Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, 2007. / Submitted by Rebeca Araujo Mendes (bekinhamendes@gmail.com) on 2009-12-21T23:59:54Z
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Previous issue date: 2007 / A síndrome do X frágil (SXF), causa comum de retardo mental (RM) hereditário ocorre por mutação no gene FMR1 pela expansão não traduzida de CGG na extremidade 5’. Na população, a expansão varia de 5 a 52 repetições. Portadores de 52 a 200 cópias (pré-mutação) produzem a proteína FMRP, porém podem transmitir a mutação à geração seguinte. Indivíduos SXF possuem mais de 200 cópias (mutação completa) e resulta em metilação e inativação do gene impedindo a expressão da FMRP. Ausência de FMRP é responsável pelo RM. Mulheres portadoras da mutação completa apresentam variação no grau de RM devido inativação aleatória do X. A SXF é identificada pelo sítio frágil (FRAXA), na região Xq27.3. A imuno-histoquímica permite observar a expressão da FMRP no bulbo de cabelo. Indivíduos afetados mostram níveis inferiores aos controles. A reação em cadeia da polimerase (PCR) representa o melhor método para triagem da SXF. Objetivos: 1) avaliar a eficácia da técnica de imuno-histoquímica em pacientes suspeitos de SXF; 2) comparar três técnicas de diagnóstico para SXF: imuno-histoquímica, citogenética e PCR. Métodos: foram selecionados 121 pacientes com suspeita SXF sendo 59 (48,8%) homens e 62 (51,2%) mulheres. Noventa indivíduos sadios, com 75-100% de expressão da FMRP foram selecionados como controles negativos da imuno-histoquímica. Resultados: de 113 pacientes que realizaram a imuno-histoquímica, 54 eram homens, com 17 positivos, 28 negativos e nove inconclusivos e 59 mulheres, com sete positivas, 27 negativas e 25 inconclusivas. Entre os 119 que realizaram a PCR, 57 homens, com 20 positivos e 37 negativos e 62 mulheres, com três negativas e 59 inconclusivas. Dos 104 pacientes analisados para citogenética, 57 homens, com 34 positivos e 23 negativos e 47 mulheres, com 27 positivas e 20 negativas. A menor expressão do FRAXA foi 3% nos homens e 5,4% nas mulheres e a maior
36% e 37,1%, respectivamente. Comparando as técnicas imuno-histoquímica e PCR (considerado o padrão-ouro) nos homens observaram-se sensibilidade 84,2%; especificidade 100%; acurácia 94,2%. Comparando citogenética com PCR nos homens, encontraram-se sensibilidade 100%; especificidade 61,1%; acurácia 74,5%. A citogenética exige profissional qualificado, cultura prolongada, tendo boa sensibilidade em homens. As vantagens da imuno-histoquímica são: rapidez, baixo custo e facilidade de obtenção das amostras de bulbo de cabelo. A PCR é técnica versátil, rápida, com alta sensibilidade e especificidade com desvantagem em relação às mulheres de não diferenciar as homozigotas normais das heterozigotas afetadas. Conclusões: Houve correlação positiva e significativa entre homens com a SXF pela imuno-histoquímica comparada à PCR e citogenética. A PCR mostrou-se eficiente como triagem da SXF nos homens quando comparada com citogenética e imuno-histoquímica devido à ausência de falso-positivos. Fragilidade cromossômica em não afetados pode ser devido à presença de outros sítios frágeis. Todas as técnicas imuno-histoquímica, citogenética e PCR, isoladamente, não detectam portadores da pré-mutação e mulheres com mutação completa. ___________________________________________________________________________________ ABSTRACT / The fragile X syndrome (FXS), common cause of hereditary mental retardation (MR) is caused by a mutation in the gene FMR1 characterized by a not translated CGG expansion in extremity 5'. In the population, this expansion varies from five to 52 repetitions. Carriers of 52 to 200 copies (premutation) show a normal production of the FMRP protein, but they can transmit an expanded mutation to the following generation. Individuals FXS have more than 200 copies (full mutation) which result in gene methylation and inactivation hindering the expression of the FMRP. Absence of FMRP is responsible for the MR. Female carrying the full mutation presents various degrees of MR due to random inactivation of the X chromosome. The FXS is identified by the presence of a fragile site (FRAXA) located in the Xq27.3 region. The immunohystochemical method allows the detection of the FMRP expression in the hair bulb. Affected individuals disclose decreased levels of FMRP than controls. The polimerase chain reaction (PCR) represents the best screening method to identify FXS affected individuals. Objectives: 1) to evaluate the effectiveness of the immunohystochemical technique in the identification of suspected FXS patients; 2) to compare three techniques used in the diagnosis of FXS: immunohystochemical, cytogenetic and PCR. Methods: Were selected 121 patients with signs and symptoms consistent with a probable diagnosis of FXS. Fifty-nine (48.8%) men and 62 (51.2%) were women. As negative controls were used 90 healthy individuals with 75 to 100% of FMRP expression. Results: Among the 113 patients that underwent immunohystochemical testing, 54 were men (17 being positive, 28 negative and nine with dubious results) and 59 were women with seven positive, 27 negative and 25 with dubious results. Among the 119 individuals that underwent PCR, 57 were men (with 20 positive and 37 negative results) and 62 women (with three negative and 59 showing dubious results). Of the 104 patients analyzed by cytogenetic testing were men (with 34 positives and 23 negatives results) and 47 were women (with 27 positive and 20 negatives results). The FRAXA lower expression was detected in 3% of men and 5.4% of women xx and the greater expression was 36% and 37.1%, respectively. Comparing the immunohystochemical technique and PCR (considered the gold-standard method) in men was observed a sensitivity of 84.2%; and a specificity of 100%; the accuracy was 94.2%. Comparing cytogenetic analysis and PCR in men, the sensitivity of the analysis was 100% and its specificity of 61.1%; accuracy was 74.5%. The cytogenetic analysis requires a qualified professional, a prolonged cell culture time its sensitivity being satisfying mainly in men. The advantages of the immunohystochemical are its shorter execution time, low cost, and facility to obtain the hair bulb samples. The PCR is a fastest technique, versatile, with high sensitivity and specificity. The disadvantage of this test being its incapacity to differentiate the normal homozigous from the affected heterozygous women. Conclusion: A positive correlation was found between FXS affected men when comparing the immunohystochemical technique to PCR and to cytogenetic analysis. The PCR was considered an efficient method in detecting the FXS in men when compared to cytogenetic analysis and immunohystochemical technique mainly due to the absence of false positive results. The presence of chromosome fragility in non affected individuals can be due to the presence of other fragile sites. Considering all the three techniques, immunohystochemical, cytogenetic and PCR, can be concluded that, itself, all are ineffective in the identification of premutation carriers and in women with full mutation.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.unb.br:10482/3090 |
| Date | 10 July 2007 |
| Creators | Queiroz, Lílian Barros |
| Contributors | Ferrari, Íris, Pratesi, Riccardo |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Source | reponame:Repositório Institucional da UnB, instname:Universidade de Brasília, instacron:UNB |
| Rights | info:eu-repo/semantics/openAccess |
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