Human Molecular Genetics - Advance Access

Integrative genomics identifies DSCR1 (RCAN1) as a novel NFAT-dependent mediator of phenotypic modulation in vascular smooth muscle cells

Thu, 19 Nov 2009 06:19:50 PST

Vascular smooth muscle cells (SMC) display remarkable phenotypic plasticity in response to environmental cues. The Nuclear Factor of Activated T-cells (NFAT) family of transcription factors plays a critical role in vascular pathology. However, known functional NFAT gene targets in vascular SMCs are currently limited. Publicly available whole-genome expression array datasets were analyzed to identify differentially expressed genes in human, mouse, and rat SMCs. Comparison between vehicle and phenotypic modulatory stimuli identified 63 species-conserved, upregulated genes. Integration of the 63 upregulated genes with an in silico NFAT-ome (a species-conserved list of gene promoters containing at least one NFAT binding site) identified 18 putative NFAT-dependent genes. Further intersection of these 18 potential NFAT target genes with a mouse in vivo vascular injury microarray identified 4 putative NFAT-dependent, injury responsive genes. In vitro validations substantiated the NFAT-dependent role of Cyclooxygenase 2 (COX2/PTGS2) in SMC phenotypic modulation and uncovered Down Syndrome Candidate Region 1 (DSCR1/RCAN1) as a novel NFAT target gene in SMCs. We show that induction of DSCR1 inhibits calcineurin/NFAT signaling through a negative feedback mechanism; DSCR1 overexpression attenuates NFAT transcriptional activity and COX2 protein expression whereas knockdown of endogenous DSCR1 enhances NFAT transcriptional activity. Our integrative genomics approach illustrates how the combination of publicly available gene expression arrays, computational databases, and empirical research methods can answer specific questions in any cell type for a transcriptional network of interest. Herein, we report DSCR1 as a novel NFAT-dependent, injury-inducible, early gene that may serve to negatively regulate SMC phenotypic switching.

Genetic evidence that raised Sex Hormone Binding Globulin (SHBG) levels reduce the risk of type 2 diabetes

Wed, 18 Nov 2009 03:30:23 PST

Epidemiological studies consistently show that circulating sex hormone binding globulin (SHBG) levels are lower in type 2 diabetes patients than non-diabetic individuals but the causal nature of this association is controversial. Genetic studies can help dissect causal directions of epidemiological associations because genotypes are much less likely to be confounded, biased, or influenced by disease processes.

Using this Mendelian randomization principle, we selected a common single nucleotide polymorphism (SNP) near the SHBG gene, rs1799941 that is strongly associated with SHBG levels. We used data from this SNP, or closely correlated SNPs, in 27,657 type 2 diabetes patients and 58,481 controls from 15 studies. We then used data from additional studies to estimate the difference in SHBG levels between type 2 diabetes patients and controls.

The SHBG SNP rs1799941 was associated with type 2 diabetes, odds ratio 0.94 (95%CI: 0.91, 0.97), p=2x10^–5, with the SHBG raising allele associated with reduced risk of type 2 diabetes. This effect was very similar to that expected [odds ratio 0.92 (95%CI: 0.88-0.96)], given the SHBG-SNP versus SHBG-levels association (SHBG levels are 0.2 standard deviations higher per copy of the A allele) and the SHBG-levels versus type 2 diabetes association (SHBG levels are 0.23 standard deviations lower in type 2 diabetic patients compared to controls). Results were very similar in men and women. There was no evidence that this variant is associated with diabetes related intermediate traits, including several measures of insulin secretion and resistance.

Our results, together with those from another recent genetic study, strengthen evidence that SHBG and sex hormones are involved in the aetiology of type 2 diabetes.

Novel Sequence Feature Variant Type Analysis of the HLA Genetic Association in Systemic Sclerosis

Wed, 18 Nov 2009 05:10:57 PST

We describe a novel approach to genetic association analyses with proteins sub-divided into biologically relevant smaller sequence features (SFs), and their variant types (VTs). SFVT analyses are particularly informative for study of highly polymorphic proteins such as the human leukocyte antigen (HLA), given the nature of its genetic variation: the high level of polymorphism, the pattern of amino acid variability, and that most HLA variation occurs at functionally important sites, as well as its known role in organ transplant rejection, autoimmune disease development, and response to infection. Further, combinations of variable amino acid sites shared by several HLA alleles (shared epitopes) are most likely better descriptors of the actual causative genetic variants. In a cohort of systemic sclerosis patients/controls, SFVT analysis shows that a combination of sequence features implicating specific amino acid residues in peptide binding pockets 4 and 7 of HLA-DRB1 explains much of the molecular determinant of risk.

Genetic structure of a unique admixed population: implications for medical research

Wed, 18 Nov 2009 18:52:31 PST

Statement: In naming population groups, we think a chief aim is to use terms that the group members use themselves, or find familiar and comfortable. The terms used in this manuscript to describe populations are as historically correct as possible and are chosen so as not to offend any population group. Two of the authors (DCP and REvdR) belong to the Coloured population, with one of the authors (REvdR) having contributed extensively to current literature on the history of the Coloured people of South Africa and served as Vice-President of the South African Institute of Race Relations. According to the 2001 South African census (http://www.statssa.gov.za/census01/HTML/CInBrief/ CIB2001.pdf), ‘Statistics South Africa continues to classify people by population group, in order to monitor progress in moving away from the apartheid-based discrimination of the past. However, membership of a population group is now based on self-perception and self-classification, not on a legal definition. Five options were provided on the questionnaire, Black African, Coloured, Indian or Asian, White and Other. Responses in the category "Other" were very few and were therefore imputed’. We have elected to use the term Bushmen rather than San to refer to the hunter-gatherer people of Southern Africa. Although they have no collective name for themselves, this decision was based on the term Bushmen (or Bossiesman) being the more familiar to the communities themselves, while the term San is the more accepted academic classification.

Understanding human genetic structure has fundamental implications for understanding the evolution and impact of human diseases. In this study, we describe the complex genetic substructure of a unique and recently admixed population arising ~350 years ago as a direct result of European settlement in South Africa. Analysis was performed using over 900 000 genome-wide single nucleotide polymorphisms in 20 unrelated ancestry-informative marker selected Coloured individuals and made comparisons with historically predicted founder populations. We show that there is substantial genetic contribution from at least four distinct population groups: Europeans, South Asians, Indonesians and a population genetically close to the isiXhosa sub-Saharan Bantu. This is in good accord with the historical record. We briefly examine the implications of determining the genetic diversity of this population, not only for furthering understanding of human evolution out of Africa, but also for genome-wide association studies using admixture mapping. In conclusion, we define the genetic structure of a uniquely admixed population that holds great potential to advance genetic-based medical research.

Drosophila models of peroxisomal biogenesis disorder: Peroxins are required for spermatogenesis and very long chain fatty acid metabolism

Chen, H., Liu, Z., Huang, X. — Tue, 17 Nov 2009 23:14:39 PST

Peroxisomes are vital eukaryotic organelles that participate in lipid metabolism, in particular the metabolism of very long chain fatty acids (VLCFA). The biogenesis of peroxisomes is regulated by a set of peroxin proteins (PEX). In humans, mutations affecting peroxin protein production or function result in devastating diseases classified as peroxisome biogenesis disorders (PBD). The way in which peroxisomal dysfunction leads to the pathophysiological consequences of PBD is not well understood. Here we report that Drosophila pex mutants faithfully recapitulate several key features of human PBD, including impaired peroxisomal protein import, elevated VLCFA levels, and growth retardation. Moreover, disruption of pex function results in spermatogenesis defects, including spermatocyte cytokinesis failure in Drosophila. Importantly, increased VLCFA levels enhance these spermatogenesis defects while reduced VLCFA levels alleviate them. Thus, regulation of proper VLCFA levels by pex genes is crucial for spermatogenesis. Together our study reveals an indispensable function of pex genes during spermatogenesis and provides a causative link between the phenotypic severity of pex mutants and VLCFA levels.

A novel form of cell type-specific partial IFN-{gamma}R1 deficiency caused by a germ line mutation of the IFNGR1 initiation codon

Mon, 16 Nov 2009 00:14:47 PST

IFN-R1 deficiency is a genetic etiology of Mendelian susceptibility to mycobacterial diseases, and includes two forms of complete recessive deficiency, with or without cell surface expression, and two forms of partial deficiency, dominant or recessive. We report here a novel form of partial and recessive Interferon receptor 1 (IFN-R1) deficiency, which is almost as severe as complete deficiency. The patient is homozygous for a mutation of the initiation codon (M1K). No detectable expression and function of IFN-R1 were found in the patient's fibroblasts. However, IFN-R1 expression was found to be impaired, but not abolished, on the EBV-transformed B cells, which could respond weakly to IFN-. The mechanism underlying this weak expression involves leaky translation initiation at both non-AUG codons and the third AUG codon at position 19. It results in the residual expression of IFN-R1 protein of normal molecular weight and function. The residual IFN- signaling documented in this novel form of partial IFN-R1 deficiency was not ubiquitous and was milder than that seen in other forms of partial IFN-R1 deficiency, accounting for the more severe clinical phenotype of the patient, which was almost as severe as that of patients with complete deficiency.

