Sequence and copy number analyses of HEXB gene in patients affected by Sandhoff disease: functional characterization of 9 novel sequence variants

• Published in: PloS one Vol. 7; no. 7; p. e41516
• Main Authors: Zampieri, Stefania, Cattarossi, Silvia, Oller Ramirez, Ana Maria, Rosano, Camillo, Lourenco, Charles Marques, Passon, Nadia, Moroni, Isabella, Uziel, Graziella, Pettinari, Antonella, Stanzial, Franco, de Kremer, Raquel Dodelson, Azar, Nydia Beatriz, Hazan, Filiz, Filocamo, Mirella, Bembi, Bruno, Dardis, Andrea
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 27.07.2012; Public Library of Science (PLoS)
• Subjects: Alternative Splicing - genetics; Analysis; Assaying; Base Sequence; beta-Hexosaminidase beta Chain - genetics; beta-Hexosaminidase beta Chain - metabolism; Biology; Clonal deletion; Copy number; Deoxyribonucleic acid; Disease; DNA; Female; Fibroblasts; Gene deletion; Genes; Genetic aspects; HEK293 Cells; Hospitals; Humans; Male; Medicine; mRNA; mRNA processing; Multiplex Polymerase Chain Reaction; Multiplexing; Mutation; Nonsense mutation; Patients; Proteins; RNA; RNA, Messenger - biosynthesis; RNA, Messenger - genetics; Sandhoff Disease - genetics; Sandhoff Disease - metabolism; Sequence Deletion; Italy; Argentina
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0041516

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.