Journal of Inherited Metabolic Disease

, Volume 33, Supplement 3, pp 191–198

Functional characterization of the novel intronic nucleotide change c.288+9C>T within the BCKDHA gene: understanding a variant presentation of maple syrup urine disease

  • Paula Fernández-Guerra
  • Rosa Navarrete
  • Kara Weisiger
  • Lourdes R. Desviat
  • Seymour Packman
  • Magdalena Ugarte
  • Pilar Rodríguez-Pombo
Research Report

Abstract

Mutations in any of the three different genes—BCKDHA, BCKDHB, and DBT—encoding for the E1α, E1β, and E2 catalytic components of the branched-chain α-ketoacid dehydrogenase complex can cause maple syrup urine disease (MSUD). Disease severity ranges from the classic to the mildest variant types and precise genotypes, mostly based on missense mutations, have been associated to the less severe presentations of the disease. Herein, we examine the consequences at the messenger RNA (mRNA) level of the novel intronic alteration c.288+9C>T found in heterozygous fashion in a BCKDHA variant MSUD patient who also carries the nucleotide change c.745G>A (p.Gly249Ser), previously described as a severe change. Direct analysis of the processed transcripts from the patient showed—in addition to a low but measurable level of normal mRNA product—an aberrantly spliced mRNA containing a 7-bp fragment of intron 2, which could be rescued when the patient’s cells were treated with emetine. This aberrant transcript with a premature stop codon would be unstable, supporting the possible activation of nonsense-mediated mRNA decay pathway. Consistent with this finding, minigene splicing assays demonstrated that the point mutation c.288+9C>T is sufficient to create a cryptic splice site and cause the observed 7-bp insertion. Furthermore, our results strongly suggest that the c.288+9C>T allele in the patient generates both normal and aberrant transcripts that could sustain the variant presentation of the disease, highlighting the importance of correct genotyping to establish genotype–phenotype correlations and as basis for the development of therapeutic interventions.