Involvement of a novel preimplantation-specific gene encoding the high mobility group box protein Hmgpi in early embryonic development

Sat, 14 Nov 2009 02:39:37 PST

Mining gene-expression-profiling data identified a novel gene that is specifically expressed in preimplantation embryos. Hmgpi, a putative chromosomal protein with two high-mobility-group boxes, is zygotically transcribed during zygotic genome activation, but is not transcribed postimplantation. The Hmgpi-encoded protein (HMGPI), first detected at the 4-cell stage, remains highly expressed in pre-implantation embryos. Interestingly, HMGPI is expressed in both the inner cell mass (ICM) and the trophectoderm, and translocated from cytoplasm to nuclei at the blastocyst stage, indicating differential spatial requirements before and after the blastocyst stage. siRNA (siHmgpi)-induced reduction of Hmgpi transcript levels caused developmental loss of preimplantation embryos and implantation failures. Furthermore, reduction of Hmgpi prevented blastocyst outgrowth leading to generation of embryonic stem cells. The siHmgpi-injected embryos also lost ICM and trophectoderm integrity, demarcated by reduced expressions of Oct4, Nanog and Cdx2. The findings implicated an important role for Hmgpi at the earliest stages of mammalian embryonic development.

Sporadic ALS has compartment-specific aberrant exon splicing and altered cell-matrix adhesion biology

Fri, 13 Nov 2009 09:48:52 PST

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive weakness from loss of motor neurons. The fundamental pathogenic mechanisms are unknown and recent evidence is implicating a significant role for abnormal exon splicing and RNA processing. Using new comprehensive genomic technologies, we studied exon splicing directly in 12 sporadic ALS and 10 control lumbar spinal cords acquired by a rapid autopsy system that processed nervous systems specifically for genomic studies. ALS patients had rostral onset and caudally advancing disease and abundant residual motor neurons in this region. We created two RNA pools, one from motor neurons collected by laser capture microdissection and one from the surrounding anterior horns. From each, we isolated RNA, amplified mRNA, profiled whole-genome exon splicing, and applied advanced bioinformatics. We employed rigorous quality control measures at all steps and validated findings by qPCR. In the motor neuron enriched mRNA pool, we found two distinct cohorts of mRNA signals, most of which were up-regulated: 148 differentially expressed genes (P ≤ 10^–3) and 411 aberrantly spliced genes (P ≤ 10^–5). The aberrantly spliced genes were highly enriched in cell adhesion (P ≤ 10^–57), especially cell–matrix as opposed to cell–cell adhesion. Most of the enriching genes encode transmembrane or secreted as opposed to nuclear or cytoplasmic proteins. The differentially expressed genes were not biologically enriched. In the anterior horn enriched mRNA pool, we could not clearly identify mRNA signals or biological enrichment. These findings, perturbed and up-regulated cell–matrix adhesion, suggest possible mechanisms for the contiguously progressive nature of motor neuron degeneration. Data deposition: GeneChip raw data (CEL-files) have been deposited for public access in the Gene Expression Omnibus (GEO), www.ncbi.nlm.nih.gov/geo, accession number GSE18920.

Life course variations in the associations between FTO and MC4R gene variants and body size

Thu, 12 Nov 2009 07:47:01 PST

The timing of associations between common genetic variants for weight or body mass index (BMI) across the life course may provide insights into the aetiology of obesity. We genotyped variants in FTO (rs9939609) and near MC4R (rs17782313) in 1240 men and 1239 women born in 1946 and participating in the MRC National Survey of Health and Development. Birth weight was recorded and height and weight were measured or self-reported repeatedly at 11 time-points between ages 2 and 53 years. Hierarchical mixed models were used to test whether genetic associations with weight or BMI standard deviation scores (SDS) changed with age during childhood and adolescence (2–20 years) or adulthood (20–53 years). The association between FTO rs9939609 and BMI SDS strengthened during childhood and adolescence (rate of change: 0.007 SDS/A-allele/year; 95% CI: 0.003–0.010, P < 0.001), reached a peak strength at age 20 years (0.13 SDS/A-allele, 0.08–0.19), and then weakened during adulthood (–0.003 SDS/A-allele/year, –0.005 to –0.001, P = 0.001). MC4R rs17782313 showed stronger associations with weight than BMI; its association with weight strengthened during childhood and adolescence (0.005 SDS/C-allele/year; 0.001–0.008, P = 0.006), peaked at age 20 years (0.13 SDS/C-allele, 0.07–0.18), and weakened during adulthood (–0.002 SDS/C-allele/year, –0.004 to 0.000, P = 0.05). In conclusion, genetic variants in FTO and MC4R showed similar biphasic changes in their associations with BMI and weight, respectively, strengthening during childhood up to age 20 years and then weakening with increasing adult age. Studies of the aetiology of obesity spanning different age groups may identify age-specific determinants of weight gain.

Mitochondrial DNA background modifies the bioenergetics of NARP/MILS ATP6 mutant cells

D'Aurelio, M., Vives-Bauza, C., Davidson, M.M., Manfredi, G. — Thu, 12 Nov 2009 07:47:00 PST

Mutations in the mitochondrial DNA (mtDNA) encoded subunit 6 of ATPase (ATP6) are associated with variable disease expression, ranging from adult onset neuropathy, ataxia and retinitis pigmentosa (NARP) to fatal childhood maternally inherited Leigh's syndrome (MILS). Phenotypical variations have largely been attributed to mtDNA heteroplasmy. However, there is often a discrepancy between the levels of mutant mtDNA and disease severity. Therefore, the correlation among genetic defect, bioenergetic impairment and clinical outcome in NARP/MILS remains to be elucidated. We investigated the bioenergetics of cybrids from five patients carrying different ATP6 mutations: three harboring the T8993G, one with the T8993C and one with the T9176G mutation. The bioenergetic defects varied dramatically, not only among different ATP6 mutants, but also among lines carrying the same T8993G mutation. Mutants with the most severe ATP synthesis impairment showed defective respiration and disassembly of respiratory chain complexes. This indicates that respiratory chain defects modulate the bioenergetic impairment in NARP/MILS cells. Sequencing of the entire mtDNA from the different mutant cell lines identified variations in structural genes, resulting in amino acid changes that destabilize the respiratory chain. Taken together, these results indicate that the mtDNA background plays an important role in modulating the biochemical defects and clinical outcome in NARP/MILS.

Pre-symptomatic development of lower motor neuron connectivity in a mouse model of severe spinal muscular atrophy

Wed, 11 Nov 2009 06:53:49 PST

The childhood motor neuron disease spinal muscular atrophy (SMA) results from reduced expression of the survival motor neuron (SMN) gene. Previous studies using in vitro model systems and lower organisms have suggested that low levels of Smn protein disrupt prenatal developmental processes in lower motor neurons, influencing neuronal outgrowth, axon branching and neuromuscular connectivity. The extent to which these developmental pathways contribute to selective vulnerability and pathology in the mammalian neuromuscular system in vivo remains unclear. Here, we have investigated the pre-symptomatic development of neuromuscular connectivity in differentially vulnerable motor neuron populations in Smn^–/–;SMN2 mice, a model of severe SMA. We show that reduced Smn levels have no detectable effect on morphological correlates of pre-symptomatic development in either vulnerable or stable motor units, indicating that abnormal pre-symptomatic developmental processes are unlikely to be a prerequisite for subsequent pathological changes to occur in vivo. Microarray analyses of spinal cord from two different severe SMA mouse models demonstrated that only minimal changes in gene expression were present in pre-symptomatic mice. In stark contrast, microarray analysis of late-symptomatic spinal cord revealed widespread changes in gene expression, implicating extracellular matrix integrity, growth factor signalling and myelination pathways in SMA pathogenesis. Taken together, these data suggest that reduced Smn levels induce SMA pathology by instigating rapidly progressive neurodegenerative pathways in lower motor neurons around the time of disease onset rather than by modulating pre-symptomatic neurodevelopmental pathways.

