Expanding the Genetic Spectrum of ANOS1 Mutations in Patients with Congenital Hypogonadotropic Hypogonadism

STUDY QUESTION: What is the prevalence and functional consequence of ANOS1 (KAL1) mutations in a group of men with congenital hypogonadotropic hypogonadism (CHH)? SUMMARY ANSWER: Three of forty-two (7.1%) patients presented ANOS1 mutations, including a novel splice site mutation leading to exon skipping and a novel contiguous gene deletion associated with ichthyosis. WHAT IS KNOWN ALREADY: CHH is characterized by lack of pubertal development and infertility, due to deficient production, secretion or action of GnRH, and can be associated with anosmia/hyposmia (Kallmann syndrome, KS) or with a normal sense of smell (normosmic CHH). Mutations in the anosmin-1 (ANOS1) gene are responsible for the X-linked recessive form of KS. STUDY DESIGN, SIZE, DURATION: This cross-sectional study included 42 unrelated men with CHH (20 with KS and 22 with normosmic CHH). PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were screened for mutations in the ANOS1 gene by DNA sequencing. Identified mutations were further investigated by RT-PCR analysis and multiplex ligation-dependent probe amplification (MLPA) analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Hemizygous mutations were identified in three (7.1%) KS cases: a novel splice acceptor site mutation (c.542-1G>C), leading to skipping of exon 5 in the ANOS1 transcript in a patient with self-reported normosmia (but hyposmic upon testing); a recurrent nonsense mutation (c.571C>T, p.Arg191*); and a novel 4.8 Mb deletion involving ANOS1 and eight other genes (VCX3B, VCX2, PNPLA4, VCX, STS, HDHD1, VCX3A and NLGN4X) in KS associated with ichthyosis. LIMITATIONS, REASONS FOR CAUTION: Objective olfactory testing was not performed in all cases of self-reported normosmia and this may have underestimated the olfactory deficits. WIDER IMPLICATIONS OF THE FINDINGS: This study further expands the spectrum of known genetic defects associated with CHH and suggests that patients with self-reported normal olfactory function should not be excluded from ANOS1 genetic testing. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Portuguese Foundation for Science and Technology. The authors have no conflicts of interest.info:eu-repo/semantics/publishedVersio

Expanding the Genetic Spectrum of ANOS1 Mutations in Patients with Congenital Hypogonadotropic Hypogonadism

STUDY QUESTION: What is the prevalence and functional consequence of ANOS1 (KAL1) mutations in a group of men with congenital hypogonadotropic hypogonadism (CHH)? SUMMARY ANSWER: Three of forty-two (7.1%) patients presented ANOS1 mutations, including a novel splice site mutation leading to exon skipping and a novel contiguous gene deletion associated with ichthyosis. WHAT IS KNOWN ALREADY: CHH is characterized by lack of pubertal development and infertility, due to deficient production, secretion or action of GnRH, and can be associated with anosmia/hyposmia (Kallmann syndrome, KS) or with a normal sense of smell (normosmic CHH). Mutations in the anosmin-1 (ANOS1) gene are responsible for the X-linked recessive form of KS. STUDY DESIGN, SIZE, DURATION: This cross-sectional study included 42 unrelated men with CHH (20 with KS and 22 with normosmic CHH). PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were screened for mutations in the ANOS1 gene by DNA sequencing. Identified mutations were further investigated by RT-PCR analysis and multiplex ligation-dependent probe amplification (MLPA) analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Hemizygous mutations were identified in three (7.1%) KS cases: a novel splice acceptor site mutation (c.542-1G>C), leading to skipping of exon 5 in the ANOS1 transcript in a patient with self-reported normosmia (but hyposmic upon testing); a recurrent nonsense mutation (c.571C>T, p.Arg191*); and a novel 4.8 Mb deletion involving ANOS1 and eight other genes (VCX3B, VCX2, PNPLA4, VCX, STS, HDHD1, VCX3A and NLGN4X) in KS associated with ichthyosis. LIMITATIONS, REASONS FOR CAUTION: Objective olfactory testing was not performed in all cases of self-reported normosmia and this may have underestimated the olfactory deficits. WIDER IMPLICATIONS OF THE FINDINGS: This study further expands the spectrum of known genetic defects associated with CHH and suggests that patients with self-reported normal olfactory function should not be excluded from ANOS1 genetic testing. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Portuguese Foundation for Science and Technology. The authors have no conflicts of interest.info:eu-repo/semantics/publishedVersio

Brain correlates of pro-social personality traits: a voxel-based morphometry study

