Copyright © 2005 by the Genetics Society of America DOI: 10.1534/genetics.105.042580 A Duplication in the Canine ␤-Galactosidase Gene GLB1 Causes Exon Skipping and GM1-Gangliosidosis in Alaskan Huskies Robert Kreutzer,* Tosso Leeb,†,1 Gundi Mu¨ller,* Andreas Moritz‡ and Wolfgang Baumga¨rtner* *Department for Pathology and †Department for Animal Breeding and Genetics, University of Veterinary Medicine, 30559 Hannover, Germany and ‡Clinic for Small Animals, Internal Medicine, Justus Liebig University, 35392 Giessen, Germany Manuscript received February 25, 2005 Accepted for publication April 28, 2005 ABSTRACT 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 ϩ1688–ϩ1706 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. HE canine lysosomal acidic ␤-galactosidase (GLB1, cally active ␤-galactosidase (D’Azzo et al. 1982; Bous- TEC 3.2.1.23) is an exoglycosidase that removes tany et al. 1993). The significance of the 22- to 24-kDa ␤-ketosidically linked galactose residues from glycopro- C-terminal GLB1 domain, encoded partially by exons teins, sphingolipids, and keratan sulfate (van der Spoel 15 and 16, is supported by the identification of several et al. 2000). GM1 -gangliosidosis is a lysosomal storage amino acid substitutions in different forms of GM1 -gan- disease, inherited as an autosomal recessive disorder, gliosidosis in canine (Wang et al. 2000; Yamato et al. predominantly caused by structural defects in the ␤-galac- 2002) and human patients (Boustany et al. 1993; Mor- tosidase gene (GLB1)(Thomas and Beaudet 1995; Cal- rone et al. 2000; van der Spoel et al. 2000). lahan 1999). Mutations in the GLB1 gene were identi- Many disease-associated mutations affect pre-mRNA fied in Portuguese waterdogs with GM1 -gangliosidosis splicing, usually causing incorrect exon assembly (Wang et al. 2000) and in Shiba inus with GM1 -ganglio- (Cartegni et al. 2002). Up to 15% of point mutations sidosis (Yamato et al. 2002). The GLB1 gene is located responsible for genetic diseases in humans cause aber- on chromosome 3p21 in humans (NCBI MapViewer, rant splicing (Krawczak et al. 1992). The most common human genome build 35.1) and on chromosome 23 in consequence of these mutations is exon skipping. In dog (Priat et al. 1998; Breen et al. 2001). Both ortholo- constitutive splicing all exons are included in the mature gous GLB1 genes contain 16 exons and share 86.5% mRNA, whereas in the skipped pattern one or more identity at the nucleotide level and 81% identity at the exons are missing. The regulation of this process is still amino acid level (Wang et al. 2000). In both species not very well understood; so far, cis-regulatory elements GLB1 is synthesized as an 85-kDa precursor protein, such as exonic splicing enhancers (ESEs) were mostly which is subsequently processed into a 64- to 66-kDa identified in individual cases (Schaal and Maniatis mature form and a 22- to 24-kDa cleavage fragment (van 1999; Black 2003; Faustino and Cooper 2003). Analy- der Spoel et al. 2000). Comparative studies carried out sis of candidate sequences demonstrated that purine- on human, mouse, and bovine GLB1 revealed that the rich motifs (GGAGA/GGGA/AGAGA) and CA-rich con- released 22- to 24-kDa proteolytic fragment remains as- sensus motifs (C)CACC(C) are frequently used as splicing sociated to the 64- to 66-kDa chain to form the catalyti- enhancer elements (Du et al. 1997; Liu et al. 1998; Romano et al. 2002; Miriami et al. 2003; Fairbrother et al. 2004). In a previous study we reported clinical and pathologi- 1Corresponding author: Department for Animal Breeding and Genet- ics, University of Veterinary Medicine, Bu¨nteweg 17p, 30559 Hann- cal findings in Alaskan huskies with GM1 -gangliosidosis over, Germany. E-mail: [email protected] (Mu¨ller et al. 1998, 2001). The objective of the present Genetics 170: 1857–1861 ( August 2005) 1858 R. Kreutzer et al. study was to identify the causative genetic defect. There- quenced to confirm their identity. The sequencing reactions fore, the GLB1 gene and GLB1 mRNA processing were were performed by MWG Biotech (Ebersberg, Germany). RNA analysis: Mutation analysis of the whole coding region investigated in a family of Alaskan huskies with GM1 - of the canine GLB1 cDNA was performed on total RNA isolated gangliosidosis. from canine skin fibroblasts using TRIzol Reagent (Invitrogen, Carlsbad, CA). The SMART-RACE method (Clontech, Palo Alto, CA) was used for cDNA synthesis. For