There are numerous different white spotting patterns in the horse, including two of particular interest tobiano and sabino. In the mouse, genetic variation in the gene KIT causes many white spotting patterns. Due to the phenotypic similarity among white spotting patterns in horses and mice, KIT was investigated as the cause of the tobiano and sabino spotting patterns in horses. Initially, the KIT cDNA sequences from horses with several spotting patterns were compared. Three single nucleotide polymorphisms (SNPs) were identified, though none were associated with a spotting pattern. Three novel splicing variants were also observed: exon 17 skipping, exon 18 skipping and alternative splicing of exon 3. Families segregating for a sabino spotting pattern (designated Sabino 1) and exon 17 skipping were discovered. Sequencing revealed a SNP (KI16+1037) within intron 16 that was completely associated with skipping of exon 17. Using a PCR-RFLP for KI16+1037, linkage was discovered for sabino spotting (LOD=9.02 for =0) and presence of the Sabino 1 allele detected in seven breeds. While all horses with this SNP exhibited the Sabino 1 phenotype, some horses with a sabino phenotype did not possess the SNP. This is most likely due to genetic heterogeneity of the phenotype. Fluorescent in situ hybridization (FISH) was used to investigate the possibility of chromosome inversion in the region of KIT. A chromosomal inversion was discovered spanning ECA3q13 to 3q21 using BAC clones containing KIT and other genes in the same region. The ECA3q inversion was completely associated with Tobiano in the eight horses tested by FISH. This inversion may disrupt regulatory sequences of the KIT gene and thereby cause tobiano spotting. Spotting patterns are important to horse breeders for aesthetic as well as economic reasons. Spotting patterns in the horse may also be an interesting scientific model. The two genetic variants discovered in this work are good examples of genetic diversity due to mechanisms other than SNPs. Study of these variants may be valuable for examining the effects of the KIT gene on health traits. In particular, the KIT gene directs many functions of the mast cell, a cell that is involved in the etiology of inflammation.

beta-Bungarotoxin (beta-Bgt), the presynaptic neurotoxin purified from the venom of Bungarus multicinctus, consists of the A chain and the B chain, cross-linked by an interchain disulfide bond. In this study, two novel beta-Bgt isotoxins were purified from Bungarus multicinctus venom by the combinations of ion-exchange chromatography and reverse phase HPLC. Amino acid sequencing, peptide mapping and mass analyses revealed that they probably contained the same A chain, but their B chain differed. Consequently, the discrepancies in their biological activity and fine structure reflected the role of B chain in intact of beta-Bgt. In Yeast-Two-Hybrid system, a potassium channel binding protein was identified to interact with the B chain of beta-Bgt. Although the recombinant potassium channel binding protein functionally bound with Ca2+, but it could not prove to bind with BM12 and BM13 as revealed by in vitro cross-linking assay. The A chain genes including A1 chain, A2 chain and A8 chain genes were amplified by PCR reaction. Their nucleotide sequences shared up to 97.5% identity. Alignment of the determined A chain genes with A chain cDNAs revealed that the A1 chain gene was organized with four exon and three intron, while A2 chain gene comprised three exons and two introns. When A2 chain is expressed, the region corresponds to the first exon of A1 chain gene is skipped instead of inclusion of intronic sequence adjacent to the second intron. The resulting A2 chain mRNA encoded a 25 residues signal peptide, which different from A1 chain mRNA with a 27 residues signal peptide. Comparative analyses on phospholipase A2 genes and cDNAs suggest that this is the first report on skipping of exon changes the signal peptide sequence of snake venom proteins.

No description available.

