Human Molecular Genetics - Advance Access

Abnormal social behaviors and altered gene expression rates in a mouse model for Potocki-Lupski Syndrome

2008-05-10

The Potocki-Lupski syndrome (PTLS) is associated with a microduplication of 17p11.2. Clinical features include multiple congenital and neurobehavioral abnormalities and autistic features. We have generated a PTLS mouse model, Dp(11)17/+, that recapitulates some of the physical and neurobehavioral phenotypes present in patients. Here we investigated the social behavior and gene expression pattern of this mouse model in a pure genetic background. Dp(11)17/+ male mice displayed normal home cage behavior but increased anxiety and increased dominant behavior in specific tests. A subtle impairment in the preference for a social target vs. an inanimate target and abnormal preference for social novelty (the preference to explore an unfamiliar mouse versus a familiar one) was also observed. Our results indicate that these animals could provide a valuable model to identify the specific gene(s) that confer abnormal social behaviors and that map within this delimited genomic deletion interval. In a first attempt to identify candidate genes and for elucidating the mechanisms of regulation of these important phenotypes we directly assessed the relative transcription of genes within and around this genomic interval. In this mouse model we found that candidates genes include not only most of the duplicated genes, but also normal-copy genes that flank the engineered interval; both categories of genes showed altered expression levels in the hippocampus of Dp(11)17/+ mice.

The primary open-angle glaucoma gene WDR36 functions in ribosomal-RNA processing and interacts with the p53 stress-response pathway

Skarie, J. M., Link, B. A. — 2008-05-10

Primary open angle glaucoma (POAG) is a genetically complex neuropathy that affects retinal ganglion cells and is a leading cause of blindness worldwide. WDR36, a gene of unknown function, was recently identified as causative for POAG at locus GLC1G. Subsequent studies found disease associated variants in control populations, leaving the role of WDR36 in this disease unclear. To address this issue we determined the function of WDR36. We studied Wdr36 in zebrafish and found it is the functional homolog of yeast Utp21. Utp21 is cell essential and functions in the nucleolar processing of 18S rRNA, which is required for ribosome biogenesis. Evidence for functional homology comes from sequence alignment, ubiquitous expression, sub-cellular localization to the nucleolus, and loss of function phenotypes that include defects in 18S rRNA processing and abnormal nucleolar morphology. Additionally, we show loss of Wdr36 function leads to an activation of the p53 stress response pathway, suggesting that co-inheritance of defects in p53-pathway genes may influence the impact of WDR36 variants on POAG. While these results overall do not provide evidence for or against a role of WDR3 6 in POAG, they do provide important baseline information for future studies.

Elucidating the Relationship between DISC1, NDEL1, and NDE1 and the Risk for Schizophrenia: Evidence of Epistasis and Competitive Binding

2008-05-10

DISC1 influences susceptibility to psychiatric disease and related phenotypes. Intact functions of DISC1 and its binding partners, NDEL1 and NDE1, are critical to neurodevelopmental processes aberrant in schizophrenia. Despite evidence of a NDEL1-DISC1 protein interaction, there have been no investigations of the NDEL1 gene or the relationship between NDEL1 and DISC1 in schizophrenia. We genotyped 6 NDEL1 SNPs in 275 Caucasian schizophrenia patients and 200 controls and tested for association and interaction between the functional SNP Ser704Cys in DISC1 and NDEL1. We also evaluated the relationship between NDE1 and DISC1 genotype and schizophrenia. Finally, in a series of in-vitro assays, we determined the binding profiles of NDEL1 and NDE1, in relation to DISC1 Ser704Cys.

We observed a single haplotype block within NDEL1; the majority of variation was captured by NDEL1 rs1391768. We observed a significant interaction between rs1391768 and DISC1 Ser704Cys, with the effect of NDEL1 on schizophrenia evident only against the background of DISC1 Ser704 homozygosity. Secondary analyses revealed no direct relationship between NDE1 genotype and SZ; however, there was an opposite pattern of risk for NDE1 genotype when conditioned on DISC1 Ser704Cys, with NDE1rs3784859 imparting a significant effect but only in the context of a Cys carrying background. In addition, we report opposing binding patterns of NDEL1 and NDE1 to Ser704 versus Cys704, at the same DISC1 binding domain. These data suggest that NDEL1 significantly influences risk for SZ via an interaction with DISC1. We propose a model where NDEL1 and NDE1 compete for binding with DISC1.

No Association between the SRD5A2 Gene A49T Missense Variant and Prostate Cancer Risk: Lessons Learned

2008-05-10

The steroid 5-alpha reductase type II gene (SRD5A2) encodes the enzyme which converts testosterone (T) to the more active androgen dihydrotestosterone (DHT). A non-synonymous single nucleotide polymorphism (SNP), A49T (rs9282858), in SRD5A2 has been implicated in prostate cancer risk; however, results have been inconsistent. In 1999 we reported a strong association between the A49T variant and prostate cancer risk among African-Americans and Latinos in the Hawaii-Los Angeles Multiethnic Cohort (MEC). We report here an updated analysis of MEC data including the five major ethnic groups of the MEC, an increased sample size, improved genotyping technology, and a comprehensive meta-analysis of the published literature. We found a non-statistically significant positive association between prostate cancer risk and carrying either the AT or TT genotype (odds ratio (OR)=1.20, 95% confidence interval (CI) 0.82-1.77) in the MEC. This finding is in contrast to our previous results of ORs of 3.28 and 2.50 for the association between prostate cancer risk and the variant in African-American and Latino men respectively; this can be accounted for by genotyping error in our earlier study. Meta-analysis of the published literature, including the current MEC data, shows a summary OR of 1.13 (95% CI 0.95-1.34) for the A49T variant with prostate cancer risk among sporadic, unselected cases. After evaluating over 6,000 cases and 6,000 controls, there is little evidence of a role for the SRD5A2 A49T variant in prostate cancer risk. Overall, this report highlights the importance of rigorous genotyping quality control measures and replication efforts in genetic association studies.

Lis1-Nde1 dependent neuronal fate control determines cerebral cortical size and lamination

Pawlisz, A. S., Mutch, C., Wynshaw-Boris, A., Chenn, A., Walsh, C. A., Feng, Y. — 2008-05-10

Neurons in the cerebral cortex originate predominantly from asymmetrical divisions of polarized radial glial or neuroepithelial cells. Fate control of neural progenitors through regulating cell division asymmetry determines the final cortical neuronal number and organization. Haplo-insufficiency of human LIS1 results in type I lissencephaly (smooth brain) with severely reduced surface area and laminar organization of the cerebral cortex. Here we show that LIS1 and its binding protein Nde1 (mNudE) regulate the fate of radial glial progenitors collaboratively. Mice with an allelic series of Lis1 and Nde1 double mutations displayed a striking dose-dependent size reduction and de-lamination of the cerebral cortex. The neocortex of the Lis1-Nde1 double mutant mice showed over 80% reduction in surface area and inverted neuronal layers. Dramatically increased neuronal differentiation at the onset of corticogenesis in the mutant led to overproduction and abnormal development of earliest-born preplate neurons and Cajal-Retzius cells at the expense of progenitors. While both Lis1 and Nde1 are known to regulate the mitotic spindle orientation, only a moderate alteration in mitotic cleavage orientation was detected in the Lis1-Nde1 double deficient progenitors. Instead, a striking change in the morphology of metaphase progenitors with reduced apical attachment to the ventricular surface and weakened lateral contacts to neighbouring cells appear to hinder the accurate control of cell division asymmetry and underlie the dramatically increased neuronal differentiation. Our data suggest that maintaining the shape and cell-cell interactions of radial glial neuroepithelial progenitors by the Lis1-Nde1 complex is essential for their self renewal during the early phase of corticogenesis.

ccm1 cell autonomously regulates endothelial cellular morphogenesis and vascular tubulogenesis in zebrafish

Hogan, B. M., Bussmann, J., Wolburg, H., Schulte-Merker, S. — 2008-05-10

Cerebral cavernous malformations (CCMs) are a prevalent class of vascular anomalies characterized by thin-walled clusters of malformed blood vessels in the brain. Heritable forms are caused by mutations in CCM1, CCM2 and CCM3 but despite the importance of these factors in vascular biology, an understanding of their molecular and cellular functions remains elusive. Here we describe the characterization of a zebrafish embryonic model of CCM. Loss of ccm1 in zebrafish embryos leads to severe and progressive dilation of major vessels, despite normal endothelial cell fate and number. Vascular dilation in ccm1 mutants is accompanied by progressive spreading of endothelial cells and thinning of vessel walls despite ultrastructurally normal cell-cell contacts. Zebrafish ccm2 mutants display comparable vascular defects. Finally, we show that ccm1 function is cell autonomous, suggesting that it is endothelial cellular morphogenesis that is regulated by CCM proteins during development and pathogenesis.

