Abstract Approximately 5–10% of all breast cancer cases are estimated to result from a hereditary predisposition to the disease. Currently no more than 25–30% of these familial cases can be explained by mutations in the known susceptibility genes, BRCA1 and BRCA2 being the major ones. Additional predisposing genes are therefore likely to be discovered. This study evaluates whether germline alterations in three BRCA1-associated genes, BACH1 (i.e. BRIP1/FANCJ), BARD1 and TOPBP1, contribute to familial breast cancer. Altogether 214 Finnish patients having breast and/or ovarian cancer were analysed for germline mutations in the BACH1 gene. Nine alterations were observed, four of which located in the protein-encoding region. The previously unidentified Pro1034Leu was considered a possible cancer-associated alteration as it appeared with two-fold higher frequency among cancer cases compared to controls. All the other observed alterations were classified as harmless polymorphisms. Mutation analysis of the BARD1 gene among 126 Finnish patients having family history of breast and/or ovarian cancer revealed seven alterations in the protein-encoding region. The Cys557Ser alteration was seen at an elevated frequency among familial cancer cases compared to controls (p = 0.005, odds ratio [OR] 4.2, 95% confidence interval [CI] 1.7–10.7). The other alterations appeared to be harmless polymorphisms. To evaluate further the possible effect of Cys557Ser on cancer risk, a large case-control study was performed, consisting of 3,956 cancer patients from the Nordic countries. The highest prevalence of Cys557Ser was found among breast and ovarian cancer patients from BRCA1/BRCA2 mutation-negative families (p < 0.001, OR 2.6, 95% CI 1.7–4.0). In contrast, no significant association with male breast cancer, ovarian, colorectal or prostate cancer was observed. The current study is the first evaluating the role of TOPBP1 mutations in familial cancer predisposition. The analysis of 125 Finnish patients having breast and/or ovarian cancer revealed one putative pathogenic alteration. The commonly occurring Arg309Cys allele was observed at a significantly higher frequency among familial cancer cases compared to controls (p = 0.002, OR 2.4, 95% CI 1.3–4.2). The other 18 alterations observed were classified as harmless polymorphisms.

Aims: The purpose of the research was to investigate the benefits of a hereditary colorectal cancer registry in the management of patients and families with Lynch syndrome. In study one, a systematic review was performed to quantify the impact of registration and screening on colorectal cancer (CRC) incidence and mortality, with comparison between familial adenomatous polyposis (FAP) and Lynch syndrome (LS). In study two, a regional Lynch syndrome registry was utilised to evaluate the uptake of predictive testing and colorectal screening among first-degree relatives (FDRs) and investigate novel methods for engaging at-risk relatives, including an enhanced role for the general practitioner (GP). In study three, the registry was used to investigate proposed associations between Lynch syndrome and prostate and bladder cancer. In study four, mismatch repair-deficient (dMMR) CRCs from Lynch syndrome patients and randomised-controlled trials (RCTs) were used to evaluate a novel prognostic biomarker, beta-2 microglobulin (B2M). Methods: An electronic database search was conducted to identify studies describing CRC incidence and/or mortality in FAP or LS, with comparison of either: 1) screened and unscreened patients or 2) patients ‘before and after’ establishment of the registry. Using the Manchester regional Lynch syndrome registry database, the uptake of predictive testing and colorectal screening among FDRs was assessed with Kaplan-Meier analysis. Novel strategies for improving engagement were explored via a patient advisory group discussion and a regional primary care questionnaire. Cases of prostate and bladder cancer in male mutation carriers and their male FDRs were identified, and cumulative and relative risks were calculated, using expected rates from cancer registry data. DNA from 350 dMMR CRC specimens from Lynch syndrome patients and RCTs were tested for B2M mutations using Sanger sequencing, and correlated with clinical outcome. Results: 43 studies were included in the systematic review (33 FAP; 10 Lynch). Registry-based screening was associated with a significant reduction in CRC incidence and in Lynch syndrome, CRC-related mortality was negligible in those undergoing surveillance. 242 Lynch syndrome families were recorded on the Manchester Lynch syndrome registry. 329 of 591 (55.7%) eligible FDRs had undergone predictive testing. Uptake was significantly lower in males and younger age groups (<25 yrs). Compliance with colorectal screening was excellent following a mutation positive predictive test but poor in untested individuals (97.3% vs 35.0%). Eight prostate cancers were identified in 821 male LS mutation carriers and male FDRs. MSH2 mutation carriers had a ten-fold increased risk of prostate cancer (RR 10.41; 95%CI 2.80, 26.65) but no association with bladder cancer was identified. 69/286 (24.1%) of dMMR CRCs contained significant B2M mutations. B2M mutations were associated with complete absence of recurrence (0/39) during follow-up in the QUASAR trial (stage II), compared with 14/77 (18.2%) in wild-type B2M (p=0.005). Conclusion: Studies consistently report that registration and screening result in a reduction of CRC incidence and mortality in FAP and LS (Level 2a evidence, Grade B recommendation). Funding and managerial support for registries should be made available. Uptake of predictive testing and colorectal screening in Lynch syndrome could be substantially improved, particularly among males and younger age groups, but this requires advances in communication with at-risk relatives. It is unlikely that GPs will actively participate without considerable support from genetics services. A trial of PSA screening in MSH2 mutation carriers from 50 years would be appropriate. B2M mutation status has potential clinical utility as a prognostic biomarker in stage II dMMR CRC.