O sistema complemento participa da imunidade natural e da imunidade adquirida. Este sistema pode ser ativado por três diferentes vias: a via clássica, a via alternativa e a via das lectinas, desencadeando uma cascata proteolítica que irá resultar na possível eliminação de microorganismos e, por sua vez, no restabelecimento da homeostasia do indivíduo. A proteína C3 possui um papel central participando das três vias deste sistema. Sua clivagem gera fragmentos que estão associados a várias funções biológicas como opsonização, quimiotaxia, anafilatoxinas, além da formação da C3-convertases, que amplificam a via de ativação efetora contra patógenos. A completa deficiência de C3 é rara, está comumente associada a repetidas infecções, ao desenvolvimento de doenças mediadas por imunocomplexos e glomerulonefrite. Neste trabalho nós investigamos as bases moleculares desta deficiência em uma paciente com histórico de consanguinidade. Nosso estudo identificou uma mutação genética responsável pela completa deleção do éxon 27, implicando na perda de 99 nucleotídeos (região 3450 até 3549 correspondente ao cDNA do C3). O sequenciamento do gene C3 da região 6690313 até 6690961 mostrou uma troca de nucleotídeos T por um C (T → C) na posição 6690626, causando a exclusão do o éxon 27 e deficiência da proteína C3 do complemento humano nessa paciente. Também confirmamos o padrão de herança autossômica recessiva desta deficiência. Verificamos ainda que os fibloblastos da paciente estimulados com LPS por 24 h foram incapazes de secretar a proteína C3, o que nos leva a suspeitar que esta proteína mutante seja rapidamente degradada ainda dentro da célula, impedindo sua liberação para o meio extracelular. / The complement system participates in the natural immunity and acquired immunity. This system can be activated by three different pathways: the classical pathway, the alternative pathway and the lectin pathway, triggering a proteolytic cascade that will possibly result in the elimination of microorganisms and, in turn, in restoring the homeostasis of the individual. The C3 protein has a central role in the activation of all pathways. Its cleavage produces fragments that are associated with various biological functions such as opsonization, chemotaxis, and production of anaphylatoxins. In addition, the C3 participates in formation of C3 convertase of the alternative pathway which amplifies the activation of effector against pathogens. The complete C3 deficiency is rare and commonly associated with recurrent infections, development of diseases mediated by immune complexes and glomerulonephritis. In this work, we investigated the molecular basis of this deficiency in a patient family with a history of consanguinity. Our study has identified a genetic mutation responsible for the complete deletion of exon 27, resulting in the loss of 99 nucleotides (region 3450 to 3549 of the cDNA corresponding to C3). The sequencing of the C3 region 6690313 to 6690961 showed a T nucleotide exchange to C (T → C) at position 6690626 causing deletion of exon 27 leading to deficiency of the human complement protein C3 in this patient. We also confirmed the autosomal recessive patters of this patient deficiency in the family. We observed that patient fibroblasts stimulated with LPS for 24 h were unable to secrete C3, which leads us to suspect that this protein is degraded within the cell preventing its release into the extracellular.

O sistema complemento participa da imunidade natural e da imunidade adquirida. Este sistema pode ser ativado por três diferentes vias: a via clássica, a via alternativa e a via das lectinas, desencadeando uma cascata proteolítica que irá resultar na possível eliminação de microorganismos e, por sua vez, no restabelecimento da homeostasia do indivíduo. A proteína C3 possui um papel central participando das três vias deste sistema. Sua clivagem gera fragmentos que estão associados a várias funções biológicas como opsonização, quimiotaxia, anafilatoxinas, além da formação da C3-convertases, que amplificam a via de ativação efetora contra patógenos. A completa deficiência de C3 é rara, está comumente associada a repetidas infecções, ao desenvolvimento de doenças mediadas por imunocomplexos e glomerulonefrite. Neste trabalho nós investigamos as bases moleculares desta deficiência em uma paciente com histórico de consanguinidade. Nosso estudo identificou uma mutação genética responsável pela completa deleção do éxon 27, implicando na perda de 99 nucleotídeos (região 3450 até 3549 correspondente ao cDNA do C3). O sequenciamento do gene C3 da região 6690313 até 6690961 mostrou uma troca de nucleotídeos T por um C (T → C) na posição 6690626, causando a exclusão do o éxon 27 e deficiência da proteína C3 do complemento humano nessa paciente. Também confirmamos o padrão de herança autossômica recessiva desta deficiência. Verificamos