Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Etude du mode d'action du facteur de transcription Hoxa1 dans la carcinogenèse mammaire

Hoxa1 belongs to the homeotic or Hox gene family coding for homeodomain proteins involved in embryogenesis and organogenesis in bilaterian animals. Studies have shown that Hoxa1 displays oncogenic activity and is overexpressed in the mammary gland in response to a deregulation of the autocrine growth hormone (GH). It has therefore been suggested that Hoxa1 plays a pivotal role in mammary carcinogenesis under those conditions of GH misregulation. While the functions of Hoxa1 have been well investigated, its mode of action under normal and pathologic situations remains poorly documented. The Hoxa1 protein interacts with Pbx transcription factors by the required intermediate of a hexapeptide motif. Replacement of conserved WM amino acids by AA in this hexapeptide (Hoxa1WM-AA mutant) results in a loss of Hoxa1 function during embryonic development. So the normal embryonic activity of Hoxa1 seemingly relies on its partnership with Pbx. Therefore we now addressed the importance of this partnership for the oncogenic activity of Hoxa1. Expression vectors for Hoxa1WM-AA and wild type Hoxa1 have been stably transfected in human breast MCF7 cells. Cellular clones have been obtained and have been validated for Hoxa1 and Hoxa1WM-AA expression, as well as for their proper nuclear localization. The different clones have been characterized for Hoxa1 transcriptional activity, as well as their proliferation, anchorage-independent growth and contact inhibition. Results show that the mutant Hoxa1WM-AA has lost its ability to stimulate oncogenic transformation of the cells. Now we also characterized the Hoxa1 and Hoxa1WM-AA expressing clones by gene profiling to identify responsive targets relevant to the Hoxa1-mediated oncogenic process. Together our data demonstrate that the hexapeptide motif of Hoxa1 is required to confer its oncogenic activity. In view of the hexapeptide importance in the interaction bewteen Hoxa1 and Pbx particularly, we suggest that Hoxa1 activity is related with its partnership with Pbx.(BIOL 3) -- UCL, 201

Bioluminescence as a functional marker of brachial regeneration in Amphiura filiformis (O.F.Muller, 1776) [Echinodermata: Ophiuroidea]

Bioluminescence is the ability to produce visible light and is generated by a biochemical reaction. Among echinoderms, many ophiuroids harbour this capability. Another amazing characteristic of echinoderms is their ability to regenerate. In this work, the luminescence capability of the brittle star Amphiura filiformis is used as an indicator of functional recovery of regenerated arms. Regeneration was experimentally induced and followed for a period of 45 to 60 days post amputation. Different pharmacological stimuli were used and both morphological and luminous parameters were analysed. Analysis shows a rise in KCl-induced luminescence between 45 and 55 days. Luminescence of regenerated arms obtained with neurotransmitter applications showed more variability. This study suggests that morphological recovery seems to be almost complete within 60 days while nervous control of luminescence is still not fully restored

Proximal to distal gradient of luminescence in the arm of Amphiura filiformis (Echinodermata-Ophiuroidea)

Bioluminescence is a widespread phenomenon. Among benthic phyla, echinoderms contain some luminous representatives. The emission of visible light originates from a biochemical reaction where chemical energy is transformed into luminous energy. Two bioluminescent systems exist to date: the luciferin- luciferase and the photoprotein one. We here report for the first time the presence of a luciferin-luciferase system in Amphiura filiformis. New assay methods have been developed to detect luciferin and luciferase contents in A. filiformis tissues. This work shows that luciferin/luciferase contents increase from arm base to the arm tip; this increase parallels the observed raise of the light intensity induced by potassium chloride (KCl) depolarisation of isolated arm. This new approach will provide a useful tool to study functional recovery of luminous capabilities during regeneration in Amphiura filiformis

Hoxa1^WT and Hoxa1^I-V relieve MCF7 cells from contact inhibition, while expressing Hoxa1^WM-AA does not.

