A deep intronic mutation inFGBcreates a consensus exonic splicing enhancer motif that results in afibrinogenemia caused by aberrant mRNA splicing, which can be corrected in vitro with antisense oligonucleotide treatment

Abstract

We previously described a novel homozygous point mutation (FGB c.115-600A>G) located deep within intron 1 of the fibrinogen beta gene (FGB), as a likely cause of afibrinogenemia. While this was the only mutation detected, its pathological mechanism was unclear. Here we show the mutation causes the inclusion of a 50-bp cryptic exon by creating a consensus heptad motif recognized by the spliceosome recruiting protein pre-mRNA splicing factor (SF2)/arginine/serine-rich alternative splicing factor (ASF) splicing factor 2/alternative splicing factor (SF2/ASF). Translation of the aberrant mRNA would result in truncation of the Bbeta chain, preventing fibrinogen synthesis. Selective introduction of a second mutation into the enhancer motif abolished the SF2/ASF binding motif and re-established normal pre-mRNA splicing. Subsequent introduction of antisense phosphorodiamidate morpholino oligonucleotides (PMOs) into transfected cells containing the mutant construct blocked the protein-RNA interaction and successfully restored normal splicing ( approximately 50% at 2 microM and approximately 90% at 10 microM). The molecular characterization of this case has revealed a unique disease mechanism, shown the importance of screening for deep intronic mutations, and provided evidence that antisense gene therapy is potentially practical for the treatment of diseases caused by this class of mutation.

CD22 EXON 12 deletion as a pathogenic mechanism of human B-precursor leukemia

“…RNA splicing requires a complex interplay of multiple RNA-binding proteins that are equipped with domains to bind sequence motifs on single stranded RNA to ensure accurate determination of exon recognition. In silico interrogation of the wild-type pre-mRNA segment derived from the 132-bp mutation-rich intronic sequence between exons 12 and 13 for accessible splicing factor binding sites resulted in identification of multiple potential binding sites for the RNA-binding proteins hnRNP-E2/ PCBP, hnRNP-I/PTB, and hnRNP-L, three members of the heterogenous nuclear ribonucleoprotein family of splicing factors that act as global regulators of alternative splicing and are abundantly expressed in infant leukemias (40)(41)(42)(43)(44)(45)(46)(47)(48)(49) (Fig. 2A.1).…”

Mucopolysaccharidosis IVA mutations in Chinese patients: 16 novel mutations

“…33 A number of nucleotides in the genome, coding or noncoding, have been found to function in the regulation of transcription, splicing and translation. Characteristics of these cis-acting regulatory elements are not clearly understood and therefore such mutations may be misjudged as missense mutation or synonymous polymorphism 34,35 or left in the unchecked region of introns 36 or interval between genes. More efforts, such as RNA-based strategy, are required to detect these unknown alleles for better understanding of GALNS gene mutations and for more precise prenatal gene diagnosis.…”

Antisense oligonucleotide treatment for a pseudoexon-generating mutation in theNPC1gene causing Niemann-Pick type C disease

“…This strategy has been used successfully to restore correct splicing in several disease models such as cystic fibrosis [Friedman et al, 1999], ocular albinism type I [Vetrini et al, 2006], afibrinogenemia [Davis et al, 2009], and congenital disorder of glycosylation type IA [Vega et al, 2009].…”