neurogenetics

, 10:49 | Cite as

Molecular and functional analysis of the HEXB gene in Italian patients affected with Sandhoff disease: identification of six novel alleles

  • Stefania Zampieri
  • Mirella Filocamo
  • Emanuele Buratti
  • Marina Stroppiano
  • Kristian Vlahovicek
  • Natalia Rosso
  • Eleonora Bignulin
  • Stefano Regis
  • Franco Carnevale
  • Bruno Bembi
  • Andrea Dardis
Original Article

Abstract

We report the molecular characterization of 12 unrelated Italian patients affected with Sandhoff disease (SD), a recessively inherited disorder caused by mutations in HEXB gene. We identified 11 different mutations of which six are novel: one large deletion of 2,406 nt, (c.299+1471_408del2406), one frameshift mutation c.965delT (p.I322fsX32), one nonsense c.1372C>T (p.Q458X), and three splicing mutations (c.299G>T, c.300-2A>G and c.512-1G>T). One allele was only characterized at the messenger RNA (mRNA) level (r = 1170_1242del). Real-time polymerase chain reaction analysis of the HEXB mRNA from fibroblasts derived from patients carrying the novel point mutations showed that the presence of the premature termination codon in the transcript bearing the mutation c.965delT triggers the nonsense-mediated decay (NMD) pathway, which results in the degradation of the aberrant mRNA. The presence of the c.299G>T mutation leads to the degradation of the mutated mRNA by a mechanism other than NMD, while mutations c.300-2A>G and c.512-1G>T cause the expression of aberrant transcripts. In our group, the most frequent mutation was c.850C>T (p.R284X) representing 29% of the alleles. Haplotype analysis suggested that this mutation did not originate from a single genetic event. Interestingly, the common 16-kb deletion mutation was absent. This work provides valuable information regarding the molecular genetics of SD in Italy and provides new insights into the molecular basis of the disease.