Orientador: Anamarli Nucci / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T09:41:22Z (GMT). No. of bitstreams: 1 Ramos_MargotGuarieiro_M.pdf: 985606 bytes, checksum: 434d1760ad15f7e85ae84d37db9d1523 (MD5) Previous issue date: 2006 / Resumo: Charcot-Marie-Tooth (CMT) está entre as neuropatias hereditárias mais comuns, com prevalência mundial de 1:2.500 indivíduos. Constitui-se de doenças geneticamente heterogêneas, caracterizadas por atrofia e fraqueza distais dos membros inferiores, podendo estender-se para os membros superiores. Objetivos: elaborar protocolos de exame podiátrico exeqüível em nosso meio. Aplicá-los e utilizar o Escore de Neuropatia para CMT para avaliação das disfunções e sua documentação em uma amostra de pacientes do Ambulatório de Doenças Neuromusculares. Descrever o fenótipo do conjunto dos pacientes estudados. Métodos: realizou-se avaliação clínico neurológica dos pacientes, teste do suporte lateral dos pés e preencheu-se o Escore de Neuropatia para CMT. Dois protocolos de avaliação podiátrica foram elaborados, com os respectivos escores. A parte comum a ambos consistiu na avaliação da impressão plantar e da função dos plantiflexores. A parte diferencial constituiu-se na utilização de goniometria para avaliação do ângulo do tornozelo e do calcanhar em um e utilização de fotos digitalizadas para leitura dos mesmos ângulos em outro. Resultados: participaram 20 pacientes com CMT, entre 7 e 53 anos (média e mediana de 29 anos), 50% de cada sexo, 90% com CMT de herança autossômica dominante. Atrofia peroneal ocorreu em 65% dos pacientes e atrofia dos músculos intrínsecos das mãos em 50%; rigidez da articulação subtalar em 20%; pés cavos em 90%; dedos em martelo em 85%; hipertrofia dos nervos periféricos em 40%. No escore de Neuropatia para CMT encontrou-se pontuação mínima de 9 e máxima de 30. Os protocolos podiátricos resultaram em escores sem diferenças estatísticas significantes. A avaliação do ângulo do tornozelo pelo método da goniometria demonstrou pé eqüino grave em 57,5% dos membros inferiores e pelas fotos digitalizadas em 50%. Setenta e cinco por cento (75%) dos pés tinham inversão do calcanhar. A avaliação da impressão plantar diagnosticou 70% de pés cavos. Houve déficit da flexão plantar em 60% dos pacientes. Conclusões: dois escores podiátricos por métodos diferentes foram elaborados, ambos factíveis e com resultados similares. A aplicação do Escore de Neuropatia para CMT mostrou 1 paciente com incapacidade leve, 10 (50%) com incapacidade moderada e 9 (45%) com incapacidade grave. O fenótipo de atrofia peroneal com pés eqüinos graves e inversão dos calcanhares foi predominante. Palavras-chave: Charcot-Marie-Tooth, avaliação podiátrica, Escore de Neuropatia para CMT / Abstract: Charcot-Marie-Tooth (CMT) is a genetically heterogeneous group of peripheral neuropathies presenting world prevalence of 1:2.500. Atrophy and weakness of the distal lower limbs are common clinical features and these signs may extend to the upper limbs. Objectives: to elaborate reliable protocols for podiatric exams, apply them and make use of the CMT neuropathy score in a sample of outpatients of the Neuromuscular Disorders Unit at Unicamp Hospital. To describe patients phenotypes by using the mentioned tools. Methods: patients were clinically examined and test block was realized. CMT neuropathy score was fulfilled. Two protocols of podiatric evaluation were produced, resulting in two distinct scores. Both protocols had the evaluation of footprint and plantarflexors functions in common and have used different means to assess ankle and heel angles. One of them has used goniometry to assess those angles and the other has made use of digitalized photos to do the same measurement. Results: 20 CMT patients from 7 to 53 years old (being both the average age and the median value 29) were studied - 50% males and 50% females, 90% with dominant autosomal CMT. Peroneal atrophy was observed in 65% of the patients and atrophy of intrinsic hand muscles in 50%; subtalar rigidity in 20%; pes cavus in 90%; claw of toes in 85%; peripheral nerves hypertrophy in 40%. CMT neuropathy score ranged from the minimal score of 9 and the maximal of 30. There were no