No description available.

No description available.

Diss. (sammanfattning) Umeå : Univ., 2005. / Härtill 5 uppsatser.

The Congenital Dyserythropoietic Anemias (CDA) are rare hereditary hemolytic disorders with large bi- to multi-nucleated erythroblasts in the bone marrow. Hemolysis is negative in a direct antiglobulin test (DAT). Based on morphology and clinical picture, three major forms of CDAs, type I, II, and III have been defined. CDA III, dominantly inherited, constitutes the rarest type with a majority of cases belonging to a family in Västerbotten, Sweden. The genetic background of CDA I and CDA II has been linked to mutations in CDAN1 and SEC23B respectively. The mutation of CDA III has been linked to 15q22 in earlier studies. In this project we have defined the causative genetic lesion in two families with CDA III. The novel mutation KIF23 c.2747C>G (p.P916R) was shown to segregate with CDA III in the Swedish and American CDA III families and was absent in 356 healthy controls. KIF23 encodes mitotic kinesin-like protein 1 (MKLP1), which plays a central role in the last step of cytokinesis. RNAi-based knock-down and rescue experiments demonstrated that the p.P916R mutation causes cytokinesis failure in HeLa cells, resulting in increasing number of bi-nuclear cells, consistent with appearance of large multinucleated erythroblasts in CDA III patients. We conclude that CDA III is caused by a mutation in KIF23, encoding MKLP1, a conserved mitotic kinesin crucial for cytokinesis. Flow cytometry with eosin-5´-maleimide (EMA), anti-CD55 and anti-CD59 is commonly used when investigating non-autoimmune hemolytic anemias. Reduced fluorescence of EMA, typically detected in hereditary spherocytosis, is also seen in CDA II, while reduction of CD55 and CD59 characterizes paroxysmal nocturnal hemoglobinuria (PNH). We studied the flow cytometric profile of EMA, CD55, and CD59 on erythrocytes in CDA III. We found no abnormality of the erythrocyte membrane in CDA III and concluded that standard flow cytometry cannot be used to discriminate between CDA III and normal controls. In CDA I and CDA II a majority of patients, including those who are not transfusion dependent, suffer from iron overload, which, according to earlier studies, is not the case in CDA III. We found that individuals of the Västerbotten CDA III family carry mutations in the hemochromatosis (HFE) gene. Three CDA III patients with heterozygous or compound HFE mutations need treatment with phlebotomy due to iron overload. One of them carries heterozygous H63D mutation, which is not reported to lead to iron overload by itself in otherwise healthy individuals. We propose that molecular genetic testing of the HFE gene is indicated in all patients with CDA, including CDA III.

L’étude des formes familiales de syndrome néphrotique (SN) ou de protéinurie glomérulaire avec lésions histologiques de hyalinose segmentaire et focale (HSF) a permis d’incriminer plus d’une vingtaine de gènes, majoritairement exprimés par le podocyte, cellule principale de la barrière de filtration glomérulaire (BFG). Parmi ces gènes, près d’une dizaine code des régulateurs du cytosquelette d’actine démontrant ainsi le rôle central de la plasticité et de l’architecture du podocyte dans le fonctionnement du filtre glomérulaire. L’ensemble de ces travaux a permis de définir une nouvelle catégorie de maladies nommées podocytopathies héréditaires. Mon projet de thèse a porté sur la caractérisation de plusieurs gènes (TTC21B, WDR73, TRIM3), dont nous avons identifié des mutations dans des cas de podocytopathies héréditaires isolées ou syndromiques. Les résultats du premier volet de ma thèse ont montré que la mutation faux sens p.P209L dans le gène TTC21B induit à l’état homozygote une nouvelle entité clinique associant à la fois une atteinte glomérulaire et une atteinte tubulaire. TTC21B code l’IFT139 (intraflagellar transport protein 139), une protéine impliquée dans le transport protéique antérograde dans le cil primaire, un organite présent à la surface de la plupart des cellules épithéliales. Ces résultats étaient inattendus car l’identification de mutations dans un gène codant une protéine ciliaire n’avait jamais été démontrée auparavant dans des cas de podocytopathies héréditaires, et surtout, il ne semblait pas exister de cil primaire à la surface des podocytes matures. Effectivement, nous avons montré que le cil primaire est présent dans les podocytes humains indifférenciés, mais disparait au cours de la différenciation. Nos résultats ont permis de comprendre l’apparente contradiction entre la survenue d’une pathologie glomérulaire relativement tardive (protéinurie et SN à l’adolescence) et l’absence de cil dans le podocyte mature. En effet, nous avons montré que la mutation p.P209L est une mutation hypomorphe qui induit des défauts mineurs dans la fonction ciliaire, alors qu’elle provoque, dans le podocyte différencié, une déstructuration importante du réseau d’actine et de microtubules du podocyte. Cette étude montre que la protéine ciliaire IFT139, par sa fonction extra-ciliaire, permet de réguler la dynamique des microtubules. Dans le deuxième volet de mon projet, en collaboration avec l’équipe de D Bonneau (Angers), nous avons identifié des mutations tronquantes dans le gène WDR73, dans deux familles non apparentées présentant un syndrome de Galloway-Mowat (SGM), pathologie de transmission autosomique récessive, très hétérogène cliniquement, associant SN et microcéphalie. Ces travaux ont permis d’identifier le premier gène impliqué dans le SGM, dans un sous-groupe de patients présentant un phénotype neurologique très homogène (microcéphalie post-natale, atrophie corticale avec atrophie cérébelleuse majeure, déficience intellectuelle très sévère), alors que l’atteinte glomérulaire est très variable. Ce gène code WDR73, une protéine à motifs WD40. Nos travaux ont montré que la protéine est exprimée dans les neurones du système nerveux central, en particulier dans les cellules de Purkinje du cervelet et dans les podocytes. Des études fonctionnelles nous ont permis de montrer que WDR73 est impliquée dans la survie cellulaire, puisqu’en son absence, une apoptose accrue est observée dans les fibroblastes de patients. De plus, elle est également nécessaire au maintien de la dynamique des microtubules dans les fibroblastes et dans les podocytes différenciés, alors qu’elle ne semble pas avoir de rôle dans la régulation de l’actine. (...) / The genetic study of familial forms of nephrotic syndrome or proteinuria with focal segmental glomerulosclerosis has permitted the identification of 30 causal genes, mainly expressed in the podocyte, which is the principal actor of the glomerular filtration barrier (GFB). Among those genes, approximately ten encode actin cytoskeleton regulators and components, thus highlighting the dramatic role of the podocyte architecture and plasticity in the function of the GFB. During the last decade, all the accumulating results, has made a new category of disease called hereditary podocytopathies. The aim of my thesis project was to characterize the effect of mutations in three candidate genes (TTC21B, WDR73, WDR73), identified by whole exome sequencing in isolated or syndromic podocytopathies. In the first part of my project, we found a homozygous missense mutation (p.P209L) in TTC21B, which encodes a ciliary gene named Intraflagellar transport protein IFT139. This protein ensures the trafficking of components from the tip to the base of the primary cilium, which is an organelle present on most mammalian epithelial cells. These results were unexpected because until now, the existence of the primary cilium was unknown. Our work demonstrates the presence of the primary cilium in the human immature podocyte that disappears once podocytes have differentiated. We also showed that IFT139 localized at the basal body and then relocalized along the complex microtubule network of differenciated cells. We showed that the hypomorphic mutation p.P209L causes minor ciliary defects in undifferentiated cells that are not responsible for the glomerular phenotype. Indeed, the glomerular lesions are rather due to drastic damage in actin and, microtubular dysregulation, found in differentiated podocytes. The second part of my thesis aimed to characterize the effects of truncating mutations identified in the WDR73 gene, found in two families. WDR73 is the first gene identified in Galloway Mowat syndrome by whole exome sequencing combined with homozygous mapping. This rare disease is defined by the association of microcephaly with nephrotic syndrome. In this study, the phenotypes of patients with WDR73 mutations are homogenous concerning neurological features, and are heterogeneous with regards to the renal defects. Thus, WDR73 mutations are responsible for a subset of particular patients affected with Galloway-Mowat syndrome. The WDR73 gene encodes WDR73, a WD-40 containing protein of unknown function. Our studies demonstrated that this protein is expressed in both neurons and podocytes in human tissues. We demonstrated that in undifferentiated cells, WDR73 is weakly expressed in the cytosol, while strong expression and relocalization to the spindle pole, microtubule asters and in the cleavage furrow occur during mitosis. Patient fibroblasts and WDR73-depleted podocytes displayed defects in nuclear morphology, which was associated with a decrease in cell survival in patient fibroblasts. Furthermore, we showed that patient fibroblasts and differentiated WDR73-depleted podocytes harbored an atypical morphology associated with a disorganized microtubule network, suggesting microtubule polymerization defects. Our functional studies demonstrated that WDR73 is crucial in both cell survival and microtubule polymerization in neurons and podocytes. The final part of my PhD work focused on the characterization of a missense mutation in the TRIM3 gene R28W identified by whole exome sequencing in a non consanguineous family with autosomal dominant focal segmental glomerulosclerosis. TRIM3 encodes TRIM3, an E3 ubiquitin-ligase that plays a role in transferrin endosomal recycling, and in microtubule trafficking via KIF21B, one of its known partners. Interestingly, the polymorphism V801M in ACTN4 co-segrates with the disease. Furthermore, mutations in this gene were already incriminated in autosomal dominant cases of HSF. (...)

L’étude des formes familiales de syndrome néphrotique (SN) ou de protéinurie glomérulaire avec lésions histologiques de hyalinose segmentaire et focale (HSF) a permis d’incriminer plus d’une vingtaine de gènes, majoritairement exprimés par le podocyte, cellule principale de la barrière de filtration glomérulaire (BFG). Parmi ces gènes, près d’une dizaine code des régulateurs du cytosquelette d’actine démontrant ainsi le rôle central de la plasticité et de l’architecture du podocyte dans le fonctionnement du filtre glomérulaire. L’ensemble de ces travaux a permis de définir une nouvelle catégorie de maladies nommées podocytopathies héréditaires. Mon projet de thèse a porté sur la caractérisation de plusieurs gènes (TTC21B, WDR73, TRIM3), dont nous avons identifié des mutations dans des cas de podocytopathies héréditaires isolées ou syndromiques. Les résultats du premier volet de ma thèse ont montré que la mutation faux sens p.P209L dans le gène TTC21B induit à l’état homozygote une nouvelle entité clinique associant à la fois une atteinte glomérulaire et une atteinte tubulaire. TTC21B code l’IFT139 (intraflagellar transport protein 139), une protéine impliquée dans le transport protéique antérograde dans le cil primaire, un organite présent à la surface de la plupart des cellules épithéliales. Ces résultats étaient inattendus car l’identification de mutations dans un gène codant une protéine ciliaire n’avait jamais été démontrée auparavant dans des cas de podocytopathies héréditaires, et surtout, il ne semblait pas exister de cil primaire à la surface des podocytes matures. Effectivement, nous avons montré que le cil primaire est présent dans les podocytes humains indifférenciés, mais disparait au cours de la différenciation. Nos résultats ont permis de comprendre l’apparente contradiction entre la survenue d’une pathologie glomérulaire relativement tardive (protéinurie et SN à l’adolescence) et l’absence de cil dans le podocyte mature. En effet, nous avons montré que la mutation p.P209L est une mutation hypomorphe qui induit des défauts mineurs dans la fonction ciliaire, alors qu’elle provoque, dans le podocyte différencié, une déstructuration importante du réseau d’actine et de microtubules du podocyte. Cette étude montre que la protéine ciliaire IFT139, par sa fonction extra-ciliaire, permet de réguler la dynamique des microtubules. Dans le deuxième volet de mon projet, en collaboration avec l’équipe de D Bonneau (Angers), nous avons identifié des mutations tronquantes dans le gène WDR73, dans deux familles non apparentées présentant un syndrome de Galloway-Mowat (SGM), pathologie de transmission autosomique récessive, très hétérogène cliniquement, associant SN et microcéphalie. Ces travaux ont permis d’identifier le premier gène impliqué dans le SGM, dans un sous-groupe de patients présentant un phénotype neurologique très homogène (microcéphalie post-natale, atrophie corticale avec atrophie cérébelleuse majeure, déficience intellectuelle très sévère), alors que l’atteinte glomérulaire est très variable. Ce gène code WDR73, une protéine à motifs WD40. Nos travaux ont montré que la protéine est exprimée dans les neurones du système nerveux central, en particulier dans les cellules de Purkinje du cervelet et dans les podocytes. Des études fonctionnelles nous ont permis de montrer que WDR73 est impliquée dans la survie cellulaire, puisqu’en son absence, une apoptose accrue est observée dans les fibroblastes de patients. De plus, elle est également nécessaire au maintien de la dynamique des microtubules dans les fibroblastes et dans les podocytes différenciés, alors qu’elle ne semble pas avoir de rôle dans la régulation de l’actine. (...) / The genetic study of familial forms of nephrotic syndrome or proteinuria with focal segmental glomerulosclerosis has permitted the identification of 30 causal genes, mainly expressed in the podocyte, which is the principal actor of the glomerular filtration barrier (GFB). Among those genes, approximately ten encode actin cytoskeleton regulators and components, thus highlighting the dramatic role of the podocyte architecture and plasticity in the function of the GFB. During the last decade, all the accumulating results, has made a new category of disease called hereditary podocytopathies. The aim of my thesis project was to characterize the effect of mutations in three candidate genes (TTC21B, WDR73, WDR73), identified by whole exome sequencing in isolated or syndromic podocytopathies. In the first part of my project, we found a homozygous missense mutation (p.P209L) in TTC21B, which encodes a ciliary gene named Intraflagellar transport protein IFT139. This protein ensures the trafficking of components from the tip to the base of the primary cilium, which is an organelle present on most mammalian epithelial cells. These results were unexpected because until now, the existence of the primary cilium was unknown. Our work demonstrates the presence of the primary cilium in the human immature podocyte that disappears once podocytes have differentiated. We also showed that IFT139 localized at the basal body and then relocalized along the complex microtubule network of differenciated cells. We showed that the hypomorphic mutation p.P209L causes minor ciliary defects in undifferentiated cells that are not responsible for the glomerular phenotype. Indeed, the glomerular lesions are rather due to drastic damage in actin and, microtubular dysregulation, found in differentiated podocytes. The second part of my thesis aimed to characterize the effects of truncating mutations identified in the WDR73 gene, found in two families. WDR73 is the first gene identified in Galloway Mowat syndrome by whole exome sequencing combined with homozygous mapping. This rare disease is defined by the association of microcephaly with nephrotic syndrome. In this study, the phenotypes of patients with WDR73 mutations are homogenous concerning neurological features, and are heterogeneous with regards to the renal defects. Thus, WDR73 mutations are responsible for a subset of particular patients affected with Galloway-Mowat syndrome. The WDR73 gene encodes WDR73, a WD-40 containing protein of unknown function. Our studies demonstrated that this protein is expressed in both neurons and podocytes in human tissues. We demonstrated that in undifferentiated cells, WDR73 is weakly expressed in the cytosol, while strong expression and relocalization to the spindle pole, microtubule asters and in the cleavage furrow occur during mitosis. Patient fibroblasts and WDR73-depleted podocytes displayed defects in nuclear morphology, which was associated with a decrease in cell survival in patient fibroblasts. Furthermore, we showed that patient fibroblasts and differentiated WDR73-depleted podocytes harbored an atypical morphology associated with a disorganized microtubule network, suggesting microtubule polymerization defects. Our functional studies demonstrated that WDR73 is crucial in both cell survival and microtubule polymerization in neurons and podocytes. The final part of my PhD work focused on the characterization of a missense mutation in the TRIM3 gene R28W identified by whole exome sequencing in a non consanguineous family with autosomal dominant focal segmental glomerulosclerosis. TRIM3 encodes TRIM3, an E3 ubiquitin-ligase that plays a role in transferrin endosomal recycling, and in microtubule trafficking via KIF21B, one of its known partners. Interestingly, the polymorphism V801M in ACTN4 co-segrates with the disease. Furthermore, mutations in this gene were already incriminated in autosomal dominant cases of HSF. (...)

Problem statement: Mutations in the genetic material BRCA I/II are linked to increased incidence of cancer among the women who carry these alterations, raising lifetime risk of breast cancer to as high as 87%. Genetic testing exists to identify these alterations, empowering women to obtain advanced screening for breast and ovarian cancer, as well as incorporate prophylactic medications and surgeries for prevention of disease. Research has shown that appropriate risk assessment methods are not being utilized among primary care providers to identify those patients who would benefit from genetic counseling and testing. Purpose: To determine if a knowledge deficit about U.S. Preventative Services Task Force BRCA risk assessment recommendations exists among the rural Arizona nurse practitioner population, and to determine the level of confidence Arizona NPs have regarding the topic. Methods: A needs assessment completed via an 18-question online survey distributed through two provider organizations in Arizona. Inclusion criteria included NP must hold an active license in Arizona, NP must practice in primary healthcare, and practice site must serve patients who reside in a rural area of Arizona. Data collection remained open for three weeks. Analysis: Descriptive statistics using quantitative analysis evaluated provider demographics, responses to basic knowledge questions and clinical scenarios, and provider self-confidence analyses. Results: Participants were able to identify inheritance patterns of BRCA mutations, but incorrectly answered the majority of knowledge questions. Regarding self-reported confidence with awareness and use of the USPSTF guideline, nearly half of participants felt that they had at least average confidence. However, only one participant was able to answer every question correctly. Most agreed that the guidelines were relevant to their current practice. These results indicate a knowledge gap among NPs who care for patients living in rural Arizona. These results may inform future research aimed at educational interventions and practice improvement initiatives that will improve understanding and use of guidelines for screening, counseling, and testing patients at high-risk of carrying a harmful BRCA-mutation. Ultimately, these results will impact outcomes of patients living in rural Arizona.

1 Abstract This thesis deals with the conditions that need to be fulfilled in order to acquire inheritance. The aim of this thesis is to provide outlook into the matter of the law of succession and thoroughly describe and further analyse individual conditions of hereditary succession, which are legal requirements that needs to be fulfilled in order to acquire inheritance. In this thesis I systematically discuss the law of succession in its general meaning, canons of inheritance, legal sources of the law of succession and also legal institutes including several new ones which are govern in the new civil code. The main part of this thesis analyses individual conditions of hereditary succession. This thesis is composed of four chapters. Chapter one deals with the general meaning of the law of succession and in this chapter I provide introduction to the law of succession and define some fundamental legal terms. In chapter two I describe the canons of inheritance. This chapter is divided into three subchapters where I discuss in my opinion the three leading canons of inheritance. First subchapter deals with the principle of personal autonomy. Second one deals with the principle of universal succession. The third subchapter deals with the principle that the predecessor's possession is transferred by law to his...

ABSTRACT INTRODUCTION: Cancer is one of the most common diseases in the developed world and both genetic and environmental factors play a role in the development of cancer. About 5-10% of all cancers are due to predisposing genes. Some of the more common inherited cancer syndromes are hereditary breast and ovarian cancer (HBOC) and two colorectal cancer syndromes, familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC). Recognition of cancer susceptibility can allow “at risk” individuals and families to participate in cancer risk assessment, genetic testing, and various cancer prevention strategies. As the public is becoming more aware of inherited cancers, it is expected that there will be an increasing demand for genetic services and testing. For this reason more GP involvement is required to assess patients and families at risk and refer them appropriately. Since the Clinical and Counselling Section, Division of Human Genetics, National Health Laboratory Service and University of the Witwatersrand, Johannesburg is establishing a cancer genetics service it woud be of great value to assess the GPs’ practice, knowledge and attitudes with regards to cancer genetics and this was therefore the aim of this study. METHODOLOGY: A quantitative, exploratory research design was chosen and GPs in the Johannesburg area were selected as subjects. After the completion of a pilot study a research package was mailed to 196 GPs. This package was sent out twice and both times the GPs were asked to respond within 3-4 weeks. The final sample consisted of 61 GPs and the data were analysed using descriptive statistics. RESULTS: Of the 61 participants more male GPs (42, 69%) than female GPs (19, 31%) responded and there were about an equal number of GPs practicing alone (29, 48) and in a multiple practice (32, 52%). Twenty two (33%) of the GPs had never had personal experience of cancer. Practices: The GPs made use of several cancer screening procedures but obtained limited information on cancer history from their patients particularly from second degree relatives and about age of onset. Very few subjects (15, 25%) reported that they assess patients’ risk for inherited cancer susceptibility and only 22 (36%) reported that they refer patients to other facilities for risk assessment and genetic testing. Knowledge: Only 32 (52%) of the GPs were aware of genetic testing facilities and 54 (86%) reported never having received advertising material to promote genetic testing for cancer susceptibility services. They also are not aware of genetic counselling facilities but do feel patients should have genetic counselling by a genetic counselor, clinical geneticist or oncologist before genetic testing. Even though genetic testing for inherited cancer susceptibility is only available at some academic institutions, mostly on a research basis, the GPs seem to be unaware of the availability of genetic testing in South Africa for colorectal cancer genes (8, 13% and 9, 15%) but 28 (46%) knew about breast cancer genes. They were not aware of the autosomal dominant inheritance of hereditary breast cancer and the percentage of individuals with breast cancer who carry the BRCA1/2 gene nor did they know the penetrance of HNPCC genes. Attitudes: The subjects’ attitudes to genetic testing for inherited cancer susceptibility were positive although they reported that they were unaware of several general factors regarding cancer genetic testing. The GPs had limited knowledge about inherited cancers and do not take an active part in cancer genetic management. However, 53 (87%) of the GPs reported interest in learning about these services and expected to play a role in cancer genetics in the future. CONCLUSION: The findings of this study suggest that there is a need to educate GPs about the basic cancer genetic concepts so that they can identify patients at risk for an inherited cancer syndrome. They need to be informed about the genetic tests currently available for the inherited cancer syndromes, and about genetic counselling and testing facilities.

A Thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy / Pterygium, a wing-shaped fibrovascular growth of the conjunctiva onto the cornea, can impair vision and be cosmetically unacceptable. Its frequency varies in Africa and postsurgical recurrence in blacks may be high. Determinants of pterygium occurrence and recurrence in rural Africans are not known. This study aimed to establish the determinants of pterygium occurrence and recurrence in rural blacks. The case controlled study comprised 230 patients and 157 controls. Interviews and eye examination were conducted; however, data from 150 patients and 150 controls were analyzed as pre-calculated. Families of 51 cases and 50 controls were studied. Surgery was done on 200 eligible patients. Those who experienced post-surgical recurrence were subclassified as cases and those who did not, controls. Immunohistochemistry was done on 59 pterygium sections and 7 controls. Family history of pterygium was present in 46 cases (30.6%) of 150, and 15 controls (10%) of 150; Odds ratio (OR) =3.93; p <0.01. Traditional eye medication was used by 79 cases (52.6%) of 150, and 60 controls (40%) of 150; OR =2.03; p <0.01. The tear film was unstable in 10 cases (6.6%) of 150, and 26 controls (17.3%) of 150; OR =0.30; p <0.01. Groups of 3-5 individuals per household were pterygium-affected in 36 pterygium families (70.5%) of 51 vs. 1 control (2%) of 50. After surgery, only 190 patients completed followup for a minimum duration of 6 months, and 52 (27.4%) experienced post-surgical recurrence. Of the 52 cases, 21 (40%) had grade 2 pterygia v. 8 post-surgical controls having grade 2 pterygia (6%) of 138; OR =9.1; p <0.01. The limbal basal epithelium expressed p53 in 11 pterygia (18.6%) of 59 v. 5 controls (71.4%) of 7; p <0.01. It expressed matrixmetalloproteinase-1 (MMP1) in 14 pterygia (23.7%) of 59 v. 5 controls (71.4%) of 7; p =0.02. MMP2 and MMP3 were detected in 16 cases (27.1%) of 59 v. 5 controls (71.4%) of 7; p =0.03. Pterygium occurred in families and was associated with traditional eye medication. Pterygium occurrence was not associated with unstable tear film, p53, and MMPs. Postsurgical recurrence was connected to grade 2 pterygia.