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Thermal nociception involves the transmission of temperature-related noxious information from the periphery to the CNS and is a heritable trait that could predict transition to persistent pain. Rodent forward genetics complement human studies by controlling genetic complexity and environmental factors, analysis of end point tissue, and validation of variants on appropriate genetic backgrounds. Reduced complexity crosses between nearly identical inbred substrains with robust trait differences can greatly facilitate unbiased discovery of novel genes and variants. We found BALB/cByJ mice showed enhanced sensitivity on the 53.5°C hot plate and mechanical stimulation in the von Frey test compared to BALB/cJ mice and replicated decreased gross brain weight in BALB/cByJ versus BALB/cJ. We then identified a quantitative trait locus (QTL) on chromosome 13 for hot plate sensitivity (LOD = 10.7; p < 0.001; peak = 56 Mb) and a QTL for brain weight on chromosome 5 (LOD = 8.7; p < 0.001). Expression QTL mapping of brain tissues identified H2afy (56.07 Mb) as the top transcript with the strongest association at the hot plate locus (FDR = 0.0002) and spliceome analysis identified differential exon usage within H2afy associated with the same locus. Whole brain proteomics further supported decreased H2AFY expression could underlie enhanced hot plate sensitivity, and identified ACADS as a candidate for reduced brain weight. To summarize, a BALB/c reduced complexity cross combined with multiple-omics approaches facilitated identification of candidate genes underlying thermal nociception and brain weight. These substrains provide a powerful, reciprocal platform for future validation of candidate variants.
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Nociceptividade , Locos de Características Quantitativas , Animais , Encéfalo , Mapeamento Cromossômico , Camundongos , Camundongos Endogâmicos BALB C , Locos de Características Quantitativas/genéticaRESUMO
Understanding the pharmacogenomics of opioid metabolism and behavior is vital to therapeutic success, as mutations can dramatically alter therapeutic efficacy and addiction liability. We found robust, sex-dependent BALB/c substrain differences in oxycodone behaviors and whole brain concentration of oxycodone metabolites. BALB/cJ females showed robust state-dependent oxycodone reward learning as measured via conditioned place preference when compared with the closely related BALB/cByJ substrain. Accordingly, BALB/cJ females also showed a robust increase in brain concentration of the inactive metabolite noroxycodone and the active metabolite oxymorphone compared with BALB/cByJ mice. Oxymorphone is a highly potent, full agonist at the mu opioid receptor that could enhance drug-induced interoception and state-dependent oxycodone reward learning. Quantitative trait locus (QTL) mapping in a BALB/c F2 reduced complexity cross revealed one major QTL on chromosome 15 underlying brain oxymorphone concentration that explained 32% of the female variance. BALB/cJ and BALB/cByJ differ by fewer than 10,000 variants, which can greatly facilitate candidate gene/variant identification. Hippocampal and striatal cis-expression QTL (eQTL) and exon-level eQTL analysis identified Zhx2, a candidate gene coding for a transcriptional repressor with a private BALB/cJ retroviral insertion that reduces Zhx2 expression and sex-dependent dysregulation of cytochrome P450 enzymes. Whole brain proteomics corroborated the Zhx2 eQTL and identified upregulated CYP2D11 that could increase brain oxymorphone in BALB/cJ females. To summarize, Zhx2 is a highly promising candidate gene underlying brain oxycodone metabolite levels. Future studies will validate Zhx2 and its site of action using reciprocal gene editing and tissue-specific viral manipulations in BALB/c substrains. SIGNIFICANCE STATEMENT: Our findings show that genetic variation can result in sex-specific alterations in whole brain concentration of a bioactive opioid metabolite after oxycodone administration, reinforcing the need for sex as a biological factor in pharmacogenomic studies. The cooccurrence of female-specific increased oxymorphone and state-dependent reward learning suggests that this minor yet potent and efficacious metabolite of oxycodone could increase opioid interoception and drug-cue associative learning of opioid reward, which has implications for cue-induced relapse of drug-seeking behavior and for precision pharmacogenetics.
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Encéfalo , Proteínas de Homeodomínio , Oxicodona , Oximorfona , Analgésicos Opioides/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Feminino , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oxicodona/farmacologia , Oximorfona/farmacologia , RecompensaRESUMO
Bioanalysis, a key supporting function for generating data for pre-clinical and clinical studies in drug development, is under the regulation of local agencies as well as global organizations to ensure the data integrity and quality in submission. As major regulatory agencies and organizations, the US Food and Drug Administration, the European Medicines Agency and the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use have been updating their industry guidance for bioanalytical method validation, to keep up with the development new modalities, technologies and regulations. This article summarizes the recent updates and any clarifications and controversies triggered by those updates. Perspectives and recommendations are given based on our own experience as well as commonly accepted practice in the bioanalytical community.
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Química Farmacêutica , Cromatografia , Química Farmacêutica/legislação & jurisprudência , Química Farmacêutica/normas , Cromatografia/métodos , Cromatografia/normas , Ensaios Clínicos como Assunto , Humanos , Preparações Farmacêuticas/sangue , Preparações Farmacêuticas/urina , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estados Unidos , United States Food and Drug AdministrationRESUMO
Diagnosing and treating asthma in paediatric patients remains challenging, with many children and adolescents remaining uncontrolled despite treatment. Selecting the most appropriate pharmacological treatment to add onto inhaled corticosteroids (ICS) in children and adolescents with asthma who remain symptomatic despite ICS can be difficult. This literature review compares the efficacy and safety of long-acting ß2-agonists (LABAs), leukotriene receptor antagonists (LTRAs) and long-acting muscarinic antagonists (LAMAs) as add-on treatment to ICS in children and adolescents aged 4-17 years.A literature search identified a total of 29 studies that met the inclusion criteria, including 21 randomised controlled trials (RCTs) of LABAs versus placebo, two RCTs of LAMAs (tiotropium) versus placebo, and four RCTs of LTRA (montelukast), all as add-on to ICS. In these studies, tiotropium and LABAs provided greater improvements in lung function than LTRAs, when compared with placebo as add-on to ICS. Although exacerbation data were difficult to interpret, tiotropium reduced the risk of exacerbations requiring oral corticosteroids when added to ICS, with or without additional controllers. LABAs and LTRAs had a comparable risk of asthma exacerbations with placebo when added to ICS. When adverse events (AEs) or serious AEs were analysed, LABAs, montelukast and tiotropium had a comparable safety profile with placebo.In conclusion, this literature review provides an up-to-date overview of the efficacy and safety of LABAs, LTRAs and LAMAs as add-on to ICS in children and adolescents with asthma. Overall, tiotropium and LABAs have similar efficacy, and provide greater improvements in lung function than montelukast as add-on to ICS. All three controller options have comparable safety profiles.
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Agonistas de Receptores Adrenérgicos beta 2/administração & dosagem , Asma/tratamento farmacológico , Progressão da Doença , Antagonistas de Leucotrienos/administração & dosagem , Pulmão/fisiologia , Brometo de Tiotrópio/administração & dosagem , Adolescente , Antiasmáticos/administração & dosagem , Asma/diagnóstico , Asma/fisiopatologia , Broncodilatadores/administração & dosagem , Criança , Pré-Escolar , Preparações de Ação Retardada/administração & dosagem , Humanos , Pulmão/efeitos dos fármacos , Ensaios Clínicos Controlados Aleatórios como Assunto/métodosRESUMO
Chronic spontaneous urticaria is challenging to manage and substantially affects quality of life. This US, non-interventional qualitative study examined patients' clinical journeys and emotional burden from symptom onset through disease management. Chronic spontaneous urticaria patients participated in interviews and completed diaries focusing on disease and treatment history/perspectives, impact on personal/family life, and relationships with physicians/other healthcare providers. Physicians were interviewed about their views on disease management and patient care. Twenty-five patients, previously or currently receiving chronic spontaneous urticaria treatment(s), and 12 physicians participated. Key stages following symptom onset were identified: Crisis (associated with feelings of torment/disorientation/shock); Searching for answers (puzzlement/frustration/anxiety); Diagnosis (relief/satisfaction/fear/isolation); and Disease management (frustration/hope/powerlessness). Findings revealed patients' perceptions and experiences of chronic spontaneous urticaria, including living with a 'skinemy', experiencing their 'own personal hell' and feeling 'like an experiment'. Awareness of unmet needs in patient care/management identified in this study may ultimately improve patient support and enhance physicians' understanding of disease burden.
