Cloning of the Canine β-Glucuronidase cDNA, Mutation Identification in Canine MPS VII, and Retroviral Vector-Mediated Correction of MPS VII Cells

GENOMICS

48, 248–253 (1998) GE975189

ARTICLE NO.

Cloning of the Canine b-Glucuronidase cDNA, Mutation Identification in Canine MPS VII, and Retroviral Vector-Mediated Correction of MPS VII Cells Jharna Ray,* Alain Bouvet,† Christopher DeSanto,* John C. Fyfe,‡ Danbin Xu,‡ John H. Wolfe,†,§ Gustavo D. Aguirre,* Donald F. Patterson,† Mark E. Haskins,†,§ and Paula S. Henthorn†1 *James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853; †Section of Medical Genetics and §Laboratory of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and ‡Department of Microbiology, Michigan State University, East Lansing, Michigan 48824 Received October 17, 1997; accepted December 22, 1997

Mucopolysaccharidosis type VII (MPS VII) is an inherited disease resulting from deficient activity of the lysosomal acid hydrolase b-glucuronidase (GUSB) and has been reported in humans, mice, cats, and dogs. To characterize canine MPS VII, we have isolated and sequenced the canine GUSB cDNA from normal and affected animals. A single nucleotide substitution was detected in the GUSB cDNA derived from MPS VII dogs. This guanosine to adenine base change at nucleotide position 559 in the canine cDNA sequence causes an arginine to histidine substitution at amino acid position 166. Introduction of the G to A substitution at position 559 in a mammalian expression vector containing the normal canine GUSB cDNA nearly eliminated the GUSB enzymatic activity, demonstrating that this mutation is the cause of canine MPS VII. A retroviral vector expressing the full-length canine bglucuronidase cDNA corrected the deficiency in MPS VII cells. q 1998 Academic Press

INTRODUCTION

The lysosomal acid hydrolase b-glucuronidase (E.C. 3.2.1.31) is a homotetrameric enzyme that plays an important role in the degradation of glucuronic acidcontaining glycosaminoglycans (Paigen, 1989). An inherited deficiency of b-glucuronidase (GUSB) activity results in the accumulation of partially degraded glycosaminoglycans in lysosomes and the clinical syndrome mucopolysaccharidosis type VII (MPS VII)(OSequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession No. AF019759. 1 To whom correspondence should be addressed at Section of Medical Genetics, University of Pennsylvania School of Veterinary Medicine, 3900 Delancey Street, Philadelphia, PA 19104-6010. Telephone: (215) 898-9601. Fax: (215) 573-2162.

0888-7543/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.

AID

GENO 5189

/

6r60$$$381

MIM 253220), as seen in humans (Sly et al., 1973), dogs (Haskins et al., 1984; 1991), mice (Birkenmeier et al., 1989), and cats (Gitzelmann et al., 1994; Fyfe et al., 1996). The normal cDNAs from human (Oshima et al., 1987), mouse (Gallagher et al., 1988), and rat (Nishimura et al., 1986) GUSB genes have been cloned and sequenced. Over 30 GUSB mutations and 1 GUSB pseudodeficiency allele have been characterized in human MPS VII patients (Tomatsu et al., 1991; Shipley et al., 1993a; Vervoort et al., 1993, 1995, 1996, 1997; Wu and Sly 1993; Yamada et al., 1995; Islam et al., 1996), and the mutation has been described in MPS VII mice (Sands and Birkenmeier, 1993). The animal homologues have been widely used in studies of the pathology and potential treatments of MPS VII (see for example Haskins et al., 1997; Moullier et al., 1995; Stramm et al., 1990; Wolfe et al., 1990, 1992, 1995). As part of the characterization of this disease, we report the normal dog GUSB cDNA sequence, the identification of a missense mutation in MPS VII affected dogs that drastically reduces normal GUSB enzymatic activity, and the use of the normal canine GUSB cDNA to correct the enzyme deficiency in MPS VII cells. MATERIALS AND METHODS Canine GUSB cDNA cloning and sequencing. A 590-bp caninespecific GUSB cDNA fragment was generated using primers BG5 and BG6, based on conserved regions of human, rat, and mouse GUSB cDNAs (Wolfe et al., 1995), to amplify aliquots of a canine testis cDNA library (Stoltzfus et al., 1992). The sequence of this fragment was consistent with its identity as a portion of the canine GUSB cDNA. This fragment was used to screen a canine testis cDNA library using standard bacteriophage l plaque hybridization methods (Sambrook et al., 1989). Two partial clones that contained GUSB cDNA inserts of 1.2 kb (clone pcGUSB23) and 1.9 kb (pcGUSB3) were isolated. Clone pcGUSB3 was missing the 5* 255 bp of the protein coding region. A second canine cDNA library was constructed from RNA isolated