significant statistical differences in the results of both podiatric protocols. The evaluation of the ankle angle by goniometry showed severe footdrop in 57,5% of the lower limbs and when the digital photos were used - 50%. Seventy-five per cent (75%) of the feet showed heel inversion. Footprint method diagnosed 70% of pes cavus. Plantarflexors failure was seen in 60% of patients. Conclusions: by using different methods, two podiatric scores were elaborated - both reliable and presenting similar results. CMT neuropathy score classified 1 patient with mild disability, 10 (50%) with moderate and 9 (45%) with severe. The predominant phenotype was peroneal atrophy associated with severe footdrop and heel inversion. Key words: Charcot-Marie-Tooth, podiatric evaluation, CMT neuropathy score / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

A complexidade da fisiologia da audição resulta da participação e interação de produtos de grande número de genes, razão pela qual a surdez hereditária exibe enorme heterogeneidade genética. Estudos moleculares nas duas últimas décadas permitiram a identificação de vários genes responsáveis por surdez; entretanto, muitos ainda restam ser identificados. A maioria dos estudos de mapeamento de genes de surdez até então conduzidos privilegiou estratégias que buscavam mutações de ponto. Outros mecanismos mutacionais, como deleções e duplicações, foram pouco investigados. Portanto, a contribuição das CNVs (Copy Number Variations) na surdez hereditária é pouco conhecida. O objetivo desse trabalho foi identificar novos genes que possam ter papel na etiologia da surdez sindrômica ou não-sindrômica por meio da investigação de microdeleções e microduplicações em pacientes com perda auditiva. Selecionamos 25 genes candidatos (CTTN, FGF3, FGF19, FOXC1, FOXF2, FOXQ1, IMMP2L, KIF5C, LRRN3, MAP1A, MYLK4, PPP3CA, SHANK2, SLC5A7, STRC, TMC1, TMC2, TMC3, TMC4, TMC5, TMC6, TMC7, TMC8, TPCN2 e TUBB2A) para a triagem de microrrearranjos por meio da técnica de MLPA (Multiplex Ligation-dependent Probe Amplification). Os genes candidatos foram selecionados a partir de rearranjos detectados em um estudo prévio realizado por meio de array-CGH (array-based Comparative Genomic Hybridization) em indivíduos com surdez sindrômica estudados em nosso laboratório, e também a partir de dados da literatura. Nossa casuística foi composta por 163 indivíduos, dos quais 74 são pacientes com surdez associada a outros sinais (sindrômicos), a maioria casos isolados, e 89 são pacientes com surdez não-sindrômica, propósitos de famílias em que segrega surdez de herança autossômica dominante ou recessiva. Desenhamos uma sonda sintética intragênica de MLPA para cada um dos genes candidatos. Foram detectadas seis deleções em TMC6 (3,7%), seis deleções e uma duplicação em STRC (4,3%) e uma duplicação em IMMP2L (0,6%). A triagem de alterações nesses três genes em 189 indivíduos fenotipicamente normais revelou quatro deleções em TMC6 (2,1%), oito deleções e três duplicações em STRC (5,8%) e três deleções em IMMP2L (1,6%). Todas as alterações em TMC6, tanto nos casos de surdez como nos controles, eram na realidade artefatos devidos a problemas de hibridação da sonda correspondente. No gene STRC, previamente já relacionado à surdez, os rearranjos nos indivíduos afetados devem se tratar de polimorfismos sem efeito fenotípico por serem muito frequentes na população. Contudo, é possível que haja nesses pacientes mutações adicionais que não puderam ser rastreadas e que poderiam contribuir ao fenótipo, em combinação com o rearranjo detectado, como já descrito em um caso da literatura. A duplicação em IMMP2L em uma paciente com surdez não-sindrômica, herdada da mãe igualmente afetada, mostrou-se a mais provavelmente relacionada ao fenótipo, pois o estudo complementar por meio de array-CGH revelou que o rearranjo inclui uma duplicação parcial da porção 3 de outro gene, DOCK4. O produto desse gene possui uma isoforma que se localiza nos estereocílios das células ciliadas e se liga a uma importante proteína relacionada à audição, a harmonina. Portanto, nossa hipótese é a de que a duplicação seja a causa da surdez na família e que DOCK4 seja um novo gene responsável por surdez. A associação