Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 104-114).

X-Linked Retinoschisis (XLRS) is a currently incurable, progressive retinal degeneration that affects approximately 1:20,000 males. Sufferers have a loss of retinal structure and visual acuity, leading to blindness. The condition is caused by mutation of the RS1 gene encoding the retinal-specific protein retinoschisin. Retinoschisin is critical in maintaining the normal, ordered retinal architecture, with deletion in mice models leading to loss of both structure and visual processing, analogous to XLRS sufferers. However, re-introduction of retinoschisin using adeno-associated viral vectors leads to complete rescue in these models. Despite the importance of retinoschisin in maintaining retinal architecture, the mechanism by which it maintains this structure remains unknown. As a result, this study aimed to structurally characterise retinoschisin and XLRS-associated point mutants R141H and H207Q to gain insight into the mechanism of retinoschisin action. To this end, retinoschisin was expressed and purified from HEK 293-EBNA cells and the structure of both monomeric and octameric retinoschisin was investigated using Small-Angle X-Ray Scattering (SAXS) and Cryo-electron microscopy (Cryo-EM). Monomeric retinoschisin was found to adopt an elongated structure that allowed for the tight association of the subunits into a planer propeller structure. However, in solution conditions the octamer also stably self-assembled into a dimer of octamers, for which the structure was solved using cryo-EM. This allowed for construction of a quasi-atomic model, enabling mapping of XLRS-associated point mutations on the complex. Two major classes of mutation were identified, in the intra-octamer and inter-octamer interfaces, suggesting a mechanism of pathology for these mutants. Observation of clustered conservative mutations at the inter-octamer interface suggested the dimer of octamers may be physiologically relevant. Furthermore, comparison of the R141H mutant to the wild-type revealed an additional mutated site in the propeller tips. Here, R141H was suggested to induce a small conformational change and alter an interaction site. Another mutant, H207Q, however, induced a destabilization of the assembled retinoschisin molecule. In conclusion, we purified and structurally characterised human retinoschisin, identifying a new hexadecameric oligomer. The structure of this allowed for identification of distinct classes of mutations on the assembled molecule and a hypothesis of the mechanism of retinoschisin action in the retina.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / X-linked hypophosphatemic rickets (XLHR) is the most common cause of heritable rickets, with an incidence of 1:20,000 live births, representing more than 80% of familial hypophosphatemic rickets. Saliva is the most easily available and accessible body fluid, which makes it one of the most sought after tools in diagnostic pathology. In this context, this thesis, constituted by 4 articles aimed to: (1) describe the main systemic manifestations, oral findings and dental management in 3 generations of an affected family; (2) analyze the mineralization pattern of enamel and dentin in patients affected by XLHR using micro-CT, and to associate enamel and dentin mineralization in primary and permanent teeth with tooth position, gender and presence/absence of this disease; (3) evaluate the peptide profile in the saliva of patients with X-linked hypophosphatemic rickets using high performance liquid chromatography; and (4) characterize salivary proteins in this condition using unidimensional electrophoresis. On study 1, oral exams, laboratorial and histologic evaluations, cone-beam computed tomographies, panoramic and periapical radiographs were performed to properly institute the most adequate treatment strategy. On study 2, teeth were collected from 5 individuals from the same family. Gender, age, tooth position (anterior/posterior) and tooth type (deciduous/permanent) were recorded for each patient. Following collection, teeth were placed in 0.1% thymol solution until Micro-CT scan. Projection images were reconstructed and analyzed. On study 3, unstimulated whole and stimulated parotid saliva were obtained from 8 individuals with (AFF) and 8 healthy individuals, both genders, without (CON) x-linked hypophosphatemic rickets aged from 8 to 66 years. Supernatants were analyzed by high performance liquid chromatography, and the salivary flow rate (ml/min) was calculated. Each major peak in the HPLC chromatogram of each sample was characterized. On study 4, unstimulated whole and stimulated parotid saliva were also obtained, being total protein concentration determined by the Bicinchoninic Acid Protein (BCA) method. Proteins were characterized according to their molecular weights within the unidimensional electrophoresis. The study 1 showed the importance of the knowledge of clinical signs and symptoms of XLHR for the correct diagnosis of this disease, and for the establishment of preventive and comprehensive dental care. On article 2, teeth of all affected patients presented dentin with a different mineralization pattern compared to the teeth of the healthy individual with dentin defects observed next to the pulp chambers. On the third article, whole and parotid salivary flows were significantly different (p = 0.001), being flow of whole saliva higher (0.518  0.282 mL/min) than parotid saliva (0.124  0.086 mL/min). Whole salivary flow rate was higher in the AFF group (0.698  0.229) than in the CON group (0.339  0.210 mL/min) (p = 0.006). Twenty-eight peaks were found in whole and 21 peaks in parotid saliva. Whole saliva of the CON group presented lower number of peaks than AFF group. In parotid saliva, peaks 17 and 28 (retention times: 24 and 39 min) were found exclusively in the AFF group, and peak 13 (retention time: 19 min) exclusively in the CON. Article 4 showed difference concerning to total protein concentration between whole and parotid saliva (p < 0.001), being higher concentration found in whole saliva (102.603  42.336 Âg/mL) than in parotid saliva (0.699  0.438 Âg/mL). Bands with 102 kDa, 48 kDa and 24 kDa presented higher intensity in whole saliva of CON group (p = 0.015, p = 0.043 and p = 0.022). In conclusion, XLHR patients presented specific characteristics in dentin mineralization and salivary proteins and peptides, which can lead to differentiate these patients from healthy individuals, improving the diagnostic field. / Raquitismo hipofosfatÃmico ligado ao cromossomo X (XLHR) à a maior causa de raquitismo hereditÃrio, com uma incidÃncia de 1:20.000 nascidos vivos, representando mais de 80% das formas de raquitismo hipofosfatÃmico familiar. A saliva à o fluido humano mais disponÃvel e de fÃcil acesso, o que faz dela uma das ferramentas mais pesquisadas no diagnÃstico de patologias. Nesse contexto, essa tese, constituÃda de 4 artigos objetivou: (1) descrever as principais manifestaÃÃes sistÃmicas, achados orais e tratamentos dentÃrios em 3 geraÃÃes de uma famÃlia afetada; (2) analisar o padrÃo de mineralizaÃÃo do esmalte e da dentina nos pacientes afetados por XLHR, utilizando microtomografia computadorizada (Micro CT), e associar a mineralizaÃÃo do esmalte e da dentina em dentes decÃduos e permanentes, segundo gÃnero e presenÃa/ausÃncia da doenÃa; (3) avaliar o perfil de peptÃdeos na saliva de pacientes com XLHR, utilizando cromatografia lÃquida de alta performance (HPLC); e (4) caracterizar proteÃnas salivares nessa condiÃÃo, utilizando eletroforese unidimensional. No estudo 1, exames orais, laboratoriais e avaliaÃÃes histolÃgicas, tomografias computadorizadas cone-beam e radiografias periapicais foram realizadas para a apropriada instituiÃÃo da estratÃgia de tratamento mais adequada. No estudo 2, dentes foram coletados de 5 indivÃduos de uma mesma famÃlia. GÃnero, idade, posiÃÃo dentÃria (anterior/posterior) e tipo dentÃrio (decÃduo/permanente) foram registrados para cada paciente. ApÃs a coleta, os dentes foram colocados em soluÃÃo de timol a 0,1% atà a anÃlise atravÃs do Micro CT. As imagens projetadas foram reconstruÃdas e analisadas. No estudo 3, saliva total nÃo estimulada e saliva de parÃtida estimulada foram obtidas de 8 indivÃduos afetados com (AFF) e 8 indivÃduos sem (CON) XLHR, de ambos os gÃneros e idades entre 8 e 66 anos. Sobrenadantes foram analisados por meio de HPLC e o fluxo salivar (mL/min) foi calculado. Os picos que se apresentaram maiores nos cromatogramas do HPLC foram caracterizados. No estudo 4, saliva total nÃo estimulada e saliva de parÃtida estimulada tambÃm foram obtidas, sendo a concentraÃÃo de proteÃnas totais determinada pelo MÃtodo do