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Adaptação Psicológica , Urticária Crônica/psicologia , Efeitos Psicossociais da Doença , Qualidade de Vida , Adulto , Idoso , Urticária Crônica/diagnóstico , Urticária Crônica/terapia , Emoções , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Pesquisa Qualitativa , Fatores de Tempo , Estados Unidos , Adulto JovemRESUMO
BACKGROUND: Adults and adolescents were included in 3 phase 3 omalizumab trials in chronic idiopathic urticaria (CIU): ASTERIA I, ASTERIA II, and GLACIAL. OBJECTIVE: To describe the baseline clinical profile of adolescent patients with CIU enrolled in the omalizumab trials to add to the limited literature available on CIU in this population. METHODS: Data for patient demographics, baseline clinical disease characteristics, medical history, and previous CIU medication information (not efficacy assessments) from phase 3 omalizumab trials were pooled and descriptive statistical analyses performed for adolescent (12 to <18 years old) and adult (≥18 years old) subgroups. Inferential analysis was inappropriate, partly because of small sample size in the adolescent subgroup. RESULTS: The pooled population of 975 patients with CIU included 39 adolescents (4.0%). Demographics of adolescents and adults with CIU were similar, but compared with adults, fewer adolescents had positive Chronic Urticaria Index test results. Baseline clinical disease characteristics were also similar between the subgroups, with the number of previous CIU medications slightly lower in adolescents compared with adults. Medical history and existing conditions in adolescents tended to be more allergy than cardiovascular related, and fewer experienced angioedema compared with adults. CONCLUSION: Pooled data indicate differences in baseline demographic and clinical characteristics between adult and adolescent patient subgroups. This finding helps augment our understanding of the clinical profile of CIU in adolescents, but larger-scale studies in this population are warranted. TRIAL REGISTRATION: ClinicalTrials.gov Identifiers: NCT01287117 (ASTERIA I), NCT01292473 (ASTERIA II), and NCT01264939 (GLACIAL).
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Antiasmáticos/uso terapêutico , Omalizumab/uso terapêutico , Urticária/diagnóstico , Urticária/tratamento farmacológico , Adolescente , Adulto , Idoso , Criança , Doença Crônica , Ensaios Clínicos Fase III como Assunto , Comorbidade , Feminino , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Masculino , Pessoa de Meia-Idade , Ensaios Clínicos Controlados Aleatórios como Assunto , Fatores de Risco , Resultado do Tratamento , Urticária/etiologia , Adulto JovemRESUMO
Uncompetitive inhibition is an effective strategy for suppressing dysregulated enzymes and their substrates, but discovery of suitable ligands depends on often-unavailable structural knowledge and serendipity. Hence, despite surging interest in mass spectrometry-based target identification, proteomic studies of substrate-dependent target engagement remain sparse. Herein, we describe the Thermal Shift Assay with ATP and RNA (TSAR) as a template for proteome-wide discovery of substrate-dependent ligand binding. Using proteomic thermal shift assays, we show that simple biochemical additives can facilitate detection of target engagement in native cell lysates. We apply our approach to rocaglates, a family of molecules that specifically clamp RNA to eukaryotic translation initiation factor 4A (eIF4A), DEAD-box helicase 3X (DDX3X), and potentially other members of the DEAD-box (DDX) family of RNA helicases. To identify unexpected interactions, we optimized a target class-specific thermal denaturation window and evaluated ATP analog and RNA probe dependencies for key rocaglate-DDX interactions. We report novel DDX targets of the rocaglate clamping spectrum, confirm that DDX3X is a common target of several widely studied analogs, and provide structural insights into divergent DDX3X affinities between synthetic rocaglates. We independently validate novel targets of high-profile rocaglates, including the clinical candidate Zotatifin (eFT226), using limited proteolysis-mass spectrometry and fluorescence polarization experiments. Taken together, our study provides a model for screening uncompetitive inhibitors using a systematic chemical-proteomics approach to uncover actionable DDX targets, clearing a path towards characterization of novel molecular clamps and associated RNA helicase targets.