Thymidine kinase 2 (H126N) knockin mice show the essential role of balanced deoxynucleotide pools for mitochondrial DNA maintenance

2008-05-08

Mitochondrial DNA (mtDNA) depletion syndrome (MDS), an autosomal recessive condition, is characterized by variable organ involvement with decreased mtDNA copy number and activities of respiratory chain enzymes in affected tissues. MtDNA depletion has been associated with mutations in nine autosomal genes, including thymidine kinase (TK2), which encodes a ubiquitous mitochondrial protein. To study the pathogenesis of TK2-deficiency, we generated mice harboring an H126N Tk2 mutation. Homozygous Tk2 mutant (Tk2^–/–) mice developed rapidly progressive weakness after age 10 days and died between ages 2-3 weeks. Tk2^–/– animals showed Tk2 deficiency, unbalanced dNTP pools, mtDNA depletion, and defects of respiratory chain enzymes containing mtDNA-encoded subunits that were most prominent in the central nervous system. Histopathology revealed an encephalomyelopathy with prominent vacuolar changes in the anterior horn of the spinal cord. The H126N TK2 mouse is the first knock-in animal model of human MDS and demonstrates that the severity of TK2 deficiency in tissues may determine the organ-specific phenotype.

A variant in the sonic hedgehog regulatory sequence (ZRS) is associated with triphalangeal thumb and deregulates expression in the developing limb

2008-05-07

A locus for triphalangeal thumb, variably associated with preaxial polydactyly, was previously identified in the Zone of Polarizing Activity Regulatory Sequence (ZRS), a long range limb-specific enhancer of the Sonic Hedgehog gene (SHH) at human chromosome 7q36.3. Here, we demonstrate that a 295T>C variant in the human ZRS, previously thought to represent a neutral polymorphism, acts as a dominant allele with reduced penetrance. We found this variant in 3 independently ascertained probands from southern England with triphalangeal thumb, demonstrated significant linkage of the phenotype to the variant (LOD = 4.1), and identified a shared microsatellite haplotype around the ZRS, suggesting that the probands share a common ancestor. An individual homozygous for the 295C allele presented with isolated bilateral triphalangeal thumb resembling the heterozygous phenotype, suggesting that the variant is largely dominant to the wild type allele. As a functional test of the pathogenicity of the 295C allele, we utilised a mutated ZRS construct to demonstrate that it can drive ectopic anterior expression of a reporter gene in the developing mouse forelimb. We conclude that the 295T>C variant is in fact pathogenic and, in southern England, appears to be the most common cause of triphalangeal thumb. Depending on the dispersal of the founding mutation, it may play a wider role in the etiology of this disorder.

Usher syndromes due to MYO7A, PCDH15, USH2A or GPR98 mutations share retinal disease mechanism

2008-05-07

Usher syndrome (USH) is a genetically heterogeneous group of autosomal recessive deaf-blinding disorders. The pathophysiology leading to the blinding retinal degeneration in USH is uncertain. There is evidence for involvement of the photoreceptor cilium, photoreceptor synapse, the adjacent retinal pigment epithelium (RPE) cells, and the Crumbs protein complex, the latter implying developmental abnormalities in the retina. Testing hypotheses has been difficult in murine USH models because most do not show a retinal degeneration phenotype. We defined the retinal disease expression in vivo in human USH using optical imaging of the retina and visual function. In MYO7A (USH1B), results from young individuals or those at early stages indicated the photoreceptor was the first detectable site of disease. Later stages showed photoreceptor and RPE cell pathology. Mosaic retinas in Myo7a-deficient shaker1 mice supported the notion that the mutant photoreceptor phenotype was cell autonomous and not secondary to mutant RPE. Humans with PCDH15 (USH1F), USH2A or GPR98 (USH2C) had a similar retinal phenotype to MYO7A (USH1B). There was no evidence of photoreceptor synaptic dysfunction and no dysplastic phenotype as in CRUMBS1 retinopathy. The results point to the photoreceptor cell as the therapeutic target for USH treatment trials, such as MYO7A somatic gene replacement therapy.

Evidence that the Gene Encoding Insulin Degrading Enzyme Influences Human Lifespan

2008-04-30

Studies in model organisms have demonstrated that components of insulin and insulin-like signaling pathways are involved in the regulation of lifespan but the relevance of those findings to humans has remained obscure. Here we provide evidence suggesting that variants of the gene encoding insulin-degrading enzyme (IDE) may be influencing human lifespan. We have employed a variety of models and diverse samples that reproducibly indicate the relative change in IDE genotype frequency across the age spectrum as well as allow the detection of association with age-at-death. A tenable molecular basis of this is suggested by the observation of genetic association with both fasting plasma insulin levels and IDE mRNA expression. Across populations the emergent genetic model is indicative of over-dominance, where heterozygotes of critical markers have increased IDE mRNA expression and insulin levels, and this is reflected in diminished heterozygosity at advanced age. A critical and replicating feature of this study is that change in IDE genotype frequency with advancing age appears to only be occurring in men, and this is supported in that insulin levels are only associated with IDE in men. Results suggest a relationship between a gene that is intimately involved in insulin metabolism and the determination of lifespan in humans, but over-dominance and gender specificity will be important parameters to consider towards clarifying the biological importance of these findings.

Activated caspase-6 and caspase-6-cleaved fragments of huntingtin specifically colocalize in the nucleus

2008-04-29

Proteolysis of mutant huntingtin is crucial to the development of Huntington disease (HD). Specifically preventing proteolysis at the capase-6 (C6) consensus sequence at amino acid (aa) 586 of mutant huntingtin prevents the development of behavioural, motor and neuropathological features in a mouse model of HD. However, the mechanism underlying the selective toxicity of the 586aa cleavage event is currently unknown. We have examined the subcellular localization of different caspase proteolytic fragments of huntingtin using neo-epitope antibodies. Our data suggest that the nucleus is the primary site of htt cleavage at aa586. Endogenously-cleaved 586aa fragments are enriched in the nucleus of immortalized striatal cells and primary striatal neurons where they co-localize with active C6. Cell stress induced by staurosporine results in the nuclear translocation and activation of C6 and an increase in 586aa fragments of huntingtin in the nucleus. In comparison, endogenous caspase-2/3-generated huntingtin 552aa fragments localize to the perinuclear region. The different cellular itineraries of endogenously-generated caspase products of huntingtin may provide an explanation for the selective toxicity of huntingtin fragments cleaved at aa586.

Rac1 mediates the osteoclast gains-in-function induced by haploinsufficiency of Nf1

2008-04-29

Epidermal Expression of the Truncated Prelamin A Causing Hutchinson-Gilford Progeria Syndrome: Effects on Keratinocytes, Hair and Skin

2008-04-28

Hutchinson-Gilford progeria syndrome (HGPS) is an accelerated aging disorder caused by point mutation in LMNA encoding A-type nuclear lamins. The mutations in LMNA activate a cryptic splice donor site, resulting in expression of a truncated, prenylated prelamin A called progerin. Expression of progerin leads to alterations in nuclear morphology, which may underlie pathology in HGPS. We generated transgenic mice expressing progerin in epidermis under control of a keratin 14 promoter. The mice had severe abnormalities in morphology of skin keratinocyte nuclei, including nuclear envelope lobulation and decreased nuclear circularity not present in transgenic mice expressing wild type human lamin A. Primary keratinocytes isolated from these mice had a higher frequency of nuclei with abnormal shape compared to those from transgenic mice expressing wild type human lamin A. Treatment with a farnesyltransferase inhibitor significantly improved nuclear shape abnormalities and induced the formation of intranuclear foci in the primary keratinocytes expressing progerin. Similarly, spontaneous immortalization of progerin-expressing cultured keratinocytes selected for cells with normal nuclear morphology. Despite morphological alterations in keratinocyte nuclei, mice expressing progerin in epidermis had normal hair grown and wound healing. Hair and skin thickness were normal even after crossing to Lmna null mice to reduce or eliminate expression of normal A-type lamins. Although progerin induces significant alterations in keratinocyte nuclear morphology that are reversed by inhibition of farnesyltransferasae, epidermal expression does not lead to alopecia or other skin abnormalities typically seen in human subjects with HGPS.

