Hereditary Hemorrhagic Telangiectasia: Breakpoint Characterization of a Novel Large Deletion in ACVRL1 Suggests the Causing Mechanism

Abstract

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia. Mutations in either ENG or ACVRL1 account for around 85% of cases, and 10% are large deletions and duplications. Here we present a large novel deletion in ACVRL1 gene and its molecular characterization in a 3 generation Italian family. We employed short tandem repeats (STRs) analysis, direct sequencing, multiplex ligation-dependant probe amplification (MLPA) analysis, and 'deletion-specific' PCR methods. STRs Analysis at ENG and ACVRL1 loci suggested a positive linkage for ACVRL1. Direct sequencing of this gene did not identify any mutations, while MLPA identified a large deletion. These results were confirmed and exactly characterized with a 'deletion-specific' PCR: the deletion size is 4,594 bp and breakpoints in exon 3 and intron 8 show the presence of short direct repeats of 7 bp [GCCCCAC]. We hypothesize, as causative molecular mechanism, the replication slippage model. Understanding the fine mechanisms associated with genomic rearrangements may indicate the nonrandomness of these events, highlighting hot spots regions. The complete concordance among MLPA, STRs analysis and 'deletion-specific PCR' supports the usefulness of MLPA in HHT molecular analysis.

Keywords: ACVRL1; Large deletion; MLPA; Short direct repeats; Slippage.

Fig. 1

The pedigree of the family. The proband (II:1) and all analysed affected family members (black-shaded) were positive for the ACVRL1 exon 3-8 deletion. MLPA results are indicated for each family member as (+) or (-) for the presence or absence of the ACVRL1 deletion, respectively. D12S1677 is an intragenic marker located in IVS9.

Fig. 2

Characterization of the deletion. a MLPA results obtained with Coffalyser. The ACVRL1 deleted exons 3-8 have a peak ratio <0.7. b Schematization of the deleted region and sequence of the PCR product encompassing the breakpoints and showing a deletion of 4,594 bp (Hg18 (NCBI 36): chr12:g.50593334-50597927). Black vertical arrows in the scheme indicate deletion breakpoints. White dashed vertical lines indicate MLPA probes position. Large horizontal arrows indicate position of primers for deletion-specific PCR. In the electropherogram, the ‘immunoglobulin heavy chain class switch repeat’ sequence is boxed.