京都大学 / 新制・課程博士 / 博士(医学) / 甲第24138号 / 医博第4878号 / 新制||医||1060(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 村川 泰裕, 教授 波多野 悦朗, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第25497号 / 医博第5097号 / 新制||医||1073(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 中島 貴子, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

As neuropatias periféricas hereditárias são um grupo heterogêneo de doenças relacionadas que afetam o sistema nervoso periférico. Elas podem ser classificadas de acordo com a velocidade de condução motora nos membros superiores (tipo 1 - CMT1, tipo 2 - CMT2 ou intermediário - iCMT), de acordo com o padrão de herança (autossômicas dominantes, autossômicas recessivas ou ligadas ao X) e quanto ao fenótipo de apresentação (neuropatias hereditária sensitivo e motora - CMT, neuropatia hereditária sensitiva - HSN ou neuropatia motora hereditária distal - dHMN). O uso das tecnologias de sequenciamento de nova geração (NGS) para diagnóstico de pacientes com neuropatia hereditária é particularmente eficiente uma vez que representa uma doença Mendeliana com mais de 90 genes diferentes relacionados. Foram avaliados 30 pacientes com diferentes subtipos de neuropatia hereditária (3 CMT1, 12 CMT2, 8 iCMT, 4 dHMN e 3 HSN). Foram identificadas 6 mutações (SH3TC2, GDAP1, MME, IGHMBP2, 2 AARS) e 7 variantes provavelmente patogênicas (KIF1A, DRP2, MME, MPZ, VRK1, SIGMAR1, FLVCR1). Com uma taxa de positividade de 43.3%. As variantes provavelmente patogênicas foram consideradas como a causa da apresentação fenotípica apresentada pelos pacientes baseado na frequência de variantes nos bancos de população normal, no efeito bioquímico das variantes sobre a estrutura proteica e pela análise in silico. No entanto, essas variantes necessitam de evidências adicionais que confirmem sua patogenicidade. Foram identificadas variantes novas nos genes MPZ, KIF1A, DRP2, IGHMBP2, VRK1, SIGMAR1 e FLVCR1 ampliando a variabilidade genotípica desses genes. A associação das mutações identificadas nos genes VRK1, KIF1A, IGHMBP2 e FLVRC1 permitiu a expansão dos fenótipos relacionados a esses genes. Mutações no gene VRK1 podem causar uma dHMN com sinais de liberação piramidal e envolviemento preferencial do compartimento posterior da perna. Transtorno do espectro autista pode ser observado em associação a mutações no gene KIF1A e mutações no gene FLVRC1 podem causar um fenótipo grave caracterizado por insensibilidade congénita a dor e acromutilações. Mutações no gene IGHMBP2 podem causar uma sobreposição entre os fenótipos SMARD1/CMT2S com disautonomia restrita ao trato gastro intestinal. Esse estudo demonstra que o uso de WES para o diagnóstico molecular de doenças geneticamente heterogêneas como as neuropatias hereditárias é uma ferramenta útil. / The hereditary peripheral neuropathies are a heterogeneous group of genetic disorders in which peripheral nervous system degeneration leads to weakness, atrophy and loss of sensation. It can be classified according motor conduction velocities in the upper limbs (type 1 - CMT1, type 2 - CMT2 or intermediate - iCMT), according to inheritance pattern (autosomal dominant, autosomal recessive or X linked) and according to the mainly group of fibres clinically involved (hereditary sensory and motor neuropathy - CMT, hereditary sensory neuropathy - HSN or distal hereditary motor neuropathy - dHMN). The use of next generation sequencing technologies (NGS) for the diagnosis of patients with genetic diseases is well established, as CMT is a Mendelian disease with more than 90 different related genes already reported. We evaluated 30 patients with all subtypes of hereditary neuropathy (3 CMT1, 12 CMT2, 8 iCMT, 4 dHMN and 3 HSN). Six mutations (SH3TC2, GDAP1, MME, IGHMBP2, 2 AARS) and 7 likely pathogenic variants (KIF1A, DRP2, MME, MPZ, VRK1, SIGMAR1, FLVCR1) were detected, leading to a positive rate of 43.3%. Likely pathogenic variants were considered based on their frequency in normal population, in silico analysis and segregation with phenotype. Despite they have strong evidences to support their causative status further evidence of their pathogenicity is required. New variants were identified in the genes MPZ, KIF1A, DRP2, IGHMBP2, VRK1, SIGMAR1 and FLVCR1 amplifying their genotypic variability. The mutations identified in VRK1, KIF1A, IGHMBP2 and FLVRC1 expanded their phenotype spectrum. Mutations in the VRK1 gene may cause dHMN with upper motor neuron signs. Autistic spectrum