A Duplication in the Canine -Galactosidase Gene GLB1 Causes Exon Skipping and GM1-Gangliosidosis in Alaskan Huskies

GM1-gangliosidosis is a lysosomal storage disease that is inherited as an autosomal recessive disorder, predominantly caused by structural defects in the -galactosidase gene (GLB1). The molecular cause of GM1-gangliosidosis in Alaskan huskies was investigated and a novel 19-bp duplication in exon 15 of the GLB1 gene was identified. The duplication comprised positions 1688– 1706 of the GLB1 cDNA. It partially disrupted a potential exon splicing enhancer (ESE), leading to exon skipping in a fraction of the transcripts. Thus, the mutation caused the expression of two different mRNAs from the mutant allele. One transcript contained the complete exon 15 with the 19-bp duplication, while the other transcript lacked exon 15. In the transcript containing exon 15 with the 19-bp duplication a premature termination codon (PTC) appeared, but due to its localization in the last exon of canine GLB1, nonsense-mediated RNA decay (NMD) did not occur. As a consequence of these molecular events two different truncated GLB1 proteins are predicted to be expressed from the mutant GLB1 allele. In heterozygous carrier animals the wild-type allele produces sufficient amounts of the active enzyme to prevent clinical signs of disease. In affected homozygous dogs no functional GLB1 is synthesized and GM1-gangliosidosis occurs. THE canine lysosomal acidic -galactosidase (GLB1, cally active -galactosidase (D’Azzo et al. 1982; Boustany et al. 1993). The significance of the 22to 24-kDa EC 3.2.1.23) is an exoglycosidase that removes C-terminal GLB1 domain, encoded partially by exons -ketosidically linked galactose residues from glycopro15 and 16, is supported by the identification of several teins, sphingolipids, and keratan sulfate (van der Spoel amino acid substitutions in different forms of GM1-ganet al. 2000). GM1-gangliosidosis is a lysosomal storage gliosidosis in canine (Wang et al. 2000; Yamato et al. disease, inherited as an autosomal recessive disorder, 2002) and human patients (Boustany et al. 1993; Morpredominantly caused by structural defects in the -galacrone et al. 2000; van der Spoel et al. 2000). tosidase gene (GLB1) (Thomas and Beaudet 1995; CalMany disease-associated mutations affect pre-mRNA lahan 1999). Mutations in the GLB1 gene were identisplicing, usually causing incorrect exon assembly fied in Portuguese waterdogs with GM1-gangliosidosis (Cartegni et al. 2002). Up to 15% of point mutations (Wang et al. 2000) and in Shiba inus with GM1-ganglioresponsible for genetic diseases in humans cause abersidosis (Yamato et al. 2002). The GLB1 gene is located rant splicing (Krawczak et al. 1992). The most common on chromosome 3p21 in humans (NCBI MapViewer, consequence of these mutations is exon skipping. In human genome build 35.1) and on chromosome 23 in constitutive splicing all exons are included in the mature dog (Priat et al. 1998; Breen et al. 2001). Both ortholomRNA, whereas in the skipped pattern one or more gous GLB1 genes contain 16 exons and share 86.5% exons are missing. The regulation of this process is still identity at the nucleotide level and 81% identity at the not very well understood; so far, cis-regulatory elements amino acid level (Wang et al. 2000). In both species such as exonic splicing enhancers (ESEs) were mostly GLB1 is synthesized as an 85-kDa precursor protein, identified in individual cases (Schaal and Maniatis which is subsequently processed into a 64to 66-kDa 1999; Black 2003; Faustino and Cooper 2003). Analymature form and a 22to 24-kDa cleavage fragment (van sis of candidate sequences demonstrated that purineder Spoel et al. 2000). Comparative studies carried out rich motifs (GGAGA/GGGA/AGAGA) and CA-rich conon human, mouse, and bovine GLB1 revealed that the sensus motifs (C)CACC(C) are frequently used as splicing released 22to 24-kDa proteolytic fragment remains asenhancer elements (Du et al. 1997; Liu et al. 1998; sociated to the 64to 66-kDa chain to form the catalytiRomano et al. 2002; Miriami et al. 2003; Fairbrother