Exoner är de sekvenser i DNA vilka rymmer koden för proteiner i människan och i alla andra organismer. Intronerna, vilka utgör utrymmet mellan exoner, består av ickekodande sekvenser och kontrollelement. Exoner tillhörande en gen måste inte alltid inkluderas i den slutliga mRNA produkten, alternativ splicing tillåter exkludering av vissa sekvenser och gör att en gen kan ha mer än en mRNA produkt, därigenom kan en gen koda för flera olika proteiner. Alternativ splicing är ett fält som snabbt utvecklas och dess relevans för många sjukdomar har blivit uppenbar. Detta arbete går igenom ett flertal av dessa sjukdomar för att sammanställa ny forskning och tydliggöra rollen av alternativ splicing i dem. De sjukdomar som undersökts är cystisk fibros, ärftlig frontotemporal dementia, systemisk lupus erythematosus, aniridi, myotonisk dystrofi, amyotrophic lateral sclerosoch familial dysautonomia. Dessa sjukdomar har involvering av alternativ splicing, de genetiska processerna bakom dem är dock mycket olika och kan visa på de många sätt alternativ splicing kan påverka cell och kroppsfunktion. Målet med arbetet är en översiktlig bild av framstegen som gjorts och vilken forskning som nu bedrivs.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(論文) 学位授与年月日:平成5年2月19日 竹中晃氏の博士論文として提出された

Thesis (PhD) - Swinburne University of Technology, Faculty of Engineering and Industrial Sciences, 2009. / A thesis submitted for the degree of Doctor of Philosophy, Faculty of Engineering and Industrial Sciences, Swinburne University of Technology, 2009. Typescript. "February 2009". Includes bibliographical references (p. 291-313)

The nuclear matrix (NM) is proposed to be a permanent network of core filaments underlying thicker fibres, present regardless of transcriptional activity. It is found to be both RNA and protein rich; indeed, numerous important nuclear proteins are components of the structure. In addition to mediating the organisation of entire chromosomes, the NM has also been demonstrated to tether telomeres via their TTAGGG repeats. In order to examine telomeric interactions with the NM, a technique known as the DNA halo preparation has been employed. Regions of DNA that are tightly attached to the structure are found within a so-called residual nucleus, while those sequences forming lesser associations produce a halo of DNA. Coupled with various FISH methodologies, this technique allowed the anchorage of genomic regions by the NM, to be analysed. In normal fibroblasts, the majority of chromosomes and telomeres were extensively anchored to the NM. Such interactions did not vary significantly in proliferating and senescent nuclei. However, a decrease in NM-associated telomeres was detected in quiescence. Since lamin A is an integral component of the NM, it seemed pertinent to examine chromosome and telomere NM-anchorage in Hutchinson-Gilford Progeria Syndrome (HGPS) fibroblasts, which contain mutant forms of lamin A. Indeed, genome tethering by the NM was perturbed in HGPS. In immortalised HGPS fibroblasts, this disrupted anchorage appeared to be rescued; the implications of this finding will be discussed. This study also suggested that telomere-NM interactions are aberrant in X-linked Emery-Dreifuss Muscular Dystrophy (X-EDMD), which is caused by mutant forms of emerin, another NM-associated protein. The positioning of selected genes in control and X-EDMD cell lines was examined in un-extracted nuclei using 2D and 3D FISH. Subtle shifts in the organisation of these genes were detected in diseased cells; however, their expression levels remained unaltered. Furthermore, in order to examine the architectural integrity of the nuclear lamina in lamin A and emerin mutant cell lines, scanning electron microscopy (SEM) was employed. This work revealed that such structures were indeed compromised in disease. The findings presented in this thesis highlight the importance of lamin A and emerin in mediating the organisation of the genome and taken together, promote the hypothesis that dysfunctional NM dynamics may well contribute to disease pathology.

Skeletal muscle is a major organ comprising 30-40% of the human body mass. The coordination of processes resulting in mature muscle requires many genes, and their loss can result in debilitating muscle disorders. Of the strategies being developed to cure muscle diseases, enhancement of the natural process of muscle cell fusion in existing or introduced myogenic cells has great therapeutic potential. In this work, we determined whether a drug that stimulates proliferation and fusion of myoblasts could alleviate murine Duchenne muscular dystrophy. We also studied the necessity of a gene that is upregulated in early fusing human myoblast cultures and its role in muscle disease development.

RNA localization, a regulated step of gene expression, is fundamentally important in development and differentiation. In multidisciplinary experiments, we discovered that RNA (mis)localization underlies the human disease myotonic dystrophy (DM). DM, the most prevalent adult muscular dystrophy, is caused independently by two alleles: DM1 is characterized by a (CTG)n expansion in the DM kinase (DMPK) gene 3' untranslated region while DM2 has a mutation in a small presumptive RNA binding protein. These analyses were guided by disease characteristics and have provided insights to DM's cytopathology, cell biology and molecular genetics. Examining muscle biopsies, it is demonstrated here that DM kinase mRNA is specifically subcellularly localized within normal human muscle and that DM kinase mRNA harboring the 3’UTR mutation (DM1) is mislocalized in DM patient muscle to cytoplasmic areas characteristic of DM disease pathology. Thus, the disease mutation alters the cellular distribution of the effected message. DMPK mRNA mislocalization causes altered DM kinase protein localization, correlates with novel phosphoprotein appearance and can account for DM’s diseased phenotype. While we were fortunate to access DM patient tissue to establish these key findings, the system does not lend itself to experimental manipulation. Hence, I established a disease- relevant tissue culture system, which recapitulates DMPK trafficking, Employing this system; I elucidate a complementary role for the DM2 gene product as a localization factor for DMPK mRNA (DM1 gene product). Comprehensive RNA-protein interaction experiments reveal the DM2 protein specifically and selectively recognizes a small, definitive area within the DMPK RNA 3'UTR. Detailed biochemical, cytological and functional experiments reveal 1) the DM2 protein colocalizes with DMPK mRNA, 2) the small area of the DMPK 3’UTR bound by pDM2 acts to properly localize a reporter construct and 3) disruption of the DM2 protein results in DMPK mRNA mislocalization. These data establish mRNA localization as a vital process underlying human disease etiology. Moreover, they reveal DM1 and DM2 gene products function in the same molecular pathway and that mutation of either causes DMPK mRNA mislocalization, leading to disease. These data have apparent application to several neuromuscular disorders and open a plethora of novel research avenues, both basic and applied. / text

Mutations in BEST1, the gene encoding for Bestrophin-1 (Best1), cause five, clinically distinct inherited retinopathies: Best vitelliform macular dystrophy (BVMD), adult-onset vitelliform macular dystrophy (AVMD), autosomal recessive bestrophinopathy (ARB), autosomal dominant vitreoretinochoroidopathy (ADVIRC), and retinitis pigmentosa (RP). Little is known regarding how BEST1 mutations cause disease and why mutations cause multiple disease phenotypes. Within the eye, Best1 is a homo-oligomeric, integral membrane protein that is exclusively localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Here, it regulates intracellular Ca2+ signaling and putatively mediates anion transport. Since defects in localization and oligomerization are known to underlie other channelopathies, we investigated how mutations causal for BVMD, AVMD, ARB, ADVIRC, and RP impact the localization and oligomerization of Best1. We generated replication-defective adenoviral vectors encoding for WT and 31 mutant forms of Best1 associated with these five diseases and expressed them in confluent, polarized Madin-Darby canine kidney and/or RPE cells. Localization was assessed via immunofluorescence and confocal microscopy. Oligomerization was examined using live-cell fluorescence resonance energy transfer (FRET) as well as reciprocal co-immunoprecipitation experiments. We report that all 31 BVMD, AVMD, ARB, ADVIRC, and RP mutants tested can reciprocally co-immunoprecipitate with and exhibit comparable FRET efficiencies to WT Best1, indicative of unimpaired oligomerization. While all RP and ADVIRC mutants were properly localized to the basolateral plasma membrane, many but not all AVMD, ARB, and BVMD mutants were mislocalized to intracellular compartments. When co-expressed with WT Best1, mislocalized mutants predominantly co-localized with WT Best1 in intracellular compartments. Studies involving four ARB truncation mutants reveal that the first 174 amino acids are sufficient to mediate oligomerization with WT Best1 and that amino acids 472-585 are not necessary for proper trafficking. We conclude that, although mislocalization is a common result of BEST1 mutation, it is not an absolute feature of any individual bestrophinopathy. Moreover, we show that some recessive mutants mislocalize WT Best1 when co-expressed, indicating that mislocalization cannot, on its own, generate a disease phenotype, and that the absence of Best1 at the plasma membrane is well tolerated.

Myotonic dystrophy is the commonest form of adult muscular dystrophy. Myotonic dystrophy 1 and 2 (DM 1 and DM 2) are autosomal dominant inherited disorders with unusual multisystem clinical features characterized by myotonia, progressive muscle weakness and wasting, cataracts, hypogonadism, frontal balding, cardiac conduction defects and diabetes. Severity varies from asymptomatic to severely affected phenotypes. DM1 presents with predominantly distal weakness whereas DM2 have predominantly proximal weakness.98% of patients identified worldwide present with DM1. DM 1 is caused by the expansion of an unstable CTG trinucleotide repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene on chromosome 19ql3.3. DM 2 is linked to the long arm of chromosome 3q21. It is caused by a tranucleotide, CCTG expansion in intron 1 of the zinc finger protein 9(ZNF9) gene that interferes with processing of a variety of RNAs. All DM mutations can be detected using a combination of the Southern Blot and Polymerase Chain reaction (PCR) techniques. Aim: This study aims to characterize the clinical spectrum and molecular features of myotonic dystrophy patients in KwaZulu - Natal between 1989 and 2005. Methodology: Patients included in this study were obtained from the database of patients diagnosed with Myotonic Dystrophy at the Department of Neurology in KwaZulu-Natal from 1989 to 2005. Patients were subjected to clinical, radiological and neurophysiological assessment. Molecular testing was performed using PCR and Southern blot. Results: Thirty-seven patients with Myotonic Dystrophy were identified. Twenty patients consented and were included into the study. Eighty-five percent of patients were of Indian descent and the remaining fifteen percent were White. No African patients were identified. Sixty-five percent were male and thirty-five percent female. Myotonia was clinically present in all patients. Ninety-five percent of patients presented with predominantly distal weakness of which 40% demonstrated mild weakness, 35% moderate weakness and 25 % severe weakness. No patients were identified with predominantly proximal wasting or weakness. Southern blotting demonstrated expanded CTG repeats (DM1) in all 20 samples analysed. The PCR analysis was unable to demonstrate expanded alleles. Conclusion: This study identified patients presenting with Myotonic dystrophy to the Department of Neurology in KwaZulu-Natal and demonstrated that Myotonic Dystrophy Type 1 remains the commonest clinical and molecular presentation. In addition it substantiated previous research findings wherein no South African of African descent was found to be affected by the disease. There have been no reported cases of Myotonic Dystrophy in African Black patients presenting to the Department of Neurology in Durban, no African Black patients have been diagnosed with Myotonic Dystrophy over the past 20 years. However ,the predominance of Indians in this study is more likely a reflection of referral bias than differing incidence amongst sections of the population. PCR analysis cannot detect trinucleotide repeat expansions beyond 200 repeats and as a result Southern Blotting remains the gold standard in obtaining a molecular diagnosis. A clinical diagnosis is sufficient and molecular confirmation is not an absolute requirement. / Thesis (M.Med)-University of KwaZulu-Natal, Durban, 2006.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 137-156).