Keywords

GM2 gangliosidosis Sandhoff disease HEXB gene Mutation analysis 

References

  1. 1.
    Leinekugel P, Michel S, Conzelmann E, Sandhoff K (1992) Quantitative correlation between the residual activity of beta-hexosaminidase A and arylsulfatase A and the severity of the resulting lysosomal storage disease. Hum Genet 88:513–523CrossRefPubMedGoogle Scholar
  2. 2.
    Gravel RA, Kaback MM, Proia RL, Sandhoff K, Suzuki K, Suzuki K (2001) The GM2 gangliosidosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The matabolic and molecular basis of inherited diseases. McGraw-Hill, New York, pp 3827–3876Google Scholar
  3. 3.
    Stenson PD, Ball EV, Mort M, Phillips AD, Shiel JA, Thomas NS, Abeysinghe S, Krawczak M, Cooper DN (2003) Human Gene Mutation Database (HGMD) 2003 update. Hum Mutat 21:577–581CrossRefPubMedGoogle Scholar
  4. 4.
    Gomez-Lira M, Perusi C, Brutti N, Farnetani MA, Margollicci MA, Rizzuto N, Pignatti PF, Salviati A (1995) A 48-bp insertion between exon 13 and 14 of the HEXB gene causes infantile-onset Sandhoff disease. Hum Mutat 6:260–262CrossRefPubMedGoogle Scholar
  5. 5.
    Gomez-Lira M, Perusi C, Mottes M, Pignatti PF, Rizzuto N, Gatti R, Salviati A (1998) Splicing mutation causes infantile Sandhoff disease. Am J Med Genet 75:330–333CrossRefPubMedGoogle Scholar
  6. 6.
    Gomez-Lira M, Sangalli A, Mottes M, Perusi C, Pignatti PF, Rizzuto N, Salviati A (1995) A common beta hexosaminidase gene mutation in adult Sandhoff disease patients. Hum Genet 96:417–422CrossRefPubMedGoogle Scholar
  7. 7.
    Gomez-Lira M, Mottes M, Perusi C, Pignatti PF, Rizzuto N, Gatti R, Salviati A (2001) A novel 4-bp deletion creates a premature stop codon and dramatically decreases HEXB mRNA levels in a severe case of Sandhoff disease. Mol Cell Probes 15:75–79CrossRefPubMedGoogle Scholar
  8. 8.
    Santoro M, Modoni A, Sabatelli M, Madia F, Piemonte F, Tozzi G, Ricci E, Tonali PA, Silvestri G (2007) Chronic GM2 gangliosidosis type Sandhoff associated with a novel missense HEXB gene mutation causing a double pathogenic effect. Mol Genet Metab 9:111–114CrossRefGoogle Scholar
  9. 9.
    Brown CA, Mahuran DJ (1993) Beta-hexosaminidase isozymes from cells cotransfected with alpha and beta cDNA constructs: analysis of the alpha-subunit missense mutation associated with the adult form of Tay-Sachs disease. Am J Hum Genet 53:497–508PubMedPubMedCentralGoogle Scholar
  10. 10.
    Neote K, McInnes B, Mahuran DJ, Gravel RA (1990) Structure and distribution of an Alu-type deletion mutation in Sandhoff disease. J Clin Invest 86:1524–1531CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Mark BL, Mahuran DJ, Cherney MM, Zhao D, Knapp S, James MN (2003) Crystal structure of human beta-hexosaminidase B: understanding the molecular basis of Sandhoff and Tay-Sachs disease. J Mol Biol 327:1093–1109CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18:2714–2723CrossRefPubMedGoogle Scholar
  13. 13.
    Zhang ZX, Wakamatsu N, Mules EH, Thomas GH, Gravel RA (1994) Impact of premature stop codons on mRNA levels in infantile Sandhoff disease. Hum Mol Genet 3:139–145CrossRefPubMedGoogle Scholar
  14. 14.
    Wakamatsu N, Kobayashi H, Miyatake T, Tsuji S (1992) A novel exon mutation in the human beta-hexosaminidase beta subunit gene affects 3′ splice site selection. J Biol Chem 267:2406–2413PubMedGoogle Scholar
  15. 15.
    Redonnet-Vernhet I, Mahuran DJ, Salvayre R, Dubas F, Levade T (1996) Significance of two point mutations present in each HEXB allele of patients with adult GM2 gangliosidosis (Sandhoff disease) homozygosity for the Ile207®Val substitution is not associated with a clinical or biochemical phenotype. Biochim Biophys Acta 1317:127–133CrossRefPubMedGoogle Scholar
  16. 16.
    Cooper DN, Krawczak M (1989) Cytosine methylation and the fate of CpG dinucleotides in vertebrate genomes. Hum Genet 83:181–188CrossRefPubMedGoogle Scholar
  17. 17.
    Bolhuis PA, Bikker H (1992) Deletion of the 5′-region in one or two alleles of HEXB in 15 out of 30 patients with Sandhoff disease. Hum Genet 90:328–239CrossRefPubMedGoogle Scholar
  18. 18.
    Neote K, Bapat B, Dumbrille-Ross A, Troxel C, Schuster SM, Mahuran DJ, Gravel RA (1988) Characterization of the human HEXB gene encoding lysosomal beta-hexosaminidase. Genomics 3:279–286CrossRefPubMedGoogle Scholar
  19. 19.
    Kleiman FE, Ramirez AO, Dodelson de Kremer R, Gravel RA, Argarana CE (1998) A frequent TG deletion near the polyadenylation signal of the human HEXB gene: occurrence of an irregular DNA structure and conserved nucleotide sequence motif in the 3′ untranslated region. Hum Mutat 12:320–329CrossRefPubMedGoogle Scholar
  20. 20.
    Maquat LE (2004) Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics. Nat Rev Mol Cell Biol 5:89–99, (review)CrossRefPubMedGoogle Scholar
  21. 21.
    Yeo G, Burge CB (2004) Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J Comput Biol 11:377–394CrossRefPubMedGoogle Scholar
  22. 22.
    Reese MG, Eeckman FH, Kulp D, Haussler D (1997) Improved splice site detection in Genie. J Comput Biol 4:311–323CrossRefPubMedGoogle Scholar
  23. 23.
    Yoshizawa T, Kohno Y, Nissato S, Shoji S (2002) Compound heterozygosity with two novel mutations in the HEXB gene produces adult Sandhoff disease presenting as a motor neuron disease phenotype. J Neurol Sci 195:129–138CrossRefPubMedGoogle Scholar
  24. 24.
    Neote K, Bapat B, Dumbrille-Ross A, Troxel C, Schuster SM, Mahuran DJ, Gravel RA (1988) Characterization of the human HEXB gene encoding lysosomal beta-hexosaminidase. Genomics 3:279–286CrossRefPubMedGoogle Scholar
  25. 25.
    Yamanaka S, Johnson ON, Norflus F, Boles DJ, Proia RL (1994) Structure and expression of the mouse beta-hexosaminidase genes, Hexa and Hexb. Genomics 21:588–596CrossRefPubMedGoogle Scholar
  26. 26.
    Muldoon LL, Neuwelt EA, Pagel MA, Weiss DL (1994) Characterization of the molecular defect in a feline model for type II GM2-gangliosidosis (Sandhoff disease). Am J Pathol 144:1109–1118PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Stefania Zampieri
    • 1
  • Mirella Filocamo
    • 2
  • Emanuele Buratti
    • 3
  • Marina Stroppiano
    • 2
  • Kristian Vlahovicek
    • 4
  • Natalia Rosso
    • 5
  • Eleonora Bignulin
    • 1
  • Stefano Regis
    • 2
  • Franco Carnevale
    • 6
  • Bruno Bembi
    • 1
    • 7
  • Andrea Dardis
    • 1
  1. 1.Unità di Malattie MetabolicheIRCCS Burlo GarofoloTriesteItaly
  2. 2.Laboratorio Diagnosi Pre-Postnatale Malattie MetabolicheIRCCS G. GasliniGenovaItaly
  3. 3.International Centre for Genetic Engineering and BiotechnologyTriesteItaly
  4. 4.Bioinformatics Unit, Molecular Biology Department, Division of BiologyFaculty of ScienceZagrebCroatia
  5. 5.Centro Studi FegatoTriesteItaly
  6. 6.UO Malattie Metaboliche-Genetica ClinicaOspedale Pediatrico “Giovanni XXIII”BariItaly
  7. 7.Regional Coordinator Centre for Rare DiseasesUniversity Hospital “Santa Maria della Misericordia”UdineItaly

Personalised recommendations