<p>MCF7 cells were transiently transfected for Hoxa1^WT, Hoxa1^I-V and Hoxa1^WM-AA, together with Pbx1a and Prep1 cofactors, and grown for three weeks. Controls included cells transfected for the potent oncogene hRAS or cells transfected for Pbx1a and Prep1 only. Foci formation was observed for hRAS, Hoxa1^WT and Hoxa1^I-V transfected cells (arrowheads) while not for CTL and Hoxa1^WM-AA cells.</p

The expression of Hoxa1^WM-AA in human mammary carcinoma cells does not result in increased cell proliferation and growth.

<p>(A) WST-1 based proliferation assays were performed for MCF7-Hoxa1^WT, MCF7-Hoxa1^I-V, MCF7-Hoxa1^WM-AA and MCF7-CTL clones. The proliferation index was determined for each clone as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025247#s4" target="_blank">Materials and Methods</a>. Results were pooled for each type of clones and represented as means ± S.D. of triplicates. *, p<0.05 (ANOVA 2). (B) Cells for MCF7-Hoxa1^WT, MCF7-Hoxa1^I-V, MCF7-Hoxa1^WM-AA and MCF7-CTL clones were inoculated, kept in culture for 16 days and counted after day 4, 7, 9, 11, 14 and 16. Growth curves represent the mean of four independent experiments.</p

The expression of Hoxa1^WM-AA in human mammary carcinoma cells does not result in increased anchorage independent cell growth.

<p>Cells were grown in soft agar and colonies were revealed by crystal violet staining (A) MCF7-Hoxa1^WT and MCF7-Hoxa1^I-V cells produced a lot of colonies in soft agar while CTL and MCF7-Hoxa1^WM-AA only provide a modest colony growth. (B) For each culture, colonies were counted in three random microscopic fields at 16X magnification. Results were pooled for each type of clones and represented as means ± S.D of triplicates. **, p<0.01; ***, p<0.001.</p

Activation of a Hoxa1 target reporter in mammary carcinoma cell clones.

<p>The Hoxa1 target reporter <i>EphA2-r42B-luc</i> is activated in cell clones for Hoxa1^WT and Hoxa1^I-V while not in Hoxa1^WM-AA clones. In each experiment, the pML-EphA2-r42B-luc reporter plasmid was transfected in combination with expression vectors for both Prep1 and Pbx1a. The constitutively active pCMV-LacZ reporter plasmid was added as a transfection control. Results were calculated by a luciferase/β-galactosidase ratio, pooled for each type of clones and represented as means ± S.D. of triplicates. *, p<0.05 and ***, p<0.001 (ANOVA 2).</p

Transcriptional activity and relative expression of Hoxa1 variants.

<p>(A) The Hoxa1 target reporter <i>EphA2-r42B-luc</i> is activated in MCF7 cells in the presence of expression vectors for Hoxa1^WT, Hoxa1^I-V while not in the presence of Hoxa1^WM-AA or of an empty (CTL) plasmid. In each experiment, the pML-EphA2-r42B-luc reporter plasmid was transfected in combination with expression vectors for both Prep1 and Pbx1a. Results were calculated by a luciferase/β-galactosidase ratio and represented as means ± S.D. of triplicates. ***, p<0.001 (ANOVA2). (B) Detection of Hoxa1 variant proteins from whole cell lysates obtained from transiently transfected MCF7 cells reveal that Hoxa1^WT, Hoxa1^I-V and Hoxa1^WM-AA proteins are equally expressed and stable. No Hoxa1 protein could be detected from MCF7 cells or from cells transfected with an empty (CTL) expression vector. Detection of constitutively expressed β-actin protein was performed as control load.</p

Characterization of MCF7 clones for the constitutive expression of Hoxa1 variants.

<p>(A) Expression of Hoxa1, Neomycin resistance (Neo), Pbx1 and β-actin genes was detected by RT-PCR. While MCF7 cells do not express Hoxa1, clones obtained the stable transfection of Hoxa1^WT, Hoxa1^I-V and Hoxa1^WM-AA coding plasmids express the Hoxa1 variants at similar levels (β-actin used as reference). All cells express the endogenous Pbx1 gene. (B) The Hoxa1 and (C) PBX1B protein immunolocalisation reveals that both proteins localize into the cell nucleus.</p