Significant contributions of the extraembryonic membranes and maternal genotype to the placental pathology in heterozygous Nsdhl deficient female embryos

Wed, 11 Nov 2009 06:53:47 PST

Mutations in the gene encoding the cholesterol biosynthetic enzyme NSDHL are associated with the X-linked male-lethal bare patches (Bpa) mouse. Mutant male embryos for several Nsdhl alleles die in midgestation with placental insufficiency. We examined here a possible role of the maternal genotype in such placental pathology. Pre-pregnancy plasma cholesterol levels were similar between wild-type (WT) and Bpa^1H/+ dams fed a standard, cholesterol-free diet. However, there was a marked decrease in cholesterol levels between embryonic day (E)8.5 and E10.5 for both genotypes. Further, there was a significant lag between E11.5 and E13.5 (P = 0.0011) in the recovery of levels in Bpa^1H/+ dams to their pre-pregnancy values. To investigate possible effects of the maternal genotype on fetal placentation, we generated transgenic mice that expressed human NSDHL and rescued the male lethality of the Bpa^1H null allele. We then compared placenta area at E10.5 in WT and Bpa^1H/+ female embryos where the mutant X chromosome was transmitted from a heterozygous mother or a rescued mutant father. In mutant conceptuses, placental areas were ~50% less than WT. Surprisingly, expression of Nsdhl in trophoblast lineages of the placenta and yolk sac endoderm, which occurs only from the maternally inherited allele in a female embryo, had the largest effect on placental area (–0.681 mm²; P < 0.0001). The maternal genotype had a smaller effect, independent of the fetal genotype (–0.283 mm²; P = 0.024). These data demonstrate significant effects of the mother and fetal membranes on pregnancy outcome, with possible implications for cholesterol homeostasis during human pregnancy.

Phosphorylation of parkin by Parkinson disease-linked kinase PINK1 activates parkin E3 ligase function and NF-{kappa}B signaling

Sha, D., Chin, L.-S., Li, L. — Wed, 11 Nov 2009 01:32:39 PST

Mutations in PTEN-induced putative kinase 1 (PINK1) or parkin cause autosomal recessive forms of Parkinson disease (PD), but how these mutations trigger neurodegeneration is poorly understood and the exact functional relationship between PINK1 and parkin remains unclear. Here, we report that PINK1 regulates the E3 ubiquitin-protein ligase function of parkin through direct phosphorylation. We find that phosphorylation of parkin by PINK1 activates parkin E3 ligase function for catalyzing K63-linked polyubiquitination and enhances parkin-mediated ubiquitin signaling through the IB kinase/nuclear factor B (NF-B) pathway. Furthermore, the ability of PINK1 to promote parkin phosphorylation and activate parkin-mediated ubiquitin signaling is impaired by PD-linked pathogenic PINK1 mutations. Our findings support a direct link between PINK1-mediated phosphorylation and parkin-mediated ubiquitin signaling and implicate the deregulation of the PINK1/parkin/NF-B neuroprotective signaling pathway in the pathogenesis of PD.

The nystagmus-associated FRMD7 gene regulates neuronal outgrowth and development

Wed, 11 Nov 2009 01:32:38 PST

Mutations in the gene encoding FERM domain-containing 7 protein (FRMD7) are recognized as an important cause of X-linked idiopathic infantile nystagmus (IIN). However, the precise role of FRMD7 and its involvement in the pathogenesis of IIN are not understood. In the present study, we have explored the role of FRMD7 in neuronal development. Using in situ hybridization and immunohistochemistry, we reveal that FRMD7 expression is spatially and temporally regulated in both the human and mouse brain during embryonic and fetal development. Furthermore, we show that FRMD7 expression is up-regulated upon retinoic acid (RA)-induced differentiation of mouse neuroblastoma NEURO2A cells, suggesting FRMD7 may play a role in this process. Indeed, we demonstrate, for the first time, that knockdown of FRMD7 during neuronal differentiation results in altered neurite development. Taken together, our data suggest that FRMD7 is involved in multiple aspects of neuronal development, and have direct importance to further understanding the pathogenesis of IIN.

Fibroblast phenotype in male carriers of FMR1 premutation alleles

Wed, 11 Nov 2009 06:53:46 PST

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder among carriers of premutation expansions (55–200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. The clinical features of FXTAS, as well as various forms of clinical involvement in carriers without FXTAS, are thought to arise through a direct toxic gain of function of high levels of FMR1 mRNA containing the expanded CGG repeat. Here we report a cellular endophenotype involving increased stress response (HSP27, HSP70 and CRYAB) and altered lamin A/C expression/organization in cultured skin fibroblasts from 11 male carriers of premutation alleles of the FMR1 gene, including six patients with FXTAS and five premutation carriers with no clinical evidence of FXTAS, compared with six controls. A similar abnormal cellular phenotype was found in CNS tissue from 10 patients with FXTAS. Finally, there is an analogous abnormal cellular distribution of lamin A/C isoforms in knock-in mice bearing the expanded CGG repeat in the murine Fmr1 gene. These alterations are evident even in mouse embryonic fibroblasts, raising the possibility that, in humans, the expanded-repeat mRNA triggers pathogenic mechanisms early in development, thus providing a molecular basis for the neurodevelopmental abnormalities observed in some children and clinical symptoms in some adults who are carriers of premutation FMR1 alleles. Cellular dysregulation in fibroblasts represents a novel and highly advantageous model for investigating disease pathogenesis in premutation carriers and for quantifying and monitoring disease progression. Fibroblast studies may also prove useful in screening and testing the efficacy of therapeutic interventions.

Frataxin interacts with Isu1 through a conserved tryptophan in its {beta}-sheet

Leidgens, S., De Smet, S., Foury, F. — Wed, 11 Nov 2009 01:32:37 PST

Friedreich's ataxia is a neurodegenerative disease caused by the low expression of frataxin, a mitochondrial iron-binding protein which plays an important, but non-essential, role in the formation of iron–sulfur (Fe/S) clusters. It has been shown that Yfh1, the yeast frataxin homologue, interacts functionally and physically with Isu1, the scaffold protein on which the Fe/S clusters are assembled. The large β-sheet platform of frataxin is a good ligand candidate for this interaction. We have generated 12 yeast mutants in conserved residues of the β-sheet protruding at the surface or buried in the protein core. The Q129A, I130A, W131A(F) and R141A mutations, which reside in surface exposed residues of the fourth and fifth β-strands, result in severe cell growth inhibition on high-iron media and low aconitase activity, indicating that Fe/S cluster biosynthesis is impaired. The null phenotype of the I130A mutant results from the high instability of the protein, pointing that this buried residue is essential for folding. In contrast, Gln-129, Trp-131 and Arg-141 residues which are spatially closely clustered define a patch important for protein function. Co-immunoprecipitation experiments using cell extracts show that W131A, unlike W131F, is the sole mutation that strongly decreases the interaction with Isu1. Therefore, Trp-131, which is the only strictly conserved frataxin residue in all sequenced species, appears as a major contributor to the interaction with Isu1 through its surface-exposed aromatic side chain.

Consortin, a trans-Golgi network cargo receptor for the plasma membrane targeting and recycling of connexins

Wed, 11 Nov 2009 01:32:36 PST

Targeting of numerous transmembrane proteins to the cell surface is thought to depend on their recognition by cargo receptors that interact with the adaptor machinery for anterograde traffic at the distal end of the Golgi complex. We report here on consortin, a novel integral membrane protein that is predicted to be intrinsically disordered, i.e. that contains large segments whose native state is unstructured. We identified consortin as a binding partner of connexins, the building blocks of gap junctions. Consortin is located at the trans-Golgi network (TGN), in tubulovesicular transport organelles, and at the plasma membrane. It directly interacts with the TGN clathrin adaptors GGA1 and GGA2, and disruption of this interaction by expression of a consortin mutant lacking the acidic cluster–dileucine (DXXLL) GGA interaction motif causes an intracellular accumulation of several connexins. RNA interference-mediated silencing of consortin expression in HeLa cells blocks the cell surface targeting of these connexins, which accumulate intracellularly, whereas partial depletion and redistribution of the consortin pool slows down the intracellular degradation of gap junction plaques. Altogether, our results show that, by studying connexin trafficking, we have identified the first TGN cargo receptor for the targeting of transmembrane proteins to the plasma membrane. The identification of consortin provides in addition a potential target for therapies aimed at diseases in which connexin traffic is altered, including cardiac ischemia, peripheral neuropathies, cataracts and hearing impairment.

Sequence accession numbers. GenBank: Human CNST cDNA, NM_152609; mouse Cnst cDNA, NM_146105.

