title: Cryptic exon activation by disruption of exon splice enhancer: novel mechanism causing 3-methylcrotonyl-CoA carboxylase deficiency.
abstract: 3-Methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine catabolism. MCC is a heteromeric mitochondrial enzyme composed of biotin-containing alpha (MCCA) and smaller beta (MCCB) subunits encoded by MCCA and MCCB, respectively. We report studies of the c.1054G-->A mutation in exon 11 of MCCB detected in the homozygous state in a patient with MCC deficiency. Sequence analysis of MCCB cDNA revealed two overlapping transcripts, one containing the normal 73 bp of exon 11 including the missense mutation c.1054G-->A (p.G352R), the other with exon 11 replaced by a 64-bp sequence from intron 10 (cryptic exon 10a) that maintains the reading frame and is flanked by acceptable splice consensus sites. In expression studies, we show that both transcripts lack detectable MCC activity. Western blot analysis showed slightly reduced levels of MCCB using the transcript containing the missense mutation, whereas no MCCB was detected with the transcript containing the cryptic exon 10a. Analysis of the region harboring the mutation revealed that the c.1054G-->A mutation is located in an exon splice enhancer sequence. Using MCCB minigene constructs to transfect MCCB-deficient fibroblasts, we demonstrate that the reduction in utilization of exon 11 associated with the c.1054G-->A mutation is due to alteration of this exon splice enhancer. Further, we show that optimization of the weak splice donor site of exon 11 corrects the splicing defect. To our knowledge, this is the first demonstration of a point mutation disrupting an exon splice enhancer that causes exon skipping along with utilization of a cryptic exon.
authors.affiliation: Division of Metabolism, University Children's Hospital, 8032 Zürich, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Stucki
authors.forename: Terttu
authors.initials: T
authors.lastname: Suormala
authors.forename: Brian
authors.initials: B
authors.lastname: Fowler
authors.forename: David
authors.initials: D
authors.lastname: Valle
authors.forename: Matthias R
authors.initials: MR
authors.lastname: Baumgartner
mesh_terms: D017398:Alternative Splicing; D000592:Amino Acid Metabolism, Inborn Errors / Q000235:genetics*; D019735:Carbon-Carbon Ligases / Q000172:deficiency* / Q000235:genetics*; D003241:Consanguinity; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D005091:Exons; D005183:Failure to Thrive / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008957:Models, Genetic; D009154:Mutation; D020125:Mutation, Missense; D017422:Sequence Analysis, DNA; D012867:Skin / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D019735:Carbon-Carbon Ligases; C021573:methylcrotonoyl-CoA carboxylase
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