Nf1 +/- Mice Have Increased Neointima Formation Via Hyperactivation of a Gleevec Sensitive Molecular Pathway

2008-04-28

Neurofibromatosis type I (NF1) is a genetic disorder caused by mutations in the NF1 tumor suppressor gene. Neurofibromin is encoded by NF1 and functions as a negative regulator of Ras activity. Somatic mutations in the residual normal NF1 allele within cancers of NF1 patients is consistent with NF1 functioning as a tumor-suppressor. However, the prevalent nonmalignant manifestations of NF1, including learning and bone disorders emphasize the importance of dissecting the cellular and biochemical effects of NF1 haploinsufficiency in multiple cell lineages. One of the least studied complications of NF1 involves cardiovascular disorders, including arterial occlusions that result in cerebral and visceral infarcts. NF1 vasculopathy is characterized by vascular smooth muscle cell (VSMC) accumulation in the intima area of vessels resulting in lumen occlusion. We recently showed that Nf1 haploinsufficiency increases VSMC proliferation and migration via hyperactivation of the Ras-Erk pathway, which is a signaling axis directly linked to neointima formation in diverse animal models of vasculopathy. Given this observation, we tested whether heterozygosity of Nf1 would lead to vasoocclusive disease in genetically engineered mice in vivo. Strikingly, Nf1+/- mice have increased neointima formation, excessive vessel wall cell proliferation and Erk activation after vascular injury in vivo. Further, this effect is directly dependent on a Gleevec sensitive molecular pathway. Therefore, these studies establish an Nf1 model of vasculopathy, which mirrors features of human NF1 vasoocclusive disease, identifies a potential therapeutic target, and provides a platform to further dissect the effect of Nf1 haploinsufficiency in cardiovascular disease.

Susceptibility locus for clinical and subclinical coronary artery disease at chromosome 9p21 in the multi-ethnic ADVANCE Study

2008-04-28

A susceptibility locus for CAD at chromosome 9p21 has been recently reported which may influence the age of onset of CAD. We sought to replicate these findings among white subjects and to examine whether these results are consistent in other racial/ethnic groups by genotyping three SNPs in the risk interval in the ADVANCE study. One or more of these SNPs was associated with clinical CAD in whites, U.S. Hispanics, and U.S. East Asians. None of the SNPs was associated with CAD in African Americans although the power to detect an OR in this group equivalent to that seen in whites was only 24% to 30%. ORs were higher in Hispanics and East Asians and lower in African Americans but in all groups the 95% confidence intervals overlapped with ORs observed in whites. The high risk alleles were also associated with increased coronary artery calcification in controls and the magnitude of these associations by racial/ethnic group closely mirrored the magnitude observed for clinical CAD. Unexpectedly, we noted significant genotype frequency differences between male and female cases (p=0.003-0.05). Consequently, men tended towards a recessive and women tended towards a dominant mode of inheritance. Finally, an effect of genotype on the age of onset of CAD was detected but only in men carrying two versus one or no copy of the high risk allele and presenting with CAD at an age>than 50 years. Further investigations in other populations are needed to confirm or refute our findings.

Premature aging in mice activates a systemic metabolic response involving autophagy induction

2008-04-28

Autophagy is a highly regulated intracellular process involved in the turnover of most cellular constituents and in the maintenance of cellular homeostasis. It is well-established that the basal autophagic activity of living cells decreases with age, thus contributing to the accumulation of damaged macromolecules during aging. Conversely, the activity of this catabolic pathway is required for lifespan extension in animal models such as Caenorhabditis elegans and Drosophila melanogaster. In this work, we describe the unexpected finding that Zmpste24-null mice, which show accelerated aging and are a reliable model of human Hutchinson-Gilford progeria, exhibit an extensive basal activation of autophagy instead of the characteristic decline in this process occurring during normal aging. We also show that this autophagic increase is associated with a series of changes in lipid and glucose metabolic pathways, which resemble those occurring in diverse situations reported to prolong lifespan. These Zmpste24^–/- mice metabolic alterations are also linked to substantial changes in circulating blood parameters, such as leptin, glucose, insulin or adiponectin which in turn lead to peripheral LKB1-AMPK activation and mTOR inhibition. On the basis of these results, we propose that nuclear abnormalities causing premature aging in Zmpste24^–/- mice trigger a metabolic response involving the activation of autophagy. However, the chronic activation of this catabolic pathway may turn an originally intended pro-survival strategy into a pro-aging mechanism and could contribute to the systemic degeneration and weakening observed in these progeroid mice.

AAV-mediated intramuscular delivery of myotubularin corrects the myotubular myopathy phenotype in targeted murine muscle and suggests a function in plasma membrane homeostasis

2008-04-28

Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital muscular disease due to mutations in the myotubularin gene (MTM1) and characterized by the presence of small myofibres with frequent occurence of central nuclei. Myotubularin is a ubiquitously expressed phosphoinositide phosphatase with a muscle specific role in man and mouse that is poorly understood. No specific treatment exists to date for patients with myotubular myopathy. We have constructed an adeno-associated virus (AAV) vector expressing myotubularin in order to test its therapeutic potential in a XLMTM mouse model. We show that a single intramuscular injection of this vector in symptomatic Mtm1-deficient mice ameliorates the pathological phenotype in the targeted muscle. Myotubularin replacement in mice largely corrects nuclei and mitochondria positioning in myofibres and leads to a strong increase in muscle volume and recovery of the contractile force. In addition, we used this AAV vector to overexpress myotubularin in wild-type skeletal muscle and get insights into its localisation and function. We show that a substantial proportion of myotubularin associates to the sarcolemma and I band, including triads. Myotubularin overexpression in muscle induces the accumulation of packed membrane saccules and presence of vacuoles that contain markers of sarcolemma and T-tubules, suggesting that myotubularin is involved in plasma membrane homeostasis of myofibres. This study provides a proof-of-principle that local delivery of an AAV vector expressing myotubularin can improve the motor capacities of XLMTM muscle and represents a novel approach to study myotubularin function in skeletal muscle.

Haemoglobin S and haemoglobin C: 'quick but costly' versus 'slow but gratis' genetic adaptations to Plasmodium falciparum malaria

2008-04-27

Progressive loss of mitochondrial DNA in thymidine kinase 2 deficient mice

2008-04-22

Deficient enzymatic activity of the mitochondrial deoxyribonucleoside kinases deoxyguanosine kinase (DGUOK) or thymidine kinase 2 (TK2) cause mitochondrial DNA (mtDNA) depletion syndromes in humans. Here we report the generation of a Tk2 deficient mouse strain and show that the mice develop essentially normal for the first week but from then on exhibit growth retardation and die within the first weeks of life. Several organs including skeletal muscle, heart, liver and spleen showed progressive loss of mtDNA without increased mtDNA mutations or structural alterations. There were no major histological changes in skeletal muscle, but heart muscle showed disorganized and damaged muscle fibers. Electron microscopy showed mitochondria with distorted cristae. The Tk2 deficient mice exhibited pronounced hypothermia and showed loss of hypodermal fat and abnormal brown adipose tissue. We conclude that Tk2 has a major role in supplying deoxyribo-nucleotides for mtDNA replication and that other pathways of deoxyribonucleotide synthesis cannot compensate for loss of this enzyme.

Re-evaluation of putative rheumatoid arthritis susceptibility genes in the post-genome wide association study era and hypothesis of a key pathway underlying susceptibility

2008-04-22

Rheumatoid arthritis (RA) is an archetypal common complex autoimmune disease with both genetic and environmental contributions to disease aetiology. Two novel RA susceptibility loci have been reported from recent genome-wide and candidate gene association studies. We, therefore, investigated the evidence for association of the STAT4 and TRAF1/C5 loci with RA using imputed data from the Wellcome Trust Case Control Consortium (WTCCC). No evidence for association of variants mapping to the TRAF1/C5 gene was detected in the 1,860 RA cases and 2,930 control samples tested in that study. Variants mapping to the STAT4 gene did show evidence for association (rs7574865, p = 0.04). Given the association of the TRAF1/C5 locus in two previous large case control series from populations of European descent and the evidence for association of the STAT4 locus in the WTCCC study, SNPs mapping to these loci were tested for association with RA in an independent UK series comprising DNA from >3,000 cases with disease and >3,000 controls and a combined analysis including the WTCCC data was undertaken. We confirm association of the STAT4 and the TRAF1/C5 loci with RA bringing to 5 the number of confirmed susceptibility loci. The effect sizes are less than those reported previously but are likely to be a more accurate reflection of the true effect size given the larger size of the cohort investigated in the current study.

Major basic protein-1 promotes fibrosis of dystrophic muscle and attenuates the cellular immune response in muscular dystrophy

2008-04-21

The immune response to dystrophin-deficient muscle promotes the pathology of Duchenne muscular dystrophy (DMD) and the mdx mouse model of DMD. In this investigation, we find that the release of major basic protein (MBP) by eosinophils is a prominent feature of DMD and mdx dystrophy and that eosinophils lyse muscle cells in vitro by the release of MBP-1. We also show that eosinophil depletions of mdx mice by injections of anti-chemokine receptor-3 reduce muscle cell lysis, although lysis of mdx muscle membranes is not reduced by null mutation of MBP-1 in vivo. However, ablation of MBP-1 expression in mdx mice produces other effects on muscular dystrophy. First, fibrosis of muscle and hearts, a major cause of mortality in DMD, is greatly reduced by null mutation of MBP-1 in mdx mice. Furthermore, either ablation of MBP-1 or eosinophil depletion causes large increases in cytotoxic T-lymphocytes (CTLs) in mdx muscles. The increase in CTLs in MBP-1-null mice does not reflect a general shift toward a Th1 inflammatory response, because the mutation had no significant effect on the expression of interferon-gamma, inducible nitric oxide synthase or tumor necrosis factor. Rather, MBP-1 reduces the activation and proliferation of splenocytes in vitro, indicating that MBP-1 acts in a more specific immunomodulatory role to affect the inflammatory response in muscular dystrophy. Together, these findings show that eosinophil-derived MBP-1 plays a significant role in regulating muscular dystrophy by attenuating the cellular immune response and promoting tissue fibrosis that can eventually contribute to increased mortality.