Nesprin 1 is critical for nuclear positioning and anchorage

Mon, 09 Nov 2009 08:01:24 PST

Nesprin 1 is an outer nuclear membrane protein that is thought to link the nucleus to the actin cytoskeleton. Recent data suggest that mutations in Nesprin 1 may also be involved in the pathogenesis of Emery-Dreifuss muscular dystrophy. To investigate the function of Nesprin 1 in vivo, we generated a mouse model in which all isoforms of Nesprin 1 containing the C-terminal spectrin-repeat region with or without KASH domain were ablated. Nesprin 1 knockout mice are marked by decreased survival rates, growth retardation and increased variability in body weight. Additionally, nuclear positioning and anchorage are dysfunctional in skeletal muscle from knockout mice. Physiological testing demonstrated no significant reduction in stress production in Nesprin 1-deficient skeletal muscle in either neonatal or adult mice, but a significantly lower exercise capacity in knockout mice. Nuclear deformation testing revealed ineffective strain transmission to nuclei in muscle fibers lacking Nesprin 1. Overall, our data show that Nesprin 1 is essential for normal positioning and anchorage of nuclei in skeletal muscle.

Mutation of the bone morphogenetic protein GDF3 causes ocular and skeletal anomalies

Mon, 09 Nov 2009 08:01:23 PST

Ocular mal-development results in heterogeneous and frequently visually disabling phenotypes that include coloboma and microphthalmia. Due to the contribution of bone morphogenetic proteins to such processes, the function of the paralogue Growth Differentiation Factor 3 was investigated. Multiple mis-sense variants were identified in patients with ocular and/or skeletal (Klippel–Feil) anomalies including one individual with heterozygous alterations in GDF3 and GDF6. These variants were characterized, individually and in combination, through integrated biochemical and zebrafish model organism analyses, demonstrating appreciable effects with western blot analyses, luciferase based reporter assays and antisense morpholino inhibition. Notably, inhibition of the zebrafish co-orthologue of GDF3 accurately recapitulates patient phenotypes. By demonstrating the pleiotropic effects of GDF3 mutation, these results extend the contribution of perturbed BMP signaling to human disease and potentially implicate multi-allelic inheritance of BMP variants in developmental disorders.

Effects of 2,4-diaminoquinazoline derivatives on SMN expression and phenotype in a mouse model for spinal muscular atrophy

Fri, 06 Nov 2009 07:02:56 PST

Proximal spinal muscular atrophy (SMA), one of the most common genetic causes of infant death, results from the selective loss of motor neurons in the spinal cord. SMA is a consequence of low levels of SMN protein. In humans, the SMN gene is duplicated; SMA results from the loss of SMN1 but SMN2 remains intact. SMA severity is related to the copy number of SMN2. Compounds which increase the expression of SMN2 could, therefore, be potential therapeutics for SMA. Ultrahigh-throughput screening recently identified substituted quinazolines as potent SMN2 inducers. A series of C5-quinazoline derivatives were tested for their ability to increase SMN expression in vivo. Oral administration of three compounds (D152344, D153249 and D156844) to neonatal mice resulted in a dose-dependent increase in Smn promoter activity in the central nervous system. We then examined the effect of these compounds on the progression of disease in SMN7 SMA mice. Oral administration of D156844 significantly increased the mean lifespan of SMN7 SMA mice by ~21-30% when given prior to motor neuron loss. In summary, the C5-quinazoline derivative D156844 increases SMN expression in neonatal mouse neural tissues, delays motor neuron loss at PND11 and ameliorates the motor phenotype of SMN7 SMA mice.

Overexpression of Hr links excessive induction of Wnt signaling to Marie Unna Hereditary Hypotrichosis

Fri, 06 Nov 2009 07:02:55 PST

Marie Unna hereditary hypotrichosis (MUHH) is a rare autosomal dominant hair disorder. Through the study of a mouse model, we identified a mutation in the 5'-untranslated region of the hairless (HR) gene in patients with MUHH in a Caucasian family. The corresponding mutation, named ‘hairpoor’, was found in mutant mice that were generated through N-ethyl-N-nitrosourea mutagenesis. Hairpoor mouse mutants display partial hair loss at an early age and progress to near alopecia, which resembles the MUHH phenotype. This mutation conferred overexpression of HR through translational derepression and, in turn, decreased the expression of Sfrp2, an inhibitor of the Wnt signaling pathway. This study indicates that the gain in function of HR also results in alopecia, as seen with the loss of function of HR, via abnormal upregulation of the Wnt signaling pathway.

A Genome-wide Association Study Identifies GLT6D1 as a Susceptibility Locus for Periodontitis

Fri, 06 Nov 2009 07:02:54 PST

Periodontitis is a widespread, complex inflammatory disease of the mouth, which results in a loss of gingival tissue and alveolar bone, with aggressive periodontitis (AgP) as its most severe form. To identify genetic risk factors for periodontitis, we conducted a genome-wide association study (GWAS) in German AgP patients. We found AgP to be strongly associated with the intronic SNP rs1537415, which is located in the glycosyltransferase gene GLT6D1. We replicated the association in a panel of Dutch generalized and localized AgP patients. In the combined analysis including 1,758 subjects, rs1537415 reached a genome-wide significance level of P = 5.51 x 10^–9, OR=1.59 (95% CI 1.36-1.86). The associated rare G allele of rs1537415 showed an enrichment of 10% in periodontitis cases (48.4% in comparison with 38.8% in controls). Fine-mapping and a haplotype analysis indicated that rs1537415 showed the strongest association signal. Sequencing identified no further associated variant. Tissue specific expression analysis of GLT6D1 indicated high transcript levels in the leukocytes, the gingiva and testis. Analysis of potential transcription factor binding sites at this locus predicted a significant reduction of GATA-3 binding affinity, and an electrophoretic mobility assay indicated a T cell specific reduction of protein binding for the G allele. Overexpression of GATA-3 in HEK293 cells resulted in allele specific binding of GATA-3, indicating the identity of GATA-3 as the binding protein. The identified association of GLT6D1 with AgP implicates this locus as an important susceptibility factor, and GATA-3 as a potential signaling component in the pathophysiology of periodontitis.

Replication of the 5 Novel Loci for Uric Acid Concentrations and Potential Mediating Mechanisms

Tue, 27 Oct 2009 08:51:23 PDT

Uric acid is the final catabolic product of purine metabolism and elevated levels are associated with diabetes and cardiovascular disease. A recent meta-analysis of genome-wide association studies totalling 28,141 participants identified 5 novel loci associated with serum uric acid levels. In our population based cohort of 7,795 subjects we replicated 4 of these 5 loci; PDZK1 (rs12129861 P=1.07x10^–3), GCKR (rs780094 P=4.83x10^–4), SLC16A9 (rs742132 P=0.047), and SLC22A11 (rs17300741 P=6.13x10^–3), but not LRRC16A (rs742132 P=0.645). Serum uric acid concentration is a complex trait, closely associated to renal uric acid handling (fractional uric acid excretion; P< 1 x 10^–300), renal function (serum creatinine; P< 1 x 10^–300), and the metabolic syndrome (including fasting insulin; P=2.48x10^–232, insulin resistance; P=2.51x10^–258, waist circumference; P< 1 x 10^–300), and systolic blood pressure (P=1.93 x 10^–219). Together these factors explain 67% of the variance in uric acid levels. Therefore, we sought to determine the potential contribution of these factors to the association of these novel loci with uric acid levels, by including them as additional explanatory variables in our analyses, and by considering them as alternative response variables. The association with the GCKR locus is attenuated by serum triglycerides and fractional uric acid excretion. We also observed the GCKR locus to be associated with total cholesterol (P= 7.52x10^–6), triglycerides (P=2.65x10^–9), fasting glucose (P=0.011), fractional uric acid excretion (P=3.36 x10^–5), and high-sensitive CRP (P=1.18x10^–3) also after adjusting for serum UA levels. We argue that GCKR locus affects serum UA levels through a factor that also affects triglycerides.

Prolyl 3-Hydroxylase 1 and CRTAP are Mutually Stabilizing in the Endoplasmic Reticulum Collagen Prolyl 3-Hydroxylation Complex

Chang, W., Barnes, A. M., Cabral, W. A., Bodurtha, J. N., Marini, J. C. — Wed, 21 Oct 2009 04:53:13 PDT

Null mutations in cartilage-associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1/LEPRE1) cause Types VII and VIII OI, respectively, two novel recessive forms of osteogenesis imperfecta with severe to lethal bone dysplasia and overmodification of the type I collagen helical region. CRTAP and P3H1 form a complex with cyclophilin B in the ER which 3-hydroxylates the Pro986 residue of 1(I) and 1(II) collagen chains. We investigated the interaction of complex components in fibroblasts from types VII and VIII OI patients. Both CRTAP and P3H1 are absent or reduced on Western blots and by immunofluorescence microscopy in cells containing null mutations in either gene. Levels of LEPRE1 or CRTAP transcripts, however, are normal in CRTAP- or LEPRE1-null cells, respectively. Stable transfection of a CRTAP or LEPRE1 expression construct into cells with null mutations for the transfected cDNA restored both CRTAP and P3H1 protein levels. Normalization of collagen helical modification in transfected CRTAP-null cells demonstrated that the restored proteins functioned effectively as a complex. These data indicate that CRTAP and P3H1 are mutually stabilized in the collagen prolyl 3-hydroxylation complex. Cyclophilin B levels were unaffected by mutations in either CRTAP or LEPRE1. Proteasomal inhibitors partially rescue P3H1 protein in CRTAP-null cells. In LEPRE1-null cells, secretion of CRTAP is increased compared to control cells and accounts for 15-20% of the decreased CRTAP detected in cells. Thus, mutual stabilization of P3H1 and CRTAP in the ER collagen modification complex is an underlying mechanism for the overlapping phenotype of type VII and VIII OI.

Murine Hippocampal Neurons Expressing Fmr1 gene Premutations Show Early Developmental Deficits and Late Degeneration

Wed, 21 Oct 2009 01:21:16 PDT

Premutation CGG repeat expansions (155-200 CGG repeats; preCGG) within the fragile X mental retardation 1 (FMR1) gene give rise to the neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS), primary ovarian insufficiency (POI) and neurodevelopmental problems. Morphometric analysis of Map2B immunofluorescence reveals that neurons cultured from heterozygous female mice with preCGG repeats in defined medium display shorter dendritic lengths and fewer branches between 7 and 21 days in vitro (DIV) compared to wild type (WT) littermates. Although the numbers of synapsin and phalloidin puncta do not differ from WT, preCGG neurons possess larger puncta. PreCGG neurons display lower viability, and express elevated stress protein as they mature. PreCGG neurons have inherently different patterns of growth, dendritic complexity, and synaptic architecture discernable early in the neuronal trajectory to maturation, and may reflect a cellular basis for the developmental component of the spectrum of clinical involvement in carriers of premutation alleles. The reduced viability of preCGG neurons is consistent with the mRNA toxicity and neurodegeneration associated with FXTAS.

THE HOMEOBOX GENE CHX10/VSX2 REGULATES RDCVF PROMOTER ACTIVITY IN THE INNER RETINA

Tue, 20 Oct 2009 01:21:02 PDT

Rod-derived Cone Viability Factor (RdCVF) is a trophic factor with therapeutic potential for the treatment of retinitis pigmentosa, a retinal disease that commonly results in blindness. RdCVF is encoded by Nucleoredoxin-like 1 (Nxnl1), a gene homologous with the family of thioredoxins that participate in the defense against oxidative stress. RdCVF expression is lost after rod degeneration in the first phase of retinitis pigmentosa, and this loss has been implicated in the more clinically significant secondary cone degeneration that often occurs. Here we describe a study of the Nxnl1 promoter using an approach that combines promoter and transcriptomic analysis. By transfection of selected candidate transcription factors, chosen based upon their expression pattern, we identified the homeodomain proteins CHX10/VSX2, VSX1, and PAX4, as well as the zinc finger protein SP3, as factors that can stimulate both the mouse and human Nxnl1 promoter. In addition, CHX10/VSX2 binds to the Nxnl1 promoter in vivo. Since CHX10/VSX2 is expressed predominantly in the inner retina, this finding motivated us to demonstrate that RdCVF is expressed in the inner as well as the outer retina. Interestingly, the loss of rods in the rd1 mouse, a model of retinitis pigmentosa, is associated with decreased expression of RdCVF by inner retinal cells as well as by rods. Based upon these results, we propose an alternative therapeutic strategy aimed at recapitulating RdCVF expression in the inner retina, where cell loss is not significant, to prevent secondary cone death and central vision loss in patients suffering from retinitis pigmentosa.

Tsc/mTORC1 signaling in oocytes governs the quiescence and activation of primordial follicles

Tue, 20 Oct 2009 01:21:00 PDT

To maintain the female reproductive lifespan, the majority of ovarian primordial follicles are preserved in a quiescent state in order to provide ova for later reproductive life. However, the molecular mechanism that maintains the long quiescence of primordial follicles is poorly understood. Here we provide genetic evidence to show that the tumor suppressor Tsc1 (tuberous sclerosis complex 1), which negatively regulates mTORC1 (mammalian target of rapamycin complex 1), functions in oocytes to maintain the quiescence of primordial follicles. In mutant mice lacking the Tsc1 gene in oocytes, the entire pool of primordial follicles is activated prematurely due to elevated mTORC1 activity in the oocyte, ending up with follicular depletion in early adulthood and causing premature ovarian failure (POF). We further show that maintenance of the quiescence of primordial follicles requires synergistic, collaborative functioning of both Tsc and PTEN (phosphatase and tensin homolog deleted on chromosome ten), and that these two molecules suppress follicular activation through distinct ways. Our results suggest that Tsc/mTORC1 signaling and PTEN/PI3K (phosphatidylinositol 3 kinase) signaling synergistically regulate the dormancy and activation of primordial follicles, and together ensure the proper length of female reproductive life. Deregulation of these signaling pathways in oocytes results in pathological conditions of the ovary, including POF and infertility.

Proteotoxic stress increases nuclear localization of ataxin-3

Reina, C. P., Zhong, X., Pittman, R. N. — Mon, 19 Oct 2009 23:26:14 PDT

Spinocerebellar ataxia type 3 (SCA3)/ Machado Joseph disease (MJD) results from expansion of the polyglutamine domain in ataxin-3 (Atx3). Atx3 is a transcriptional co-repressor, as well as a deubiquitinating enzyme that appears to function in cellular pathways involved in protein homeostasis. In this study we show that interactions of Atx3 with VCP and hHR23B are dynamic and modulated by proteotoxic stresses. Heat shock, a general proteotoxic stress, also induced wild-type and pathogenic Atx3 to accumulate in the nucleus. Mapping studies showed that two regions of Atx3, the Josephin domain and the C-terminus, regulated heat shock-induced nuclear localization. Heat shock-induced nuclear localization of Atx3 was not affected by a casein kinase-2 inhibitor or by mutating a predicted nuclear localization signal. However, serine-111 of Atx3 was required for nuclear localization of the Josephin domain and regulated nuclear localization of full-length Atx3. Atx3 null cells were more sensitive to toxic effects of heat shock suggesting that Atx3 had a protective function in the cellular response to heat shock. Importantly, we found that oxidative stress also induced nuclear localization of Atx3; both wild-type and pathogenic Atx3 accumulated in the nucleus of SCA3 patient fibroblasts following oxidative stress. Heat shock and oxidative stress are the first processes identified that increase nuclear localization of Atx3. Observations in this study provide new and important insights for understanding SCA3 pathology as the nucleus is likely a key site for early pathogenesis.

PHF8, a gene associated with cleft lip/palate and mental retardation, encodes for an N{varepsilon}-dimethyl lysine demethylase

Mon, 19 Oct 2009 23:26:14 PDT

Mutations of human PHF8 cluster within its JmjC encoding exons and are linked to mental retardation (MR) and a cleft lip/palate phenotype. Sequence comparisons, employing structural insights, suggest that PHF8 contains the double stranded β-helix fold and ferrous iron binding residues that are present in 2-oxoglutarate dependent oxygenases. We report that recombinant PHF8 is an Fe(II) and 2-oxoglutarate dependent N-methyl lysine demethylase, which acts on histone substrates. PHF8 is selective in vitro for N-di- and mono-methylated lysine residues and does not accept trimethyl substrates. Clinically observed mutations to the PHF8 gene cluster in exons encoding for the double stranded β-helix fold and will therefore disrupt catalytic activity. The PHF8 missense mutation c.836C>T is associated with mild MR, mild dysmorphic features, and either unilateral or bilateral cleft lip and cleft palate in two male siblings. In the c.836C>T encoded F279S variant of PHF8, a conserved hydrophobic pocket is modified; assays with both peptides and intact histones reveal this variant to be catalytically inactive. The dependence of PHF8 activity on oxygen availability is interesting because the occurrence of fetal cleft lip has been demonstrated to increase with maternal hypoxia in mice studies. Cleft lip and other congenital anomalies are also linked indirectly to maternal hypoxia in humans, including from maternal smoking and maternal hyptertension treatment. Our results will enable further studies aimed at defining the molecular links between developmental changes in histone methylation status, congenital disorders, and mental retardation.