ainda que os fibloblastos da paciente estimulados com LPS por 24 h foram incapazes de secretar a proteína C3, o que nos leva a suspeitar que esta proteína mutante seja rapidamente degradada ainda dentro da célula, impedindo sua liberação para o meio extracelular. / The complement system participates in the natural immunity and acquired immunity. This system can be activated by three different pathways: the classical pathway, the alternative pathway and the lectin pathway, triggering a proteolytic cascade that will possibly result in the elimination of microorganisms and, in turn, in restoring the homeostasis of the individual. The C3 protein has a central role in the activation of all pathways. Its cleavage produces fragments that are associated with various biological functions such as opsonization, chemotaxis, and production of anaphylatoxins. In addition, the C3 participates in formation of C3 convertase of the alternative pathway which amplifies the activation of effector against pathogens. The complete C3 deficiency is rare and commonly associated with recurrent infections, development of diseases mediated by immune complexes and glomerulonephritis. In this work, we investigated the molecular basis of this deficiency in a patient family with a history of consanguinity. Our study has identified a genetic mutation responsible for the complete deletion of exon 27, resulting in the loss of 99 nucleotides (region 3450 to 3549 of the cDNA corresponding to C3). The sequencing of the C3 region 6690313 to 6690961 showed a T nucleotide exchange to C (T → C) at position 6690626 causing deletion of exon 27 leading to deficiency of the human complement protein C3 in this patient. We also confirmed the autosomal recessive patters of this patient deficiency in the family. We observed that patient fibroblasts stimulated with LPS for 24 h were unable to secrete C3, which leads us to suspect that this protein is degraded within the cell preventing its release into the extracellular.

Les Epidermolyses Bulleuses Dystrophiques (EBD) sont des génodermatoses rares et sévères transmises sur un mode autosomique récessif (EBDR) ou dominant (EBDD), dues à une perte de l’adhésion dermo-épidermique. Les patients atteints d’EBD souffrent de décollements bulleux cutanéo-muqueux qui menacent le pronostic fonctionnel et vital dans les formes les plus graves. Toutes les formes d’EBD sont dues à des mutations du gène COL7A1 codant pour le collagène VII qui est le constituant des fibres d’ancrage assurant l’adhésion de l’épiderme au derme. Il n’existe actuellement pas de thérapie satisfaisante des EBD. La première partie de ma thèse visait à démontrer la faisabilité d’une approche thérapeutique par saut d’exon des EBDR. Cette stratégie consiste à exciser l’exon porteur de la mutation durant le processus d’épissage, afin de restaurer l’expression d’une protéine fonctionnelle. Les exons 73 et 80 de COL7A1 sont particulièrement intéressants car ils sont le siège de mutations récurrentes et que leur excision préserve le cadre ouvert de lecture. Nous avons dans un premier temps démontré le caractère non indispensable des séquences codées par ces exons in vivo en utilisant un modèle de xénogreffe de peau humaine EBDR reconstruite, génétiquement modifiée à l’aide de vecteurs rétroviraux exprimant l'ADNc de COL7A1 délété des séquences des exons 73 ou 80. Puis, j’ai pu établir que la transfection d’oligoribonucléotides antisens (AONs) dirigés contre certaines séquences régulatrices de l’épissage permettait d’induire le saut en phase de ces exons dans des cellules primaires de patients EBDR, avec une efficacité atteignant 90% de saut d’exon. Les analyses par western blot et immunocytofluorescence après transfection ont permis de mettre en évidence une réexpression significative du collagène VII (jusqu’à 25%) dans les cellules de trois patients EBDR. Enfin, j’ai pu démontrer la réexpression du collagène VII in vivo, après injection de différentes doses d’AONs dans des peaux équivalentes générées avec des cellules de patients et greffées sur des souris immunodéficientes. Dans la seconde partie de ma thèse, j’ai étudié une famille EBD particulière, dont les deux enfants atteints présentaient une EBD beaucoup plus sévère que leur mère et leur grand père maternel, atteints d’une forme modérée d’EBDD. Le séquençage des 118 exons de COL7A1 et des régions d’épissage adjacentes a permis d’identifier une seule mutation dominante c.6698G>A (p.Gly2233Asp) dans l’exon 84, à l’état hétérozygote chez les quatre sujets. A partir de l’étude des transcrits paternels, j’ai pu identifier une nouvelle mutation c.2587+40G>A dans l’intron 19 de COL7A1, qui active un site donneur cryptique dans l’intron 19, entraînant sa rétention partielle et la formation d’un codon stop prématuré. La confirmation de la présence de cette seconde mutation, récessive, dans