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Asma/tratamento farmacológico , Broncodilatadores/farmacocinética , Brometo de Tiotrópio/farmacocinética , Broncodilatadores/administração & dosagem , Broncodilatadores/sangue , Criança , Método Duplo-Cego , Feminino , Humanos , Masculino , Brometo de Tiotrópio/administração & dosagem , Brometo de Tiotrópio/sangue , Resultado do TratamentoRESUMO
PURPOSE: The treatment of complex neurological diseases often requires the administration of large therapeutic drugs, such as antisense oligonucleotide (ASO), by lumbar puncture into the intrathecal space in order to bypass the blood-brain barrier. Despite the growing number of ASOs in clinical development, there are still uncertainties regarding their dosing, primarily around their distribution and kinetics in the brain following intrathecal injection. The challenge of taking measurements within the delicate structures of the central nervous system (CNS) necessitates the use of non-invasive nuclear imaging, such as positron emission tomography (PET). Herein, an emergent strategy known as "pretargeted imaging" is applied to image the distribution of an ASO in the brain by developing a novel PET tracer, [18F]F-537-Tz. This tracer is able to undergo an in vivo "click" reaction, covalently binding to a trans-cyclooctene conjugated ASO. PROCEDURES: A novel small molecule tracer for pretargeted PET imaging of ASOs in the CNS is developed and tested in a series of in vitro and in vivo experiments, including biodistribution in rats and non-human primates. RESULTS: In vitro data and extensive in vivo rat data demonstrated delivery of the tracer to the CNS, and its successful ligation to its ASO target in the brain. In an NHP study, the slow tracer kinetics did not allow for specific binding to be determined by PET. CONCLUSION: A CNS-penetrant radioligand for pretargeted imaging was successfully demonstrated in a proof-of-concept study in rats, laying the groundwork for further optimization.
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Química Click , Compostos Radiofarmacêuticos , Animais , Ratos , Química Click/métodos , Compostos Radiofarmacêuticos/química , Distribuição Tecidual , Oligonucleotídeos Antissenso/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismoRESUMO
The human immunological mechanisms defining the clinical outcome of SARS-CoV-2 infection remain elusive. This knowledge gap is mostly driven by the lack of appropriate experimental platforms recapitulating human immune responses in a controlled human lung environment. Here, we report a mouse model (i.e., HNFL mice) co-engrafted with human fetal lung xenografts (fLX) and a myeloid-enhanced human immune system to identify cellular and molecular correlates of lung protection during SARS-CoV-2 infection. Unlike mice solely engrafted with human fLX, HNFL mice are protected against infection, severe inflammation, and histopathological phenotypes. Lung tissue protection from infection and severe histopathology associates with macrophage infiltration and differentiation and the upregulation of a macrophage-enriched signature composed of 11 specific genes mainly associated with the type I interferon signaling pathway. Our work highlights the HNFL model as a transformative platform to investigate, in controlled experimental settings, human myeloid immune mechanisms governing lung tissue protection during SARS-CoV-2 infection.
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COVID-19 , Animais , COVID-19/genética , Modelos Animais de Doenças , Humanos , Imunidade Inata , Pulmão/patologia , Macrófagos , Camundongos , SARS-CoV-2RESUMO
INTRODUCTION: In pediatric patients with asthma, measurements of forced expiratory volume in 1 s (FEV1) may be normal or may not correlate with symptom severity. Forced expiratory flow at 25-75% of the vital capacity (FEF25-75%) is a potentially more sensitive parameter for assessing peripheral airway function. This post hoc analysis compared FEF25-75% with FEV1 as an endpoint to assess bronchodilator responsiveness in children with asthma. METHODS: Change from baseline in trough FEF25-75% and trough FEV1 following treatment with either tiotropium (5 µg or 2.5 µg) or placebo Respimat® was analyzed in four phase III trials in children (aged 6-11 years) and adolescents (aged 12-17 years) with symptomatic moderate (VivaTinA-asthma® and PensieTinA-asthma®) and mild (CanoTinA-asthma® and RubaTinA-asthma®) asthma. Data from all treatment arms were pooled and correlations between FEF25-75% and FEV1 were calculated and analyzed. RESULTS: A total of 1590 patients were included in the analysis. Tiotropium Respimat® consistently improved FEF25-75% and FEV1 versus placebo, although in adolescents with severe asthma, the observed improvements were not statistically significant. Improvements in FEF25-75% response with tiotropium versus placebo were largely more pronounced than improvements in FEV1. Statistical assessment of the correlation of FEV1 and FEF25-75% showed moderate-to-high correlations (Pearson's correlation coefficients 0.73-0.80). CONCLUSIONS: In pediatric patients, FEF25-75% may be a more sensitive measure to detect treatment response, certainly to tiotropium, than FEV1 and should be evaluated as an additional lung function measurement.
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The burden of uncontrolled asthma in children and adolescents is high. Treatment options for pediatric patients (aged under 18 years) with asthma are largely influenced by the Global Initiative for Asthma recommendations. Algorithms for adolescents (12-18 years) and adults are identical, but recommendations for children aged under 6 years and 6-11 years differ. Although the goals of treatment for pediatric patients with asthma are similar to those for adults, relatively few new therapies have been approved for this patient population within the last decade. Designing clinical trials involving children presents several challenges, notably that children are often less able to perform lung function tests, and traditional endpoints used in clinical trials with adults, such as forced expiratory volume in 1 second, asthma exacerbations and questionnaires, have limitations associated with their use in children. There are also ethical considerations related to the performance of longer placebo-controlled exacerbation trials. This review considers additional clinical endpoints to those traditionally reported, including forced expiratory flow at 25%-75% of forced vital capacity, which may help shed light on which treatments are most effective for use in pediatric patients with asthma. The pros and cons of specific and potentially clinically relevant endpoints are considered, along with device considerations and patient preferences that may enhance adherence and quality of life. Recent advances in the management of children and adolescents, including the US Food and Drug Administration and European Medicines Agency approval of tiotropium in patients with asthma aged 6 years and over, are also discussed.
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BACKGROUND: Tiotropium add-on therapy has demonstrated efficacy and safety in 6- to 17-year-olds with symptomatic asthma despite treatment with inhaled corticosteroids (ICSs), with or without additional controllers. Pediatric patients often have a significant allergic component to their asthma. OBJECTIVE: To explore whether responses to tiotropium add-on were influenced by patients' type 2 status, assessed by serum IgE levels and blood eosinophil counts. METHODS: Data from 2 phase III trials in symptomatic moderate asthma (CanoTinA-asthma; RubaTinA-asthma) and 2 phase III trials in symptomatic severe asthma (VivaTinA-asthma; PensieTinA-asthma) were pooled by severity. Patients were treated with tiotropium 5 µg, tiotropium 2.5 µg, or placebo (2 puffs once daily via Respimat inhaler), as add-on to ICSs, with or without additional controllers. Modeling of efficacy outcomes was performed over the whole range of baseline IgE levels and blood eosinophil counts, and the treatment effect of the tiotropium doses was presented graphically. RESULTS: Improvements with tiotropium in peak FEV1 within 3 hours postdose, trough FEV1, forced expiratory flow at 25% to 75% of the pulmonary volume, and FEV1/forced vital capacity responses were generally consistent across the range of baseline IgE levels and blood eosinophil counts. Risk of exacerbations and improvement in Asthma Control Questionnaire responder rates with tiotropium were also largely independent of IgE levels or eosinophil counts. CONCLUSIONS: This exploratory analysis suggests that improvements with tiotropium as add-on to ICSs, with or without additional controllers, in 6- to 17-year-olds with symptomatic asthma do not vary according to systemic markers of T2 inflammation, namely, total IgE and blood eosinophil counts.
