsatellite markers D1S412 and D1S413 on chromosome 1q31.3. These markers flanked CRB1. Our results excluded linkage of all the other arRP loci/genes tested. Sequencing of the 12 coding exons and splice sites of CRB1 disclosed a homozygous missense mutation in exon 7 at nucleotide c. 2291G>A, resulting in an arginine to histidine substitution (p.R764H). CONCLUSIONS: R764H is a novel mutation associated with CRB1-related arRP. Previously, an R764C mutation was reported. Extending the mutation spectrum of CRB1 with additional families is important for genotype-phenotype correlations and characterization of the scope of mutation.