disorder may be observed in association with mutations in the KIF1A gene and mutations in the FLVRC1 gene may cause a severe phenotype characterized by congenital insensitivity to pain and acromutilations. Mutations in the IGHMBP2 gene may cause an overlap between SMARD1 and CMT2S phenotypes with organ specific dysautonomia. This study demonstrates that WES is a powerful tool for molecular diagnosis of hereditary neuropathies. Additionally, this study provides new information on the mutations in the VRK1, KIF1A and FLVRC1 genes by adding new mutations and increasing the phenotypic variability of the neuropathies associated with these genes.This study demonstrates WES is a powerful tool for molecular diagnosis of hereditary neuropathies.

Pas de résumé / Chronic mucocutaneous candidiasis (CMC) is seen in human patients with a variety of conditions and refers to recurrent or persistent infection of the skin, nails and/or mucosae by commensal Candida species. Its pathogenesis had long remained elusive, until human genetic studies of rare patients with inherited forms of idiopathic CMC (whether isolated or syndromic), incriminated impaired interleukin (IL)-17A/F immunity. The first genetic etiologies of idiopathic isolated CMC, autosomal dominant (AD) IL-17F and autosomal recessive (AR) IL-17 receptor A (IL-17RA) deficiencies, were reported in 2011 in a multiplex and in a sporadic case, respectively. Using Whole Exome Sequencing (WES), we identified 26 novel patients bearing 15 different homozygous mutations in the IL17RA gene. The mutations identified are either nonsense; missense; frameshift deletions; frameshift insertions; or non-coding essential splice site mutations. Interestingly, 2 alleles encode for surface expressed receptors, whereas all the other tested alleles are not detected at the surface of the patient’s cells (fibroblasts or leucocytes). IL-17RA deficiency is a fully penetrant AR disease, with early onset symptoms, usually within the first year of life. CMC is always present. In addition, 17 patients present with staphylococcal skin infections, and some patients with pyogenic infections of the respiratory tract, including pneumonia. Interestingly, tuberculosis occurred in two unrelated BCG-vaccinated patients. The response to IL-17A and IL-17F homo- and heterodimers is abrogated in fibroblasts, as well as the response to IL-17E/IL-25 in T cells. Human IL-17RA is thus essential for mucocutaneous immunity against Candida and Staphylococcus, but otherwise largely redundant. AR IL-17RA deficiency should be considered in children or adults with CMC, cutaneous staphylococcal disease, or both. In a separate project, I investigated a female child patient born to consanguineous parents who suffered from CMC, recurrent viral infections, disseminated BCG disease and biliary cryptosporidiosis, suggestive of combined immunodeficiency, and who is homozygous for a mutation in REL, encoding the NF-kB protein c-REL. Sanger sequencing confirmed that the patient is homozygous and that both parents are heterozygous for the mutation, consistent with an AR inheritance. The candidate mutation is a nucleotide substitution localized in an acceptor splice site; is not reported in available public databases; and is predicted to be damaging in silico. The mutation disrupts mRNA splicing and is loss-of expression. The patient shows normal counts of lymphoid subsets, with the exception of diminished frequencies of memory CD4+ T, Th2, Th1*, and memory B cells. The patient’s T cells fail to proliferate in response to recall antigens. Naïve CD4+ T cells produce little IL-2 and respond poorly to polyclonal stimulation, a phenotype reverted by exogenous IL-2. Memory CD4+ T cells also produce little amounts of IL-2, and strongly diminished amounts of key effector cytokines (IFN-γ, IL-4, IL-17A and IL-21). The patient exhibited with no detectable specific antibody response following vaccination. Survival and therefore proliferation of naïve B cells are compromised leading to poor generation of plasma cell, and immunoglobulins secretion. The patient shows normal counts of myeloid cells, and frequencies of dendritic cell subsets. IL-12 production is abolished in whole blood in response to BCG+IFN-γ and B-EBV cells in response to mitogens. Although further investigation is needed to fully characterize the patient’s phenotype, these results strongly suggest that the patient suffers from AR complete c-REL deficiency.