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Asma/tratamento farmacológico , Broncodilatadores/uso terapêutico , Brometo de Tiotrópio/uso terapêutico , Administração por Inalação , Adolescente , Corticosteroides/uso terapêutico , Asma/sangue , Asma/imunologia , Asma/fisiopatologia , Criança , Método Duplo-Cego , Eosinófilos/efeitos dos fármacos , Feminino , Volume Expiratório Forçado , Humanos , Imunoglobulina E/sangue , Contagem de Leucócitos , Masculino , Fenótipo , Resultado do Tratamento , Capacidade VitalRESUMO
Optimization of isoquinolinone PI3K inhibitors led to the discovery of a potent inhibitor of PI3K-γ (26 or IPI-549) with >100-fold selectivity over other lipid and protein kinases. IPI-549 demonstrates favorable pharmacokinetic properties and robust inhibition of PI3K-γ mediated neutrophil migration in vivo and is currently in Phase 1 clinical evaluation in subjects with advanced solid tumors.
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BACKGROUND: Cough commonly occurs as a symptom of seasonal allergic rhinitis (SAR), an inflammatory condition of the nasal mucous membranes that results in rhinorrhea, nasal stuffiness/congestion, nasal itching, and sneezing. Mometasone furoate nasal spray (MFNS, Nasonex, Schering, Kenilworth, NJ), an anti-inflammatory nasal corticosteroid, has been shown to be safe and effective in reducing the nasal inflammation of SAR. OBJECTIVE: To examine the effectiveness of MFNS in relieving SAR-associated cough, in addition to nasal symptoms. METHODS: This was a multicenter, randomized, double-blind study. Patients 12 years of age or older with > or = 1-year history of SAR symptoms, positive skin test to a prevailing seasonal allergen, moderate nasal symptoms, and moderate cough were treated for 14 days with MFNS 200 microg daily (n = 122) or placebo (n = 123). RESULTS: The group treated with MFNS showed significant improvement in the daytime cough severity score at endpoint compared with placebo (P = 0.049). Improvement in the nighttime cough severity score showed a trend in favor of MFNS treatment. Treatment with MFNS significantly improved total nasal symptoms in both the daytime and nighttime compared with placebo at endpoint (P < or = 0.017). Overall daytime symptom scores (cough + total nasal) improved significantly compared with placebo at endpoint (P = 0.005). Overall nighttime symptom scores improved significantly compared with placebo at endpoint (P = 0.028). Treatments were well tolerated, with no significant differences in the incidence of adverse events. CONCLUSIONS: MFNS is effective and well tolerated in the treatment of daytime cough associated with SAR.
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Antialérgicos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Tosse/tratamento farmacológico , Descongestionantes Nasais/uso terapêutico , Obstrução Nasal/tratamento farmacológico , Pregnadienodiois/uso terapêutico , Prurido/tratamento farmacológico , Rinite Alérgica Sazonal/complicações , Espirro/efeitos dos fármacos , Administração Intranasal , Adolescente , Adulto , Aerossóis , Idoso , Antialérgicos/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Criança , Tosse/etiologia , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Furoato de Mometasona , Descongestionantes Nasais/administração & dosagem , Obstrução Nasal/etiologia , Pregnadienodiois/administração & dosagem , Prurido/etiologia , Rinite Alérgica Sazonal/tratamento farmacológico , Segurança , Resultado do TratamentoRESUMO
Lymphedema-distichiasis (LD) syndrome is a clinically variable autosomal dominant disorder. The disorder is caused by mutations in the forkhead transcription factor FOXC2 gene on chromosome band 16q24.3. Here, we report the sequence of the FOXC2 gene in a German-Irish family with LD in six affected relatives over three generations and identify a single adenine base pair insertion at nt 1006--1007. This insertion creates a frameshift mutation that predicts a premature stop at codon 462. In addition to LD, four of the affected family members have renal disease and three have diabetes mellitus (DM), not usually seen in the LD syndrome. Polymorphisms of FOXC2 in diabetics have been studied in different populations. Our sequence analysis of the 5' untranslated region (UTR) C-512T shows the homozygous T allele in all family members tested. The sequencing data in this family suggests the possibility of a novel phenotype-haplotype. This novel phenotype, LD/renal disease/type 2 diabetes, might be the result of a combination of the nt 1006--1007 insA and the upstream UTR homozygous T polymorphism.