first-strand cDNA MATERIALS AND METHODS synthesis 100 ng total RNA and SuperScript II reverse tran- scriptase (Invitrogen) were used. Second-strand synthesis was Animals: A large pedigree of Alaskan huskies segregating performed using 10 ␮l first-strand cDNA and Advantage GC for GM1 -gangliosidosis was available (Mu¨ller et al. 2001). PCR polymerase mix (Clontech). The double-stranded cDNA Affected and related heterozygous carrier animals previously obtained was purified with the QIAquick PCR purification classified by clinical, biochemical, and pathological investiga- kit (QIAGEN, Hilden, Germany). In subsequent 25-␮l PCR tions were used for nucleic acid isolation (Figure 1). RNA reactions 100 ng double-stranded cDNA, primers Ex1_F and from DH82 cells (ATCC CRL-10389) and canine primary skin Ex16_R (Table 1), and the KOD Hot Start polymerase were fibroblasts from two healthy control dogs (Airedale terrier C 1 used. The resulting RT-PCR products were cloned and se- and dachshund C 2) were used as controls in RT-PCR experi- quenced (two independent clones each) or directly se- ments. quenced. DNA analysis: Genomic DNA was isolated from canine skin Sequence analysis and bioinformatics: Sequences were as- fibroblast cell cultures using the Puregene kit (Biozym, Ger- sembled and analyzed for mutations with Sequencher 4.2 many). All enzymatic reactions were carried out in a DNA (Genecodes, Ann Arbor, MI). To predict the possible localiza- programmable thermal controller (PTC-200; MJ Research, tion of SR-protein-specific putative ESEs, a web-based tool, the Cambridge, MA). PCR reactions were performed using prim- ESEfinder Release 2.0, was used (http://rulai.cshl.edu/tools/ ers Ex15_F and Ex15_R (Table 1) and the KOD Hot Start ESE/). The default threshold values were set as the median DNA polymerase (Novagen, Darmstadt, Germany). The PCR of the highest score for each sequence in a set of 30 randomly amplifies a 182-bp product within exon 15 of the GLB1 gene chosen 20-nucleotide sequences previously used for functional (Table 1). The resulting PCR products were cloned and se- SELEX (Cartegni et al. 2003). RESULTS At the beginning of the study a large pedigree of Alaskan huskies segregating for GM1 -gangliosidosis was available (Figure 1A). We had previously measured the biochemical activity of GLB1 in these dogs, which con- firmed that the affected dogs had very little GLB1 activity (Mu¨ller et al. 1998, 2001). Therefore, we started to investigate whether mutations in the GLB1 gene are causative for this defect. We amplified full-length cDNAs from dogs with the three different postulated genotypes Figure 1.—Animals and results of the mutation analysis. (A) Alaskan husky pedigree used in this study (modified from Mu¨ller et al. 2001). (B) RT-PCR amplification of the complete coding region of GLB1 mRNAs with primers Ex1_F and Ex16_R. Samples are numbered according to the pedigree in A. The sizes of the RT-PCR products are indicated beneath the lanes. Note that in the heterozygous dog (1) three different mRNA species are expressed. The agarose gel does not resolve the 2052-bp and the 2071-bp bands; however, several clones from each band were sequenced to confirm the data. M, 250- bp ladder. (C) Mutation analysis of exon 15 on mRNA. Primers Ex15_F and Ex15_R were used for RT-PCR to detect the 19- bp duplication on the mRNA/cDNA level. The presence of the 19-bp duplication in exon 15 of the GLB1 gene leads to a 202-bp product while the wild-type product is only 183 bp. M, 100-bp ladder; neg., negative control without cDNA; DH, DH82 cells from a normal dog; C 1 and C 2, normal control dogs. (D) Mutation analysis on genomic DNA. PCR was again performed with primers Ex15_F and Ex15_R on genomic DNA. C 1, normal control animal; M, 100-bp ladder. (E) Sche- matic of the 19-bp duplication in exon 15 of the canine GLB1 gene that leads to GM1 -gangliosidosis in Alaskan huskies. The position of the diagnostic PCR primers Ex15_F and Ex15_R for this mutation is indicated. Canine GLB1 Mutation 1859 TABLE 1 Primers used for amplification of canine GLB1 fragments Product Ј Ј a Primer Nucleotide sequence (5 -3 ) Position size (bp) TM Ex15_F AAC ACT GAG GAT GCA GTA CGC AGC ϩ1546–ϩ1569 183 68Њ Ex15_R TCC AGG AAA CTG GAT AAA GGT GTC ϩ1705–ϩ1728 Ex1_F AGG GAC GTG GCG ACG GCG ATG Ϫ18–ϩ3 2052 68Њ Ex16_R ACT CCA ACG GGT CAC AGT GTT TC 2012–2034 a ϩ1 corresponds to the adenosine of the translation start codon.