Bases genéticas y celulares de neuropatías periféricas hereditarias

Tesis doctoral; 208 págs.Capítulo I: CARACTERIZACIÓN DE LAS BASES GENÉTICAS DE NUEVAS FORMAS DE NEUROPATÍAS RECURRENTES HEREDITARIAS Las neuropatías recurrentes hereditarias, en las que los enfermos presentan grados parciales de recuperación, no son habituales y normalmente se deben a mutaciones en los genes PMP22, SEPT9 o SCN9A. Contamos con dos extensas familias (fCMT‐129 y fCMT‐ 266) en las que los pacientes presentan neuropatías recurrentes hereditarias y en las que previamente habíamos descartado la implicación de los genes candidatos conocidos. Mediante cartografiado genómico y análisis de ligamiento hemos identificado dos nuevos loci, uno en el cromosoma 21q21 en la fCMT‐129 y otro en el cromosoma 17p13.3 en la fCMT‐266, hasta ahora no relacionados con este grupo de enfermedades. Los haplotipos construidos con marcadores pertenecientes a las regiones identificadas, cosegregan perfectamente con la enfermedad en cada familia. En la fCMT‐129 hemos descartado la presencia de mutaciones en los exones de todos los genes codificantes de proteínas o miRNAs contenidos en la región candidata, así como la presencia de grandes deleciones y/o duplicaciones. En la fCMT‐266 hemos identificado la mutación SMYD4 p.K82E en la región candidata como principal candidata de la neuropatía. Hemos caracterizado la expresión de SMYD4 en el sistema nervioso periférico así como su relevancia en dicho tejido empleando Drosophila melanogaster como organismo modelo. Nuestros hallazgos indican que hay nuevos loci genéticos implicados en neuropatías recurrentes hereditarias, y ponen de manifiesto la heterogeneidad genética y clínica asociada a este grupo de enfermedades. Capítulo II: BÚSQUEDA DE MODIFICADORES GENÉTICOS EN LA NEUROPATÍA DE CHARCOT MARIE TOOTH CMT2K DEBIDA A MUTACIONES DOMINANTES EN EL GEN GDAP1. Las mutaciones en el gen GDAP1 causan diferentes formas de la neuropatía de Charcot‐Marie‐Tooth (CMT). Las mutaciones con herencia dominante en este gen conducen a CMT2K, que se caracteriza por la presencia de variabilidad clínica intrafamiliar debido a penetrancia incompleta y expresividad variable, sugiriendo la existencia de modificadores genéticos. Hemos identificado y caracterizado el gen JPH1 como modificador genético y funcional de GDAP1. Ambos genes constituyen un cluster conservado de genes funcionalmente relacionados en vertebrados. La sobreexpresión de JPH1 rescata los defectos en el mecanismo del SOCE (store‐operated calcium entry) presente en células deficientes en GDAP1. En un cribado mutacional del gen JPH1 en una serie de 29 pacientes CMT2K portadores de la mutación GDAP1 p.R120W hemos identificado la mutación JPH1 p.R213P en un paciente con un cuadro clínico agravado. La combinación de las mutaciones GDAP1 p.R120W y JPH1 p.R213P en células provoca defectos más acusados en la homeostasis del calcio. Niveles alterados de JPH1 en pacientes CMT2K también podrían contribuir en las consecuencias fenotípicas de las mutaciones en GDAP1, lo que hemos comprobado usando D. melanogaster como organismo modelo. Es la primera vez que se describe un modificador genético en formas de CMT2. La investigación empleando los modelos de D. melanogaster nos ha permitido demostrar el posible papel de las proteínas JPH como modificadores de otra enfermedad neurológica, como es la enfermedad de Huntington, así como establecer por primera vez una relación entre los genes JPH y la ruta de señalización de Notch.Durante la realización de la presente Tesis Doctoral, el autor ha disfrutado en el Instituto de Biomedicina de Valencia (IBV‐CSIC) y en el Centro de Investigación Príncipe Felipe de Valencia (CIPF) de una beca predoctoral FPU (Ref. AP2009‐0642) concedida por el Ministerio de Educación y de un contrato asociado a proyecto de investigación (Ref. ISCIII‐IRDiRC 2011) financiado por el Instituto de Salud Carlos III y el International Rare Diseases Research Consortium (IRDiRC) en el CIBER de Enfermedadades Raras. El trabajo se ha enmarcado dentro de los proyectos “Bases genéticas y fisiopatología celular de las neuropatías periféricas hereditarias” (Ref. PS09/00095) e “Investigación traslacional y mecanismos de enfermedad en neuropatías periféricas hereditarias” (Ref. PI12/00453), concedidos por el Instituto de Salud Carlos III; “Identificación de genes responsables de enfermedades raras mediante secuenciación masiva: el modelo de una neuropatía recurrente hereditaria” (Ref. AP‐207/11), concedido por la Conselleria de Sanitat de la Generalitat Valenciana; “TREAT‐CMT: Translational Research, Experimental Medicine and Therapeutics on Charcot‐Marie‐Tooth disease” (Ref. ISCIII‐IRDiRC 2011), concedido por el Instituto de Salud Carlos III, International Rare Diseases Research Consortium (IRDiRC).Peer reviewe