Ãcido BicinconÃnico (BCA). ProteÃnas foram caracterizadas de acordo com o peso molecular atravÃs de eletroforese unidimensional. O estudo 1 mostrou a importÃncia do conhecimento dos sinais e sintomas clÃnicos do XLHR para o correto diagnÃstico dessa doenÃa, e para o estabelecimento de atendimento odontolÃgico preventivo e abrangente. No artigo 2, os dentes de todos os pacientes afetados apresentaram dentina com padrÃo de mineralizaÃÃo diferente comparado aos dentes de indivÃduos saudÃveis, sendo os defeitos na dentina observados prÃximo Ãs cÃmaras pulpares. No artigo 3, os fluxos salivares da saliva total e de parÃtida foram significativamente diferentes (p=0,001), sendo o fluxo de saliva total maior (0,518  0,282 mL/min) do que o de saliva de parÃtida (0,124  0,086 mL/min). O fluxo salivar da saliva total foi maior no grupo AFF (0,698  0,229) que no grupo CON (0,339  0,210 mL/min) (p = 0,006). Vinte e oito picos foram encontrados em saliva total e 21 em saliva de parÃtida. A saliva total do grupo CON apresentou menor nÃmero de picos que a do grupo AFF. Na saliva de parÃtida, os picos 17 e 28 (tempos de retenÃÃo: 24 e 39 min) foram encontrados exclusivamente no grupo AFF e o pico 13 (tempo de retenÃÃo: 19 min) no CON. Artigo 4 demonstrou diferenÃa relacionada à concentraÃÃo de proteÃnas totais entre saliva total e de parÃtida (p < 0,001), sendo a maior concentraÃÃo encontrada na saliva total (102,603  42,336 Âg/mL) que na saliva de parÃtida (0,699  0,438 Âg/mL). Bandas com 102 kDa, 48 kDa e 24 kDa apresentaram maior intensidade na saliva total do grupo CON (p = 0,015, p = 0,043 e p = 0,022). Em conclusÃo, pacientes com XLHR apresentaram caracterÃsticas especÃficas relacionadas à mineralizaÃÃo dentinÃria e proteÃnas e peptÃdeos salivares que podem levar à diferenciaÃÃo desses pacientes de indivÃduos saudÃveis, avanÃando no campo diagnÃstico.

There are different conformational states of proteins characterized by different Gibbs free energy levels, manifested in folding-unfolding dynamics, for example. Recently, a set of protein states, which require relatively small amount of folding energies, emerged as subjects of intensive research, and proteins or regions characterized by the presence of these states have been termed as ‘Intrinsically Disordered Proteins’ (IDP) and ‘Intrinsically Disordered Protein Regions’ (IDPR), respectively. Predisposition for intrinsic disorder of a query protein is encoded in its amino acid sequence and composition, and can be rather accurately predicted using several intrinsic disorder algorithms. Since pathology of many human diseases can be driven by proteins characterized by high intrinsic disorder scores, research on various disease-associated proteins is often started with the analysis of their intrinsic disorder propensities. In this work, I utilized computational approaches based on the concept of intrinsic disorder to address three health-related issues. To this end, I developed a novel computational platform for disorder-based drug discovery and applied this tool for finding inhibitors of the cancer-related MBD2-NuRD complex, utilized molecular dynamic simulations to explain the effects of mutations on the functionality of the X-linked protoporphyria-related protein ALAS, and used bioinformatics tools to examine the effects ofcardiomyopathy-related mutations in cardiac troponin. Since the complex between the Methyl-CpG-binding domain protein 2 (MBD2) and the Nucleosome Remodeling Deacetylase complex (NuRD) specifically binds to the mCpG-island and blocks tumor suppressor gene expression, finding an inhibitor of this MBD2-NuRD complex is hypothesized to be important for the development of novel anti-cancer drugs. I found that the site, which is responsible for the MBD2 interaction with thetranscriptional repressor p66-α (p66α, which is a part of the NuRD complex), is characterized by a specific disorder-to-order transition pattern, this pattern showed a remarkable similarity to the disorder-to-order pattern of the Myc transcription factor binding site for the Max transcription factor. Importantly, several inhibitors of the Myc-Max interaction targeting the disorder-to-order transition site of Myc were previously described. By applying molecular docking at the disorder-to-order transition site of MBD2, two compounds were identified and further evaluated through molecular