DNA instability in low-risk myelodysplastic syndromes: refractory anemia with or without ring sideroblasts

Novotna, B., Neuwirtova, R., Siskova, M., Bagryantseva, Y. — 2008-04-21

We tested genomic instability in patients with myelodysplastic syndrome (MDS) by the comet assay and verified the suitability of this approach as a tool for analysis of ineffective hematopoiesis in refractory anemia (RA) and RA with ring sideroblasts (RARS). Erythroid and myeloid cell population from bone marrow aspirates of 20 RA, 14 RARS and 15 control subjects were separated by differential expression of glycophorin A and subjected to comet assay. The extent of DNA migration was measured in single cells (200 cells/bone marrow fraction/ subject). The results were in agreement with the concept of increased apoptosis in low-risk MDS subtypes. The RA samples had a significantly higher DNA instability than controls in glycophorin A positive cells and the extent of DNA breakage correlated with the degree of cytopenia. Although RARS had an even higher rate of genomic instability in bone marrow cells than RA, there was no clear relationship to peripheral cytopenia. This suggests an additional DNA instability of non-apoptotic origin. Whether this increase is associated with an increased repair of oxidative damage in DNA arising due to iron deposits in ring sideroblasts remains to be formally proven. Comet assay provides a promising tool for investigation of difference between RA and RARS pathobiology.

Molecular dissection of ALS-associated toxicity of SOD1 in transgenic mice using an exon-fusion approach

2008-04-18

Mutations in Cu,Zn superoxide dismutase (SOD1) are associated with amyotrophic lateral sclerosis (ALS). Among more than 100 ALS-associated SOD1 mutations, premature termination codon (PTC) mutations exclusively occur in exon 5, the last exon of SOD1. The molecular basis of ALS-associated toxicity of the mutant SOD1 is not fully understood. Here, we show that nonsense-mediated mRNA decay (NMD) underlies clearance of mutant mRNA with a PTC in the non-terminal exons. To further define the crucial ALS-associated SOD1 fragments, we designed and tested an exon-fusion approach using an artificial transgene SOD1^T116X that harbors a PTC in exon 4. We found that the SOD1^T116X transgene with a fused exon could escape NMD in cellular models. We generated a transgenic mouse model that overexpresses SOD1^T116X. This mouse model developed ALS-like phenotype and pathology. Thus, our data have demonstrated that a "mini-SOD1" of only 115 amino acids is sufficient to cause ALS. This is the smallest ALS-causing SOD1 molecule currently defined. This proof of principle result suggests that the exon-fusion approach may have potential not only to further define a shorter ALS-associated SOD1 fragment, thus providing a molecular target for designing rational therapy, but also to dissect toxicities of other proteins encoded by genes of multiple exons through a "gain of function" mechanism.

Variants in the Estrogen Receptor Alpha Gene and its mRNA Contribute to Risk for Schizophrenia

2008-04-18

Estrogen modifies human emotion and cognition and impacts symptoms of schizophrenia. We hypothesized that variation in the estrogen receptor alpha (ESR1) gene and in cortical ESR1 mRNA is associated with schizophrenia. In a small case-control genetic association analysis of postmortem brain tissue, genotype CC (rs2234693) and haplotypes containing the C allele of a single nucleotide polymorphism (SNP) in intron1 (PvuII) were more frequent in African American schizophrenics (p=0.01 to 0.001). In a follow-up family-based association analysis, we found overtransmission of PvuII allele C and a PvuII C-containing haplotype (p=0.01-p=0.03) to African American and Caucasian patients with schizophrenia. Schizophrenics with the "at risk" PvuII genotype had lower ESR1 mRNA levels in the frontal cortex. Eighteen ESR1 splice variants and decreased frequencies of the wild-type ESR1 mRNA were detected in schizophrenica. In one patient, a unique ESR1 transcript with a genomic insert encoding a premature stop codon and a truncated ESR1 protein lacking most of the estrogen binding domain was the only transcript detected. Using a luciferase assay, we found that mRNA encoding a truncated ESR1 significantly attenuates gene expression at estrogen-response elements (ERE) demonstrating a dominant negative function. An intron 6 SNP [rs2273207(G)] was associated with an ESR1 splice variant missing exon seven. The T allele of another intron 6 SNP was part of a 3’ haplotype less common in schizophrenia [rs2273206(T), rs2273207(G), rs2228480(G)]. Thus, variation in the ESR1 gene is associated with schizophrenia and the mechanism of this association may involve alternative gene regulation and transcript processing.

Iron-dependent regulation of frataxin expression: implications for treatment of Friedreich ataxia

Li, K., Besse, E. K., Ha, D., Kovtunovych, G., Rouault, T. A. — 2008-04-17

Friedreich ataxia (FA) is a progressive neurodegenerative disease caused by expansion of a trinucleotide repeat within the first intron of the gene that encodes frataxin. In our study, we investigated the regulation of frataxin expression by iron and demonstrated that frataxin mRNA levels decrease significantly in multiple human cell lines treated with the iron chelator, desferal. In addition, frataxin mRNA and protein levels decrease in fibroblast and lymphoblast cells derived from both normal controls and from patients with FA when treated with desferal. Lymphoblasts and fibroblasts of FA patients have evidence of cytosolic iron depletion, as indicated by increased levels of iron regulatory protein 2 (IRP2). We postulate that this inferred cytosolic iron depletion occurs as frataxin-deficient cells overload their mitochondria with iron, a downstream regulatory effect that has been observed previously when mitochondrial iron-sulfur cluster assembly is disrupted. The mitochondrial iron overload and presumed cytosolic iron depletion potentially further compromise function in frataxin deficient cells by decreasing frataxin expression. Thus, our results imply that therapeutic efforts should focus on an approach that combines iron removal from mitochondria with a treatment that increases cytosolic iron levels to maximize residual frataxin expression in FA patients.

Sphingosine kinase 1/S1P receptor signaling axis controls glial proliferation in mice with Sandhoff disease

Wu, Y.-P., Mizugishi, K., Bektas, M., Sandhoff, R., Proia, R. L. — 2008-04-17

Sphingosine-1-phosphate (S1P) is a lipid signaling molecule produced by sphingosine kinase in response to a wide number of stimuli. By acting through a family of widely expressed G protein-coupled receptors, S1P regulates diverse physiological processes. Here we examined the role of S1P signaling in neurodegeneration using a mouse model of Sandhoff disease, a prototypical neuronopathic lysosomal storage disorder. When sphingosine kinase 1 (Sphk1) was deleted in Sandhoff disease mice, a milder disease course occurred, with decreased proliferation of glial cells and less-pronounced astrogliosis. A similar result of milder disease course and reduced astroglial proliferation was obtained by deletion of the gene for the S1P₃ receptor, a G protein-coupled receptor enriched in astrocytes. Our studies demonstrate a functional role of S1P synthesis and receptor expression in astrocyte proliferation leading to astrogliosis during the terminal stages of neurodegeneration in Sandhoff disease mice. Because astrocyte responses are widely involved in many types of neurodegeneration, the Sphk1/S1P receptor signaling axis may be generally important during the pathogenesis of neurodegenerative diseases.

UNCOUPLING OF CHONDROCYTE DIFFERENTIATION AND PERICHONDRIAL MINERALIZATION UNDERLIES THE SKELETAL DYSPLASIA IN TRICHO-RHINO-PHALANGEAL SYNDROME

2008-04-17

Tricho-rhino-phalangeal syndrome (TRPS) is an autosomal dominant craniofacial and skeletal dysplasia that is caused by mutations involving the TRPS1 gene. Patients with TRPS have short stature, hip abnormalities, cone-shaped epiphyses, and premature closure of growth plates reflecting defects in endochondral ossification. The TRPS1 gene encodes for the transcription factor TRPS1 that has been demonstrated to repress transcription in vitro. To elucidate molecular mechanisms underlying skeletal abnormalities in TRPS, we analyzed Trps1 mutant mice (Trps1GT mice). Analyses of growth plates demonstrated delayed chondrocyte differentiation and accelerated mineralization of perichondrium in Trps1 mutant mice. These abnormalities were accompanied by increased Runx2 and Ihh expression and increased Ihh signaling. We demonstrated that Trps1 physically interacts with Runx2 and represses Runx2-mediated transactivation. Importantly, generation of Trps1^GT/+;Runx2^+/- double heterozygous mice rescued the opposite growth plate phenotypes of single mutants demonstrating the genetic interaction between Trps1 and Runx2 transcription factors. Collectively, these data suggest that skeletal dysplasia in TRPS is caused by dysregulation of chondrocyte and perichondrium development partially due to loss of Trps1 repression of Runx2.