de IMMP2L com surdez é menos provável devido ao grande número de CNVs não patogênicas já descritas que incluem partes desse gene. Estudos complementares são necessários para mapear a duplicação com mais precisão. Além disso, o rastreamento de mutações em DOCK4 em outras famílias com surdez pode vir a confirmar o possível papel desse gene na etiologia da surdez. / Several genes contribute to the complexity of physiology of hearing. Consequently, hereditary deafness is extremely heterogeneous from the genetic point of view. In the last two decades, several genes responsible for hereditary hearing loss have been identified, but a large number of genes remains to be found, as evidenced by the unexplained cases of inherited deafness. The search for point mutations in candidate genes after mapping based on linkage studies has been the main strategy in the identification of such genes. Other mutation mechanisms, such as deletions and duplications, have been rarely investigated, and the contribution of DNA copy number variants (CNVs) to hearing loss is not well known. This study aimed at identifying novel genes, which might play a role in the etiology of syndromic and non-syndromic deafness, through the search of gene microdeletions and microduplications. We selected 25 candidate genes (CTTN, FGF3, FGF19, FOXC1, FOXF2, FOXQ1, IMMP2L, KIF5C, LRRN3, MAP1A, MYLK4, PPP3CA, SHANK2, SLC5A7, STRC, TMC1, TMC2, TMC3, TMC4, TMC5, TMC6, TMC7, TMC8, TPCN2 and TUBB2A) based on their involvement in microimbalances detected by Array-based Comparative Genomic Hybridization (aCGH) in a previous study of a Brazilian sample of individuals with syndromic hearing loss from our laboratory and others reported in the literature. We studied 163 subjects, 74 of them presenting syndromic deafness, the majority were isolated cases, and 89 being probands of families in which nonsyndromic deafness had an autosomal dominant or recessive mode of inheritance. Gene deletions or duplications were screened by Multiplex Ligant-dependent Probe Amplification (MLPA) using one synthetic intragenic probe designed for each candidate gene. We detected six deletions in TMC6 (3,7%), six deletions and one duplication in STRC (4,3%), and one duplication in IMMP2L (0,6%). The screening of imbalances in these genes in a control sample of 189 hearing individuals revealed four deletions in TMC6 (2,1%), eight deletions and three duplications in STRC (5,8%) and three deletions in IMMP2L (1,6%). The imbalances found in TMC6, both in affected and control individuals, were in fact artifacts due to problems in the hybridization of the corresponding probe. As to the STRC gene, previously related to deafness, the imbalances are more likely to be 4 polymorphisms with no phenotypic effect. However, the possibility remains that additional undetected mutations in affected individuals contribute to their phenotype, in combination with the microrearrangement, as already reported in the literature. The duplication in IMMP2L in a non-syndromic patient, and also present in her affected mother, is most likely causative of deafness, since a complementary study performed with aCGH revealed that the rearrangement included a partial duplication of the 3 end of another gene, DOCK4. An isoform of the DOCK4 protein localizes to the stereocilia in the inner ear and interacts with harmonin, a protein already known to be involved in hearing. We hypothesize that this duplication may be the cause of deafness in the family and, this being the case, DOCK4 appears as a novel deafness gene. The causal association between IMMP2L and deafness is less plausible, because of the large number of reported non-pathogenic CNVs that include parts of this gene. Further studies are required to precisely map this duplication. In addition, the screening of mutations in DOCK4 in other families with hearing impairment is required to evaluate its possible role in the etiology of deafness.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第21535号 / 理博第4442号 / 新制||理||1638(附属図書館) / 京都大学大学院理学研究科数学・数理解析専攻 / (主査)准教授 加藤 周, 教授 重川 一郎, 教授 並河 良典 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

Purpose: This study assessed effectiveness of five tools recommended by the US Preventive Services Task Force (USPSTF), designed to help primary care clinicians determine which unaffected patients to refer to genetics specialists for breast cancer risk assessment based on concerning family history. Design: This descriptive secondary analysis included 85 women aged 40-74. All participants had a first-degree female relative previously diagnosed with breast cancer who also had uninformative negative BRCA1/2 tests. Methods: Each pedigree was evaluated using the five tools including the Family History Screen-7 (FHS-7), Pedigree Assessment Tool (PAT), Manchester Scoring System, Referral Screening Tool (RST), and Ontario-Family History Assessment Tool (Ontario-FHAT). All five tools were applied to each study participant. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated to describe each tool’s ability to identify women with elevated risk as calculated by the Claus model. Receiver operating curves (ROC) were also plotted. Differences between areas under the curve (AUCs) for all possible pairs of tools were estimated through logistic regression to assess for differences in tool performance. Results: Claus calculations identified 14 women out of 85 whose lifetime risk of breast cancer was elevated at > 15%. Only two tools, the Ontario-FHAT and FHS-7, identified all 14 women with elevated risk, a sensitivity of 100%. The FHS-7 tool flagged all 85 participants, meaning its specificity was zero. The Ontario-FHAT flagged 59 participants as needing referral (specificity 36.2%) and had a negative predictive value (NPV) of 100%, indicating that if a woman was not found to need a referral to a genetics professional, it is likely she did not have an elevated lifetime risk of developing breast cancer. AUC values were not significantly different between tools (all p values > .05), and thus were not helpful in discriminating between the tools. Conclusion: In this population, the Ontario-FHAT out-performed other tools in terms of sensitivity and negative predictive value; however, low specificity and positive predictive value must be balanced against these findings. Thus, the Ontario-FHAT can help determine which women would benefit from referral to a genetics specialist.

Die vorliegende Untersuchung beschäftigt sich mit dem hereditären Angioödem (HAE) als seltene, erbliche Erkrankung. Beim HAE kommt es zu rezidivierenden Schwellungen an der Haut, des Larynx und der intestinalen Organe. Ziel der vorliegenden prospektiven, offenen Studie war die klinische Charakterisierung von HAE-Patienten aus dem mitteldeutschen Raum einschließlich der Erfassung und des Vergleiches der Lebensqualität (LQ). Es wurden 46 HAE-Patienten aus dem mitteldeutschen Raum um Einwilligung in die Studie gebeten. 21 selbstausgefüllte Lebensqualitätsfragebögen sowie 19 standardisierte, 20-30-Minütige Telefoninterviews und die retrospektiv erfassten Krankendaten konnten ausgewertet werden. Der Vergleich der Lebensqualität von HAE-Patienten mit der Lebensqualität von Patienten mit chronischen Erkrankungen und Hautkrankheiten mit dem SF-36 und FLQA-d erfolgte im Studienzeitraum erstmalig. Die Studie konnte die Merkmale der Erkrankung des hereditären Angioödems bei Patienten aus dem mitteldeutschen Raum aufzeigen. Zwischen den HAE-Typ I und II-Patienten bestanden Unterschiede bezüglich der Vorzeichen und der Organmanifestationen. Auf die Therapie hatten diese Unterschiede keinen Einfluss. Insgesamt zeigte sich auch nach Anwendung der Lebensqualitätsfragebögen: SF-36 und FLQA-d wie schwierig die Erfassung der Lebensqualität bei HAE-Patienten ist. Zum einen sind die Fragebögen nicht spezifisch für HAE-Patienten und zum anderen sind die Ergebnisse dieser Studie durch die kleine Gruppengröße und der einzeitigen Erfassung in der Übertragbarkeit eingeschränkt. In zukünftigen Studien sollte dem Bereich der sozialen Funktionsfähigkeit bei der Erfassung der Lebensqualität Beachtung geschenkt werden. Der im Juli 2012 von Prior et al veröffentlichte, krankheitsspezifische Lebensqualitätsfragebogens HAE-QoL (111) sollte weiterentwickelt sowie die erhobenen Daten mittels einer prospektiven Studie bezüglich der Lebensqualität vor und nach einer bestimmten Therapie überprüft werden.