Bases genéticas y celulares de neuropatías periféricas hereditarias

Bases genéticas y celulares de neuropatías periféricas hereditarias Capítulo I: CARACTERIZACIÓN DE LAS BASES GENÉTICAS DE NUEVAS FORMAS DE NEUROPATÍAS RECURRENTES HEREDITARIAS Las neuropatías recurrentes hereditarias, en las que los enfermos presentan grados parciales de recuperación, no son habituales y normalmente se deben a mutaciones en los genes PMP22, SEPT9 o SCN9A. Contamos con dos extensas familias (fCMT-129 y fCMT-266) en las que los pacientes presentan neuropatías recurrentes hereditarias y en las que previamente habíamos descartado la implicación de los genes candidatos conocidos. Mediante cartografiado genómico y análisis de ligamiento hemos identificado dos nuevos loci, uno en el cromosoma 21q21 en la fCMT-129 y otro en el cromosoma 17p13.3 en la fCMT-266, hasta ahora no relacionados con este grupo de enfermedades. Los haplotipos construidos con marcadores pertenecientes a las regiones identificadas, cosegregan perfectamente con la enfermedad en cada familia. En la fCMT-129 hemos descartado la presencia de mutaciones en los exones de todos los genes codificantes de proteínas o miRNAs contenidos en la región candidata, así como la presencia de grandes deleciones y/o duplicaciones. En la fCMT-266 hemos identificado la mutación SMYD4 p.K82E en la región candidata como principal candidata de la neuropatía. Hemos caracterizado la expresión de SMYD4 en el sistema nervioso periférico así como su relevancia en dicho tejido empleando Drosophila melanogaster como organismo modelo. Nuestros hallazgos indican que hay nuevos loci genéticos implicados en neuropatías recurrentes hereditarias, y ponen de manifiesto la heterogeneidad genética y clínica asociada a este grupo de enfermedades. Capítulo II: BÚSQUEDA DE MODIFICADORES GENÉTICOS EN LA NEUROPATÍA DE CHARCOT MARIE TOOTH CMT2K DEBIDA A MUTACIONES DOMINANTES EN EL GEN GDAP1. Las mutaciones en el gen GDAP1 causan diferentes formas de la neuropatía de Charcot-Marie-Tooth (CMT). Las mutaciones con herencia dominante en este gen conducen a CMT2K, que se caracteriza por la presencia de variabilidad clínica intrafamiliar debido a penetrancia incompleta y expresividad variable, sugiriendo la existencia de modificadores genéticos. Hemos identificado y caracterizado el gen JPH1 como modificador genético y funcional de GDAP1. Ambos genes constituyen un cluster conservado de genes funcionalmente relacionados en vertebrados. La sobreexpresión de JPH1 rescata los defectos en el mecanismo del SOCE (store-operated calcium entry) presente en células deficientes en GDAP1. En un cribado mutacional del gen JPH1 en una serie de 29 pacientes CMT2K portadores de la mutación GDAP1 p.R120W hemos identificado la mutación JPH1 p.R213P en un paciente con un cuadro clínico agravado. La combinación de las mutaciones GDAP1 p.R120W y JPH1 p.R213P en células provoca defectos más acusados en la homeostasis del calcio. Niveles alterados de JPH1 en pacientes CMT2K también podrían contribuir en las consecuencias fenotípicas de las mutaciones en GDAP1, lo que hemos comprobado usando D. melanogaster como organismo modelo. Es la primera vez que se describe un modificador genético en formas de CMT2. La investigación empleando los modelos de D. melanogaster nos ha permitido demostrar el posible papel de las proteínas JPH como modificadores de otra enfermedad neurológica, como es la enfermedad de Huntington, así como establecer por primera vez una relación entre los genes JPH y la ruta de señalización de Notch