dynamics simulations. Anti-leukemia and anti-metastasis effectiveness of these compounds was demonstrated in dedicated in vitro and in vivo experiments conducted by our collaborators. In relation to the defective protein associated with the X-linked protoporphyria (XLPP), the hepta-variant of mouse erythroid 5-aminolevulinate synthase (mALAS2), previously shown to be characterized by a remarkable acceleration of the reaction rate, was investigated through molecular dynamics simulations. In this study, a loop to β-strand transition was observed, and this observation was crucial for a better understanding of the previously described rate-enhancing effects of seven simultaneous variations in the active loop site of this protein. Finally, a wide spectrum of bioinformatics tools was applied to carefully analyze a potential role of intrinsic disorder in a set of cardiomyopathy-related mutations in the components of human cardiac troponin. This analysis revealed that, in comparison with the wild type troponin, chains containing the disease-associated mutations were typically characterized by a local decrease in intrinsic disorder propensity. These mutations affected some disorder-based protein-protein interaction sites and caused remarkable rearrangements of the complex pattern of post-translational modifications. Therefore, this work illustrates that inclusion of the protein intrinsic disorder analysis into the arsenal of techniques used by the biomedical researchers represents an important and promising approach that provides novel inputs for the better understanding of protein behavior in relation to human disease at the molecular level. Techniques and methods developed and utilized in this study will significantly contribute to future biomedical research.

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

Estudamos uma família com cinco homens (dois falecidos) afetados por deficiência mental (DM) não-sindrômica em duas gerações, num padrão de herança ligada ao cromossomo X. A análise do padrão de inativação do cromossomo X, com base na metilação do gene AR, evidenciou que a mulher portadora obrigatória tinha desvio completo de inativação nos leucócitos, uma característica freqüente em portadoras de mutações do cromossomo X relacionadas com DM. Para o mapeamento da DM, genotipamos 28 locos de microssatélites ao longo do cromossomo X e delimitamos um segmento de cerca de 32 Mb, entre os marcadores DXS986 e DXS8067, compartilhado pelos afetados e pela portadora obrigatória, mas não pelo homem normal ou pelas possíveis portadoras que não tinham desvio do padrão de inativação do cromossomo X. Na busca do gene mutado, analisamos, por seqüenciamento direto, genes mapeados no intervalo compartilhado e já relacionados a DM ou que tivessem expressão em cérebro e leucócitos. Nos afetados e na portadora obrigatória, encontramos a mutação c.845C&#8594;T no gene ACSL4, que resulta na substituição do aminoácido histidina, conservado na família de sintetases de acil-CoA humanas e em diversos outros organismos, por tirosina (p.H323Y da isoforma cérebro-específica). Tratando-se de mutação que altera um aminoácido evolutivamente conservado em gene já relacionado com DM, que segregava com a DM na família, não tendo sido encontrada em amostra controle de 160 indivíduos do sexo masculino, concluímos que era a causa da DM na família. Mutações de ponto no gene ACSL4 foram relacionadas com a DM não sindrômica em três famílias descritas na literatura. O gene ACSL4 codifica a acil-coA sintetase 4 da família das sintetases de cadeia longa, que catalisa a formação de ésteres acil-coA a partir de ácidos graxos de cadeia longa. Sua expressão já foi documentada em vários tecidos, incluindo o cérebro e dados recentes mostraram que a proteína é essencial para a formação normal de espinhos dendríticos. A nova mutação do gene ACSL4 que descrevemos como causa de DM vem reforçar a relação alterações desse gene e a DM de herança ligada ao X. O padrão de inativação do X totalmente desviado foi mais uma vez observado em mulher portadora da mutação, indicando a importância da expressão desse gene em leucócitos. A presença de dificuldades de aprendizado na portadora da mutação concorda com o observado nas três famílias da literatura em que o estudo das portadoras foi relatado, indicando o efeito de mutações do gene ACSL4 sobre a função