Of the five personality dimensions described by the Big Five Personality Model (Costa and McCrae 1992), Extraversion and Agreeableness are the traits most commonly associated with a pro-social orientation. In this study we tested whether a pro-social orientation, as expressed in terms of Extraversion and Agreeableness, is associated with a specific grey matter phenotype. Fifty-two healthy participants underwent magnetic resonance imaging (MRI) and completed the NEO-Five Factor Inventory (NEO-FFI), a self-report measure of the Big Five personality traits. Voxel-based morphometry (VBM) was used to investigate the correlation between brain structure and the personality traits of Agreeableness and Extraversion. We found that Extraversion was negatively correlated with grey matter density in the middle frontal and orbitofrontal gyri while Agreeableness was negatively correlated with grey matter density in the inferior parietal, middle occipital and posterior cingulate gyri. No positive correlations were found. These results suggest that pro-social personality traits seem to be associated with decreases in grey matter density in more frontal regions for Extraversion, and more posterior regions for Agreeableness.This research was funded by the Portuguese Foundation for Science and Technology (FCT): PIC/IC/83290/2007, which is supported by FEDER (POFC - COMPETE), and postdoctoral grant number: SFRH/BPD/75014/2010

Understanding the role of growth factors in modulating stem cell tenogenesis

Current treatments for tendon injuries often fail to fully restore joint biomechanics leading to the recurrence of symptoms, and thus resulting in a significant health problem with a relevant social impact worldwide. Cell-based approaches involving the use of stem cells might enable tailoring a successful tendon regeneration outcome. As growth factors (GFs) powerfully regulate the cell biological response, their exogenous addition can further stimulate stem cells into the tenogenic lineage, which might eventually depend on stem cells source. In the present study we investigate the tenogenic differentiation potential of human- amniotic fluid stem cells (hAFSCs) and adipose-derived stem cells (hASCs) with several GFs associated to tendon development and healing; namely, EGF, bFGF, PDGF-BB and TGF-β1. Stem cells response to biochemical stimuli was studied by screening of tendon-related genes (collagen type I, III, decorin, tenascin C and scleraxis) and proteins found in tendon extracellular matrix (ECM) (Collagen I, III, and Tenascin C). Despite the fact that GFs did not seem to influence the synthesis of tendon ECM proteins, EGF and bFGF influenced the expression of tendon-related genes in hAFSCs, while EGF and PDGF-BB stimulated the genetic expression in hASCs. Overall results on cellular alignment morphology, immunolocalization and PCR analysis indicated that both stem cell source can be biochemically induced towards tenogenic commitment, validating the potential of hASCs and hAFSCs for tendon regeneration strategies.Authors thank the Portuguese Foundation for Science and Technology (FCT) for the research project BIBS (PTDC/CVT/102972/2008) and for the post-doc fellowship grant: SFRH/BPD/86775/2012. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

Background: Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).Methods/Principal Findings: The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.Conclusions/Significance: Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.This work received financial support from the Ministry of Science and Technology of Argentina [PICT 2011-0207 to AGS] and the National Scientific and Technical Research Council in Argentina (CONICET) [PIP 112 2011-010-0974 to AGS]. Work related to evaluation of biological samples was partially sponsored by the Pan-American Health Organization (PAHO) [Small Grants Program PAHO-TDR]; the Drugs and Neglected Diseases Initiative (DNDi, Geneva, Switzerland), Wellcome Trust (London, United Kingdom), SANOFI-AVENTIS (Buenos Aires, Argentina) and the National Council for Science and Technology in Mexico (CONACYT) [FONSEC 161405 to JMR]

Retinoic acid regulates avian lung branching through a molecular network

Retinoic acid (RA) is of major importance during vertebrate embryonic development and its levels need to be strictly regulated otherwise congenital malformations will develop. Through the action of specific nuclear receptors, named RAR/RXR, RA regulates the expression of genes that eventually influence proliferation and tissue patterning. RA has been described as crucial for different stages of mammalian lung morphogenesis, and as part of a complex molecular network that contributes to precise organogenesis; nonetheless, nothing is known about its role in avian lung development. The current report characterizes, for the first time, the expression pattern of RA signaling members (stra6, raldh2, raldh3, cyp26a1, rar alpha, and rar beta) and potential RA downstream targets (sox2, sox9, meis1, meis2, tgf beta 2, and id2) by in situ hybridization. In the attempt of unveiling the role of RA in chick lung branching, in vitro lung explants were performed. Supplementation studies revealed that RA stimulates lung branching in a dose-dependent manner. Moreover, the expression levels of cyp26a1, sox2, sox9, rar beta, meis2, hoxb5, tgf beta 2, id2, fgf10, fgfr2, and shh were evaluated after RA treatment to disclose a putative molecular network underlying RA effect. In situ hybridization analysis showed that RA is able to alter cyp26a1, sox9, tgf beta 2, and id2 spatial distribution; to increase rar beta, meis2, and hoxb5 expression levels; and has a very modest effect on sox2, fgf10, fgfr2, and shh expression levels. Overall, these findings support a role for RA in the proximal-distal patterning and branching morphogenesis of the avian lung and reveal intricate molecular interactions that ultimately orchestrate branching morphogenesis.The authors would like to thank Ana Lima for slide sectioning and Rita Lopes for contributing to the initiation of this project. This work has been funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the Project POCI-01-0145-FEDER-007038; and by the Project NORTE-01-0145- FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio