Univ., Diss.-2005 u.d.T.: Fumagalli, Manuel Angelo: Die immanenten Schranken des pränatalen Familienrechtsverhältnisses--Hamburg, 2004. / Literaturverz. S. 303 - 328.

O diabetes mellitus tipo MODY (maturity-onset diabetes of the young) é caracterizado por defeito na secreção de insulina, herança autossômica dominante, hiperglicemia de início precoce e anticorpos anti-células beta negativos. Até o momento, já foram descritas mutações em 14 genes diferentes. A confirmação do diagnóstico de MODY é feita por estudo genético-molecular, tradicionalmente pelo método de Sanger. Diante da grande heterogeneidade genética de MODY, acrescida da dificuldade de estudo de alguns genes por seu grande tamanho e ausência de hotspots, o sequenciamento em larga escala (SLE) mostra-se promissor para uma análise genética custo-efetiva na suspeita de MODY. No Brasil, existem poucos estudos genéticos de rastreamento de MODY e uma alta prevalência de casos sem mutações identificadas nos genes testados (MODY X). Os objetivos deste estudo foram: 1) analisar simultaneamente todos os genes associados a MODY em uma coorte de pacientes com suspeita clínica, utilizando um painel de SLE; 2) avaliar a patogenicidade das variantes alélicas identificadas de acordo com os critérios da Sociedade Americana de Genética Médica (ACMG). Foram selecionados 80 casos com fenótipo de MODY e análise prévia negativa dos 2 genes mais prevalentes, GCK e HNF1A, pelo método de sequenciamento de Sanger. Estes casos foram analisados pelo método de SLE, direcionado para regiões gênicas alvo, por meio de um painel customizado, com sequenciamento simultâneo de 51 genes nucleares e do genoma mitocondrial. As mutações identificadas foram correlacionadas com o fenótipo e foi realizada a segregação familiar. Uma cobertura de no mínimo 20x foi obtida em 98% das regiões alvo. Dos 80 pacientes avaliados, foram detectadas variantes patogênicas/potencialmente patogênicas em 16 casos (20%), confirmando o diagnóstico genético de MODY. Em 15 dos 80 pacientes foram identificadas 16 variantes de significado incerto, restando ainda 42 casos com diagnóstico molecular não esclarecido. Dos 16 casos confirmados geneticamente: 6 foram no gene GCK, 1 no HNF1A, 1 no HNF4A, 1 no HNF1B, 6 em genes raros associados a MODY (1 no ABCC8, 1 no KCNJ11, 1 no PDX1, 2 no PAX4, 1 no NEUROD1), e 1 no NEUROG3, gene associado a diabetes neonatal. Dentre estas 16 variantes, 2 não haviam sido descritas previamente. As 6 mutações no GCK não tinham sido detectadas na análise prévia por: a) 4 casos falso negativos no sequenciamento por Sanger (3 devido ao fenômeno genético de allelic dropout e 1 por erro na leitura do eletroferograma); b) 2 erros na hipótese clínica inicial do subtipo de MODY (baseada no padrão glicêmico e na resposta terapêutica dos pacientes), levando ao sequenciamento prévio de outro gene. A variante no HNF1A não foi detectada previamente por erro na leitura do eletroferograma (caso falso negativo no Sanger). Uma variante foi identificada no gene HNF4A, que não tinha sido sequenciado anteriormente e apresenta fenótipo semelhante ao do HNF1A. O paciente com variante no HNF1B não apresentava relato prévio de cistos renais ou malformações genito-urinárias e por isso não tinha sido considerada a hipótese clínica de MODY5. Além disso, o SLE confirmou o diagnóstico genético de 6 pacientes com variantes em genes de MODY considerados raros, que habitualmente não são sequenciados na rotina de Sanger e ainda detectou uma variante em um gene de diabetes neonatal (sendo necessário maiores estudos para estabelecer uma relação causal com MODY). Em 13 dos 16 casos índices diagnosticados, os familiares encontravam-se disponíveis para exame genético e a co-segregação foi concordante em 8 famílias. Todos os probandos avaliados apresentavam características clínico-laboratoriais típicas de MODY. Os achados deste estudo mostraram que o SLE foi capaz de aumentar a acurácia no diagnóstico de MODY, permitindo a confirmação molecular de 20% dos casos antes negativos e reduzindo, assim, o número de casos MODY X no Brasil. A abordagem genética por painel de SLE para diagnosticar casos com suspeita clínica de MODY mostrou-se promissora para elucidar as bases genéticas desse tipo de diabetes monogênico / Diabetes mellitus type MODY (maturity-onset diabetes of the young) is characterized by defects in insulin secretion, autosomal dominant inheritance, early onset of hyperglycemia, and negative anti-beta cell antibodies. To date, mutations in 14 genes are associated with MODY. The definitive diagnosis relies on genetic tests, traditionally by Sanger sequencing. However, given the genetic heterogeneity of this condition, added to the difficulty of studying some genes due to their large size and lack of hotspots, large-scale sequencing (LSS) seems promising for cost-effective genetic analysis on suspicion of MODY. In Brazil, there are few cohorts screened for MODY and a high prevalence of MODY X (unclear genetic diagnosis). This study aimed to analyze simultaneously all MODY genes in a cohort of clinically suspected patients using a LSS panel; and to evaluate the pathogenicity of identified allelic variants according to the criteria of the American College of Medical Genetics and Genomics (ACMG). We selected 80 subjects with MODY phenotype and negative previous analysis of the 2 most prevalent genes, GCK and HNF1A, by Sanger sequencing method. These cases were analyzed by LSS method, with simultaneous sequencing of target genes. We designed a customized panel, including 51 nuclear genes and the mitochondrial genome. The identified mutations were correlated to the phenotype and family segregation was evaluated. At least 20x coverage was obtained in 98% of the targeted regions. Of 80 evaluated subjects, pathogenic/probably pathogenic variants were detected in 16 cases (20%), confirming the genetic diagnosis of MODY. In 15 of 80 patients, 16 variants of uncertain significance were identified, remaining 42 cases with unexplained molecular diagnosis. Of the 16 genetically confirmed cases: 6 were in the GCK gene, 1 in HNF1A, 1 in HNF4A, 1 in HNF1B, and 6 in rare genes associated with MODY (1 in ABCC8, 1 in KCNJ11, 1 in PDX1, 2 in PAX4 and 1 in NEUROD1), and 1 in NEUROG3, a gene associated with neonatal diabetes. Of these 16 variants, 2 had not been previously described. Those 6 variants in GCK were not detected in the prior analysis because of: a) 4 false negative cases in Sanger sequencing (allelic dropout had occurred in 3 cases and one variant was overlooked, due to electropherogram interpretation failure); b) 2 errors in the initial clinical hypothesis of the MODY subtype (based on the glycemic pattern and therapeutic response), leading to the prior sequencing of another gene. The variant in HNF1A was not previously identified due to misinterpretation in electropherogram (Sanger false negative case). One variant were detected in the HNF4A gene, not formerly sequenced, and had a similar phenotype to that of HNF1A. The patient with HNF1B variant did not have a previous report of renal cysts or genito-urinary malformations and therefore the clinical hypothesis of MODY5 was not considered. In addition, LSS confirmed the genetic diagnosis of 6 patients harboring variants in MODY genes considered to be rare, which are not usually sequenced in the Sanger routine, and also detected one variant in a neonatal diabetes gene (further studies are necessary to establish a causal relationship with MODY). Relatives were available for genetic testing in 13 of these 16 index cases diagnosed and co-segregation was concordant in 8 families. All probands evaluated showed typical clinical and laboratory characteristics of MODY. These study findings showed that targeted-LSS could increase accuracy in MODY diagnosis, enabling molecular confirmation of 20% of previous negative cases and thus reducing the number of MODY X cases in Brazil. The genetic approach of LSS panel to diagnose cases with clinical suspicion of MODY has shown promise for elucidating the genetic basis of this type of monogenic diabetes

Preimplantation genetic diagnosis (PGD) refers to the testing of embryos produced through in vitro fertilization (IVF) in order to identify those unaffected by a specific genetic disorder or chromosomal abnormality. In this study, different methodologies were examined and developed for performance of PGD. Investigation of various whole genome amplification (WGA) methods identified multiple displacement amplification as a reliable method for genotyping single cells. Furthermore, this technology was shown to be compatible with subsequent analysis using single nucleotide polymorphism (SNP) microarrays. Compared to conventional methods used in this study to perform single cell diagnosis (e.g. multiplex PCR), WGA techniques were found to be advantageous since they streamline the development of PGD protocols for couples at high risk of transmitting an inherited disorder and simultaneously offer the possibility of comprehensive chromosome screening (CCS). This study also aimed to develop a widely applicable protocol for accurate typing of the human leukocyte antigen (HLA) region with the purpose of identifying embryos that will be HLA-identical to an existing sibling affected by a disorder that requires haematopoietic stem cell transplantation. Additionally, a novel microarray platform was developed that, apart from accurate CCS, was capable of reliably determining the relative quantity of mitochondrial DNA in polar bodies removed from oocytes and single cells biopsied from embryos. Mitochondria are known to play an important role in oogenesis and preimplantation embryogenesis and their measurement may therefore be of clinical relevance. Moreover, real-time PCR was used for development of protocols for CCS, DNA fingerprinting of sperm samples and embryos and the relative quantitation of telomere length in embryos (since shortened telomeres might be associated with reduced viability). As well as considering the role of genetics in terms of oocyte and embryo viability assessment and the diagnosis of inherited genetic disorders, attention was given to a specific gene (Phospholipase C zeta) of relevance to male infertility. A novel mutation affecting the function of the resulting protein was discovered highlighting the growing importance of DNA sequence variants in the diagnosis and treatment of infertility.

With pre-implantation genetic diagnosis (PGD), genetic testing and selective transfer of embryos is possible. In the future, germ-line gene therapy (GLGT) applied to embryos before implantation, in order to introduce missing genes or replace mutant ones, may be possible. The objective of this dissertation is to analyse moral aspects of these technologies, as described by eighteen British, Italian and Swedish gynaecologists and geneticists. The objective is systematised into three parts: research interviews and qualitative analysis, philosophical analysis, and elaboration of a framework that supports the combination of analytic methods. PGD was described as positive since it enabled some couples at risk for a genetic disease to have a child without the disease. PGD was described as in different senses ‘better’ than methods for prenatal diagnosis and selective termination of pregnancy. It was also described as positive since it provided couples at risk with one more option, even if it did not result in the birth of a healthy child. However, interviewees were concerned about the difficulty of defining and evaluating genetic disease. They were also concerned about patients’ choices, and about exaggerated use or misuse. Whereas PGD gave rise to ambivalence in terms of how to understand, describe and evaluate it, GLGT was often described as unrealistic or undesirable. The results of the qualitative analysis are used in a philosophical analysis of the concepts of choice, autonomous choice, ambivalence, trust and ambivalence in trust relations. A set of distinct characteristics of each concept are elaborated. The results of the philosophical analysis are used in the discussion of the results of the qualitative analysis. The study shows that the technologies imply both ‘new’ ways to perform ‘old’ medical practices and ‘new’ practices. Old moral questions are reformulated. New moral questions are added. Against the background of this, the concept of genetic identity is discussed. Key words: empirical ethics, pre-implantation genetic diagnosis, germ-line gene therapy, qualitative research, philosophical analysis, medical progress, genetic disease, choice, autonomous choice, ambivalence, trust, genetic identity.

The scope of this dissertation focuses on issues related to the quality of life of children suffering with spinal muscular atrophy (SMA) and their carers and the associated social impact on families affected by this progressive and incurable disease. It describes ethical aspects of help to families with SMA and serious decisions in relation to the need to connect to artificial ventilation. Spinal Muscular Atrophy - SMA is a motoneuron disease i.e. disease of neurons, which are responsible for conscious movements of muscles e.g. running, head movement and swallowing. The prevalence is approximately 1 newborn for 6000 live births and approximately 1 person of 40 people is the carrier of the disease. SMA affects all the bone muscles i.e. proximal muscles are often affected the most. Everyone affected is in some point in life, depending on stage and type, reliant on mechanical or electrical wheelchair, in many cases also on artificial ventilation and permanent 24hr care. Families affected by this illness accept the fact of this progressive and incurable illness differently, this dissertation reflects upon such different perceptions on quality of life of the affected children, the carers. It forms a contribution in building a foundation for organising multi-discipline teams of experts with sole purpose of therapeutical interventions, to support the child and his/hers family. The World Health Organization (WHO) defines palliative care as "improving quality of life of patients facing life-threatening illnesses, and their families, through the prevention and relief of suffering by early identification and treatment of pain and other problems, whether physical, psychological, social or spiritual." Palliative care prepares families for these situations and should be provided along with whatever treatment options families choose. This dissertation is a comprehensive information base to support children affected by SMA and their families in early care in Czech Republic and in the process of inclusive educational integration into mainstream society.

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Using hauntology as a linchpin, this dissertation explores the undertheorized connection between intersex and disability. Building on important feminist research in the fields of intersex, queer, disability, crip, and hauntology studies, I ask, how do we understand and reconcile the contested meanings, responses to, and effects of intersex? Intersex is “a perpetually shifting phantasm” (Holmes 2002: 175), yet intersex is typically represented and treated as innate disorder, disability, or disease by medical professionals. That said, many intersex people appear to distance from disability. By engaging intersex studies with feminist disability and crip theories, however, I demonstrate that an intersex politic and intersex studies must be rooted in a disability politic and disability studies. Through a feminist disability and crip lens, I conduct a textual and critical discourse analysis of three case studies of interphobic violence or, what I term, “compulsory dyadism,” meaning the instituted cultural mandate that people cannot have intersex traits or house the “spectre of intersex” (Sparrow 2013: 29); such a spectre must be exorcised. The three case studies include nonconsensual medical interventions, sport sex testing, and employing reproductive technologies to select against intersex variations. My analyses of these case studies produce three important observations. First, intersex is presently and effectively being integrated into conventional notions of disability; second, ableist logics underpin interphobic violence; and third, compulsory dyadism is intertwined with, or is an iteration of, compulsory able-bodiedness. In recognizing this interconnection, theorizing intersex and disability together is not merely beneficial, doing so is necessary. Ultimately, my dissertation interrogates and extends questions of the ever-shifting categorization of body-minds, culturally mandated ways of being, and (the haunting effects of) pathologization. I apply pressure to the academic field of intersex studies as well as intersex activist and advocate communities to center disability in discussions concerning intersex human rights and interphobia.