Mutations which alter splicing in the human hypoxanthine-guanine phosphoribosyltransferase gene

143

109

The mechanism of exon skipping induced by nonsense mutations has not been well elucidated. We now report results of in vitro splicing studies which disclosed that a particular example of exon skipping is due to disruption of a splicing enhancer sequence located within the exon. A nonsense mutation (E1211X) due to a G to T transversion at the 28th nucleotide of exon 27 (G3839T) was identified in the dystrophin gene of a Japanese Becker muscular dystrophy case. Partial skipping of the exon resulted in the production of truncated dystrophin mRNA, although the consensus sequences for splicing at both ends of exon 27 were unaltered.To determine how E1211X induced exon 27 skipping, the splicing enhancer activity of purine-rich region within exon 27 was examined in an in vitro splicing system using chimeric doublesex gene pre-mRNA. The mutant sequence containing G3839T abolished splicing enhancer activity of the wild-type purine-rich sequence for the upstream intron in this chimeric pre-mRNA. An artificial polypurine oligonucleotide mimicking the purine-rich sequence of exon 27 also showed enhancer activity that was suppressed by the introduction of a T nucleotide. Furthermore, the splicing enhancer activity was more markedly inhibited when a nonsense codon was created by the inserted T residue. This is the first evidence that partial skipping of an exon harboring a nonsense mutation is due to disruption of a splicing enhancer sequence. ( J. Clin. Invest. 1997. 100:2204-2210.) Key words: dilated cardiomyopathy • reading frame • in vitro splicing • pre-mRNA • polypurine

Section: Discussionmentioning

confidence: 85%

Disruption of the splicing enhancer sequence within exon 27 of the dystrophin gene by a nonsense mutation induces partial skipping of the exon and is responsible for Becker muscular dystrophy.

Shiga¹,

Takeshima²,

Sakamoto³

et al. 1997

143

109

“…In the 5Ј-splicing consensus sequence, the dinucleotide GT is the best conserved among mammals, and mutations affecting this sequence were reported to cause the inactivation of several genes associated with human diseases including phenylalanine hydroxylase (19), procollagen COL3A1 (20), and hypoxanthine guanine phosphoribosil-transferase (21). In these genes, a G to A mutation in the GT splicing recognition sequence consistently results in skipping of the entire exon preceding the mutation, supporting the current theory of exon definition for higher eukaryotes in which the splicing sites are recognized as exonic pairs as opposed to the mechanism proposed for lower eukaryotes in which the splicing sites are recognized as intronic pairs (22,23).…”

Section: Discussionmentioning

confidence: 99%

Molecular basis of the human dihydropyrimidine dehydrogenase deficiency and 5-fluorouracil toxicity.

Wei¹,

McLeod²,

McMURROUGH³

et al. 1996

319

168

Dihydropyrimidine dehydrogenase (DPD) deficiency constitutes an inborn error in pyrimidine metabolism associated with thymine-uraciluria in pediatric patients and an increased risk of toxicity in cancer patients receiving 5-fluorouracil (5-FU) treatment. The molecular basis for DPD deficiency in a British family having a cancer patient that exhibited grade IV toxicity 10 d after 5-FU treatment was analyzed. A 165-bp deletion spanning a complete exon of the DPYD gene was found in some members of the pedigree having low DPD catalytic activity. Direct sequencing of lymphocyte DNA from these subjects revealed the presence of a G to A point mutation at the 5 Ј -splicing site consensus sequence (GT to AT) that leads to skipping of the entire exon preceding the mutation during pre-RNA transcription and processing. A PCR-based diagnostic method was developed to determine that the mutation is found in Caucasian and Asian populations. This mutation was also detected in a Dutch patient with thymine-uraciluria and completely lacking DPD activity. A genotyping test for the G to A splicing point mutation could be useful in predicting cancer patients prone to toxicity upon administration of potentially toxic 5-FU and for genetic screening of heterozygous carriers and homozygous deficient subjects. ( J. Clin. Invest. 1996. 98:610-615.)

“…The latter exonic mutation created a strong palindromic sequence (TGGGCTTGGAGAGCCCAA GAAAT/gt), which would result in an alteration of the premRNA secondary structure in this region. Steingrimsdottir et al (33) analyzed many mutants at the human hypoxanthine guanine phosphoribosyltransferase gene and detected 34 mutations that resulted in aberrant splicing. Among them, a mutation located 13 nucleotides from the 5' splice site of intron 8 caused exon 8 skipping in 90% of hypoxanthine guanine phosphoribosyltransferase transcripts.…”

Section: Discussionmentioning

confidence: 99%

Identification of a novel exonic mutation at -13 from 5' splice site causing exon skipping in a girl with mitochondrial acetoacetyl-coenzyme A thiolase deficiency.

Fukao

Yamaguchi²,

Wakazono³

et al. 1994

We identified a novel exonic mutation which causes exon skipping in the mitochondrial acetoacetyl-CoA thiolase (T2) gene from a girl with T2 deficiency (GK07). GKO7 is a compound heterozygote; the maternal allele has a novel G to T transversion at position 1136 causing Gly379 to Val substitution (G379V) of the T2 precursor. In case of in vivo expression analysis, cells transfected with this mutant cDNA showed no evidence of restored T2 activity. The paternal allele was associated with exon 8 skipping at the cDNA level. At the gene level, a C to T transition causing Glnr2 to termination codon (Q272STOP) was identified within exon 8, 13 bp from the 5' splice site of intron 8 in the paternal allele. The mRNA with Q272STOP could not be detected in GKO7 fibroblasts, presumably because pre-mRNA with Q272STOP was unstable because of the premature termination. In vivo splicing experiments revealed that the exonic mutation caused partial skipping of exon 8. This substitution was thought to alter the secondary structure of T2 pre-mRNA around exon 8 and thus impede normal splicing. The role of exon sequences in the splicing mechanism is indicated by the exon skipping which occurred with an exonic mutation. (J. Clin. Invest. 1994. 93:1035-1041.) Key words: 8-ketothiolase deficiency * exon skipping -molecular basis * in vivo splicing experiment * organic aciduria