Abstract Novel genetic aetiologies for epileptic encephalopathies and movement disorders have been discovered by using next-generation sequencing methods. The phenotypic and genotypic variability in these conditions is very wide. The aim of this study was to discover novel genetic causes and phenotypes of childhood-onset drug-resistant epilepsy and epileptic or developmental encephalopathies that occur separately or together with movement disorders, and familial movement disorders. Furthermore, the use of whole-exome sequencing (WES) as a diagnostic tool in clinical practice was evaluated. Altogether, 12 children with undefined aetiology, who fulfilled the inclusion criteria, were included in the study. GABRG2 gene was identified as a genetic cause of epileptic encephalopathies. Novel GABRG2-associated phenotypes included progressive neurodegeneration, epilepsy in infancy with migrating focal seizures, and autism spectrum disorder. New pathogenic variants, GABRG2 p.P282T and p.S306F, were discovered. The pathogenic NACC1 variant caused focal epilepsy, developmental disability, bilateral cataracts, and dysautonomia. The novel phenotype associated with the NACC1 p.R298W variant included hyperkinetic movement disorder. SAMD9L was found to be the genetic cause for the familial movement disorder. The phenotype associated with the novel SAMD9L p.I891T variant was very variable. Neuroradiological findings included cerebellar atrophy and periventricular white matter changes. After publication of these results, SAMD9L was reported to be one of the most common genetic aetiologies of childhood bone marrow failure and myelodysplastic syndrome. The pathogenic homozygous MTR variant was found to cause early-onset epileptic encephalopathy that occurred together with movement disorder and haematological disturbances. Drug resistant seizures responded to cofactor and vitamin treatments. Whole-exome sequencing for 10 patients with drug-resistant epilepsy or epileptic or developmental encephalopathy provided a genetic diagnosis for two patients (20%). This study confirmed that, for epileptic encephalopathies and movement disorders in which the genetic causes and phenotypes are heterogeneous and sometimes treatable, WES is a useful tool for diagnostics and in the search for novel aetiologies, which might turn out to be more common than expected. / Tiivistelmä Uusien sekvensointimenetelmien käyttöönotto on mahdollistanut epileptisten enkefalopatioiden ja liikehäiriöiden uusien geneettisten syiden löytymisen. Näissä sairausryhmissä geenien ja ilmiasujen vaihtelevuus on suurta. Tutkimuksen tarkoituksena oli löytää uusia geneettisiä syitä ja ilmiasuja lapsuusiällä alkavissa vaikeahoitoisissa epilepsioissa ja epileptisissä tai kehityksellisissä joko itsenäisesti tai yhdessä liikehäiriön kanssa esiintyvissä enkefalopatioissa sekä perheittäin esiintyvissä liikehäiriöissä. Lisäksi selvitettiin eksomisekvensoinnin käyttökelpoisuutta kliinisessä diagnostiikassa näiden potilasryhmien kohdalla. Tutkimukseen osallistui yhteensä 12 sisäänottokriteerit täyttävää lasta, joiden sairauden syy oli jäänyt tuntemattomaksi. GABRG2-geenin mutaatiot aiheuttivat epileptisiä enkefalopatioita, joiden uutena ilmiasuna oli etenevä taudinkuva, johon liittyivät aivojen rappeutuminen, migroiva imeväisiän paikallisalkuinen epilepsia sekä autismikirjon häiriö. Tutkimuksessa löydettiin uusia GABRG2-mutaatioita: p.P282T ja p.S306F. NACC1-geenin mutaatio aiheutti epilepsian, kehitysvammaisuuden, molemminpuolisen kaihin ja autonomisen hermoston toiminnan häiriön. Hyperkineettinen liikehäiriö oli uusi NACC1 p.R298W -mutaatioon liittyvä ilmiasu. SAMD9L-geenin mutaatio aiheutti perheessä esiintyvän liikehäiriön. Neurologinen ja hematologinen ilmiasu olivat hyvin vaihtelevia. Aivojen kuvantamislöydöksiin sisältyi pikkuaivojen rappeutumista ja valkoisen aivoaineen muutoksia aivokammioiden ympärillä. Näiden tutkimustulosten julkaisemisen jälkeen SAMD9L-geenin mutaatioiden on todettu olevan yksi yleisimmistä perinnöllisistä luuytimen vajaatoiminnan ja myelodysplasian syistä. Homotsygoottinen MTR-geenin mutaatio aiheutti varhain alkaneen epileptisen enkefalopatian, liikehäiriön ja hematologisen häiriön. Kofaktori- ja vitamiini hoidot vähensivät epileptisiä kohtauksia, joihin tavanomainen lääkitys ei tehonnut. Geneettiset syyt ja ilmiasut ovat epileptisissä enkefalopatioissa ja liikehäiriöissä hyvin vaihtelevia, ja osaan on olemassa spesifi hoito. Eksomisekvensointi on käyttökelpoinen diagnostiikan ja uusien geneettisten syiden etsimisen apuna. Tässä tutkimuksessa eksomisekvensoinnin avulla kymmenestä potilaasta kahdelle (20%) saatiin varmistettua geneettinen diagnoosi.