A nonsense mutation in the LIMP-2 gene associated with progressive myoclonic epilepsy and nephrotic syndrome

2008-04-17

The main clinical features of two siblings from a consanguineous marriage were progressive myoclonic epilepsy without intellectual impairment and a nephrotic syndrome with a strong accumulation of C1q in capillary loops and mesangium of kidney. Biochemical analysis of one of the patients revealed a normal β-glucocerebrosidase activity in leukocytes but a severe enzymatic deficiency in cultured skin fibroblasts. This deficiency suggested a defect in the intracellular sorting pathway of this enzyme. Sequence analysis of the gene encoding LIMP-2 (SCARB2), the sorting receptor for β-glucocerebrosidase confirmed this hypothesis. A homozygous nonsense mutation in codon 178 of SCARB2 was found in the patient whereas her healthy parents were heterozygous for the mutation. Besides lacking immunodetectable LIMP-2, patient fibroblasts also had decreased amounts of β-glucocerebrosidase which was mainly located in the endoplasmic reticulum, as assessed by its sensitivity to Endo H. This is the first report of a mutation in the SCARB2 gene associated with a human disease which, contrary to earlier proposals, shares no features with Charcot-Marie-Tooth disease both at the clinical and neurophysiological levels.

Interaction between a novel TGFB1 haplotype and CFTR genotype is associated with improved lung function in cystic fibrosis

2008-04-17

Cystic fibrosis (CF), the most common lethal single gene disorder in Caucasians, is due to mutations in the CFTR gene. Twin and sibling analysis indicates that modifier genes, rather than allelic variation in CFTR, are responsible for most of the variability in severity of lung disease, the major cause of mortality in CF patients. We used a family-based approach to test for association between lung function and two functional SNPs (rs1800469, "-509" and rs1982073, "codon 10") in the 5’ region of transforming growth factor-beta1 (TGFB1), a putative CF modifier gene. Quantitative transmission disequilibrium testing of 472 CF patient-parent-parent trios revealed that both TGFB1 SNPs showed significant transmission distortion when patients were stratified by CFTR genotype. Although lung function and nutritional status are correlated in CF patients, there was no evidence of association between the TGFB1 SNPs and variation in nutritional status. Additional tagging SNPs (rs8179181, rs2278422, rs8110090, rs4803455, and rs1982072) that capture most of the diversity in TGFB1 were also typed but none showed association with variation in lung function. However, a haplotype composed of the -509 C and codon 10 T alleles along with the C allele of the 3’ SNP rs8179181 was highly associated with increased lung function in patients grouped by CFTR genotype. These results demonstrate that TGFB1 is a modifier of CF lung disease and reveal a previously unrecognized beneficial effect of TGFB1 variants upon the pulmonary phenotype.

ABCB1 (MDR1) Genetic Variants are Associated with Methadone Doses Required for Effective Treatment of Heroin Dependence

2008-04-17

Methadone is a mu-opioid receptor agonist used for treating opiate dependence. The range of effective methadone doses is broad. Part of the large inter-individual variability in efficacy may be accounted for by genetic factors. Methadone is a substrate of the transporter P-glycoprotein 170 (P-gp) that is encoded by the ABCB1 (MDR1) gene. Thus P-gp variants may play a role in methadone absorption and distribution. We assessed the association between ABCB1 polymorphisms and methadone dose requirements in 98 methadone maintained patients. The stabilizing methadone doses were normally distributed with a mean and median dose of 160 mg/day (range 30-280 mg/day). Statistical analysis showed significant difference in genotype frequencies between the "higher" (>150 mg/day) and "lower" (≤150 mg/day) methadone dose groups for SNP 1236C>T (rs1128503) (experiment-wise p=0.0325). Furthermore, individuals bearing the 3- locus genotype pattern TT-TT-TT (rs1045642, rs2032582 and rs1128503) have an approximately 5-fold chance of requiring the "higher" methadone dose, while individuals heterozygous for these three SNPs have an approximately 3-fold chance of stabilizing at the "lower" methadone dose (point-wise p-value=0.026). These data suggest that specific ABCB1 variants may have clinical relevance by influencing the methadone dose required to prevent withdrawal symptoms and relapse in this population.

A functional promoter variant in IL12B predisposes to cerebral malaria

2008-04-15

The role of the Th1 pathway in the pathogenesis of severe malaria is unclear. We recently reported that a polymorphism increasing IFNG transcription is associated with protection against cerebral malaria. Interleukin-12 is required for Th1 cell differentiation, which is characterized by the production of interferon-. We investigated 21 markers in IL12-related genes, including IL12A and IL12B encoding the two IL-12 (IL12p70) subunits, IL12p35 and IL12p40. We performed a family-based association study using a total sample set of 240 nuclear families. The IL12Bpro polymorphism was associated with susceptibility to CM. The CTCTAA allele and the GC/CTCTAA genotype are over-transmitted to children with CM (P=.0002, P=.00002, respectively). We estimated the OR for risk of CM in heterozygous children to be 2.11 ([95% CI, 1.49-2.99]; P < .0001). Although the CTCTAA allele had a dominant effect on CM susceptibility, this effect is much stronger in heterozygous children, consistent with functional effects of this allele in heterozygous form. Heterozygosity for this polymorphism has been associated with reduced expression of the gene encoding IL12p40 and a low level of IL12p70 production. These results, together with findings from immunological studies of low IFN- and IL-12 levels in CM, support a protective role for the Th1 pathway in CM.

Analysis of Cerebellar Function in Ube3a Deficient Mice Reveals Novel Genotype Specific Behaviors

Heck, D., Zhao, Y., Roy, S., LeDoux, M. S., Reiter, L. T. — 2008-04-15

Angelman syndrome (AS) is a childhood-onset neurogenetic disorder characterized by functionally severe developmental delay with mental retardation, deficits in expressive language, ataxia, appendicular action tremors and unique behaviors such as inappropriate laughter and stimulus-sensitive hyperexcitibility. Most cases of AS are caused by mutations which disrupt expression of maternal UBE3A. Although some progress has been made in understanding hippocampal-related memory and learning aspects of the disorder using Ube3a deficient mice, the numerous motoric abnormalities associated with AS (ataxia, action tremor, dysarthria, dysphagia, sialorrhea, and excessive chewing/mouthing behaviors) have not been fully explored with mouse models. Here we use a novel quantifiable analysis of fluid consumption and licking behavior along with a battery of motor tests to examine cerebellar and other motor system defects in Ube3a deficient mice. Mice with a maternally-inherited Ube3a deficiency (Ube3a^m-/p+) show defects in fluid consumption behavior which are different from Ube3a^m-/p- mice as well. The rhythm of fluid licking and number of licks per visit were significantly different among the three groups and indicate that not only was fluid consumption dependent on Ube3a expression in the cerebellum, but may also depend on low levels of Ube3a expression in other brain regions. Additional neurological testing revealed defects in both Ube3a^m-/p+ and Ube3a^m-/p- mice in rope climbing, grip strength, gait, and a raised-beam task. Long term observation of fluid consumption behavior is the first phenotype reported that differentiates between mice with a maternal loss of function vs. complete loss of Ube3a in the brain. The neuronal and molecular mechanisms underlying mouse fluid consumption defects specifically associated with maternally inherited Ube3a deficiency may reveal important new insights into the pathobiology of AS in humans.

Functional Characterization of Genetic Variants in NPC1L1 Supports the Sequencing Extremes Strategy to Identify Complex Trait Genes

Fahmi, S., Yang, C., Esmail, S., Hobbs, H. H., Cohen, J. C. — 2008-04-15

Resequencing genes in individuals at extremes of the population distribution constitutes a powerful and efficient strategy to identify sequence variants associated with complex traits. An excess of sequence variants at one extreme relative to the other that is not due to chance or to population stratification constitutes evidence for genetic association and implies the presence of functionally significant sequence variants. Recently, we reported that nonsynonymous sequence variants in Niemann-Pick Type C1-Like 1 (NPC1L1), an intestinal cholesterol transporter, were significantly more common among individuals with low cholesterol absorption than in those with high cholesterol absorption. To determine if sequence variations identified in individuals with low cholesterol absorption affect protein function, we performed studies in cultured cells and in families. Expression of the mutant proteins in Chinese Hamster Ovarian (CHO)-K1 cells revealed that a majority (14 of 20) of the variants identified in low absorbers were associated with very low levels of NPC1L1 protein. In two extended families, mean cholesterol absorption levels, as measured using stable isotopes, were significantly lower in family members with the sequence variants than in those without the variant. These data indicate that the excess of sequence variations in individuals with extreme phenotypes reflects an enrichment of functionally significant variants. These findings are consistent with in silico predictions that some sequence variations found in healthy individuals are as deleterious to protein function as mutations that, in other genes, cause monogenic diseases. Such sequence variations may explain a significant fraction of quantitative phenotypic variation in humans.

