Usefulness of MLPA in the detection of SHOX deletions

SHOX haploinsufficiency causes a wide spectrum of short stature phenotypes, such as Leri-Weill dyschondrosteosis (LWD) and disproportionate short stature (DSS). SHOX deletions are responsible for approximately two thirds of isolated haploinsufficiency; therefore, it is important to determine the most appropriate methodology for detection of gene deletion. In this study, three methodologies for the detection of SHOX deletions were compared: the fluorescence in situ hybridization (FISH), microsatellite analysis and multiplex ligation-dependent probe amplification (MLPA). Forty-four patients (8 LWD and 36 DSS) were analyzed. The cosmid LLNOYCO3`M`34F5 was used as a probe for the FISH analysis and microsatellite analysis were performed using three intragenic microsatellite markers. MLPA was performed using commercial kits. Twelve patients (8 LWD and 4 DSS) had deletions in SHOX area detected by MLPA and 2 patients generated discordant results with the other methodologies. In the first case, the deletion was not detected by FISH. In the second case, both FISH and microsatellite analyses were unable to identify the intragenic deletion. In conclusion, MLPA was more sensitive, less expensive and less laborious; therefore, it should be used as the initial molecular method for the detection of SHOX gene deletion. (C) 2010 Elsevier Masson SAS. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo - FAPESP[05/04726-0]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico - CNPq[301477/2009-4]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico - CNPq[301339/2008-9]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico - CNPq[300982/2009-7

Use of nonradioactive labeling to detect large gene rearrangements in 21-hydroxylase deficiency Uso de marcação não radiativa para identificação de grandes rearranjos gênicos na deficiência da 21-hidroxilase

PURPOSE: To establish the Southern blotting technique using hybridization with a nonradioactive probe to detect large rearrangements of CYP21A2 in a Brazilian cohort with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH-21OH). METHOD: We studied 42 patients, 2 of them related, comprising 80 non-related alleles. DNA samples were obtained from peripheral blood, digested by restriction enzyme Taq I, submitted to Southern blotting and hybridized with biotin-labeled probes. RESULTS: This method was shown to be reliable with results similar to the radioactive-labeling method. We found CYP21A2 deletion (2.5%), large gene conversion (8.8%), CYP21AP deletion (3.8%), and CYP21A1P duplication (6.3%). These frequencies were similar to those found in our previous study in which a large number of cases were studied. Good hybridization patterns were achieved with a smaller amount of DNA (5 mug), and fragment signs were observed after 5 minutes to 1 hour of exposure. CONCLUSIONS: We established a non-radioactive (biotin) Southern blot/hybridization methodology for CYP21A2 large rearrangements with good results. Despite being more arduous, this technique is faster, requires a smaller amount of DNA, and most importantly, avoids problems with the use of radioactivity.<br>OBJETIVO: Padronizar a técnica de Southern blotting usando hibridização com material não radioativo para detectar grandes rearranjos no gene CYP21A2 em uma amostra da população brasileira com hiperplasia adrenal congênita. MÉTODO: Foram estudados 42 pacientes, 2 dos quais aparentados, totalizando 80 alelos não relacionados. As amostras de DNA foram obtidas de sangue periférico, digeridas com enzima de restrição Taq I, realizado Southern blotting e hibridizadas com sonda marcada com biotina. RESULTADOS: O método se mostrou eficaz, com resultados similares aos encontrados ao utilizar a metodologia com material radioativo. Foram encontradas 2,5% de deleção do CYP21A2, 8,8% de grandes conversões, 3,8% de deleção do CYP21A1P e 6,3% de duplicação do CYP21A1P. Estas freqüências foram similares às encontradas em nosso estudo prévio, onde um número significante de casos foi estudado. Um bom padrão de hibridização foi alcançado utilizando menor quantidade de DNA (5mg) e a emissão de sinais foi observada entre 5 minutos e 1 hora de exposição. CONCLUSÕES: Padronizamos uma técnica de Southern blotting/ hibridização com material não radioativo (biotina) para a pesquisa de grandes rearranjos no gene CYP21A2 com bons resultados. Apesar de ser mais trabalhoso, este método é mais rápido, utiliza menores quantidades de DNA e, principalmente, evita problemas com o uso de radioatividade