Review of recurrently mutated genes in craniosynostosis supports expansion of diagnostic gene panels

Craniosynostosis, the premature fusion of the cranial sutures, affects ~1 in 2000 children. Although many patients with a genetically determined cause harbor a variant in one of just seven genes or have a chromosomal abnormality, over 60 genes are known to be recurrently mutated, thus comprising a long tail of rarer diagnoses. Genome sequencing for the diagnosis of rare diseases is increasingly used in clinical settings, but analysis of the data is labor intensive and involves a trade-off between achieving high sensitivity or high precision. PanelApp, a crowd-sourced disease-focused set of gene panels, was designed to enable prioritization of variants in known disease genes for a given pathology, allowing enhanced identification of true-positives. For heterogeneous disorders like craniosynostosis, these panels must be regularly updated to ensure that diagnoses are not being missed. We provide a systematic review of genetic literature on craniosynostosis over the last 5 years, including additional results from resequencing a 42-gene panel in 617 affected individuals. We identify 16 genes (representing a 25% uplift) that should be added to the list of bona fide craniosynostosis disease genes and discuss the insights that these new genes provide into pathophysiological mechanisms of craniosynostosis

Characterization and In Vivo Anti-Inflammatory Efficacy of Copal (Dacryodes peruviana (Loes.) H.J. Lam) Essential Oil

Essential oils are natural aromatic substances that contain complex mixtures of many volatile compounds frequently used in pharmaceutical and cosmetic industries. Dacryodes peruviana (Loes.) H.J. Lam is a native species from Ecuador whose anti-inflammatory activity has not been previously reported, thus the aim of this study was to evaluate the anti-inflammatory activity of D. peruviana essential oil. To that end, essential oil from D. peruviana fruits was isolated by hydrodistillation and characterized physically and chemically. The tolerance of the essential oil was analyzed by cytotoxicity studies using human keratinocytes. The anti-inflammatory activity was evaluated by an arachidonic acid-induced edema model in mouse ear. The predominant compounds in D. peruviana essential oil were α-phellandrene, limonene, and α-pinene, with the three compounds reaching approximately 83% of the total composition. Tolerance studies showed high biocompatibility of this essential oil with human keratinocytes. In vivo studies demonstrated a moisturizing effect and an alleviation of several events occurred during the inflammatory process after topical treatment with D. peruviana essential oil such as decline in skin edema; reduction in leukocytic infiltrate; and decrease in inflammatory cytokines TNFα, IL-8, IL-17A, and IL-23. Therefore, this essential oil could be an attractive treatment for skin inflammation

Therapeutic applications of essential oils from native and cultivated ecuadorian plants: Cutaneous candidiasis and dermal anti-inflammatory activity