Variation in WNT Genes is Associated with Nonsyndromic Cleft Lip with or without Cleft Palate

2008-04-14

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect. Genetic and environmental factors have been causally implicated and studies have begun to delineate genetic contributions. The Wnt genes are involved in regulating midface development and upper lip fusion and are therefore strong candidates for an etiological role in NSCLP. Furthermore, the clf1 region in A/WyN clefting susceptible mice contains the Wnt3 and Wnt9B genes. To assess the role of the Wnt family of genes in NSCLP, we interrogated seven Wnt genes (Wnt3, Wnt3A, Wnt5A, Wnt7A, Wnt8A, Wnt9B, and Wnt11) in our well defined NSCLP dataset. Thirty-eight single nucleotide polymorphisms were genotyped in 132 multiplex NSCLP families and 354 simplex parent-child trios. In the entire dataset, SNPs in three genes, Wnt3A (p=0.006), Wnt 5A (p=0.002) Wnt11 (p=0.0001) were significantly associated with NSCLP after correction for multiple testing. When stratified by ethnicity, the strongest associations were found for SNPs in Wnt3A (p=0.0007), Wnt11 (p=0.0012) and Wnt8A (p=0.0013). Multiple haplotypes in Wnt genes were associated with NSCLP, and gene-gene interactions were observed between Wnt3A and both Wnt3 and Wnt5A (p=0.004 and p=0.039, respectively). This data suggests that alteration in Wnt gene function may perturb formation and/or fusion of the facial processes and predispose to NSCLP.

The molecular mechanism underlying Roberts syndrome involves loss of ESCO2 acetyltransferase activity

2008-04-14

Roberts syndrome/SC phocomelia (RBS) is an autosomal recessive disorder with growth retardation, craniofacial abnormalities and limb reduction. Cellular alterations in RBS include lack of cohesion at the heterochromatic regions around centromeres and the long arm of the Y chromosome, reduced growth capacity, and hypersensitivity to DNA damaging agents. RBS is caused by mutations in ESCO2, which encodes a protein belonging to the highly conserved Eco1/Ctf7 family of acetyltransferases that is involved in regulating sister chromatid cohesion. We identified ten new mutations expanding the number to 26 known ESCO2 mutations. We observed that these mutations result in complete or partial loss of the acetyltransferase domain except for the only missense mutation that occurs in this domain (c.1615T>G, W539G). To investigate the mechanism underlying RBS we analyzed ESCO2 mutations for their effect on enzymatic activity and cellular phenotype. We found that ESCO2 W539G results in loss of autoacetyltransferase activity. The cellular phenotype produced by this mutation causes cohesion defects, proliferation capacity reduction and mitomycin C sensitivity equivalent to those produced by frameshift and nonsense mutations associated with decreased levels of mRNA and absence of protein. We found decreased proliferation capacity in RBS cell lines associated with cell death, but not with increased cell cycle duration, which could be a factor in the development of phocomelia and cleft palate in RBS. In summary, we provide the first evidence that loss of acetyltransferase activity contributes to the pathogenesis of RBS, underscoring the essential role of the enzymatic activity of the Eco1p family of proteins.

The S18Y polymorphic variant of UCH-L1 confers an antioxidant function to neuronal cells

Kyratzi, E., Pavlaki, M., Stefanis, L. — 2008-04-14

A number of studies have associated the S18Y polymorphic variant of UCH-L1 with protection from sporadic Parkinson's Disease (PD). The mechanism involved in this protective function is unknown, but has generally been assumed to be linked to the ubiquitin-proteasome system (UPS). In the current study, we have investigated the effects of overexpression of UCH-L1 and its variants, including S18Y, in neuronal cells. We find that S18Y, but not WT, UCH-L1 confers a specific anti-oxidant protective function when expressed at physiologic levels in human neuroblastoma cells and primary cortical neurons. In contrast, neither WT, nor S18Y UCH-L1 appear to directly impact the proteasome, although they both lead to stabilization of free ubiquitin. Lack of WT mouse UCH-L1 in neurons derived from gad mice led to a decrease of free ubiquitin, but no overall decrease in UPS function or enhanced sensitivity to oxidative stress. We conclude that the S18Y variant of UCH-L1 confers a novel anti-oxidant function that is not present in the WT form and that this function may underlie the protective effects of this variant in certain PD populations. Our results furthermore provide indirect evidence for the importance of oxidative stress as a pathogenetic factor in certain forms of sporadic PD.

Novel mutations in LHX3 are associated with hypopituitarism and sensorineural hearing loss

2008-04-10

Homozygous loss-of-function mutations in the transcription factor LHX3 have been associated with hypopituitarism with structural anterior pituitary defects and cervical abnormalities with or without restricted neck rotation. We report two novel recessive mutations in LHX3 in four patients from two unrelated pedigrees. Clinical evaluation revealed that all four patients exhibit varying degrees of bilateral sensorineural hearing loss, which has not been previously reported in association with LHX3 mutations, in addition to hypopituitarism including ACTH deficiency and an unusual skin and skeletal phenotype in one family. Furthermore, re-evaluation of three patients previously described with LHX3 mutations showed they also exhibit varying degrees of bilateral sensorineural hearing loss.

We have investigated a possible role for LHX3 in inner ear development in humans using in situ hybridization of human embryonic and fetal tissue. LHX3 is expressed in defined regions of the sensory epithelium of the developing inner ear in a pattern overlapping that of SOX2, which precedes the onset of LHX3 expression and is known to be required for inner ear and pituitary development in both mice and humans. Moreover, we show that SOX2 is capable of binding to and activating transcription of the LHX3 proximal promoter in vitro. This study therefore extends the phenotypic spectrum associated with LHX3 mutations to encompass variable sensorineural hearing loss, and suggests a possible interaction between LHX3 and SOX2 likely to be important for development of both the inner ear and the anterior pituitary in human embryonic development.

De novo CoA biosynthesis is required to maintain DNA integrity during development of the Drosophila nervous system

2008-04-10

In a forward genetic screen in Drosophila melanogaster, aimed to identify genes required for normal locomotor function, we isolated dPPCS (the second enzyme of the Coenzyme A biosynthesis pathway). The entire Drosophila CoA synthesis route was dissected, annotated and additional CoA mutants were obtained (dPANK/fumble) or generated (dPPAT-DPCK). Drosophila CoA mutants suffer from neurodegeneration, altered lipid homeostasis, and the larval brains display increased apoptosis. Also, de novo CoA biosynthesis is required to maintain DNA integrity during the development of the central nervous system. In humans, mutations in the PANK2 gene, the first enzyme in the CoA synthesis route, are associated with Pantothenate Kinase-Associated Neurodegeneration (PKAN). Currently, the pathogenesis of this neurodegenerative disease is poorly understood. We provide the first comprehensive analysis of the physiological implications of mutations in the entire CoA biosynthesis route in an animal model system. Surprisingly, our findings reveal a major role of this conserved pathway in maintaining DNA and cellular integrity, explaining how impaired CoA synthesis during CNS development can elicit a neurodegenerative phenotype.

Endocannabinoid receptor 1 gene variations increase risk for obesity and modulate body mass index in European populations

2008-04-08

Background

The therapeutic effects of cannabinoid receptor blockade on obesity associated phenotypes underline the importance of the endocannabinoid pathway on the energy balance. Using a staged-approach, we examined the contribution of the endocannabinoid receptor 1 gene (CNR1) on obesity and Body Mass Index in the European population.

Methods

With the input of CNR1 exons and 3' and 5' regions sequencing and HapMap database, we selected and genotyped 26 tag-SNPs in 1,932 obese cases and 1,173 non-obese controls of French European origin. Variants that showed significant associations (P<0.05) with obesity after correction for multiple-testing were further tested in two additional European cohorts including 2,645 individuals. For the identification of the potential causal variant(s), we further genotyped SNPs in high linkage disequilibrium with the obesity-associated variants.