SUMOylation attenuates the aggregation propensity and cellular toxicity of the polyglutamine expanded ataxin-7

Mon, 19 Oct 2009 23:26:12 PDT

Post-translational modification by SUMO (small ubiquitin-like modifier) was proposed to modulate the pathogenesis of several neurodegenerative diseases. Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder, whose pathology is caused by an expansion of a polyglutamine stretch in the protein ataxin-7 (ATXN7). Here, we identified ATXN7 as new target for SUMOylation in vitro and in vivo. The major SUMO acceptor site was mapped to lysine 257, which is part of an evolutionarily conserved consensus SUMOylation motif. SUMOylation did not influence the subcellular localization of ATXN7 nor its interaction with components of the TFTC/STAGA complex. Expansion of the polyglutamine stretch did not impair the SUMOylation of ATXN7. Furthermore, SUMO1 and SUMO2 colocalized with ATXN7 in a subset of neuronal intranuclear inclusions in the brain of SCA7 patients and SCA7 knock-in mice. In a COS-7 cellular model of SCA7, in addition to diffuse nucleoplasmic staining we identified two populations of nuclear inclusions: homogenous or non homogenous. Non homogenous inclusions showed significantly reduced colocalization with SUMO1 and SUMO2, but were highly enriched in Hsp70, 19S proteasome and ubiquitin. Interestingly, they were characterized by increased staining with the apoptotic marker caspase-3 and by disruption of PML nuclear bodies. Importantly, preventing the SUMOylation of expanded ATXN7 by mutating the SUMO site increased both the amount of SDS-insoluble aggregates and of caspase-3 positive non homogenous inclusions, which act toxic to the cells. Our results demonstrate an influence of SUMOylation on the multistep aggregation process of ATXN7 and implicate a role for ATXN7 SUMOylation in SCA7 pathogenesis.

Analysis of mouse models of cytochrome c oxidase deficiency due to mutations in Sco2

Fri, 16 Oct 2009 01:43:51 PDT

Mutations in SCO2, a protein required for the proper assembly and functioning of cytochrome c oxidase (COX; complex IV of the mitochondrial respiratory chain), cause a fatal infantile cardioencephalomyopathy with COX deficiency. We have generated mice harboring a Sco2 knock-out (KO) allele and a Sco2 knock-in (KI) allele expressing an E->K mutation at position 129 (E129K), corresponding to the E140K mutation found in almost all human SCO2-mutated patients. Whereas homozygous KO mice were embryonic lethals, homozygous KI and compound heterozygous KI/KO mice were viable, but had muscle weakness; biochemically, they had respiratory chain deficiencies as well as complex IV assembly defects in multiple tissues. There was a concomitant reduction in mitochondrial copper content, but the total amount of copper in examined tissues was not reduced. These mouse models should be of use in further studies of Sco2 function, as well as in testing therapeutic approaches to treat the human disorder.

The effect of food intake on gene expression in human peripheral blood

Fri, 16 Oct 2009 01:43:50 PDT

Human gene expression traits have been shown to be dependent on gender, age, and time of day in blood and other tissues. However, other factors that may impact gene expression have not been systematically explored. For example, in studies linking blood gene expression to obesity related traits, whether the fasted or fed state will be the most informative is an open question. Here we employed a two-arm cross-over design to perform a genome-wide survey of gene expression in human peripheral blood to address explicitly this type of question. We were able to distinguish expression changes due to individual and time-specific effects from those due to food intake. We demonstrate that the transcriptional response to food intake is robust by constructing a classifier from the gene expression traits with >90% accuracy classifying individuals as being in the fasted or fed state. Gene expression traits that were best able to discriminate the fasted and fed states were more heritable and achieved greater coherence with respect to pathways associated with metabolic traits. The connectivity structure among gene expression traits was explored in the context of coexpression networks. Changes in the connectivity structure were observed between the fasted and fed states. We demonstrate that differential expression and differential connectivity are two complementary ways to characterize changes between fasted and fed states. Both gene sets were significantly enriched for genes associated with obesity related traits. Our results suggest that the pair of fasted/fed blood expression profiles provide more comprehensive information about an individual's metabolic states.

EFFICIENT INTRACEREBRAL DELIVERY OF AAV5 VECTOR ENCODING HUMAN ARSA IN NON-HUMAN PRIMATE

Fri, 16 Oct 2009 01:43:49 PDT

Metachromatic leukodystrophy is a lethal neurodegenerative disease caused by a deficiency in the lysosomal arylsulfatase A (ARSA) enzyme leading to the accumulation of sulfatides in glial and neuronal cells. We previously demonstrated in ARSA deficient mice that intracerebral injection of a serotype 5 adeno-associated viral vector (AAV) encoding human ARSA corrects the biochemical, neuropathological and behavioral abnormalities. However, before considering a potential clinical application, scaling-up issues should be addressed in large animals. Therefore, we performed intracerebral injection of the same AAV vector (total dose of 3.8x10¹¹ or 1.9x10¹² vector genome, 3 sites of injection in the right hemisphere, 2 deposits per site of injection) into three selected areas of the centrum semiovale white matter, or in the deep grey matter nuclei (caudate nucleus, putamen, thalamus) of 6 non-human primates to evaluate vector distribution, as well as expression and activity of human ARSA. The procedure was perfectly tolerated, without any adverse effect or change in neurobehavioral examination. AAV vector was detected in a brain volume of 12-15 cm³ that corresponded to 37-46% of the injected hemisphere. ARSA enzyme was expressed in multiple interconnected brain areas over a distance of 22-33 mm. ARSA activity was increased by 12-38% in a brain volume that corresponded to 50-65% of injected hemisphere. These data provide substantial evidence for potential benefits of brain gene therapy in patients with metachromatic leukodystrophy.

MDA5 and PTPN2, two candidate genes for type 1 diabetes, modify pancreatic {beta}-cell responses to the viral by-product double stranded RNA

Colli, M. L., Moore, F., Gurzov, E. N., Ortis, F., Eizirik, D. L. — Tue, 13 Oct 2009 04:14:14 PDT

β-cell destruction in type 1 diabetes (T1D) is at least in part consequence of a "dialog" between β-cells and immune system. This dialog may be affected by the individual's genetic background. We presently evaluated whether modulation of MDA5 and PTPN2, two candidate genes for T1D, affects β-cell responses to double-stranded RNA (dsRNA), a by-product of viral replication. These genes were selected following comparison between known candidate genes for T1D and genes expressed in pancreatic β-cells, as identified in previous array analysis. INS-1E cells and primary FACS-purified rat β-cells were transfected with siRNAs targeting MDA5 or PTPN2, and subsequently exposed to intracellular synthetic dsRNA (PIC). Real time RT-PCR, Western blot and viability assays were performed to characterize gene/protein expression and viability. PIC increased MDA5 and PTPN2 mRNA expression, which was inhibited by the specific siRNAs. PIC triggered apoptosis in INS-1E and primary β-cells and this was augmented by PTPN2 knockdown, while inhibition of MDA5 did not modify PIC-induced apoptosis. In contrast, MDA5 silencing decreased PIC-induced cytokine and chemokine expression, while inhibition of PTPN2 induced minor or no changes in these inflammatory mediators. These findings indicate that changes in MDA5 and PTPN2 expression modify β-cell responses to dsRNA. While MDA5 regulates inflammatory signals, PTPN2 may function as a defence mechanism against pro-apoptotic signals generated by dsRNA. These two candidate genes for T1D may thus modulate β-cell apoptosis and/or local release of inflammatory mediators in the course of a viral infection by acting, at least in part, at the pancreatic β-cell level.

Genome-wide analysis of allelic expression imbalance in human primary cells by high throughput transcriptome resequencing

Tue, 13 Oct 2009 01:33:50 PDT

Many disease associated variants identified by genome-wide association (GWA) studies are expected to regulate gene expression. Allele specific expression (ASE) quantifies transcription from both haplotypes using individuals heterozygous at tested SNPs. We performed deep human transcriptome-wide resequencing (RNA-seq) for ASE analysis and expression quantitative trait locus (eQTL) discovery. We resequenced double poly(A) selected RNA from primary CD4⁺ T-cells (n=4 individuals, both activated and untreated conditions) and developed tools for paired end RNA-seq alignment and ASE analysis. We generated an average of 20 million uniquely mapping 45 base reads per sample. We obtained sufficient read depth to test 1,371 unique transcripts for ASE. Multiple biases inflate the false discovery rate which we estimate to be approximately 50% for random SNPs. However, after controlling for these biases and considering the subset of SNPs that pass HapMap QC, 4.6% of heterozygous SNP-sample pairs show evidence of imbalance (p<0.001). We validated four findings by both bacterial cloning and Sanger sequencing assays. We also found convincing evidence for allelic imbalance at multiple reporter exonic SNPs in CD6 for two samples heterozygous at the multiple sclerosis associated variant rs17824933, linking GWA findings with variation in gene expression. Finally, we show in CD4⁺ T-cells from a further individual that high throughput sequencing of genomic DNA and RNA-seq following enrichment for targeted gene sequences by sequence capture methods offers an unbiased means to increase the read depth for transcripts of interest, and therefore a method to investigate the regulatory role of many disease associated genetic variants.

