Sequence and Copy Number Analyses of HEXB Gene in Patients Affected by Sandhoff Disease: Functional Characterization of 9 Novel Sequence Variants

PLoS One. 2012;7(7):e41516. doi: 10.1371/journal.pone.0041516. Epub 2012 Jul 27.

Abstract

Sandhoff disease (SD) is a lysosomal disorder caused by mutations in the HEXB gene. To date, 43 mutations of HEXB have been described, including 3 large deletions. Here, we have characterized 14 unrelated SD patients and developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay to investigate the presence of large HEXB deletions. Overall, we identified 16 alleles, 9 of which were novel, including 4 sequence variation leading to aminoacid changes [c.626C>T (p.T209I), c.634C>A (p.H212N), c.926G>T (p.C309F), c.1451G>A (p.G484E)] 3 intronic mutations (c.1082+5G>A, c.1242+1G>A, c.1169+5G>A), 1 nonsense mutation c.146C>A (p.S49X) and 1 small in-frame deletion c.1260_1265delAGTTGA (p.V421_E422del). Using the new MLPA assay, 2 previously described deletions were identified. In vitro expression studies showed that proteins bearing aminoacid changes p.T209I and p.G484E presented a very low or absent activity, while proteins bearing the p.H212N and p.C309F changes retained a significant residual activity. The detrimental effect of the 3 novel intronic mutations on the HEXB mRNA processing was demonstrated using a minigene assay. Unprecedentedly, minigene studies revealed the presence of a novel alternative spliced HEXB mRNA variant also present in normal cells. In conclusion, we provided new insights into the molecular basis of SD and validated an MLPA assay for detecting large HEXB deletions.

Publication types

  • Clinical Trial
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing / genetics*
  • Base Sequence*
  • Female
  • HEK293 Cells
  • Humans
  • Male
  • Multiplex Polymerase Chain Reaction
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / genetics
  • Sandhoff Disease / genetics*
  • Sandhoff Disease / metabolism
  • Sequence Deletion*
  • beta-Hexosaminidase beta Chain / genetics*
  • beta-Hexosaminidase beta Chain / metabolism

Substances

  • RNA, Messenger
  • HEXB protein, human
  • beta-Hexosaminidase beta Chain