Functional analysis of splicing mutations and of an exon 2 polymorphic variant ofSERPING1/C1NH

Abstract

Several sequence changes have been reported in hereditary angioedema patients in intron 2 of the SERPING1/C1NH gene, but their consequences on splicing have not been determined. We examined in cell transfection assays the consequences at the mRNA level of splicing mutations affecting either the +3 or the +5 donor site positions, or the conserved canonical splicing signals of exon 2, using mutant C1 inhibitor minigene constructs. Both intron 2 mutations, c.51+3A>G and c.51+5G>A, resulted in marked exon 2 skipping in these assays, but also yielded a large fraction of normal transcripts. We show that the c.51+3A>G mutation cosegregates with low C1 inhibitor protein levels in one family. Moreover, the second base of exon 2 of the SERPING1/C1NH gene is the site of a polymorphic variant, which has been proposed as a modifier of disease severity. We found that the c.-21C allele at this position yields low but significant levels of exon 2 skipping in transfected Hep G2 or Hep 3B cells, suggesting that this allele may contribute, at the RNA level, to more severe forms of angioedema. Furthermore, we describe a previously not detected alternative splicing of exon 3, found in peripheral blood cell mRNA but not in the liver or in hepatoma cell lines and we show that, in cultured monocytes of a patient carrying the c.51+3A>G mutation, this alternative splicing is shifted from exon 3 exclusion to skipping of both exons 2 and 3. The latter finding suggests that mutations affecting splicing of exon 2 of the SERPING1/C1NH gene may have different consequences in monocytes versus other cell types.

Analysis of SERPING1 expression on hereditary angioedema patients: Quantitative analysis of full-length and exon 3 splicing variants

“…3 shows the patterns of both transcripts in controls and patients bearing different types of mutations as seen on agarose gel. This novel splicing variant has also been observed by other authors in peripheral blood cells from healthy controls but it was reported to be absent in hepatocytes from either patients and controls, indicating that its expression is restricted to monocytes [15].…”

“…Furthermore, the description of an mRNA variant without exon 3, which is expressed in normal peripheral blood cells [14,15], prompted us to quantify the distribution of both isoforms (fulllength and exon 3-skipping) with two transcript-specific amplicons in the control population and in HAE patients bearing different kinds of mutations. Our starting hypothesis was to determine a possible role for the exon 3-skipping isoform in the regulation of C1NH expression by comparing the mRNA splicing patterns found in healthy controls and HAE patients carrying different types of mutations.…”

A large fraction of unclassified variants of the mismatch repair genesMLH1andMSH2is associated with splicing defects

“…The pCAS minigene was obtained by cloning into the EcoRI and BamHI sites of the pcDNA3.1(-) vector (Invitrogen, CergyPontoise, France) an EcoRI-BamHI fragment of the SERPING1/ CINH minigene, [Duponchel et al, 2006] as shown in Fig. 1A.…”

“…1A. It contains the last 124 bp of intron 1, exon 2, intron 2, and exon 3 of the SERPING1/CINH gene, fused to 123 bp of exon 4 [Duponchel et al, 2006]. An MluI site was generated in the intron of this construct 118 bp downstream of the natural BamHI site, and the MluI site at position 229 of the pcDNA3.1 vector was inactivated by site directed mutagenesis.…”

Biological effects of four PSEN1 gene mutations causing Alzheimer disease with spastic paraparesis and cotton wool plaques

“…PCR products were cloned into a pCDNA3.1 vector containing exons 2 and 3 of the SERPING1/CINH gene (Duponchel et al, 2006) separated by their natural intron in which we had inserted appropriate cloning sites.…”