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Journal of Inherited Metabolic Disease

, Volume 37, Issue 1, pp 43–52 | Cite as

A frequent splicing mutation and novel missense mutations color the updated mutational spectrum of classic galactosemia in Portugal

  • Ana I. Coelho
  • Ruben Ramos
  • Ana Gaspar
  • Cláudia Costa
  • Anabela Oliveira
  • Luísa Diogo
  • Paula Garcia
  • Sandra Paiva
  • Esmeralda Martins
  • Elisa Leão Teles
  • Esmeralda Rodrigues
  • M. Teresa Cardoso
  • Elena Ferreira
  • Sílvia Sequeira
  • Margarida Leite
  • Maria João Silva
  • Isabel Tavares de Almeida
  • João B. Vicente
  • Isabel RiveraEmail author
Original Article

Abstract

Classic galactosemia is an autosomal recessive disorder caused by deficient galactose-1-phosphate uridylyltransferase (GALT) activity. Patients develop symptoms in the neonatal period, which can be ameliorated by dietary restriction of galactose. Many patients develop long-term complications, with a broad range of clinical symptoms whose pathophysiology is poorly understood. The high allelic heterogeneity of GALT gene that characterizes this disorder is thought to play a determinant role in biochemical and clinical phenotypes. We aimed to characterize the mutational spectrum of GALT deficiency in Portugal and to assess potential genotype-phenotype correlations. Direct sequencing of the GALT gene and in silico analyses were employed to evaluate the impact of uncharacterized mutations upon GALT functionality. Molecular characterization of 42 galactosemic Portuguese patients revealed a mutational spectrum comprising 14 nucleotide substitutions: ten missense, two nonsense and two putative splicing mutations. Sixteen different genotypic combinations were detected, half of the patients being p.Q188R homozygotes. Notably, the second most frequent variation is a splicing mutation. In silico predictions complemented by a close-up on the mutations in the protein structure suggest that uncharacterized missense mutations have cumulative point effects on protein stability, oligomeric state, or substrate binding. One splicing mutation is predicted to cause an alternative splicing event. This study reinforces the difficulty in establishing a genotype-phenotype correlation in classic galactosemia, a monogenic disease whose complex pathogenesis and clinical features emphasize the need to expand the knowledge on this “cloudy” disorder.

Keywords

Mutational Spectrum Galactosemia Splice Mutation Exonic Splice Enhancer Prediction Server 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

We wish to acknowledge the patients and families enrolled in this study. This work was supported by SPDM Grant to IR, SFRH/BD/48259/2008 FCT Grant to AIC, and PEst-OE/SAU/UI4013/2011.

Conflict of interest

None.

Supplementary material

10545_2013_9623_MOESM1_ESM.ppt (130 kb)
Supplementary Fig. 1 Sequence alignment of GALT proteins. Protein sequences retrieved from NCBI BLAST and aligned with Clustal X for Windows (Thompson et al 1997). GALT protein sequence accession numbers: Human (SwissProt P079023); Rat, Rattus norvegicus (SwissProt Q03249.3); Drosophila, Drosophila melanogaster (SwissProt Q9VMA2.2); E_coli, Escherichia coli str. K12 substr. MG1655 (SwissProt P09148); Salmonella, Salmonella enterica subsp. enterica (NCBI ZP_03217106.1); Klebsiella, Klebsiella pneumoniae subsp. pneumoniae NTUH-K2044 (NCBI YP_006637266.1); Erwinia, Erwinia billingiae Eb661 (NCBI YP_003740707.1); H_influenziae, Haemophilus influenziae Rd KW20 (SwissProt P31764.2); H_haemolyticus; Haemophilus haemolyticus M19107 (GenBank EGT77758.1); Saccharomyces, Saccharomyces cerevisiae (GenBank AAA34627.1). Yellow boxes highlight the active site residues His184-Pro185-His186 and the substrate stabilizing residue Q188; red boxes highlight the mutations herein analyzed in detail (PPT 130 kb)
10545_2013_9623_MOESM2_ESM.ppt (660 kb)
Supplementary Fig. 2 Structural model of human GALT with highlighted mutations. GALT dimer composed by a monomer consisting of a structural model of human GALT (green cartoon) and a monomer of E. coli GALT (orange ribbon, PDB code 1GUP, B chain). Red sticks, GALT mutations herein studied. Green sticks, His184-Pro185-His186 active site and Q188 residue (human GALT numbering), which stabilizes bound UDP-glucose (yellow sticks). Figure generated with Pymol (PPT 660 kb)
10545_2013_9623_MOESM3_ESM.ppt (880 kb)
Supplementary Fig. 3 Structural impact of the p.F171C mutation in human GALT. Structural model of human GALT (green cartoon) and opposing monomer of E. coli GALT (orange ribbon, PDB code 1GUP, B chain). F171 is represented in red and the substituting C171 is in magenta. Orange sticks, tyrosine 339 from opposing monomer (human GALT numbering). Figure generated with Pymol (PPT 879 kb)
10545_2013_9623_MOESM4_ESM.ppt (920 kb)
Supplementary Fig. 4 Structural impact of the p.G175D mutation in human GALT. Structural model of human GALT (green cartoon) and opposing monomer of E. coli GALT (orange ribbon, PDB code 1GUP, B chain). G175 is represented in red and the substituting D175 is in yellow. Figure generated with Pymol (PPT 919 kb)
10545_2013_9623_MOESM5_ESM.ppt (892 kb)
Supplementary Fig. 5 Structural impact of the p.P185S mutation in human GALT. Structural model of human GALT (green cartoon) and opposing monomer of E. coli GALT (orange ribbon, PDB code 1GUP, B chain). P185 is represented in red and the substituting S185 is in pink. Figure generated with Pymol (PPT 892 kb)
10545_2013_9623_MOESM6_ESM.ppt (926 kb)
Supplementary Fig. 6 Structural impact of the p.S192G mutation in human GALT. Structural model of human GALT (green cartoon) and opposing monomer of E. coli GALT (orange ribbon, PDB code 1GUP, B chain). S192 is represented in red and the substituting G192 is in light grey. Figure generated with Pymol (PPT 926 kb)
10545_2013_9623_MOESM7_ESM.ppt (1010 kb)
Supplementary Fig. 7 Structural impact of the p.R259W mutation in human GALT. Structural model of human GALT (green cartoon) and opposing monomer of E. coli GALT (orange ribbon, PDB code 1GUP, B chain). R259 is represented in red and the substituting W259 is in orange, as well as the residues E271 and T268 from the mutant GALT structural model. Figure generated with Pymol (PPT 1010 kb)
10545_2013_9623_MOESM8_ESM.ppt (1.1 mb)
Supplementary Fig. 8 Structural impact of the p.P295T mutation in human GALT. Structural model of human GALT (green cartoon) and opposing monomer of E. coli GALT (orange ribbon, PDB code 1GUP, B chain). P295 is represented in red and the substituting T295 is in blue. Figure generated with Pymol (PPT 1081 kb)
10545_2013_9623_MOESM9_ESM.ppt (1.1 mb)
Supplementary Fig. 9 Structural impact of the p.R333G mutation in human GALT. Structural model of human GALT (green cartoon) and opposing monomer of E. coli GALT (orange ribbon, PDB code 1GUP, B chain). R333 is represented in red and the substituting G333 is in pale cyan. Figure generated with Pymol (PPT 1154 kb)
10545_2013_9623_MOESM10_ESM.docx (25 kb)
Table S1 Sequence of oligonucleotides used for the amplification of GALT gene and cDNA (DOCX 24 kb)
10545_2013_9623_MOESM11_ESM.doc (66 kb)
Table S2 Structural and functional effects of GALT mutations predicted by bioinformatics tools. A structural model of human GALT was generated by the SWISS-MODEL server using the E. coli GALT crystallographic structure as template (PDB code 1GUP), and uploaded to the prediction servers to analyze the effect of the selected mutations. As controls of the effect of previously unstudied mutations, the prevalent disease-causing mutations S135L, Q188R and K285N were also analyzed (DOC 65 kb)
10545_2013_9623_MOESM12_ESM.doc (42 kb)
Table S3 Structural functional effects of GALT mutations predicted by inspection of structural models. Structural models of human GALT (WT and selected mutants) were generated by the SWISS-MODEL server using the E. coli GALT crystallographic structure as template (PDB code 1GUP). Conservation was determined by aligning selected sequences in Clustal X (Supplementary Fig. S1). H-bond distances were calculated in Pymol (DOC 42 kb)

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Copyright information

© SSIEM and Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Ana I. Coelho
    • 1
    • 2
  • Ruben Ramos
    • 1
    • 2
  • Ana Gaspar
    • 3
  • Cláudia Costa
    • 3
  • Anabela Oliveira
    • 4
  • Luísa Diogo
    • 5
  • Paula Garcia
    • 5
  • Sandra Paiva
    • 5
  • Esmeralda Martins
    • 6
  • Elisa Leão Teles
    • 7
  • Esmeralda Rodrigues
    • 7
  • M. Teresa Cardoso
    • 7
  • Elena Ferreira
    • 8
  • Sílvia Sequeira
    • 9
  • Margarida Leite
    • 1
    • 2
  • Maria João Silva
    • 1
    • 2
  • Isabel Tavares de Almeida
    • 1
    • 2
  • João B. Vicente
    • 1
    • 2
  • Isabel Rivera
    • 1
    • 2
    Email author
  1. 1.Metabolism & Genetics Group, Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of PharmacyUniversity of LisbonLisbonPortugal
  2. 2.Department of Biochemistry and Human Biology, Faculty of PharmacyUniversity of LisbonLisbonPortugal
  3. 3.Department of PediatricsHospital Santa MariaLisbonPortugal
  4. 4.Department of MedicineHospital Santa MariaLisbonPortugal
  5. 5.Metabolic Clinics, Pediatric HospitalCHUCCoimbraPortugal
  6. 6.Department of PediatricsHospital Santo AntónioPortoPortugal
  7. 7.Metabolic Diseases Unit, Integrated Pediatric HospitalHospital São JoãoPortoPortugal
  8. 8.Hospital Centre, FunchalMadeiraPortugal
  9. 9.Department of PediatricsHospital D. EstefâniaLisbonPortugal

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