Die Charcot Marie Tooth Erkrankung Typ 1A (CMT1A) stellt die häufigste Unterform aller hereditären Neuropathien dar (Skre, 1974). Es handelt sich hierbei um eine autosomal- dominant vererbte Erkrankung beruhend auf einer Duplikation des Abschnittes auf Chromosom 17, welche das Gen für das periphere Myelinprotein 22kDa beinhaltet. Diese Duplikation resultiert in einer erhöhten Gendosis des pmp22, durch welche es zu einer verstärkten Bildung des Peripheren Myelin Proteins 22kDa unter anderem in Schwannzellen kommt. Es ist noch nicht abschließend geklärt in wieweit diese erhöhte Expression zu der fortschreitenden Demyelinisierung und einer fehlgesteuerten Remyelinisierung peripherer Nerven führt. Die zugrundeliegenden Pathomechanismen sind Gegenstand aktueller Forschung. Bei der CMT1A sind sowohl die efferenten als auch die afferenten Nerven betroffen. Durch die im Anschluss an demyelinisierende Prozesse stattfindende fehlgesteuerte Remyelinisierung zur Kompensation der Schädigung kommt es zu einer Verdickung der Nerven mit einer typischen Bildung von Zwiebelschalen. Diese entstehen durch die konzentrische Anlagerung überzähliger, Promyeliniserenden Schwannzellen. Im Verlauf kommt es bei einer demyelinisierenden Erkrankung auch zu einem Untergang der zugehörigen Neuronen. Klinisch imponieren diese neurodegenerativen Veränderungen als eine distal betonte, langsam progrediente Muskelschwäche und - atrophie mit variabler Ausprägung. Zusätzlich können bei den Patienten sensible Ausfälle und Schmerzen auftreten. Die klinische Manifestation ist durch das Ausmaß der Demyelinisierung und des darauffolgenden Axonverlusts bestimmt. Die Duplikation des Genes PMP22 führt bei unterschiedlichen Patienten trotz identischer genetischer Ursache zu einer starken Variabilität der Ausprägung der klinischen Symptome. Diese, und ebenfalls Unterschiede in Erstmanifestationsalter und Krankheitsverlauf, zeigen sich sogar innerhalb von Familien und bei eineiigen Zwillingen. Der ursächliche Pathomechanismus dieser Variabilität ist unbekannt. Aktuell wird der Schweregrad der Erkrankung eines Patienten meist anhand des CMT Neuropathie Scores ermittelt und abgebildet. In der klinischen Beurteilung ist eine schleichende Verschlechterung zu beobachten. Diese langsame Progression der Erkrankung zeigte sich, auch im Rahmen klinischer Studien, in einer Veränderung des CMTNS von ca. 0,23- 0,68 Punkten pro Jahr (Verhamme et al., 2009, Shy et al., 2008, Micallef et al., 2009, Pareyson et al., 2011). Durch die Erkrankung hervorgerufene motorische Einschränkungen äußern sich im Alltag durch eine verminderte Feinmotorik. Die verstärkte Atrophie der Extensoren im Vergleich zu den Flexoren führen zu einem Steppergang oder einem Schleifen der Füße, resultieren aber nur selten in einer absoluten Gehbehinderung. Die Erkrankung verläuft nicht letal. Aktuelle Studien zeigten allerdings bei CMT1A Patienten/innen mit einem langen Krankheitsverlauf eine verminderte Lebenserwartung (Vaeth et al., 2017). Die Ursache hierfür konnte aktuell noch nicht geklärt werden. Trotz intensiver Forschung gibt es noch keine wirksame kausale Therapie. Einige Substanzen (z.B. Ascorbinsäure, Onapriston, Neurotrophin-3 oder PXT3003) zeigten in Tiermodellen vielversprechende Ergebnisse. Teilweise fand bereits eine Erprobung der Substanz in humanen Studien statt. Leider erreichte bisher keine der untersuchten Substanzen ausreichend gute Ergebnisse bei einer vertretbaren klinischen Sicherheit, um eine Zulassung für die Behandlung von CMT1A zu erlangen. Eine Studie mit Ascorbinsäure, welche im Tiermodell zur Verbesserung der Myelinisierung führen konnte, zeigte bei klinischen Studien am Menschen keine signifikanten Veränderungen des klinischen Krankheitsverlaufs. Einige weitere Substanzen, welche bereits in Voruntersuchungen gute Resultate zeigten, befinden sich zurzeit in klinischer Erprobung bei CMT1A Patienten. Es könnte postuliert werden, dass eine fehlende Signifikanz in klinischen Studien auch durch die Schwierigkeiten bei der Zusammenstellung einer geeigneten Patientenkohorte, begründet liegt. Einerseits steht auf Grund der geringen Prävalenz der Neuropathie jedem Zentrum nur ein kleines Patientenkollektiv zur Verfügung. Andererseits besteht durch die beschriebene Variabilität der Erkrankung eine weitere Herausforderung darin, homogene und vergleichbare Kohorten zu bilden. Hinzu kommt, dass der bislang in der klinischen Routine verwendete CMTNS, in der Auswertung von durchgeführten Studien teilweise eine geringe Sensitivität zeigte und die einzelnen Parameter Ceiling Effekte im oberen und unteren Bereich der Skala aufwiesen. All diese Parameter einzeln und in