Genetic control of the alternative pathway of complement in humans and age-related macular degeneration

Tue, 13 Oct 2009 01:33:50 PDT

Activation of the alternative pathway of complement is implicated in common neurodegenerative diseases including age-related macular degeneration (AMD). We explored the impact of common variation in genes encoding proteins of the alternative pathway on complement activation in human blood and in AMD. Genetic variation across the genes encoding complement factor H (CFH), factor B (CFB), and component 3 (C3) was determined. The influence of common haplotypes defining transcriptional and translational units on complement activation in blood was determined in a quantitative genomic association study. Individual haplotypes in CFH and CFB were associated with distinct and novel effects on plasma levels of precursors, regulators, and activation products of the alternative pathway of complement in human blood. Further, genetic variation in CFH thought to influence cell surface regulation of complement did not alter plasma complement levels in human blood. Plasma markers of chronic activation (split-products Ba and C3d) and an activating enzyme (factor D) were elevated in AMD subjects. Most of the elevation in AMD was accounted for by the genetic variation controlling complement activation in human blood. Activation of the alternative pathway of complement in blood is under genetic control and increases with age. The genetic variation associated with increased activation of complement in human blood also increased the risk of AMD. Our data are consistent with a disease model in which genetic variation in the complement system increases AMD risk by a combination of systemic complement activation and abnormal regulation of complement activation in local tissues.

Mutations in NOTCH3 cause the formation and retention of aggregates in the endoplasmic reticulum, leading to impaired cell proliferation

Tue, 13 Oct 2009 00:33:54 PDT

Mutations in the human NOTCH3 gene cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), but the pathogenic mechanisms of the disorder remain unclear. We investigated the cytotoxic properties of mutant Notch3 using stable cell lines with inducible expression of either wild-type or two mutants p.R133C and p.C185R. We found that both mutants of Notch3 were prone to aggregation and retained in the endoplasmic reticulum (ER). The turnover rates of the mutated Notch3 proteins were strikingly slow, with half-lives greater than 6 days, whereas wild-type Notch3 was rapidly degraded, with a half-life of 0.7 days. The expression of mutant Notch3 also impaired cell proliferation compared with wild-type Notch3. In addition, cell lines expressing mutant Notch3 were more sensitive to proteasome inhibition resulting in cell death. These findings suggest that prolonged retention of mutant Notch3 aggregates in the ER decreases cell growth and increases sensitivity to other stresses. It is also possible that the aggregate-prone property of mutant Notch3 contributes to a pathogenic mechanism underlying CADASIL.

Identical oligomeric and fibrillar structures captured from the brains of R6/2 and knock-in mouse models of Huntington's disease

Mon, 12 Oct 2009 22:54:43 PDT

Huntington's disease (HD) is a late onset neurodegenerative disorder that is characterised neuropathologically by the presence of neuropil aggregates and nuclear inclusions. However, the profile of aggregate structures that are present in the brains of HD patients or of HD mouse models and the relative contribution of specific aggregate structures to disease pathogenesis is unknown. We have used the Seprion ligand to develop a highly sensitive ELISA-based method for quantifying aggregated polyglutamine in tissues from HD mouse models. We used a combination of electron microscopy, atomic force microscopy and SDS-PAGE to investigate the aggregate structures isolated by the ligand. We found that the oligomeric, proto-fibrillar and fibrillar aggregates extracted from the brains of R6/2 and HdhQ150 knock-in mice were remarkably similar. Using atomic force microscopy, we determined that the nanometre globular oligomers isolated from the brains of both mouse models have dimensions identical to those generated from recombinant huntingtin exon 1 proteins. Finally, antibodies that detect exon 1 Htt epitopes differentially recognise the ligand-captured material on SDS-PAGE gels. The Seprion-ligand ELISA provides an assay with good statistical power for use in preclinical pharmacodynamic therapeutic trials or to assess the effects of the genetic manipulation of potential therapeutic targets on aggregate load. This, together with the ability to identify a spectrum of aggregate species in HD mouse tissues, will contribute to our understanding of how these structures relate to the pathogenesis of HD and whether their formation can be manipulated for therapeutic benefit.

The asthma-associated ORMDL3 gene product regulates endoplasmic reticulum-mediated calcium signaling and cellular stress

Thu, 08 Oct 2009 22:43:49 PDT

Alterations of protein folding or Ca²⁺ levels within the endoplasmic reticulum (ER) result in the unfolded-protein response (UPR), a process considered as an endogenous inducer of inflammation. Thereby, understanding how genetic factors modify UPR is particularly relevant in chronic inflammatory diseases such as asthma. Here we identified that ORMDL3, the only genetic risk factor recently associated to asthma in a genome wide study, alters ER-mediated Ca²⁺ homeostasis and facilitates the UPR. Heterologous expression of human ER-resident transmembrane ORMDL3 protein increased resting cytosolic Ca²⁺ levels and reduced ER-mediated Ca²⁺ signaling, an effect reverted by co-expression with the sarco-endoplasmic reticulum Ca²⁺ pump (SERCA). Increased ORMDL3 expression also promoted stronger activation of UPR transducing molecules and target genes while siRNA-mediated knock-down of endogenous ORMDL3 potentiated ER Ca²⁺ release and attenuated the UPR. In conclusion, our findings are consistent with a model in which ORMDL3 binds and inhibits SERCA resulting in a reduced ER Ca²⁺ concentration and increased UPR. Thus, we provide a first insight into the molecular mechanism explaining the association of ORMDL3 with proinflammatory diseases.

Impaired Replication Dynamics at the FRA3B Common Fragile Site

Thu, 08 Oct 2009 07:04:40 PDT

Chromosomal common fragile sites (CFSs) are genetically unstable regions of the genome that are induced by conditions that impair DNA replication. In this report, we show that treatment with the DNA polymerase inhibitor, aphidicolin (APH), slows the replication rate throughout S phase. To investigate the unusual sensitivity of CFSs to APH-induced replication stress, we examined replication dynamics within a 50 kb region of the most frequently-expressed CFS, FRA3B. We mapped four origins of replication, ori 1, 2, 3, and 4, using two independent methods. In untreated cells, we detected significantly less newly-replicated DNA at FRA3B ori 1-3, as compared to three control origins located within non-fragile regions (NCFSs). In APH-treated cells, all FRA3B and control origins tested were active; however, there was a significant increase of nascent strand DNA at the control origins and, to a lesser extent, at the FRA3B ori 1-3. Based on these observations and the theoretical modeling of the nascent strand abundance assay developed in this study, we hypothesize that CFS origins may be less efficient, and that APH treatment slows replication fork movement near these origins to a greater extent, resulting in impaired DNA replication and, ultimately, leading to the genetic instability characteristic of CFSs.

Human retinopathy-associated ciliary protein Retinitis Pigmentosa GTPase Regulator (RPGR) regulates cilia-dependent vertebrate development

Thu, 08 Oct 2009 07:04:39 PDT

Dysfunction of primary cilia is associated with tissue-specific or syndromic disorders. RPGR is a ciliary protein, mutations in which can lead to retinitis pigmentosa (RP), cone-rod degeneration, respiratory infections, and hearing disorders. Though RPGR is implicated in ciliary transport, the pathogenicity of RPGR mutations and the mechanism of underlying phenotypic heterogeneity are still unclear. Here we have utilized genetic rescue studies in zebrafish to elucidate the effect of human disease-associated mutations on its function. We show that rpgr is expressed predominantly in the retina, brain, and gut of zebrafish. In the retina, RPGR primarily localizes to the sensory cilium of photoreceptors. Antisense morpholino (MO)-mediated knockdown of rpgr function in zebrafish results in reduced length of Kupffer's vesicle (KV) cilia and is associated with ciliary anomalies including shortened body-axis, kinked tail, hydrocephaly, and edema but does not affect retinal development. These phenotypes can be rescued by wild type (WT) human RPGR. Several of the RPGR mutants can also reverse the MO-induced phenotype, suggesting their potential hypomorphic function. Notably, selected RPGR mutations observed in XLRP (T99N, E589X) or syndromic RP (T124fs, K190fs, and L280fs) do not completely rescue the rpgr-MO phenotype, indicating a more deleterious effect of the mutation on the function of RPGR. We propose that RPGR is involved in cilia-dependent cascades during development in zebrafish. Our studies provide evidence for a heterogenic effect of the disease-causing mutations on the function of RPGR.