intelectual mesmo em heterozigose. A ausência de correlação entre o padrão de inativação do cromossomo X em células do sangue periférico e o comprometimento intelectual foi confirmada. Na família estudada, a identificação da mutação permitiu o aconselhamento genético. / We studied a family with five men (two of them deceased) affected by nonsyndromic mental retardation in two generations, in a pattern of X-linked inheritance (MRX). The study aimed at identifying the causative mutation. The obligate female carrier showed completely skewed inactivation of the X chromosome, based on the methylation status of the AR gene in peripheral blood in leukocytes, a common feature in carriers of X-linked mutations that cause mental retardation. We genotyped 28 microsatellite loci mapped throughout the X chromosome and delimited a 32 Mb segment, between markers DXS986 and DXS8067, that was shared by the affected males and obligate carrier, but was not present in a normal man or in two women who did not show skewed X-inactivation. We searched for the causative mutation by sequencing genes mapped to this candidate interval that had been associated with MR and/or were expressed in brain and leukocytes. In the affected men and obligate carrier, we found a c.845C&#8594;T mutation in the ACSL4 gene, resulting in the amino acid tyrosine substituting for a histidine (p.H323Y in brain isoform), which is conserved in the acyl-CoA synthetase family in humans and others organisms. This mutation was not found in a control sample of 160 men. Previously, point mutations in the ACSL4 gene had been identified as the cause of MRX in three families. ACSL4 encodes the acyl-CoA synthetase long-chain family member 4, which catalyzes the formation of acyl-CoA esters from long-chain fatty acids. It is expressed in several tissues, and in brain it is essential for the normal formation of dendritic spines. The novel mutation here described confirmed the causal association of ACSL4 mutations with non-syndromic mental retardation. The completely skewed Xinactivation, also observed in the previously described carriers, supported a functional role for this gene in peripheral blood leukocytes. The intellectual impairment present in the carrier in the family here reported is in accordance with previous findings pointing to the effect on intellectual abilities of ACSL4 mutations in heterozygosis. The absence of correlation between the pattern of X-inactivation in leukocytes and mental status was confirmed.

Isolated noncompaction of the left ventricular myocardium (INVM) is characterized by the presence of numerous prominent trabeculations and deep intertrabecular recesses within the left ventricle, sometimes also affecting the right ventricle and interventricular septum. Familial occurrence of this disorder was described previously. We present a family in which 6 affected individuals demonstrated X-linked recessive inheritance of this trait. Affected relatives presented postnatally with left ventricular failure and arrhythmias, associated with the pathognomonic echocardiographic findings of INVM. The usual findings of Barth syndrome (neutropenia, growth retardation, elevated urinary organic acids, low carnitine levels, and mitochondrial abnormalities) were either absent or found inconsistently. Fetal echocardiograms obtained between 24-30 weeks of gestation in 3 of the affected males showed a dilated left ventricle in one heart, but were not otherwise diagnostic of INVM in any of the cases. Four of the affected individuals died during infancy, one is in cardiac failure at age 8 months, and one is alive following cardiac transplant at age 9 months. The hearts from infants who died or underwent transplantation appeared, on gross examination, to be enlarged, with coarse, deep ventricular trabeculations and prominent endocardial fibroelastosis. Histologically, there were loosely organized fascicles of myocytes in subepicardial and midmyocardial zones of both ventricles, and the myocytes showed thin, often angulated fibers with prominent central clearing and reduced numbers of filaments. Markedly elongated mitochondria were present in some ventricular myocytes from one specimen, but this finding was not reproducible. Genetic linkage analysis has localized INVM to the Xq28 region, where other myopathies with cardiac involvement have been located.