l’ADN des deux enfants a ainsi permis d’expliquer les différences phénotypiques observées, les deux enfants atteints étant hétérozygotes composites pour une mutation dominante et une mutation récessive de COL7A1. Cette mutation récessive constitue la mutation intronique la plus distante des sites consensus d’épissage de COL7A1 et souligne l’importance de l’étude des ARNm pour la recherche de mutations dans le cadre des EBD. Dans une dernière partie de ma thèse, j’ai débuté la caractérisation d’un modèle murin knock-in d’EBDR développé par notre laboratoire, qui mime certaines des caractéristiques phénotypiques des patients EBDR. Mon travail a permis de démontrer in vivo la faisabilité de l’approche par saut d’exons pour COL7A1. Cette première étape importante conduit à développer des études de preuve de principe et de toxicologie dans des modèles animaux, dans la perspective d’une transition vers la clinique. Il illustre également les variations pathologiques d’épissage pouvant faire l’objet d’approches thérapeutiques similaires. / Dystrophic Epidermolysis Bullosa (DEB) is a group of rare and severe genetic skin disorders, inherited in a dominant (DDEB) or recessive (RDEB) manner, and characterised by loss of adhesion between the epidermis and the underlying dermis. DEB patients suffer from severe blistering of the skin and mucosae after mild traumas, and in the most severe forms, DEB can be life-threatening. DEB is caused by mutations in the COL7A1 gene encoding type VII collagen that assembles into anchoring fibrils forming key dermo-epidermal adhesion structures. To date, there is no specific treatment for DEB. The first part of my thesis was to develop exon skipping as a therapeutic approach for RDEB. In this work, exon skipping strategy consists in modulating the splicing of a premessenger RNA to induce the skipping of a mutated exon and lead to the synthesis of a shorter but functional protein. Exons 73 and 80 of COL7A1 are of particular interest since they carry many recurrent mutations and their excision preserves the open reading frame. In first instance, we have demonstrated the dispensability of these exons for type VII collagen function in an in vivo xenograft model using RDEB cells transduced with retroviral vectors containing COL7A1 cDNAs, deleted of the sequences of exon 73 or 80. I have subsequently transfected primary RDEB keratinocytes and fibroblasts with antisense oligoribonucleotides (AONs) targeting key splicing regulatory elements of these exons, and achieved efficient skipping of these exons (up to 90%). Western blot and immunocytofluorescence experiments demonstrated significant type VII collagen re-expression (up to 25% of the normal amount) in cells from three RDEB patients. Finally, I have generated skin equivalents with cells of these patients, grafted them on immunodeficient mice and injected different doses of AONs in the grafts, and I have demonstrated type VII collagen re-expression in vivo. In the second part of my thesis, I have studied the case of a particular DEB family, in which two affected children presented a DEB much more severe than their mother and maternal grandfather, suffering from a mild form of DDEB. Sequencing of the 118 exons ofCOL7A1 and of their flanking splice sites, lead to the identification of a single dominant mutation c.6698G>A (p.Gly2233Asp) in exon 84, at the heterozygous state in the four individuals. By carrying out analyses on the paternal transcripts, I have identified a novel c.2587+40G>A recessive mutation in intron 19, which activates a cryptic donor splice site in this intron, leading to its partial retention and to the formation of a premature termination codon. I confirmed the presence of this second, recessive, mutation in the DNA of the two affected children, thus providing an explanation for the observed intrafamilial phenotypic variability: the two affected offsprings being compound heterozygotes for a dominant mutation and a recessive mutation in COL7A1. This novel mutation is the deepest intronic mutation found in COL7A1 so far, and emphasizes the importance of studying COL7A1 at the transcripts level to unravel intronic mutations, understand their molecular consequences and their involvement in the development of the disease. In the last part of my thesis, I have started the characterisation of a knock-in murine model of RDEB generated by our laboratory, which mimics some of the phenotypic characteristics of RDEB patients. My thesis work provided the in vivo demonstration of the feasibility of an exon skipping therapeutic approach for COL7A1. This first important step leads to development of proof of concept studies and toxicological studies in different animal models, with the aim of a clinical translation. It also illustrates the pathological splicing alterations that could benefit from similar approaches.

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24996号 / 医博第5030号 / 新制||医||1069(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 滝田 順子, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

L’épissage en cis des précurseurs d’ARN messager (pre-ARNm) est une stratégie intéressante afin de réparer des gènes dont la régulation transcriptionnelle est déterminante pour la fonction de la protéine. Les mutations touchant le gène dysferline (DYSF) sont liées au développement de dystrophies musculaires: la dystrophie musculaire des ceintures de type 2B et la myopathie distale de Miyoshi. Une stratégie à modifier l’épissage en cis des pre-ARNm du gène DYSF est le procédé SMaRT (pour spliceosome-mediated mRNA trans-splicing), une technique de réparation de l'ARN messager au moyen d'un complexe de trans-épissage appelé PTM (pour pre-mRNA trans-splicing molecule). Le procédé SMaRT utilisant le complexe PTM permet le remplacement d’importantes portions de pre-ARNm tout en préservant l’intégrité totale du transcrit. Dans un soucis d’obtenir un trans-epissage efficace, seuls les introns codant pour les pre-ARNm de DYSF présentant de forts signaux d’épissage répartis de façon disparate ainsi que des tailles très différentes furent ciblées par les PTMs dans des myoblasts humains ne possédant pas de dysferlin. Le trans-épissage de deux introns ciblés du gène DYSF engendra une formation correcte de la protéine dysferlin dans des mutants DYSF-/- de souris. Les niveaux de protéine fonctionnelle furent toutefois modérés, mais similaires aux taux de récupération obtenus par des stratégies précédentes de trans-épissage ciblant d’autres gènes. Néanmoins, parmi les introns ciblés avec succès dans cette étude et dans des essais précédents, des critères concordants ont pu être identifiés afin de faciliter le choix des introns à cibler pour de futures stratégies de trans-épissage. / RNA-based therapy is an approach to cure genetic disorders with no intervention into endogenous spatiotemporal gene regulation. I established two approaches for the dysferlin gene, (i) spliceosome-mediated pre-mRNA trans-splicing (SmaRT) and (ii) exon-skipping, in order to rescue dysferlin mutations leading to Limb Girdle Muscular Dystrophy 2B and Miyoshi Myopathy. SmaRT permits the correction of numerous mutations of a gene by a single pre-mRNA trans-splicing molecule (PTM) by exchanging multiple exons of a gene for a healthy mRNA sequence. The PTM binds to intronic sequence and competes with the endogenous pre-mRNA for the binding of the spliceosome. I designed PTMs to exchange the 3’ part of the dysferlin messenger and determined two functioning PTMs bytransduction of human myoblasts and intramuscular injection in wild-type and DYSF-/- mice and could show dysferlin protein rescue in DYSF-/- mice.By exon-skipping exons carrying mutations can be excised from pre-mRNA in masking exon or intron internal sequences defining the exon in the splicing process. I employed antisense oligonucleotides (AONs) of tricyclo-DNA in order to excise dysferlin exon 32. It was shown to be particularly feasible for systemic application, making it suitable for diseases affecting different compartments of skeletal muscle and other organs. The dysferlin exon 32 has been shown to be dispensable for known functions of the dysferlin protein. I designed tc-DNA AONs leading to efficient skipping in patient myoblasts and in wild-type mice following intramuscular injection. I am collaborating to investigate effects of exon 32 skipping in an Exon-32-STOP mouse model.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19229号 / 医博第4028号 / 新制||医||1011(附属図書館) / 32228 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 良輔, 教授 妻木 範行, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM