Abstract
844ins68 is a frequent polymorphism of the cystathionine beta-synthase gene (CBS) that consists of a 68-bp insertion duplicating the 3' splice site of intron 7 and the 5'-end of exon 8. The presence of two identical 3' splice sites spaced by 68 bp should lead to either a selection of the proximal site or to at least two alternatively spliced CBS mRNA variants. Instead, an accurate selection of the distal 3' splice site is observed in the 844ins68 carriers. The duplication has generated a gene re-arrangement at the 3' splice site where two GGGG runs have been brought close to each other. Using a minigene system, we have investigated the effect this peculiar configuration might have on the selection of the 3' splice site of intron 7 in the CBS gene. Minimal disruption of the G runs resulted in a dramatic shift toward the proximal 3' splice site selection with inclusion of the 68-bp insertion and a consequent change of the reading frame. The insertional event created this peculiar configuration of two G repeats close to each other that subsequently acquired the ability to strongly bind heterogeneous nuclear ribonucleoprotein (hnRNP) H1, a specific trans-acting factor. The interaction of hnRNP H1 with G runs within the 844ins68 context might interfere with the recruitment of splicing factors to the proximal 3' splice site thus favoring the selection of the distal 3' splice site. Our results therefore suggest the possibility that the insertion was an evolutionary event that allowed the rescue of the wild-type sequence, so preserving protein function.
Highlights
The identification of the 844ins68 insertion of the cystathionine -synthase gene was initially reported in a patient affected by homocystinuria due to CBS1 deficiency [1]
It has been shown that the distal 3Ј splice site is exclusively selected, such that the 68-bp insertion is skipped in the mature mRNA derived from the 844ins68 allele [2]
The analysis of the splicing pattern by RT-PCR of these constructs after transient transfections in the Hep3B cell line showed that both the wild-type and 844ins68 constructs reproduce the splicing pattern found in vivo, with activation of the distal 3Ј splice site (Fig. 1B)
Summary
The identification of the 844ins68 insertion of the cystathionine -synthase gene was initially reported in a patient affected by homocystinuria (online Mendelian Inheritance in Man no. 236200) due to CBS1 deficiency [1]. Peculiar 3Ј Splice Site Selection in CBS 844ins68 Insertion were sequenced to confirm their identity and to exclude the presence of other mutations.
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