MLPA-based copy number variation analysis in patients with Abdominal Aortic Aneurysm: identification of a novel mutation in the human TGFbeta-R1 gene

Background. Abdominal aortic aneurysm (AAA) is a multifactorial condition associated with a strong genetic component. The transforming growth factor beta (TGFB) signalling pathway regulates several cell functions including proliferation, differentiation and vascular remodelling. TGFB receptor 1 and 2 (TGFBR1 and TGFBR2) gene sequencing identified point mutations associated with AAA manifestation. Whether heterozygous exon deletions and duplications may also be associated with AAA is presently unknown. Aim. To screen TGFBR1 and TGFBR2 genes for genomic rearrangements, in a cohort of AAA patients, by using the Multiplex Ligation-dependent Probe Amplification (MLPA) technique. Methods. 50 patients undergoing elective open AAA repair referred to the Unit of Vascular Surgery of the Centro Cardiologico Monzino were enrolled. Genomic DNA was extracted from peripheral blood and MLPA analysis was performed by using an ordinary PCR thermal cycler for the hybridization and amplification steps, and a capillary sequencer for amplicons separation. TGFBR1 protein levels in tissue samples collected during surgery from aneurysmatic abdominal aortic wall were assessed by western blot. Results. MLPA identified a new mutation in TGFBR1, consisting of a heterozygous deletion of exon 1 and 2, encoding for the receptor extracellular domain, and partially of the promoter region, in one out of the 50 patients screened. This mutation does not affect the protein expression levels, which were comparable to those detected in AAA patients who did not carry the new mutation. No rearrangements were identified in the TGFBR2 gene. Conclusions Application of the MLPA analysis to the screening of novel genomic rearrangements allowed the identification of a new heterozygous deletion in the TGFBR1 gene in a patient affected by AAA. Whether this mutation may affect the receptor functional activity, despite the apparently unaltered TGFBR1 protein levels, is currently matter of ongoing investigations.