RESUMO
Mutations in the gene TBX5 cause Holt-Oram syndrome (HOS), an autosomal dominant disorder characterized by anterior (i.e., radial ray) upper limb malformations and congenital heart defects and/or cardiac conduction anomalies. The detection rate for TBX5 mutations in HOS patients has been given as 30-35% in most reports. However, a detection rate of 74% was reported when strict clinical inclusion criteria for HOS were applied prior to TBX5 analysis. Still, in a significant proportion of typical HOS cases no mutation can be found within the TBX5 coding region and flanking intronic sequences. One explanation could be that large but submicroscopic deletions of TBX5 could cause HOS, yet only one such TBX5 deletion has been reported to date. We developed a quantitative Real Time PCR strategy to detect large, submicroscopic deletions in TBX5. Using this assay, we screened a total of 102 TBX5 mutation negative patients and discovered two novel intragenic deletions. One deletion of 7756 bp removes exon 6 and a considerable part of the neighboring intronic sequences, and the other of 3695 bp removes exon 9 with the stop codon and the 3'UTR completely as well as a part of the preceding intron 8. We conclude that quantitative Real Time PCR is a reliable method to detect submicroscopic deletions within TBX5. However, such deletions explain only approximately 2% of the TBX5 mutational spectrum in HOS cases. In addition, we also present eight novel TBX5 mutations (three nonsense, one splice mutation, four short deletions) as detected by direct sequencing in 21 families not previously analyzed for mutations.