Troubleshooting the search for potential pathogenic small INDELs (1
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1. 57 Congresso Brasileiro de Gen tica 73 Resumos do 57 Congresso Brasileiro de Gen tica 30 de agosto a 2 de setembro de 2011 Centro de Convenc es do Hotel Monte Real Resort Aguas de Lind ia SP Brasil www sbg org br ISBN 978 85 89109 06 2 Troubleshooting the search for potential pathogenic small INDELs 1 10bp in neuropsychiatric disorders Lemos RR Souza MBR Oliveira DF Pimentel LF Oliveira JRM 2 Keizo Asami Laboratory LIKA Federal University of Pernambuco Recife PE Neuropsychiatry Department Federal University of Pernambuco Recife PE robertalemos775 hotmail com Keywords INDELs Sequencing Neuropsychiatric disorder Single Nucleotide Polymorphisms SNPs and Insertion or Deletions INDELs have been increasingly recognized as a major type of genetic variations with potential impact in protein activities and gene expression levels observed in complex genetic traits like neuropsychiatric diseases However previous studies have pointed various method ological difficulties to reliably detect and validate short INDELs in a large scale manner Various approaches have been developed with bioinformatics tools and molecular techniques for identification and association of these varia tions and our group recently designed a bioinformatics pipeline to detect new potential variations in genes selected from microarrays studies performed with brain samples from patients with Alzheimer s disea2. rate of false positives in case of experiments lacking careful checking of double strand sequencing Financial Support CNPg PROPESQ UFPE CAPES and FACEPE
3. se AD and mood disorders dysthymia and bipolar affective disorder BPD We started screening this panel of variations in Brazil ian population samples affected by AD and mood disorders to confirm the variations predicted by our original method The DNA was amplified by PCR and sequenced in a Mega Bace 1000 and fragments were aligned by us ing the software CLCbio Workbench Combined version 3 6 2 So far INDELs predicted In sico ranging from 1 to 10bp were not confirmed in any samples Some false positives were consistently identified but latter ruled out after verification at the complementary strand We performed preliminary experiments with DNA pooling and they still need methodological adjustments for a better interpretation for the yield and sensitivity but are potentially ap plicable to save time and resources in such large scale screening Two genetic variations at the genes COX2 and IL1A were confirmed in AD samples respectively rs2745557 and rs17561 The rs14068 SNP located at exon 2 of the GABRA5 gene was validated in samples of patients with dysthymia and BPD Our group is currently deal ing with different approaches that might optimize sequencing and bioinformatics analyses of shorts INDELs to broaden our research capabilities of identifying those intriguing genetic variations Reporting these pitfalls might contribute with other groups that are planning to search for such interesting genetic variation and call the attention for the high
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