Autism spectrum disorder (ASD) is a neurodevelopment disorder characterized by impairment in communication skills, behavior and social interactions that affects around 1-2% worldwide. To date the etiology of ASD has not yet been fully understood, but in the last 18 years many advances have been made to understand the genetic component related to the development of the clinical phenotype. With the advent of genomic scan analysis such as chromosome analysis by microarray and whole exome sequencing (WHE) many advances have been made to understand the pathophysiology of the disease. About 10-15% of the cases can be explained by large losses or gains (deletions or duplications greater than 1000 base pairs) of the genetic material, which generally involve the disruption of one or more genes. Next generation sequencing methodologies were fundamental in the description of point mutations and small insertions and deletions associated with ASD. The WES has allowed many discoveries to be made about new candidate genes and mechanisms for the development of the disease. It is now claimed that de novo (non-inherited) and likely gene disruptive mutations, such as loss-of-function and non-synonymous changes with high prediction of damage by computational tools, in genes related to neurodevelopment are a major contributor to the disease mechanism. However, these mutations, in addition to not explaining the majority of cases, are rarely recurrent in the population, which makes it difficult to establish a definitive molecular diagnosis for most patients. WES is already a practice in clinical genetics laboratories and demonstrates high effectiveness for diseases that follow a Mendelian pattern of inheritance, and have an established genetic cause. In clinical practice WES is requested for cases of ASD, despite having different modes of inheritance and having more than 1,000 genes associated with the disease. Due to these characteristics the analysis of WES for ASD is a major challenge for the clinical laboratory. This study proposes the construction of a computerized WES analysis routine that can test different candidate genes for their sensitivity and specificity for the detection of affected individuals. The proposed approach consists in the counting of variants separated by their possible protein damage and population frequency for each individual from affected and control groups, this study analyzed 168 WES, being 49 with ASD and 119 controls. After counting formulation, these values are subjected to a sequence of statistical tests, seeking a significant difference in the amount of mutations of all the variants alone, loss-of-function or damaging missense mutations, and the application of models of multivariate analysis such as: logistic regression, decision tree, neural network, vector support machine and principal component analysis for the elaboration of more complex models for disease development. A total of 21 lists of genes were tested, of which 19 presented at least one significant result, and the analysis of variants alone was the one that obtained the largest number of significant events. From apparently protective variants (higher number in the control group), such as the missense variants in RAS/MAPK pathway as variants of stopgain with population frequency above 0.05 in chromatin genes in greater number in individuals with ASD. None of the multivariate analysis models had significant discrimination results between the two groups. Due to the small sample size, the results of this study should be interpreted with limitations, and it is necessary to replicate these scenarios in other databases. However, these findings suggest that different types and frequencies of variants may have distinct contributions to disease