title: Sequence and copy number analyses of HEXB gene in patients affected by Sandhoff disease: functional characterization of 9 novel sequence variants.
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.
authors: Regional Coordinator Center for Rare Diseases, University Hospital Santa Maria della Misericordia, Udine, Italy.
authors: Stefania
authors: S
authors: Zampieri
authors: Silvia
authors: S
authors: Cattarossi
authors: Ana Maria
authors: AM
authors: Oller Ramirez
authors: Camillo
authors: C
authors: Rosano
authors: Charles Marques
authors: CM
authors: Lourenco
authors: Nadia
authors: N
authors: Passon
authors: Isabella
authors: I
authors: Moroni
authors: Graziella
authors: G
authors: Uziel
authors: Antonella
authors: A
authors: Pettinari
authors: Franco
authors: F
authors: Stanzial
authors: Raquel Dodelson
authors: RD
authors: de Kremer
authors: Nydia Beatriz
authors: NB
authors: Azar
authors: Filiz
authors: F
authors: Hazan
authors: Mirella
authors: M
authors: Filocamo
authors: Bruno
authors: B
authors: Bembi
authors: Andrea
authors: A
authors: Dardis
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D001483:Base Sequence; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D060885:Multiplex Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012497:Sandhoff Disease / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D054821:beta-Hexosaminidase beta Chain / Q000235:genetics* / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; C513386:HEXB protein, human; D054821:beta-Hexosaminidase beta Chain