Kombination ergeben erschwerte Rahmen- bedingungen für eine erfolgreiche Erforschung von wirksamen Therapien für die CMT1A. Wie auch beim Menschen zeigte sich auch bei dem am häufigsten verwendeten Tiermodell der CMT Ratte (Sereda et al., 1996) eine Variabilität der Krankheitsausprägung und des Manifestationszeitpunktes. Bei anderen Erkrankungen werden bereits erfolgreich Biomarker zur genauere Beurteilung der Erkrankungsschwere eingesetzt. Für CMT1A existieren bisher keine derartigen Biomarker in der klinischen Routine. Daher wurden zur Identifikation in einer ersten Studie Hautbiopsien von CMT1A Ratten untersucht. Mittels mRNA Genexpressions- analysen konnten hierbei sechs potenzielle Biomarker gefunden werden, welche sich auch in einer kleinen Kohorte von humanen CMT1A Patienten/innen für die Eignung als diagnostisches Mittel bestätigen ließen (Fledrich et al., 2012). In der vorliegenden Forschungsarbeit wurde eine weitere Validierung der potenziellen Biomarker zur Diagnostik der Krankheitsschwere durchgeführt. Zusätzlich wurden weitere Marker analysiert, die Hinweise auf den Verlauf der Progression liefern können. Die Validierung erfolgte in einem ersten Schritt an einer paneuropäischen und amerikanischen Kohorte von insgesamt 266 genetisch gesicherten, klinisch gut charakterisierten Patienten mit CMT1A. Für die mRNA Expressionsanalyse wurden Gene analysiert, welche in einer, dieser Dissertation vorangegangenen Untersuchung bei CMT1A Patienten und im Tiermodell eine signifikant veränderte Expressionen aufwiesen (ANPEP, BGN, CDA, CTSA, CRISP3, ENPP1, FN1, FN3KRP, GRIA1, GSTT1, GSTT2, GSTA4, MUCL1, PPARG, SPRR1A und NRG1-I). Den für diese Studie ausgewählten Patienten wurde eine Hautbiopsie an der Fingerkuppe entnommen, mRNA extrahiert, aufbereitet und mithilfe quantitativer Realtime- PCR analysiert. Die Ergebnisse der Expressionsanalysen wurden mittels stabiler Housekeeping Gene und Kallibratoren normalisiert und bezüglich der klinischen Parameter wie Alter, BMI, Geschlecht und untersuchendes Zentrum kontrolliert ausgewertet. Die Expressionsergebnisse wurde mit den aktuell verwendeten klinischen Scores als Marker für die Krankheitsschwere korreliert. Dabei wurden sowohl der ursprüngliche Neuropathie Score für CMT, seine Erweiterungen und Subscores sowie weitere klinische Parameter, wie beispielweise der 9HPT oder der T10MW verwendet. In vorherigen Untersuchungen hatten sich variierende Sensitivitäten der einzelnen Parameter der Scores gezeigt. Daher wurden die Untersuchungsparameter einzeln und in unterschiedlichen Kombination analysiert. Von den 16 untersuchten potenziellen Biomarker zeigten acht Gene (CDA, CTSA, GRIA1, ENPP1, ANPEP, FN3KRP, GSTT2 und PPARG) eine signifikante Korrelation mit der Erkrankungsschwere. Mit einer Sensitivität von 90% und einer Spezifität von 76,1% kann die Kombination dieser acht Gene die Patienten anhand ihres Schweregrades in mild, moderat und schwer betroffen einteilen. Um anschließend die Biomarker für eine Progressionsdetektion zu validieren, erfolgte in einer kleineren Patientenkohorte nach einem Zeitraum von zwei bis drei Jahren eine zweite klinische Untersuchung und erneute Biopsieentnahme. Die klinische Progression der Patienten/innen wurde auch hier durch einen erhöhten CMTNS Punktwert oder die Veränderung von sekundären Parametern ermittelt. Sechs Gene (CDA, CTSA, ENPP1, GSTT2, PPARG und NRG1-I) zeigten eine signifikante Veränderung Ihrer Expression über den untersuchten Zeitraum. Die Kombination dieser sechs Gene korreliert signifikant mit der Veränderung der Erkrankungsschwere, welche durch die Veränderung des CMTNS abgebildet wird. Es zeigte sich eine Sensitivität von 63,2% und eine Spezifität von 100%. Insgesamt konnte bestätigt werden, dass Genexpressionsanalysen aus Hautbiopsien und die daraus identifizierten Biomarker für die Bestimmung der Krankheitsschwere geeignet sind. Gleichzeitig konnte gezeigt werden, dass die kutane Expression von individuellen Genen über den definierten Zeitraum eine signifikante Veränderung aufweist, welche mit dem Verlauf der Erkrankung korreliert und damit ebenfalls einen geeigneten Marker für die Progression der Erkrankung bilden kann. Die fünf Gene (CDA, CTSA, ENPP1, GSTT2, PPARG), die sowohl bei der Erkrankungsschwere- als auch bei der Progressionsbeurteilung identifiziert werden konnten, stellen damit ein aussichtsreiches Set von Biomarkern dar, welche im Verlauf im klinischen Alltag und in Studien weiter validiert werden sollten. In Zukunft kann die Implementierung geeigneter Biomarker in klinische Studien entscheidend dazu beitragen, die Entwicklung von erfolgreichen Therapien zu ermöglichen und weiter voranzutreiben.