development depending on the genes analyzed, rather than complex relationships between variants of the same gene list / O transtorno do espectro autista (TEA) é um distúrbio do neurodesenvolvimento caracterizado por uma incapacidade de comunicação comportamento e interações sociais que afeta em torno de 1-2% da população mundial. Até o momento a etiologia do TEA ainda não é totalmente compreendida, mas nos últimos 18 anos muitos avanços foram feitos para entender o componente genético relacionado ao desenvolvimento do quadro clínico. Com o advento das análises de varredura genômica como a análise cromossômica por microarray e o sequenciamento completo de exoma (SCE) muitos avanços foram feitos para a compreensão da fisiopatologia da doença. Em torno de 10-15% dos casos podem ser explicados por grandes perdas ou ganhos (deleções ou duplicações superiores a 1000 pares de bases) do material genético, que geralmente envolvem a disrupção de um ou mais genes. As metodologias de sequenciamento de nova geração foram fundamentais para a descrição das mutações de ponto e pequenas inserções e deleções associadas ao TEA. O SCE permitiu que muitas descobertas fossem feitas sobre novos genes candidatos e mecanismos para o desenvolvimento da doença. Atualmente afirma-se que as alterações de novo (não herdadas) e de maior probabilidade de ruptura gênica, como as mutações de perda-de-função e as alterações não-sinônimas com alta predição de dano por ferramentas computacionais, em genes de susceptibilidade a doenças do neurodesenvolvimento sejam um grande contribuidor para o mecanismo da doença. Entretanto essas mutações, além de não explicar a totalidade dos casos raramente são recorrentes na população, o que dificulta o estabelecimento de um diagnóstico molecular definitivo para a maioria dos pacientes. O SCE já é uma prática nos laboratórios clínicos de genética e demonstra uma alta efetividade para as doenças que seguem um padrão de herança mendeliano, e têm uma causa genética estabelecida. Na prática clínica o SCE é solicitado para os casos de TEA, apesar de ter diferentes modos de herança e terem mais de 1,000 genes associados à doença. Devido a estas características o SCE para os casos de TEA são um grande desafio para o laboratório clínico. Este estudo propõem a construção de uma rotina computacional de análise do SCE que possa testar diferentes genes candidatos quanto à sua sensibilidade e especificidade para a detecção dos indivíduos afetados. A abordagem proposta é a contagem de variantes separadas por seu possível dano à proteína e frequência populacional para cada indivíduo de grupos afetado e controle em 168 indivíduos com SCE, sendo 49 com TEA e 119 controles. Após a formulação da contagem esses valores são submetidos a uma sequência de testes estatísticos, buscando diferença significativa em quantidade de mutações de todas as variantes isoladamente, das mutações de perda-de-função, ou não-sinônimas danosas como um conjunto e a aplicação de modelos de análise multivariada como: regressão logística, árvore de decisão, rede neural, máquinas de suporte de vetor e análise de componente principal para a elaboração de modelos mais complexos para o desenvolvimento na doença. Ao todo foram testadas 21 listas de genes, destas, 19 apresentaram ao menos um resultado significativo, sendo a análise de variantes isoladamente a que obteve maior número de eventos significativos. Desde variantes aparentemente protetoras (maior número no grupo controle), como as variantes não-sinônimas em via de RAS/MAPK quanto variantes de perda de códon de parada com frequência populacional acima de 0.05 em genes de cromatina em maior número nos indivíduos com TEA. Nenhum dos modelos de análise multivariada obteve resultados significativos na discriminação entre os dois grupos. Devido ao pequeno número amostral os resultados deste estudo devem ser interpretados com limitações, sendo necessária a replicação deste cenário em outros bancos de dados. Entretanto, estes achados sugerem que diferentes tipos e frequências de variantes podem ter contribuições distintas para o desenvolvimento da doença a depender dos genes analisados, mais de que relações complexas entre as variantes de uma mesma lista de genes