Results

Of twenty five successfully genotyped CNR1 SNPs, 12 showed nominal evidence of association with childhood obesity, class I & II and/or class III adult obesity (1.16P<0.04). Intronic SNPs rs806381 and rs2023239, which resisted correction for multiple testing were further associated with higher BMI in both Swiss obese subjects and Danish individuals. The genotyping of all know variants in partial linkage disequilibrium (r²>0.5) with these two SNPs in the initial case-control study, identified two better associated SNPs (rs6454674 and rs10485170).

Conclusions

Our study of 5,750 subjects shows that CNR1 variations increase the risk for obesity and modulate BMI in our European population. As CB1 is a drug target for obesity, a pharmacogenetic analysis of the endocannabinoid blockade obesity treatment may be of interest to identify best responders.

A sensitized mutagenesis screen identifies Gli3 as a modifier of Sox10 neurocristopathy

2008-04-07

Haploinsufficiency for the transcription factor SOX10 is associated with the pigmentary deficiencies of Waardenburg syndrome (WS) and is modeled in Sox10 haploinsufficient mice (Sox10^LacZ^/+). As genetic background affects WS severity in both humans and mice, we established an N-ethyl-N-nitrosourea (ENU) mutagenesis screen to identify modifiers that increase the phenotypic severity of Sox10^LacZ^/+ mice. Analysis of 230 pedigrees identified three modifiers, named Modifier of Sox10 neurocristopathies (Mos1, Mos2, and Mos3). Linkage analysis confirmed their locations on mouse chromosomes 13, 4, and 3 respectively, within regions distinct from previously identified WS loci. Positional candidate analysis of Mos1 identified a truncation mutation in a hedgehog-signaling mediator, GLI-Kruppel family member 3 (Gli3). Complementation tests using a second allele of Gli3 (Gli3^Xt-J) confirmed that a null mutation of Gli3 causes the increased hypopigmentation in Sox10^LacZ^/+; Gli3^Mos1^/+ double heterozygotes. Early melanoblast markers (Mitf, Sox10, Dct, and Si) are reduced in Gli3^Mos1^/Mos1 embryos, indicating that loss of GLI3 signaling disrupts melanoblast specification. In contrast, mice expressing only the GLI3 repressor have normal melanoblast specification, indicating that the full-length GLI3 activator is not required for specification of neural crest to the melanocyte lineage. This study demonstrates the feasibility of sensitized screens to identify disease modifier loci and implicates GLI3 and other Hedgehog (HH) signaling components as modifiers of human neurocristopathies.

Sirtuin Inhibition Protects from the Polyalanine Muscular Dystrophy Protein PABPN1

2008-04-07

Oculopharyngeal muscular dystrophy (OPMD) is caused by polyalanine expansion in nuclear protein PABPN1 and characterized by muscle degeneration. Druggable modifiers of proteotoxicity in degenerative diseases, notably the longevity modulators sirtuins, may constitute useful therapeutic targets. However, the modifiers of mutant PABPN1 are unknown. Here, we report that longevity and cell metabolism modifiers modulate mutant PABPN1 toxicity in the muscle cell. Using PABPN1 nematodes that show muscle cell degeneration and abnormal motility, we found that increased dosage of the sirtuin and deacetylase sir–2.1/ SIRT1 exacerbated muscle pathology, an effect dependent on the transcription factor daf-16/FoxO and fuel sensor aak–2/AMPK, while null mutants of sir–2.1, daf-16 and aak-2 were protective. Consistently, the Sir2 inhibitor sirtinol was protective whereas the Sir2 and AMPK activator resveratrol was detrimental. Furthermore, rescue by sirtinol was dependent on daf-16 and not aak-2 whereas aggravation by resveratrol was dependent on aak-2 and not daf-16. Finally, the survival of mammalian cells expressing mutant PABPN1 was promoted by sirtinol and decreased by resveratrol. Altogether, our data identify Sir2 and AMPK inhibition as therapeutic strategies for muscle protection in OPMD, extending the value of druggable proteins in cell maintenance networks to polyalanine diseases.

Bladder tumour derived somatic TSC1 missense mutations cause loss of function via distinct mechanisms

Pymar, L. S., Platt, F. M., Askham, J. M., Morrison, E. E., Knowles, M. A. — 2008-04-07

More than 50% of transitional cell carcinomas of the bladder show loss of heterozygosity (LOH) of a region spanning the TSC1 locus at 9q34 and mutations of TSC1 have been identified in 14.5% of tumours. These comprise nonsense mutations, splicing mutations, small-deletions and missense mutations. Missense mutations are only rarely found in the germline in TSC disease. Therefore we have examined 6 somatic missense mutations found in bladder cancer to determine whether these result in loss of function. We describe loss of function via distinct mechanisms. Five mutations caused mutually exclusive defects at mRNA and protein levels. Of these, two mutations caused pre-mRNA splicing errors that were predicted to result in premature protein truncation and three resulted in markedly reduced stability of exogenous TSC1 protein. Primary tumours with aberrant TSC1 pre-mRNA splicing were confirmed as negative for TSC1 expression by immunohistochemistry. Expression was also significantly reduced in a tumour with a TSC1 missense mutation resulting in diminished protein half-life. A single TSC1 missense mutation identified in a tumour with retained heterozygosity of the TSC1 region on chromosome 9 caused an apparently TSC2- and mTOR-independent localisation defect of the mutant protein. We conclude that although TSC1 missense mutations do not play a major role in causation of TSC disease, they represent a significant proportion of somatic loss of function mutations in bladder cancer.

Therapeutic benefit derived from RNAi-mediated ablation of IMPDH1 transcripts in a murine model of autosomal dominant retinitis pigmentosa (RP10)

2008-04-04

Mutations within the inosine 5’-monophosphate dehydrogenase 1 (IMPDH1) gene cause the RP10 form of autosomal dominant retinitis pigmentosa (adRP), an early-onset retinopathy resulting in extensive visual handicap owing to progressive death of photoreceptors. Apart from the prevalence of RP10, estimated to account for 5-10% of cases of adRP in America and Europe, two observations render this form of RP an attractive target for gene therapy. Firstly, we show that while recombinant adeno-associated viral (rAAV)-mediated expression of mutant human IMPDH1 protein in the mouse retina results in an aggressive retinopathy modelling the human counterpart, expression of a normal human IMPDH1 gene under similar conditions has no observable pathological effect on retinal function, indicating that over-expression of a therapeutic replacement gene may be relatively well tolerated. Secondly, complete absence of IMPDH1 protein in mice with a targeted disruption of the gene results in relatively mild retinal dysfunction, suggesting that significant therapeutic benefit may be derived even from the suppression-only component of an RNAi-based gene therapy. We show that AAV-mediated co-expression in the murine retina of a mutant human IMPDH1 gene together with shRNA validated in vitro and in vivo, targeting both human and mouse IMPDH1, substantially suppresses the negative pathological effects of mutant IMPDH1, at a point where, in the absence of shRNA, expression of mutant protein in the RP10 model essentially ablates all photoreceptors in transfected areas of the retina. These data strongly suggest that an RNAi-mediated approach to therapy for RP10 holds considerable promise for human subjects.

Striatal and nigral pathology in a lentiviral rat model of Machado-Joseph disease

2008-04-03

Machado-Joseph disease (MJD) is a fatal, dominant neurodegenerative disorder. MJD results from polyglutamine repeat expansion in the MJD-1 gene, conferring a toxic gain of function to the ataxin-3 protein. In this study we aimed at overexpressing ataxin-3 in the rat brain using lentiviral vectors, to generate an in vivo MJD genetic model and, to study the disorder in defined brain regions: substantia nigra, an area affected in MJD, cortex and striatum, regions not previously reported to be affected in MJD.

Lentiviral vectors encoding mutant or wild-type human ataxin-3 were injected in the brain of adult rats and the animals were tested for behavioral deficits and neuropathological abnormalities. Striatal pathology was confirmed in transgenic mice and human tissue.

In substantia nigra, unilateral overexpression of mutant ataxin-3 led to: apomorphine-induced turning behavior; formation of ubiquitinated ataxin-3 aggregates; -synuclein immunoreactivity; and loss of dopaminergic markers (TH and VMAT2). No neuropathological changes were observed upon wild-type ataxin-3 overexpression. Mutant ataxin-3 expression in striatum and cortex, resulted in accumulation of misfolded ataxin-3, and within striatum, loss of neuronal markers. Striatal pathology was confirmed by observation in MJD transgenic mice of ataxin-3 aggregates and substantial reduction of DARPP-32 immunoreactivity and, in human striata, by ataxin-3 inclusions, immunoreactive for ubiquitin and -synuclein.

This study demonstrates the use of lentiviral vectors encoding mutant ataxin-3 to produce a model of Machado-Joseph disease and brings evidence of striatal pathology, suggesting that this region may contribute to dystonia and chorea observed in some MJD patients and may represent a target for therapies.