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23777号 / 医博第4823号 / 新制||医||1057(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 江藤 浩之, 教授 寺田 智祐, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Este trabalho teve o objetivo de estimar a frequência de deficiência mental causada por mutações no cromossomo X entre pacientes do sexo masculino, que constituem casos isolados de deficiência mental. A estratégia adotada foi a determinação do padrão de inativação do cromossomo X nas mães dos afetados, com base (a) nas indicações de que desvios extremos do padrão casual de inativação do cromossomo X têm alta probabilidade de estar relacionados à presença de mutações do cromossomo X e (b) na observação de que a frequência desses desvios está significantemente aumentada em mulheres certamente portadoras de mutações que causam deficiência mental de herança ligada ao X. A vantagem seletiva das células que possuem o alelo não mutado no cromossomo X ativo é uma explicação para tais desvios extremos da inativação do cromossomo X, raramente encontrados na população geral. Selecionamos 115 meninos portadores de deficiência mental moderada a grave associada a outros sinais clínicos, não característicos de síndrome conhecida e que tinham cariótipos normais e teste negativo para a síndrome do cromossomo X frágil; suas genitoras concordaram com a participação no estudo. Esses pacientes foram encaminhados ao Serviço de Aconselhamento Genético do Laboratório de Genética Humana, Departamento de Genética e Biologia Evolutiva, por diferentes serviços médicos, para diagnóstico e orientação quanto a riscos de recorrência na família. O padrão de inativação do cromossomo X nas mães dos afetados foi investigado, com base na metilação diferencial dos alelos do gene AR (Androgen Receptor gene) , no cromossomo X ativo e inativo. As mães de 100 desses meninos se revelaram heterozigóticas quanto à repetição polimórfica CAG do gene AR, requisito do teste para determinar o padrão de inativação do cromossomo X. Onze mulheres (11%) apresentaram desvios extremos do padrão de inativação do X (&ge; 98:2), frequência significativamente maior (P = 0.0001; teste exato de Fisher) do que aquela que a literatura registra, em estudo utilizando o mesmo ensaio, entre mulheres adultas da população geral (0,017; IC 95% = 0,007 0,034). A raridade de desvios tão extremos na população geral permite admitir que as mães dos afetados que apresentam tais desvios sejam portadoras de mutação no cromossomo X, que causa a deficiência mental em seus filhos. Sendo assim, estimamos em 11% a frequência de deficiência mental em nossa amostra de 100 meninos casos isolados de deficiência mental (IC 95% = 0,056 0,188), sem incluir a síndrome do X frágil, responsável por 2,5% a 3% da deficiência mental no sexo masculino. Essa nossa estimativa para a proporção de deficiência mental moderada grave ligada ao X entre indivíduos do sexo masculino com DM é da mesma ordem de grandeza daquelas relatadas na literatura, baseadas (a) na frequência da síndrome do X frágil em coortes de homens com deficiência mental e entre famílias com deficiência mental de herança ligada ao X ou (b) nas inferências da prevalência de deficiência mental e de deficiência mental causada por mutações no cromossomo X na população geral masculina. Entretanto, a frequência por nós determinada deve ser uma subestimativa, considerando que os desvios extremos do padrão de inativação ocorrem em apenas um terço das portadoras obrigatórias de mutações que causam deficiência mental com herança ligada ao X. Com base nos resultados deste estudo, consideramos indicada a avaliação do padrão de inativação do cromossomo X em mães de indivíduos do sexo masculino, casos isolados de deficiência mental. A detecção de desvio extremo da inativação deve ser considerada indicativa de deficiência mental de herança ligada ao X, constituindo subsídio para o aconselhamento genético da família e podendo levar á identificação da mutação causadora da deficiência mental. / Nearly a third of obligate carriers of mutations causative of X-linked mental retardation (XLMR) have been reported to have extreme X-inactivation skewing in peripheral blood cells, compared to their non-carrier relatives. Selective advantage of cells with the non-mutated allele on the active X chromosome would explain this skewing. Based on these findings, we used the pattern of X-inactivation in mothers of mentally retarded boys, as a parameter to evaluate the frequency of XLMR among non-familial cases. To determine the X-inactivation pattern in these women, we investigated the methylation status of the AR (Androgen Receptor) alleles in blood cells. We selected 115 boys with moderate to severe mental retardation of unknown cause, who had normal karyotypes and tested negative for fragile X syndrome; the mothers of 100 of these boys were found to be heterozygous for the polymorphic CAG repeat of the AR gene, a requisite of the X-inactivation assay. Eleven women (11%) had extremely skewed X-inactivation (&ge; 98:2), a frequency significantly higher (P = 0.0001; Fisher exact test) than the frequency reported for adult women from the general population (1.7%; 95% CI = 0.007 0,034). Assuming that every mother with extremely skewed X-inactivation is a carrier of an X-chromosome mutation that causes mental retardation in her son, the frequency of XLMR in our sample of 100 boys is 11% (95% CI = 0,056 0,188), the fragile X syndrome being excluded. Although these figures are quite in agreement with previous estimations of the frequency of XLMR among mentally retarded men, they might be an underestimation, when it is taken into account that only about a third of obligate carriers of XLMR mutations have highly skewed X inactivation.