Molecular and physiological responses to desiccation indicate the abscisic acid pathway is conserved in the peat moss, Sphagnum

Mosses of the genus Sphagnum are the main components of peatlands, a major carbon-storing ecosystem. Changes in precipitation patterns are predicted to affect water relations in this ecosystem, but the effect of desiccation on the physiological and molecular processes in Sphagnum is still largely unexplored. Here we show that different Sphagnum species have differential physiological and molecular responses to desiccation but, surprisingly, this is not directly correlated with their position in relation to the water table. In addition, the expression of drought responsive genes is increased upon water withdrawal in all species. This increase in gene expression is accompanied by an increase in abscisic acid (ABA), supporting a role for ABA during desiccation responses in Sphagnum. Not only do ABA levels increase upon desiccation, but Sphagnum plants pre-treated with ABA display increased tolerance to desiccation, suggesting that ABA levels play a functional role in the response. In addition, many of the ABA signalling components are present in Sphagnum and we demonstrate, by complementation in Physcomitrium patens, that Sphagnum ABI3 is functionally conserved. The data presented here, therefore, support a conserved role for ABA in desiccation responses in Sphagnum

Genome-Wide Association Study of Circulating Estradiol, Testosterone, and Sex Hormone-Binding Globulin in Postmenopausal Women

Genome-wide association studies (GWAS) have successfully identified common genetic variants that contribute to breast cancer risk. Discovering additional variants has become difficult, as power to detect variants of weaker effect with present sample sizes is limited. An alternative approach is to look for variants associated with quantitative traits that in turn affect disease risk. As exposure to high circulating estradiol and testosterone, and low sex hormone-binding globulin (SHBG) levels is implicated in breast cancer etiology, we conducted GWAS analyses of plasma estradiol, testosterone, and SHBG to identify new susceptibility alleles. Cancer Genetic Markers of Susceptibility (CGEMS) data from the Nurses’ Health Study (NHS), and Sisters in Breast Cancer Screening data were used to carry out primary meta-analyses among ∼1600 postmenopausal women who were not taking postmenopausal hormones at blood draw. We observed a genome-wide significant association between SHBG levels and rs727428 (joint β = -0.126; joint P = 2.09×10–16), downstream of the SHBG gene. No genome-wide significant associations were observed with estradiol or testosterone levels. Among variants that were suggestively associated with estradiol (P<10–5), several were located at the CYP19A1 gene locus. Overall results were similar in secondary meta-analyses that included ∼900 NHS current postmenopausal hormone users. No variant associated with estradiol, testosterone, or SHBG at P<10–5 was associated with postmenopausal breast cancer risk among CGEMS participants. Our results suggest that the small magnitude of difference in hormone levels associated with common genetic variants is likely insufficient to detectably contribute to breast cancer risk

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

Methylprednisolone Blocks Autoantibody-Induced Tissue Damage in Experimental Models of Bullous Pemphigoid and Epidermolysis Bullosa Acquisita through Inhibition of Neutrophil Activation

Corticosteroids are regularly used to treat autoimmune diseases, such as bullous pemphigoid (BP). In BP, autoantibodies bind to type XVII collagen (COL17), located at the dermal-epidermal junction. A crucial role of neutrophils in experimental BP has been established. Specifically, reactive oxygen species and proteolytic granule enzymes mediate tissue injury. Therefore, we investigated the effects of methylprednisolone (MP) on neutrophils, which are likely to be affected by topical treatment. First, MP inhibited dermal-epidermal separation ex vivo in cryosections of the human skin induced by co-incubation of BP autoantibodies with neutrophils from healthy volunteers. Next, MP inhibited neutrophil activation in vitro induced by immune complexes (ICs) of COL17 and autoantibodies. This neutrophil activation was associated with phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and Akt. In turn, inhibition of ERK1/2, p38 MAPK, or Akt phosphorylation inhibited neutrophil activation by IC in vitro and dermal-epidermal separation ex vivo. In addition, we observed an increase of p38 MAPK phosphorylation in dermal infiltrates of BP patients. Treatment of mice with either MP or inhibitors of p38-MAPK or ERK1/2 phosphorylation impaired induction of autoantibody- or irritant-induced neutrophil-dependent inflammation. We here identify the inhibition of Akt, ERK1/2, and p38 MAPK phosphorylation as molecular mechanisms to promote MP's therapeutic effects.Journal of Investigative Dermatology advance online publication, 4 April 2013; doi:10.1038/jid.2013.91

