Copyright © 2005 by the Genetics Society of America DOI: 10.1534/genetics.105.042580

A Duplication in the Canine ␤-Galactosidase 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 are predicted to be expressed from the mutant GLB1 allele. In heterozygous carrier animals the wild-type allele produces sufficient amounts of the active 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 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 3p21 in humans (NCBI MapViewer, rant splicing (Krawczak et al. 1992). The most common 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 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 , 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 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 start codon. The reference sequence for the numbering was taken from the canine GLB1 cDNA sequence published by Wang et al. (2000).

(homozygous mutant, heterozygous, and homozygous C-terminal region of GLB1. This causes the loss of post- wild type). Surprisingly, when the RT-PCR products translational processing sites including the proteolytic were separated on agarose gels, a homozygous mutant cleavage site, necessary for transformation of precursor and a heterozygous animal seemingly showed the same into mature acidic GLB1 protein. pattern of two different bands, while only the homozy- In the other aberrant transcript variant, which con- gous wild-type animal showed a single band of the ex- tains exon 15 with the duplication, the insertion of 19 pected size (Figure 1B). Sequencing of these RT-PCR products revealed that the smaller band seen in the affected and heterozygous dogs corresponded to a tran- script, in which exon 15 was missing. Sequencing of the larger RT-PCR product in the affected dog showed that this band corresponded to a full-length GLB1 transcript; however, 19 bp within the region encoded by exon 15 were duplicated in transcripts from the affected dog. This duplication was additionally confirmed by direct RT-PCR and PCR analyses of exon 15 (Figure 1, C and D). The duplication corresponded to positions ϩ1688– ϩ1706 in the canine GLB1 cDNA and had the sequence 5Ј-TCCCAGACTTGCCCCAGGA-3Ј (Figure 1E). To en- sure that the observed mutation is indeed the causative pathological mutation, a sample of 30 normal dogs of different breeds was tested for the presence of the 19- bp duplication. The duplication was not present on any of the 60 studied. Our results indicate that the 19-bp duplication in exon 15 leads to exon skipping in a portion of the GLB1 transcripts and two different mRNA species are Figure 2.—Schematic of the splicing events in dogs with expressed from the mutant allele (Figure 2). The results the GLB1 mutation. In the top part of the figure the genomic of the affected dog were confirmed in the heterozygous organization of the 16-exon GLB1 gene is depicted. Note that dog, where the presence of three different mRNA spe- exon and intron sizes are not drawn to scale. The newly identi- fied 19-bp duplication is located in the 255-bp exon 15. In the cies was established after DNA sequencing. Thus the middle part the 3Ј-end of the primary transcript is represented. DNA sequencing of the transcripts rectified the unex- The primary transcript is spliced into two different mRNAs pected agarose gel picture from the heterozygous dog, (bottom). One mRNA corresponds to the expected message where only two bands had been discernible initially (Fig- with exon 15 containing 19 extra nucleotides from the geno- ure 1B). The heterozygous dog expressed three differ- mic duplication. The other mRNA species lacks the entire exon 15. In the mRNA containing exon 15 the 19-bp duplica- ent GLB1 mRNAs. In addition to the normal GLB1 tion leads to a frameshift that causes the apparition of a PTC mRNA from the wild-type allele, heterozygous dogs also in exon 16. As the PTC is located in the last exon the mRNA express mRNAs lacking exon 15 and full-length mRNAs is not subject to NMD and is expressed in amounts similar to with the 19-bp duplication from the mutant allele. that of the wild-type RNA. The frameshift mutation leads to The 19-bp duplication within exon 15 is predicted to the truncation of 78 amino acids from the C terminus of the GLB protein (indicated by shading of the truncated part of have dramatic consequences for the GLB1 protein. The the ORF). The transcript variant without exon 15 leads to the exon skipping of the 255-bp exon 15 leads to the dele- translation of a protein lacking 85 amino acids encoded by tion of 85 amino acids in the functionally important this exon. 1860 R. Kreutzer et al. additional nucleotides leads to a frameshift and the mature transcript. If the binding site with the wrong apparition of a premature termination codon (PTC). distance to the 3Ј-end of exon 15 is occupied this would As the PTC resides in the last exon, the transcript is not lead to exon skipping. As the sequences in the two subject to nonsense-mediated decay (NMD). The band duplicated 19-bp segments are identical the chance that intensities of the gels with the RT-PCR products suggest a potential binding protein binds to the correct binding that it is present in roughly comparable amounts to the site is 50%, which corresponds to the experimental ob- transcript lacking exon 15. Because of the PTC, the servation that the amounts of the two transcripts are transcript containing exon 15 with the 19-bp duplication roughly equal in fibroblasts. gives rise to a protein that is 78 amino acids shorter In conclusion, we describe a novel 19-bp mutation in than the wild-type GLB1 and additionally has an entirely the canine GLB1 gene that causes interesting aberra- different C-terminal sequence. Both transcripts from tions of the normal splicing process. The molecular the mutated allele are thus likely to encode completely elucidation of GM1 -gangliosidosis in Alaskan huskies nonfunctional GLB1 proteins. provides another model for human GM1 -gangliosidosis.