Abstract: Essential oils are a complex mixture of aromatic substances whose pharmacological actions, including antimicrobial, antioxidant, anticancer, and anti-inflammatory activities, have been widely reported. This study aimed to evaluate the anti-Candida and dermal anti-inflammatory activity of essential oils from native and cultivated Ecuadorian plants. Essential oils from Bursera graveolens, Dacryodes peruviana, Mespilodaphne quixos, and Melaleuca armillaris were isolated by hydrodistillation and were characterized physically and chemically. Its tolerance was analyzed by in vitro and in vivo studies. The antifungal activity was studied against Candida albicans, Candida glabrata, and Candida parapsilosis, whereas the anti-inflammatory effect was evaluated by a mouse ear edema model. The main compounds were limonene, -phellandrene, (E)-methyl cinnamate, and 1,8-cineole, respectively. All essential oils showed high tolerability for skin application, antifungal activity against the three Candida strains, and anti-inflammatory efficacy by decreasing edema and overexpression of pro-inflammatory cytokines. Dacryodes peruviana essential oil showed the highest antifungal activity. On the other hand, Dacryodes peruviana and Melaleuca armillaris showed the greatest anti-inflammatory potential, decreasing edema by 53.3% and 65.25%, respectively, and inhibiting the overexpression of TNF- , IL-8, IL-17A, and IL-23. The results suggest that these essential oils could be used as alternative therapies in the treatment of both cutaneous candidiasis and dermal inflammation

Therapeutic applications of essential oils from native and cultivated ecuadorian plants: Cutaneous candidiasis and dermal anti-inflammatory activity

Abstract: Essential oils are a complex mixture of aromatic substances whose pharmacological actions, including antimicrobial, antioxidant, anticancer, and anti-inflammatory activities, have been widely reported. This study aimed to evaluate the anti-Candida and dermal anti-inflammatory activity of essential oils from native and cultivated Ecuadorian plants. Essential oils from Bursera graveolens, Dacryodes peruviana, Mespilodaphne quixos, and Melaleuca armillaris were isolated by hydrodistillation and were characterized physically and chemically. Its tolerance was analyzed by in vitro and in vivo studies. The antifungal activity was studied against Candida albicans, Candida glabrata, and Candida parapsilosis, whereas the anti-inflammatory effect was evaluated by a mouse ear edema model. The main compounds were limonene, -phellandrene, (E)-methyl cinnamate, and 1,8-cineole, respectively. All essential oils showed high tolerability for skin application, antifungal activity against the three Candida strains, and anti-inflammatory efficacy by decreasing edema and overexpression of pro-inflammatory cytokines. Dacryodes peruviana essential oil showed the highest antifungal activity. On the other hand, Dacryodes peruviana and Melaleuca armillaris showed the greatest anti-inflammatory potential, decreasing edema by 53.3% and 65.25%, respectively, and inhibiting the overexpression of TNF- , IL-8, IL-17A, and IL-23. The results suggest that these essential oils could be used as alternative therapies in the treatment of both cutaneous candidiasis and dermal inflammation

Therapeutic applications of essential oils from native and cultivated ecuadorian plants: Cutaneous candidiasis and dermal anti-inflammatory activity

Abstract: Essential oils are a complex mixture of aromatic substances whose pharmacological actions, including antimicrobial, antioxidant, anticancer, and anti-inflammatory activities, have been widely reported. This study aimed to evaluate the anti-Candida and dermal anti-inflammatory activity of essential oils from native and cultivated Ecuadorian plants. Essential oils from Bursera graveolens, Dacryodes peruviana, Mespilodaphne quixos, and Melaleuca armillaris were isolated by hydrodistillation and were characterized physically and chemically. Its tolerance was analyzed by in vitro and in vivo studies. The antifungal activity was studied against Candida albicans, Candida glabrata, and Candida parapsilosis, whereas the anti-inflammatory effect was evaluated by a mouse ear edema model. The main compounds were limonene, -phellandrene, (E)-methyl cinnamate, and 1,8-cineole, respectively. All essential oils showed high tolerability for skin application, antifungal activity against the three Candida strains, and anti-inflammatory efficacy by decreasing edema and overexpression of pro-inflammatory cytokines. Dacryodes peruviana essential oil showed the highest antifungal activity. On the other hand, Dacryodes peruviana and Melaleuca armillaris showed the greatest anti-inflammatory potential, decreasing edema by 53.3% and 65.25%, respectively, and inhibiting the overexpression of TNF- , IL-8, IL-17A, and IL-23. The results suggest that these essential oils could be used as alternative therapies in the treatment of both cutaneous candidiasis and dermal inflammation