References

  1. Aevarsson AE, Chuang JL, Wynn RM, Turley S, Chuang DT, Hol WG (2000) Crystal structure of human branched-chain alpha-ketoacid dehydrogenase and the molecular basis of multienzyme complex deficiency in maple syrup urine disease. Struct Fold Des 8:277–291CrossRefGoogle Scholar
  2. Baralle D, Baralle M (2005) Splicing in action: assessing disease causing sequence changes. J Med Genet 42:737–748PubMedCrossRefGoogle Scholar
  3. Baralle D, Lucassen A, Buratti E (2009) Missed threads. The impact of pre-mRNA splicing defects on clinical practice. EMBO Rep 10:810–816PubMedCrossRefGoogle Scholar
  4. Chen X, Truong TT, Weaver J, Bove BA, Cattie K, Armstrong BA, Daly MB, Godwin AK (2006) Intronic alterations in BRCA1 and BRCA2: effect on mRNA splicing fidelity and expression. Hum Mutat 27:427–435PubMedCrossRefGoogle Scholar
  5. Chuang DT, Shih VE (2001) Maple syrup urine diesease (branched chain ketoaciduria). In: Scriver CR (ed) The metabolic and molecular basis of inherited disease. McGraw-Hill, New York, pp 1971–2005Google Scholar
  6. Chuang DT, Chuang JL, Wynn RM (2006) Lessons from genetic disorders of branched-chain amino acid metabolism. J Nutr 136:243S–249SPubMedGoogle Scholar
  7. Desmet FO, Hamroun D, Lalande M, Collod-Beroud G, Claustres M, Beroud C (2009) Human splicing finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res 37:e67PubMedCrossRefGoogle Scholar
  8. Dursun A, Henneke M, Ozgul K, Gartner J, Coskun T, Tokatli A, Kalkanoglu HS, Demirkol M, Wendel U, Ozalp I (2002) Maple syrup urine disease: mutation analysis in Turkish patients. J Inherit Metab Dis 25:89–97PubMedCrossRefGoogle Scholar
  9. Flaschker N, Feyen O, Fend S, Simon E, Schadewaldt P, Wendel U (2007) Description of the mutations in 15 subjects with variant forms of maple syrup urine disease. J Inherit Metab Dis 30:903–909PubMedCrossRefGoogle Scholar
  10. Henneke M, Flaschker N, Helbling C, Muller M, Schadewaldt P, Gartner J, Wendel U (2003) Identification of twelve novel mutations in patients with classic and variant forms of maple syrup urine disease. Hum Mutat 22:417PubMedCrossRefGoogle Scholar
  11. Mitsubuchi H, Owada M, Endo F (2005) Markers associated with inborn errors of metabolism of branched-chain amino acids and their relevance to upper levels of intake in healthy people: an implication from clinical and molecular investigations on maple syrup urine disease. J Nutr 135:1565S–1570SPubMedGoogle Scholar
  12. Montfort M, Chabas A, Vilageliu L, Grinberg D (2006) Analysis of nonsense-mediated mRNA decay in mutant alleles identified in Spanish Gaucher disease patients. Blood Cells Mol Dis 36:46–52PubMedCrossRefGoogle Scholar
  13. Nellis MM, Danner DJ (2001) Gene preference in maple syrup urine disease. Am J Hum Genet 68:232–237PubMedCrossRefGoogle Scholar
  14. Nellis MM, Kasinski A, Carlson M, Allen R, Schaefer AM, Schwartz EM, Danner DJ (2003) Relationship of causative genetic mutations in maple syrup urine disease with their clinical expression. Mol Genet Metab 80:189–195PubMedCrossRefGoogle Scholar
  15. Quental S, Macedo-Ribeiro S, Matos R, Vilarinho L, Martins E, Teles EL, Rodrigues E, Diogo L, Garcia P, Eusebio F, Gaspar A, Sequeira S, Furtado F, Lanca I, Amorim A, Prata MJ (2008) Molecular and structural analyses of maple syrup urine disease and identification of a founder mutation in a Portuguese Gypsy community. Mol Genet Metab 94:148–156PubMedCrossRefGoogle Scholar
  16. Rodriguez-Pombo P, Navarrete R, Merinero B, Gomez-Puertas P, Ugarte M (2006) Mutational spectrum of maple syrup urine disease in Spain. Hum Mutat 27:715PubMedCrossRefGoogle Scholar
  17. Shapiro MB, Senapathy P (1987) RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res 15:7155–7174PubMedCrossRefGoogle Scholar
  18. Skandalis A, Uribe E (2004) A survey of splice variants of the human hypoxanthine phosphoribosyl transferase and DNA polymerase beta genes: products of alternative or aberrant splicing? Nucleic Acids Res 32:6557–6564PubMedCrossRefGoogle Scholar
  19. Spurdle AB, Couch FJ, Hogervorst FB, Radice P, Sinilnikova OM (2008) Prediction and assessment of splicing alterations: implications for clinical testing. Hum Mutat 29:1304–1313PubMedCrossRefGoogle Scholar
  20. Weischenfeldt J, Lykke-Andersen J, Porse B (2005) Messenger RNA surveillance: neutralizing natural nonsense. Curr Biol 15:R559–R562PubMedCrossRefGoogle Scholar
  21. Wynn RM, Davie JR, Chuang JL, Cote CD, Chuang DT (1998) Impaired assembly of E1 decarboxylase of the branched-chain alpha-ketoacid dehydrogenase complex in type IA maple syrup urine disease. J Biol Chem 273:13110–13118PubMedCrossRefGoogle Scholar

Copyright information

© SSIEM and Springer 2010

Authors and Affiliations

  • Paula Fernández-Guerra
    • 1
    • 3
  • Rosa Navarrete
    • 1
    • 3
  • Kara Weisiger
    • 2
  • Lourdes R. Desviat
    • 1
    • 3
  • Seymour Packman
    • 2
  • Magdalena Ugarte
    • 1
    • 3
  • Pilar Rodríguez-Pombo
    • 1
    • 3
  1. 1.Centro de Diagnóstico de Enfermedades Moleculares, Dpto. Biol. Mol., Centro Biología Molecular-SO UAM-CSICUniversidad Autónoma de Madrid, Campus CantoblancoMadridSpain
  2. 2.Division of Medical Genetics, Department of PediatricsUniversity of California–San Fancisco (UCSF)San FranciscoUSA
  3. 3.Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER)Instituto de Salud Carlos IIIMadridSpain

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