Knowledge of the causative mutation for GM1 -ganglio- DISCUSSION sidosis in Alaskan huskies will also allow genetic testing of these dogs to avoid accidental matings of carrier In this study a novel 19-bp duplication in exon 15 animals. Thus breeders will be enabled to eliminate this of the canine GLB1 gene was identified as a causative disease from their breeding lines. mutation for GM1 -gangliosidosis in Alaskan huskies. The ϩ ϩ The authors thank Werner Hecht for very useful advice. This work mutation corresponded to nucleotides 1688– 1706 of was supported by a German Research Council [Deutsche Forschungs- the GLB1 cDNA. The GLB1 mutation causing GM1 -gan- gemeinschaft (DFG)] grant from the Graduate College (GK 455 “Mo- gliosidosis in Alaskan huskies is thus different from ear- lecular Veterinary Medicine”) and by DFG grant Ba815/7-1. lier described GLB1 mutations in Portuguese waterdogs and Shiba inus (Wang et al. 2000; Yamato et al. 2002) The Portuguese waterdog mutation is a point mutation LITERATURE CITED (R60H) whereas the Shiba inu mutation represents a 1-bp deletion leading to a frameshift and PTC. Berget, S. M., 1995 Exon recognition in vertebrate splicing. J. Biol. Chem. 270: 2411–2414. The newly identified GLB1 mutation causes very pecu- Bianca, J. L., and K. J. Hertel, 2002 A general role for splicing liar aberrations in RNA processing. In animals carrying enhancers in exon definition. RNA 8: 1230–1241. the 19-bp duplication normal and abnormal splicing of Black, D. L., 2003 Mechanisms of alternative pre-messenger RNA splicing. Annu. Rev. Biochem. 72: 291–336. GLB1 pre-mRNA coexist. The 19-bp duplication inter- Boustany,R.M.,W.H.Qian and K. Suzuki, 1993 Mutations in ␤ feres with the recognition of exon 15 leading to skipping acid -galactosidase cause GM1 -gangliosidosis in American pa- of this exon in a fraction of all transcripts, while the tients. Am. J. Hum. Genet. 53: 881–888. Breen, M., S. Jouquand, C. Renier, C. S. Mellersh, C. Hitte et remaining transcripts are normally spliced. Exon recog- al., 2001 Chromosome-specific single- FISH probes allow nition is becoming more and more accepted as an im- anchorage of an 1800-marker integrated radiation-hybrid/link- portant mechanism in the splicing of mammalian tran- age map of the domestic dog genome to all chromosomes. Ge- nome Res. 11: 1784–1795. scripts (Berget 1995). The mechanism by which exons Callahan, J. W., 1999 Molecular basis of GM1 -gangliosidosis and are recognized or defined is not completely understood. Morquio disease type B. Structure-function studies of lysosomal So far, it seems clear that there is an interaction between ␤-galactosidase like protein. Biochim. Biophys. Acta 1455: 83–105. Cartegni, L., S. L Chew and A. R. Krainer, 2002 Listening to splicing factors across exons and that these interactions silence and understanding nonsense: exonic mutations that affect are often mediated by auxiliary proteins such as SR- splicing. Nat. Genet. 3: 285–298. proteins that bind to the exon sequence itself (Berget Cartegni, L., J. Wang, Z. Zhu, M. Q. Zhang and A. R. Krainer, 2003 ESEfinder: a web resource to identify exonic splicing enhancers. 