Functional definition of the mutation cluster region of adenomatous polyposis coli in colorectal tumours

Kohler, E. M., Derungs, A., Daum, G., Behrens, J., Schneikert, J. — 2008-04-02

The mutation cluster region (MCR) of ADENOMATOUS POLYPOSIS COLI (APC) is located within the central part of the open reading frame, overlapping with the region encoding the 20 amino acid repeats (20R) that are β-catenin binding sites. Each mutation in the MCR leads to the synthesis of a truncated APC product expressed in a colorectal tumour. The MCR extends from the 3' border of the first 20R coding region to approximately the middle of the third 20R coding region, reflecting both positive and negative selections of the N- and C-terminal halves of the APC protein in colon cancer cells, respectively. In contrast, the second 20R escapes selection and can be either included or excluded from the truncated APC products found in colon cancer cells. To specify the functional outcome of the selection of the mutations, we investigated the β-catenin binding capacity of the first three 20R in N-terminal APC fragments. We found in co-immunoprecipitation and intracellular co-localization experiments that the second 20R is lacking any β-catenin binding activity. Similarly, we also show that the tumor-associated truncations abolish the interaction of β-catenin with the third 20R. Thus, our data provide a functional definition of the MCR: the APC fragments typical of colon cancer are selected for the presence of a single functional 20R, the first one, and are therefore equivalent relative to β-catenin binding.

The loss of methyl-CpG binding protein 1 leads to autism-like behavioral deficits

2008-04-01

Methyl-CpG binding proteins (MBDs) are central components of DNA methylation-mediated epigenetic gene regulation. Alterations of epigenetic pathways are known to be associated with several neurodevelopmental disorders, particularly autism. Our previous studies showed that the loss of Mbd1 led to reduced hippocampal neurogenesis and impaired learning in mice. However, whether MBD1 regulates the autism-related cognitive functions remains unknown. Here we show that Mbd1 mutant (Mbd1^–/–) mice exhibit several core deficits frequently associated with autism, including reduced social interaction, learning deficits, anxiety, defective sensory motor gating, depression, and abnormal brain serotonin activity. Furthermore, we find that Mbd1 can directly regulate the expression of Htr2c, one of the serotonin receptors, by binding to its promoter, and the loss of Mbd1 led to elevated expression of Htr2c. Our results therefore demonstrate the importance of epigenetic regulation in mammalian brain development and cognitive functions. Understanding how the loss of Mbd1 could lead to autism-like behavioral phenotypes would reveal much-needed information about the molecular pathogenesis of autism.

Genetic interaction between Bardet-Biedl syndrome genes and implications for limb patterning

2008-04-01

BBS is a pleiotropic genetically heterogeneous disorder characterized by obesity, retinopathy, polydactyly, cognitive impairment, renal and cardiac anomalies, as well as hypertension and diabetes. Multiple genes are known to independently cause BBS. These genes do not appear to code for the same functional category of proteins; yet mutation of each results in a similar phenotype. Gene knockdown of different bbs genes in zebrafish show strikingly overlapping phenotypes including defective melanosome transport and disruption of the ciliated Kupffer's vesicle. Here, we demonstrate that individual knockdown of bbs1 and bbs3 results in the same prototypical phenotypes as previously reported for other BBS genes. We utilize the zebrafish system to comprehensively determine whether simultaneous pair-wise knockdown of bbs genes reveals genetic interactions between BBS genes. Using this approach, we demonstrate eight genetic interactions between a subset of BBS genes. The synergistic relationships between distinct combinations are not due to functional redundancy but indicate specific interactions within a multi-subunit BBS complex. In addition, we utilize the zebrafish model system to investigate limb development. Human polydactyly is a cardinal feature of BBS not reproduced in Bbs-mouse models. We evaluated zebrafish fin bud patterning and observed altered Sonic hedgehog (SHH) expression and subsequent changes to fin skeletal elements. The SHH fin bud phenotype was also used to confirm specific genetic interactions between BBS genes. This study reveals an in vivo requirement for BBS function in limb bud patterning. Our results provide important new insights into the mechanism and biological significance of BBS

APOE/C1/C4/C2 hepatic control region polymorphism influences plasma apoE and LDL cholesterol levels

2008-03-31

We characterized 102 kb of chromosome 19 containing the apolipoprotein (APO) E/C1/C4/C2 cluster and two flanking genes for common DNA variants associated with plasma LDL-C level. DNA variants were identified by comparing sequences of 48 haploid hybrid cell lines. We genotyped participants (1,943 Whites and 2,046 African-Americans) of the Coronary Artery Risk Development in Young Adults (CARDIA) study for 115 variants. After controlling for the effects of the APOE 2/3/4 polymorphism, a single nucleotide polymorphism (SNP), rs35136575, in the downstream hepatic control region 2 (HCR-2) was associated with LDL-C in Caucasians (p=0.0004), accounting for 1% of variation. We genotyped rs35136575 in the Atherosclerosis Risk in Communities (ARIC) cohort (3,679 African-Americans and 10,427 Whites) and in the Genetic Epidemiology Network of Arteriopathy (GENOA) sibships (1,381 African-Americans in 592 sibships, 1,116 Caucasians in 503 sibships and 1,378 Mexican-Americans in 416 sibships), finding association with LDL-C level in ARIC Caucasians (p=0.0064). Lower plasma LDL-C was observed with the rare allele. Plasma apoE level was strongly associated with HCR-2 variant genotype in all three GENOA samples (p≤0.002), indicating an effect on apoE concentration. Patterns of association for plasma apolipoprotein (apo) A-I, apoB, LDL-C, HDL-C, total cholesterol and triglyceride levels with rs35136575 in the population-based samples evaluated in this study suggest a pleiotropic effect that may be context-dependent.

Missense mutations in the forkhead domain of FOXL2 lead to subcellular mislocalisation, protein aggregation and impaired transactivation

2008-03-27

Mutations of the FOXL2 gene have been shown tocause blepharophimosis syndrome (BPES), characterised by an eyelid malformation associated with premature ovarian failure or not. Recently, polyalanine expansions and truncating FOXL2 mutations have been shown to lead to protein mislocalisation, aggregation and altered transactivation. Here, we study the molecular consequences of 17 naturally-occurring FOXL2 missense mutations. Most of them map to the conserved DNA-binding forkhead domain (FHD). The subcellular localisation and aggregation pattern of the mutant FOXL2 proteins in COS-7 cells was variable and ranged from a diffuse nuclear distribution like the wild-type to extensive nuclear aggregation often in combination with cytoplasmic mislocalisation and aggregation. We also studied the transactivation capacity of the mutants in FOXL2 expressing granulosa-like cells (KGN). Several mutants led to a loss-of-function, while others are suspected to induce a dominant negative effect. Interestingly, one mutant that is located outside the FHD (S217F), appeared to be hypermorphic and had no effect on intracellular protein distribution. This mutation gives rise to a mild BPES phenotype. In general, missense mutations located in the FHD lead to classical BPES and cannot be correlated with expression of the ovarian phenotype. However, a potential predictive value of localisation and transactivation assays in the making of genotype-phenotype correlations is proposed. This is the first study to demonstrate that a significant number of missense mutations in the FHD of FOXL2 lead to mislocalisation, protein aggregation and altered transactivation, and to provide insights into the pathogenesis associated with missense mutations of FOXL2 in human disease.

Diverse driving forces underlie the invariant occurrence of the T42A, E139D, I282V and T468M SHP2 amino acid substitutions causing Noonan and LEOPARD syndromes

2008-03-27

Missense PTPN11 mutations cause Noonan and LEOPARD syndromes (NS and LS), two developmental disorders with pleiomorphic phenotypes. PTPN11 encodes SHP2, an SH2 domain-containing protein tyrosine phosphatase functioning as a signal transducer. Generally, different substitutions of a particular amino acid residue are observed in these diseases, indicating that the crucial factor is the residue being replaced. For a few codons, only one substitution is observed, suggesting the possibility of specific roles for the residue introduced. We analyzed the biochemical behavior and ligand-binding properties of all possible substitutions arising from single-base changes affecting codons 42, 139, 279, 282 and 468 to investigate the mechanisms underlying the invariant occurrence of the T42A, E139D and I282V substitutions in NS and the Y279C and T468M changes in LS. Our data demonstrate that the isoleucine-to-valine change at codon 282 is the only substitution at that position perturbing the stability of SHP2's closed conformation without impairing catalysis, while the threonine-to-alanine change at codon 42, but not other substitutions of that residue, promotes increased phosphopeptide binding affinity. The recognition specificity of the C-SH2 domain bearing the E139D substitution differed substantially from its wild type counterpart acquiring binding properties similar to those observed for the N-SH2 domain, revealing a novel mechanism of SHP2's functional dysregulation. Finally, while functional selection does not seem to occur for the substitutions at codons 279 and 468, we point to deamination of the methylated cytosine at nucleotide 1403 as the driving factor leading to the high prevalence of the T468M change in LS.