Wolfram syndrome is a genetic disorder characterized by diabetes and neurodegeneration. Two causative genes have been identified so far, WFS1 and WFS2, both encoding endoplasmic reticulum (ER) localized transmembrane proteins. Since WFS1 is involved in the ER stress pathway, Wolfram syndrome is considered an ER disease. Despite the underlying importance of ER dysfunction in Wolfram syndrome, the molecular mechanism linking ER to the death of β cells and neurons has not been elucidated. The endoplasmic reticulum (ER) is an organelle that forms a network of enclosed sacs and tubes that connect the nuclear membrane and other organelles including Golgi and mitochondria. ER plays critical functions in protein folding, protein transport, lipid metabolism, and calcium regulation. Dysregulation of ER function disrupts normal cell metabolism and activates an array of anti-survival pathways, eventually leading to disease state. Here we show that calpain is involved in both prototypes of Wolfram syndrome. Calpain 2 activity is negatively regulated by WFS2 protein, and hyper-activation of calpain 2 by WFS2-knockdown leads to cell death. Calpain hyper-activation is also present in WFS1 loss of function cells due to the high cytosolic calcium. Extensive calpain activation exists in the Wolfram syndrome mouse model as well as in patient cells. A compound screen targeting ER homeostasis reveals that dantrolene, a ryanodine receptor inhibitor, can prevent cell death in cell models of Wolfram syndrome. Our results demonstrate that the pathway leading to calpain activation provides potential therapeutic targets for Wolfram syndrome and other ER diseases.

Protruding from the apical surface of nearly every cell in our body lies a specialized sensory organelle—the primary cilium. Eukaryotic cells use these ubiquitous structures to monitor the extracellular environment, defects in which result in an ever-growing list of human maladies termed ciliopathies including obesity, retinal degeneration and polycystic kidney disease. The sensory functions of primary cilia rely on the unique complement of receptors concentrated within the ciliary membrane. Vital to the proper functioning of the cilium is the cell's ability to target specific proteins to the ciliary membrane yet little is known how a cell achieves this highly polarized distribution. IFT20, a subunit of the intraflagellar transport particle is localized to the Golgi complex that is hypothesized to sort proteins to the ciliary membrane. We show that IFT20 is anchored to the Golgi complex by the golgin protein GMAP-210 and mice lacking GMAP210 die at birth with a pleiotropic phenotype that includes growth restriction and heart defects. Cilia on GMAP210 mutant cells have reduced amounts of the membrane protein polycystin-2 localized to them suggesting IFT20 and GMAP-210 function together in the sorting or transport of proteins to the ciliary membrane. To better understand the mechanism of ciliary protein trafficking, we identify a ciliary targeting sequence (CTS) contained within fibrocystin, the gene mutated in autosomal recessive polycystic kidney disease, and investigate a series of proteins required for the delivery of this sequence to the primary cilium. We demonstrate the small G protein Rab8 interacts with the CTS of fibrocystin and controls the ciliary levels of the CTS. Arf4 is another small G protein deemed a key regulator of ciliary protein trafficking. We show Arf4 binds the CTS of fibrocystin but is not absolutely required for trafficking of the fibrocystin CTS to cilia. Arf4 mutant mice are embryonic lethal and die at mid-gestation likely due to defects in the non-ciliated visceral endoderm, where the lack of Arf4 caused defects in cell structure and apical protein localization. This suggests Arf4 is not only important for the efficient transport of fibrocystin to cilia, but also plays critical roles in non-ciliary processes. Together this work aims to elucidate the mechanisms of protein targeting to the ciliary membrane.