Disruption of TWIST1 translation by 5' UTR variants in Saethre-Chotzen syndrome

Saethre-Chotzen syndrome (SCS), one of the most common forms of syndromic craniosynostosis (premature fusion of the cranial sutures), results from haploinsufficiency of TWIST1, caused by deletions of the entire gene or loss-of-function variants within the coding region. To determine whether non-coding variants also contribute to SCS, we screened 14 genetically undiagnosed SCS patients using targeted capture sequencing, and identified novel single nucleotide variants (SNVs) in the 5' untranslated region (UTR) of TWIST1 in two unrelated SCS cases. We show experimentally that these variants, which create translation start sites in the TWIST1 leader sequence, reduce translation from the main open reading frame (mORF). This is the first demonstration that non-coding SNVs of TWIST1 can cause SCS, and highlights the importance of screening the 5' UTR in clinically diagnosed SCS patients without a coding mutation. Similar 5' UTR variants, particularly of haploinsufficient genes, may represent an under-ascertained cause of monogenic disease. This article is protected by copyright. All rights reserved

A de novo substitution in BCL11B leads to loss of interaction with transcriptional complexes and craniosynostosis

Craniosynostosis, the premature ossification of cranial sutures, is a developmental disorder of the skull vault, occurring in approximately 1 in 2250 births. The causes are heterogeneous, with a monogenic basis identified in ~25% of patients. Using whole-genome sequencing, we identified a novel, de novo variant in BCL11B, c.7C>A, encoding an R3S substitution (p.R3S), in a male patient with coronal suture synostosis. BCL11B is a transcription factor that interacts directly with the nucleosome remodelling and deacetylation complex (NuRD) and polycomb-related complex 2 (PRC2) through the invariant proteins RBBP4 and RBBP7. The p.R3S substitution occurs within a conserved amino-terminal motif (RRKQxxP) of BCL11B and reduces interaction with both transcriptional complexes. Equilibrium binding studies and molecular dynamics simulations show that the p.R3S substitution disrupts ionic coordination between BCL11B and the RBBP4-MTA1 complex, a subassembly of the NuRD complex, and increases the conformational flexibility of Arg-4, Lys-5 and Gln-6 of BCL11B. These alterations collectively reduce the affinity of BCL11B p.R3S for the RBBP4-MTA1 complex by nearly an order of magnitude. We generated a mouse model of the BCL11B p.R3S substitution using a CRISPR-Cas9-based approach, and we report herein that these mice exhibit craniosynostosis of the coronal suture, as well as other cranial sutures. This finding provides strong evidence that the BCL11B p.R3S substitution is causally associated with craniosynostosis and confirms an important role for BCL11B in the maintenance of cranial suture patency

Pathogenic variants in the paired-related homeobox 1 gene (PRRX1) cause craniosynostosis with incomplete penetrance

Purpose Studies previously implicated PRRX1 in craniofacial development, including demonstration of murine Prrx1 expression in the pre-osteogenic cells of the cranial sutures. We investigated the role of heterozygous missense and loss-of-function variants in PRRX1 associated with craniosynostosis. Methods Trio-based genome, exome or targeted sequencing were used to screen PRRX1 in patients with craniosynostosis; immunofluorescence analyses were used to assess nuclear localization of wild-type and mutant proteins. Results Genome sequencing identified 2 of 9 sporadically affected individuals with syndromic/multisuture craniosynostosis who were heterozygous for rare/undescribed variants in PRRX1. Exome or targeted sequencing of PRRX1 revealed a further 9/1449 patients with craniosynostosis harboring deletions or rare heterozygous variants within the homeodomain. By collaboration, seven additional individuals (four families) were identified with putatively pathogenic PRRX1 variants. Immunofluorescence analyses showed that missense variants within the PRRX1 homeodomain cause abnormal nuclear localization. Of patients with variants considered likely pathogenic, bicoronal or other multi-suture synostosis was present in 11/17 (65% of the cases). Pathogenic variants were inherited from unaffected relatives in many instances, yielding a 12.5% penetrance estimate for craniosynostosis. Conclusion This work supports a key role for PRRX1 in cranial suture development and shows that haploinsufficiency of PRRX1 is a relatively frequent cause of craniosynostosis