Comparative analysis of methylation-specific PCR(MSP), Southern blot (SB) and FISH in molecular genetic diagnosis of patients with clinical picture suggestive of Prader-Willi or Angelman syndromes

Introdução: Prader-Willi (SPW) e Angelman (SA) são síndromes clinicamente distintas, causadas pela perda de expressão de genes na região cromossômica 15q11.2-q13, de origem paterna ou materna, respectivamente. Ambas compartilham os mesmos métodos diagnósticos. Nossos objetivos foram: a) analisar por PCR metilação-específica (MSP) pacientes com suspeita clínica de SPW/SA; b) comparar resultados de diferentes metodologias de diagnóstico molecular; c) aplicar a técnica MSP na rotina assistencial de pacientes encaminhados ao Serviço de Genética Médica/Hospital de Clínicas de Porto Alegre (SGM/HCPA). Métodos: Foram analisados 123 pacientes com suspeita clínica de SPW (n = 71) ou SA (n = 52) por MSP. Desses, 79 possuíam análise prévia por hibridação in situ fluorescente (FISH) e/ou Southern blot (SB). Resultados: Foram detectados 21 casos positivos – 15 de SPW (12,19%) e 6 de SA (4,88%). Nove pacientes tiveram etiologia molecular determinada, sendo sete com diagnóstico de SPW (quatro dissomias uniparentais – UPD15 materna – e três deleções na região 15q11-13) e dois com diagnóstico de SA (um com UPD15 paterna e um com deleção na região 15q11-13). Foram observados resultados equivalentes entre MSP e SB e resultados discrepantes entre MSP e FISH (n = 4) Foram padronizados dois protocolos de MSP para confirmação dos resultados e controle interno de qualidade. Conclusão: O perfil de detecção de cada técnica varia de acordo com o mecanismo etiológico presente. A análise por MSP detecta alterações no padrão de metilação geradas por deleção, UPD e defeitos de imprinting, sem identificar o mecanismo etiológico responsável. Contudo, mostrou ser eficiente para confirmação do diagnóstico clínico e screening dos pacientes com suspeita clínica sugestiva de SPW e SA. Diante de resultados positivos, é importante a identificação do mecanismo molecular subjacente para correlação genótipo-fenótipo e determinação do risco de recorrência familiar, fundamental para o aconselhamento genético.Introduction: Prader-Willi (PWS) and Angelman (AS) are clinically different syndromes caused by loss of expression of genes located on the chromosome 15q11.2-q13, of paternal or maternal origin, respectively. Both syndromes have the same diagnostic methods. The aims of the present study were: a) to perform a molecular analysis of 123 patients with clinical findings suggestive of PWS or AS using methylation-specific PCR (MSP); b) to compare the results obtained using different molecular diagnostic methodologies; c) to standardize MSP to be used in the routine care of patients at Medical Genetics Service/Hospital de Clínicas de Porto Alegre (SGM/HCPA). Methods: 123 patients with clinical findings suggestive of PWS (n = 71) or AS (n = 52) were analyzed by MSP. 79 had undergone previous laboratory analysis by fluorescence in situ hybridization (FISH) and/or Southern blot (SB). Results: MSP detected 21 positive cases – 15 PWS (12,19%) and 6 AS (4,88%). Molecular etiology was determined in 9 patients only – 7 were diagnosed with PWS (4 had uniparental disomy – maternal UPD15 – and 3 had deletions at 15q11-13) and 2 were diagnosed with AS (1 of paternal UPD15 and 1 deletion at 15q11-13) Comparing both methodologies, it was possible to observe concordant results between MSP and SB and discordant results between MSP and FISH (n = 4). We standardized two MSP methods in order to confirm the results and for internal quality control. Conclusion: The resulting profile of each technique varies according to the existing etiological mechanism. The methylation analysis by MSP technique detects changes on methylation pattern caused by deletion, UPD and imprinting defects, but it does not identify the responsible etiologic mechanism. Nevertheless, it is effective to confirm the suggestive clinical diagnosis of PWS/AS and to be used as a screening protocol. If positive results are observed, it is important to identify the underlying molecular mechanism to determine genotype-phenotype correlations and the risk of familial recurrence, which is essential for genetic counseling