A mouse model offers novel insight into the myopathy and tendinopathy that is often associated with pseudoachondroplasia and multiple epiphyseal dysplasia

Tue, 06 Oct 2009 04:39:00 PDT

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are relatively common skeletal dysplasias belonging to the same bone dysplasia family. PSACH is characterized by generalised epi-metaphyseal dysplasia, short-limbed dwarfism, joint laxity and early onset osteoarthritis. MED is a milder disease with radiographic features often restricted to the epiphyses of the long bones. PSACH and some forms of MED result from mutations in cartilage oligomeric matrix protein (COMP), a pentameric glycoprotein found in cartilage, tendon, ligament and muscle. PSACH-MED patients often have a mild myopathy characterized by mildly increased PCK levels, a variation in myofiber size and/or small atrophic fibers. In some instances patients are referred to neuromuscular clinics prior to the diagnosis of an underlying skeletal dysplasia; however, the myopathy associated with PSACH-MED has not previously been studied.

In this study we present a detailed study of skeletal muscle, tendon and ligament from a mouse model of mild PSACH harbouring a COMP mutation. Mutant mice exhibited a progressive muscle weakness associated with an increased number of muscle fibers with central nuclei at the perimysium and at the myotendinous junction. Furthermore, the distribution of collagen fibril diameters in the mutant tendons and ligaments was altered towards thicker collagen fibrils and the tendons became more lax in cyclic strain tests.

We hypothesise that the myopathy in PSACH-MED originates from an underlying tendon and ligament pathology that is a direct result of structural abnormalities to the collagen fibril architecture. This is the first comprehensive characterisation of the musculoskeletal phenotype of PSACH-MED and is directly relevant to the clinical management of these patients.

Dual Effects of Superovulation: Loss of maternal and paternal imprinted methylation in a dose-dependent manner

Market-Velker, B. A., Zhang, L., Magri, L. S., Bonvissuto, A. C., Mann, M. R.W. — Sun, 04 Oct 2009 20:44:28 PDT

Superovulation or ovarian stimulation is currently an indispensable assisted reproductive technology (ART) for human subfertility/infertility treatment. Recently, increased frequencies of imprinting disorders have been correlated with ARTs. Significantly, for Angelman and Beckwith-Wiedemann Syndromes, patients have been identified where ovarian stimulation was the only procedure used by the couple undergoing ART. In many cases, increased risk of genomic imprinting disorders has been attributed to superovulation in combination with inherent subfertility. To distinguish between these contributing factors, carefully controlled experiments are required on spontaneously-ovulated, in vivo-fertilized oocytes, and their induced-ovulated counterparts, thereby minimizing effects of in vitro manipulations. To this end, effects of superovulation on genomic imprinting were evaluated in a mouse model, where subfertility is not a confounding issue. This work represents the first comprehensive examination of the overall effects of superovulation on imprinted DNA methylation for four imprinted genes in individual blastocyst stage embryos. We demonstrate that superovulation perturbed genomic imprinting of both maternally- and paternally-expressed genes; loss of Snrpn, Peg3 and Kcnq1ot1, and gain of H19 imprinted methylation were observed. This perturbation was dose-dependent, with aberrant imprinted methylation more frequent at the high hormone dosage. Superovulation is thought to primarily affect oocyte development; thus, effects were expected to be limited to maternal alleles. Our study revealed that maternal as well as paternal H19 methylation was perturbed by superovulation. We postulate that superovulation has dual effects during oogenesis, disrupting acquisition of imprints in growing oocytes, as well as maternal-effect gene products subsequently required for imprint maintenance during preimplantation development.

Alteration of the Unfolded Protein Response Modifies Neurodegeneration in a Mouse Model of Marinesco-Sjogren Syndrome

Zhao, L., Rosales, C., Seburn, K., Ron, D., Ackerman, S. L. — Sat, 03 Oct 2009 03:04:46 PDT

Endoplasmic reticulum (ER) stress has been linked to the onset and progression of many diseases. SIL1 is an adenine nucleotide exchange factor of the essential ER lumen chaperone HSPA5/BiP that senses ER stress and is involved in protein folding. Mutations in the Sil1 gene have been associated with Marinesco-Sjögren syndrome, hallmarks of which include ataxia and cerebellar atrophy. We have previously shown that loss of SIL1 function in mouse results in ER stress, ubquitylated protein inclusions, and degeneration of specific Purkinje cells in the cerebellum. Here we report that overexpression of HYOU1/ORP150, an exchange factor that works in parallel to SIL1, prevents ER stress and rescues neurodegeneration in Sil1^–/– mice, whereas decreasing expression of HYOU1 exacerbates these phenotypes. In addition, loss of DNAJC3/p58^IPK, a co-chaperone that promotes ATP hydrolysis by BiP, ameliorates ER stress and neurodegeneration in Sil1^–/– mice. These findings suggest that alterations in the nucleotide exchange cycle cause ER stress and neurodegeneration in Sil1-deficient mice. Our results present the first evidence of important genetic modifiers of Marinesco-Sjögren syndrome, and provide additional pathways for therapeutic intervention for this, and other ER stress-induced, diseases.

PRKCSH/80K-H, the protein mutated in polycystic liver disease, protects polycystin-2/TRPP2 against HERP-mediated degradation

Sat, 03 Oct 2009 03:04:45 PDT

Autosomal dominant polycystic liver disease (PCLD) is caused by mutations of either PRKCSH or Sec63, two proteins associated with the endoplasmic reticulum (ER). Both proteins are involved in carbohydrate processing, folding and translocation of newly synthesized glycoproteins. It is postulated that defective quality control of proteins initiates endoplasmic reticulum-associated degradation (ERAD), which disrupts hepatic homeostasis in patients with PRKCSH or Sec63 mutations. However, the precise molecular mechanisms are not known. Here we show that over-expression or depletion of PRKCSH in zebrafish embryos leads to pronephric cysts, abnormal body curvature and situs inversus. Identical phenotypic changes are induced by depletion or over-expression of TRPP2. Increased PRKCSH levels ameliorate developmental abnormalities caused by over-expressed TRPP2, while excess TRPP2 can compensate the loss PRKCSH, indicating that the proteins share a common signaling pathway. PRKCSH binds the C-terminal domain of TRPP2, and both proteins co-localize within the ER. Furthermore, PRKCSH interacts with Herp, and inhibits Herp-mediated ubiquitination of TRPP2. Our findings suggest that PRKCSH functions as a chaperone-like molecule, which prevents ERAD of TRPP2. Dysequilibrium between TRPP2 and PRKCSH may lead to cyst formation in PCLD patients with PRKCSH mutations, and thereby account for the overlapping manifestations observed in PCLD and autosomal dominant polycystic kidney disease.

A MURINE MODEL OF DENYS-DRASH SYNDROME REVEALS NOVEL TRANSCRIPTIONAL TARGETS OF WT1 IN PODOCYTES

Wed, 30 Sep 2009 23:33:09 PDT

The Wilms tumor-suppressor gene WT1, a key player in renal development, also has a crucial role in maintenance of the glomerulus in the mature kidney. However, molecular pathways orchestrated by WT1 in podocytes, where it is highly expressed, remain unknown. Their defects are thought to modify the cross-talk between podocytes and other glomerular cells and ultimately lead to glomerular sclerosis, as observed in diffuse mesangial sclerosis (DMS) a nephropathy associated with WT1 mutations.

To identify podocyte WT1 targets, we generated a novel DMS mouse line, performed gene expression profiling in isolated glomeruli, and identified excellent candidates that may modify podocyte differentiation and growth factor signalling in glomeruli. Scel, encoding sciellin, a protein of the cornified envelope in the skin, and sulf1, encoding a 6-O endosulfatase, are shown to be expressed in wild type podocytes and to be strongly down-regulated in mutants. Co-expression of Wt1, Scel and Sulf1 was also found in a mesonephric cell line, and siRNA-mediated knockdown of WT1 decreased Scel and Sulf1 mRNAs and proteins. By ChIP we show that Scel and Sulf1 are direct WT1 targets. Cyp26a1, encoding an enzyme involved in the degradation of retinoic acid, is shown to be up-regulated in mutant podocytes. Cyp26a1 may play a role in the development of glomerular lesions but does not seem to be regulated by WT1. These results provide novel clues in our understanding of normal glomerular function and early events involved in glomerulosclerosis.

This article was published online in error and has been removed by the publisher.

Thu, 03 May 2007 04:26:38 PDT