1995; Bianca and Hertel 2002). The in silico analysis Nucleic Acids Res. 31: 3568–3571. of potential binding sites for SR-proteins in exon 15 of D’Azzo, A., A. T. Hoogeveen, A. J. Reuser, D. Robinson and H. the canine GLB1 gene revealed that the 19-bp duplica- Galjaard, 1982 Molecular defect in combined GLB1 and neur- tion leads to the presence of potential additional bind- aminidase deficiency in man. Proc. Natl. Acad. Sci. USA 79: 4535– 4539. ing sites for SF2/ASF, SC35, and SRp40, respectively. Du, K., Y. Peng, L. E. Greenbaum, B. A. Haber and R. Taub, 1997 Thus, it seems conceivable that the precise binding of HRS/SRp-40 mediated inclusion of fibronectin EIIIB exon, a one or more of these proteins is essential for the correct possible cause of increased EIIIB expression in proliferating liver. Mol. Cell. Biol. 17: 4096–4104. exon recognition of exon 15. This implies a hypothetical Fairbrother, W. G., D. Holste, C. B. Burge and P. A. Sharp, explanation for the peculiar observation that two differ- 2004 Single nucleotide polymorphism-based validation of exo- ent mRNAs are produced from the mutant allele: In nic splicing enhancers. PLoS Biol. 2: E268. Faustino, N. A., and T. A. Cooper, 2003 Pre-mRNA splicing and pre-mRNAs with the 19-bp duplication a splicing factor human disease. Genes Dev. 17: 419–437. might bind to one of two alternative binding sites. How- Krawczak, M., J. Reiss and D. N. Cooper, 1992 The mutational ever, only one of the two alternative binding sites is in spectrum of single base-pair substitutions in mRNA splice junc- Ј tions of human genes: causes and consequences. Hum. Genet. the correct distance to the 3 -end of exon 15 and only 90: 41–54. binding to this site leads to inclusion of exon 15 in the Liu, H. X., M. Zhang and A. R. Krainer, 1998 Identification of Canine GLB1 Mutation 1861

functional exonic splicing enhancer motifs recognized by individ- polymorphism of the cystathionine ␤-synthase gene. J. Biol. Chem. ual SR proteins. Genes Dev. 12: 1998–2012. 277: 43821–43829. Miriami, E., H. Margalit and R. Sperling, 2003 Conserved se- Schaal, T. D., and T. Maniatis, 1999 Selection and characteriza- quence elements associated with exon skipping. Nucleic Acids tion of pre-mRNA splicing enhancers: identification of novel SR Res. 31: 1974–1983. protein-specific enhancer sequences. Mol. Cell. Biol. 19: 1705– Morrone, A., T. Bardelli, M. A. Donati, M. Giorgi, M. Di Rocco 1719. et al., 2000 GLB1 gene mutations affecting the lysosomal enzyme Thomas, G. H., and A. L. Beaudet, 1995 Lysosomal disorders, pp. 2529–2561 in The Metabolic and Molecular Bases of Inherited Diseases, and the -binding protein in GM1 -gangliosidosis patients with cardiac involvement. Hum. Mutat. 15: 354–366. Ed. 7, edited by C. R. Scriver, A. L. Beaudet, W. S. Sly and D. Mu¨ller, G., W. Baumga¨rtner, A. Moritz, A. Sewell and B. Kuster- Valle. McGraw Hill, New York. mann-Kuhn, 1998 Biochemical findings in a breeding colony van der Spoel, A., E. Bonten and A. d’Azzo, 2000 Processing of lysosomal ␤-galactosidase. J. Biol. Chem. 275: 10035–10040. of Alaskan huskies suffering from GM1 -gangliosidosis. J. Inherit. Metab. Dis. 21: 430–431. Wang, Z. H., B. Zeng, H. Shibuya, G. S. Johnson, J. Alroy et al., 2000 Isolation and characterization of the normal canine GLB1 Mu¨ller, G., S. Alldinger, A. Moritz, A. Zurbriggen, N. Kirchhof gene and its mutation in a dog model of GM -gangliosidosis. J. et al., 2001 GM -gangliosidosis in Alaskan huskies: clinical and 1 1 Inherit. Metab. Dis. 23: 593–606. pathologic findings. Vet. Pathol. 38: 281–290. Yamato, O., D. Endoh, A. Kobayashi, Y. Masuoka, M. Yonemura Priat, C., C. Hitte, F. Vignaux, C Renier, Z. Jiang et al., 1998 A et al., 2002 A novel mutation in the gene for canine acid beta- whole-genome radiation hybrid map of the dog genome. Geno- galactosidase that causes GM1 -gangliosidosis in Shiba dogs. J. mics 54: 361–378. Inherit. Metab. Dis. 25: 525–526. Romano, M., R. Marcucci, E. Buratti, Y. M. Ayala, G. Sebastio et al., 2002 Regulation of 3Ј splice site selection in the 844ins68 Communicating editor: G. Gibson