Academic journal article Genetics

A Duplication in the Canine [Beta]-Galactosidase Gene GLB1 Causes Exon Skipping and GM^sub 1^-Gangliosidosis in Alaskan Huskies

Academic journal article Genetics

A Duplication in the Canine [Beta]-Galactosidase Gene GLB1 Causes Exon Skipping and GM^sub 1^-Gangliosidosis in Alaskan Huskies

Article excerpt

ABSTRACT

GM^sub 1^-gangliosidosis is a lysosomal storage disease that is inherited as an autosomal recessive disorder, predominantly caused by structural defects in the β-galactosidase gene (GLB1). The molecular cause of GM^sub 1^-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 G^sub M1^-gangliosidosis occurs.

THE canine lysosomal acidic â-galactosidase (GLBl, EC 3.2.1.23) is an exoglycosidase that removes â-ketosidically linked galactose residues from glycoproteins, sphingolipids, and keratan sulfate (VAN DER SPOEL et al. 2000). GMj-gangliosidosis is a lysosomal storage disease, inherited as an autosomal recessive disorder, predominantly caused by structural defects in the â-galactosidase gene (GLBl) (THOMAS and BEAUDET 1995; CALLAHAN 1999). Mutations in the GLBl gene were identified in Portuguese waterdogs with GMi-gangliosidosis (WANG et al. 2000) and in Shiba inus with GMi-gangliosidosis (YAMATO et al. 2002). The GLBl gene is located on chromosome 3p21 in humans (NCBI MapViewer, human genome build 35.1) and on chromosome 23 in dog (PRIAT et al. 1998; BREEN et al. 2001). Both orthologous GLBl genes contain 16 exons and share 86.5% identity at the nucleotide level and 81% identity at the amino acid level (WANG et al. 2000). In both species GLBl is synthesized as an 85-kDa precursor protein, which is subsequently processed into a 64- to 66-kDa mature form and a 22- to 24-kDa cleavage fragment (VAN DER SPOEL et al. 2000). Comparative studies carried out on human, mouse, and bovine GLBl revealed that the released 22- to 24-kDa proteolytic fragment remains associated to the 64- to 66-kDa chain to form the catalytically active â-galactosidase (D'Azzo et al. 1982; BousTANY et al 1993). The significance of the 22- to 24-kDa C-terminal GLBl domain, encoded partially by exons 15 and 16, is supported by the identification of several amino acid substitutions in different forms of GMj-gangliosidosis in canine (WANG et al. 2000; YAMATO et al. 2002) and human patients (BOUSTANY et al. 1993; MORRONE et al 2000; VAN DER SPOEL et al 2000).

Many disease-associated mutations affect pre-mRNA splicing, usually causing incorrect exon assembly (CARTEGNI et al. 2002). Up to 15% of point mutations responsible for genetic diseases in humans cause aberrantsplicing (KRAWCZAK etal. 1992). The mostcommon consequence of these mutations is exon skipping. In constitutive splicing all exons are included in the mature mRNA, whereas in the skipped pattern one or more exons are missing. The regulation of this process is still not very well understood; so far, as-regulatory elements such as exonic splicing enhancers (ESEs) were mostly identified in individual cases ( SCHAAL and MANIATIS 1999; BLACK 2003; FAUSTINO and COOPER 2003). Analysis of candidate sequences demonstrated that purinerich motifs (GGAGA/GGGA/AGAGA) and CA-rich consensus motifs (C)CACC(C) are frequently used as splicing enhancer elements (Du et al. 1997; LIU et al. …

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