Variant interpretation in molecular autopsy: a useful dilemma

Sudden cardiac death (SCD) in adolescents and young adults may be the first manifestation of an inherited arrhythmic syndrome. Thus identification of a genetic origin in sudden death cases deemed inconclusive after a comprehensive autopsy and may help to reduce the risk of lethal episodes in the remaining family. Using next-generation sequencing (NGS), a large number of variants of unknown significance (VUS) are detected. In the majority of cases, there is insufficient evidence of pathogenicity, representing a huge dilemma in current genetic investigations. Misinterpretation of such variants may lead to inaccurate genetic diagnoses and/or the adoption of unnecessary and/or inappropriate therapeutic approaches. In our study, we applied current (ACMG) recommendations for variant classification in post-mortem genetic screening of a cohort of 56 SCD victims. We identified a total 53 rare protein-altering variants (MAF < 0.2%) classified as VUS or worse. Twelve percent of the cases exhibited a clinically actionable variant (pathogenic, likely pathogenic or VUS - potentially pathogenic) that would warrant cascade genetic screening in relatives. Most of the variants detected by means of the post-mortem genetic investigations were VUS. Thus, genetic testing by itself might be fairly meaningless without supporting background data. This data reinforces the need for an experienced multidisciplinary team for obtaining reliable and accountable interpretations of variant significance for elucidating potential causes for SCDs in the young. This enables the early identification of relatives at risk or excludes family members as genetic carriers. Also, development of adequate forensic guidelines to enable appropriate interpretation of rare genetic variants is fundamental

Anti-p200 pemphigoid patients’ sera induce dermal-epidermal separation ex vivo.

<p>Sera from anti-p200 pemphigoid patients recruit neutrophils to the dermal-epidermal junction (DEJ) and induce dermal-epidermal separation (DES) in cryosections of human skin. Anti-p200 pemphigoid sera (anti-p200 pemphigoid; b, e), but not from sera of healthy volunteers (HV; c, f), recruited leukocytes to the DEJ after 1 h of incubation (b, c) and induced DES after 3 h (e, f). As positive control for DES, IgG from a patient with bullous pemphigoid was used (BP a, d). Recruited neutrophils are marked by arrows, base of the split is marked by black triangles. All sections were stained with hematoxylin and eosin. Magnification, x200.</p

Human IgG specific to laminin γ1 is not pathogenic ex vivo.

<p>Using recombinant forms of the C-terminus of laminin γ1 (hLAMC1-cterm) and full length laminin γ1 (LAMC1-FL) IgG specific for hLAMC1-cterm (a, c; lane 3) and LAMC1-FL (b, lane 3; c lane 5 ) was generated from anti-p200 pemphigoid serum (a, b, c; lane 2), as well as serum depleted from anti-hLAMC1-cterm (a, c, lane 4) and LAMC1-FL reactivity (b, lane 4; c lane 6) reactivity, respectively, as shown by immunoblotting with recombinant hLAMC1-cterm (a), LAMC1-FL (b), and extract of human dermis<u>-</u>(c). Interestingly, serum depleted from anti-hLAMC1-cterm and LAMC1-FL reactivity, respectively, (a, b; lane 4) still labeled the p200 protein in dermal extract (c, lane 4, lane 6). Monoclonal antibody against LAMC1 (a, b, c; lane 1) and serum from a healthy volunteer (a, b; lane 5; c lane 7) were used as controls. Arrows indicate the positions of the proteins and bars the molecular weight markers (a, 34 kD and 26 kD; b, 200 kD; c, 200 kD). hLAMC1-cterm-specific (h, i) and LAMC1-FL-specific patients IgG (l, m) and the monoclonal anti-LAMC1 antibody (d, e) labeled the dermal-epidermal junction (DEJ) by indirect immunofluorescence (IF) microscopy but did not induce dermal-epidermal separation (DES). In contrast, serum depleted from reactivity against hLAMC1-cterm (j, k), and LAMC1-FL (n, o), respectively, as well as patient serum (f, g) resulted in DES (black triangles mark base of the split). While untouched patient serum (f) as well as serum depleted from anti-hLAMC1-cterm (j) and LAMC1-FL reactivity (n), respectively, stained the DEJ of human skin in a linear pattern, the monoclonal anti-hLAMC1-antibody (d), hLAMC1-cterm-specific patient<u>-</u>IgG (h), and hLAMC1-FL-specific patient IgG showed an additional staining of basal keratinocytes. Serum from a healthy volunteer was used as control (p, q). Magnification: x200.</p

Passive transfer of rabbit anti-mLAMC1-cterm IgG into adult mice is not pathogenic.

<p>Rabbit IgG against the murine laminin γ1 C-terminus (mLAMC1-cterm) was not pathogenic when passively transferred into adult C57BL/6 and BALB/c mice. Injection of 15 mg rabbit anti-mLAMC1-cterm IgG every second day for 10 days did not result in clinical or histopathological (a, e) lesions on day 12. Linear deposition of rabbit IgG at the dermal-epidermal junction (DEJ) was only observed in 2 of 5 C57BL/6 (b) and one of 5 BALB/c mice (f), while staining of murine C3 was negative in all mice (c, g). At day 12, in sera of all 10 mice, rabbit IgG labeled the basal keratinocytes at the DEJ of normal mouse skin (d, h) and reacted with recombinant mLAMC1-cterm by ELISA (i) and immunoblotting (j, C57BL/6, lanes 1; BALB/c, lane 2) and the 200 kDa p200 protein in extract of murine dermis (k, C57BL/6 lane 1; BALB/c, lane 2). Normal mouse sera (j and k, C57BL/6, lane 3; BALB/c, lane 4) were used as controls.</p