248

02-11-98 08:43:22

gnma

249

CANINE MPS VII GENE DEFECT from normal dog pooled kidney proximal tubular epithelial cells (States et al., 1990), using Trizol (Life Technologies, Bethesda, MD) according to the manufacturer’s instructions. Total RNA was sent to Stratagene (La Jolla, CA) for construction of the library in the bacteriophage l vector, Lambda ZAP II, using both random octamer and oligo(dT) priming of the cDNA synthesis reaction. This library was screened with the pcGUSB3 cDNA insert. Of multiple hybridizing clones, the two longest clones had 2.15-kb inserts. The sequence of the 5* and 3* ends of one clone, pcGUSB2, confirmed that it contained a full-length GUSB cDNA. DNA sequence was determined from both strands of the cDNA clones by the dideoxy termination cycle sequencing method on an ABI 373A Automated Sequencer (Applied Biosystems, Inc., Foster City, CA) using primers dispersed throughout the full-length cDNA. Sequences of primers used in sequencing are available upon request. DNA sequences were assembled and analyzed using DNASTAR software (DNASTAR, Madison WI) and have been submitted to GenBank (Accession No. AF019759). Position 1 of the canine b-glucuronidase cDNA refers to the first nucleotide of the full-length cDNA. By this system, the A residue of the first ATG of the open reading frame is at position 63. Reverse-transcription–polymerase chain reaction (RT-PCR) of cDNA from MPS VII dogs. RNA was extracted from cultured retinal pigment epithelial (RPE) cells isolated from both a mixed breed shepherd–beagle MPS VII affected and a normal dog (Chomczynski and Sacchi, 1987). The cDNA was reverse-transcribed from 1 mg of total RNA using MuLV reverse-transcriptase (2.5 U) and random hexamers (2.5 mM) in a total volume of 20 ml using an RNA/PCR kit as recommended by the manufacturer (Perkin–Elmer, Foster City, CA). Primers for PCR were designed from the coding region of normal canine GUSB sequence (Table 1). PCRs were performed in a volume of 100 ml containing 50 mM KCl, 10 mM Tris–HCl (pH 8.3), 2 mM MgCl2 , 0.2 mM concentrations of each of deoxynucleotide triphosphate, and 0.4 mM concentrations of each primer. Taq DNA polymerase was added after a hot start for 3 min at 947C. PCRs were carried out for 35 cycles at an annealing temperature of 547C for fragments 2 through 5 and 607C for fragment 1 (see Table 1) for 1 min, polymerization temperature of 727C for 1.5 min, and a heat-denaturation temperature of 947C for 1 min in a thermal cycler (Thermolyne, Dubuque, IA). Conformation sensitive gel electrophoresis (CSGE) analysis. A set of six overlapping fragments, covering 98% of the coding region of GUSB cDNA (Table 1), was amplified by RT-PCR from the total RNA isolated from homozygous normal and MPS VII affected dog RPE. The CSGE method for detection of single-base mutations by heteroduplex formation (Ganguly et al., 1993; Ray et al., 1994) was used to search for base differences between corresponding normal and MPS VII affected fragments. Aliquots of the RT-PCR products from normal and MPS VII RNA were incubated at 987C for 5 min to separate DNA strands and then annealed at 687C for 60 min. The treated samples were concentrated to 5 ml by rotary vacuum evaporation and mixed with 5 ml of 20% ethylene glycol–30% formamide containing 0.25% xylene cyanol and bromphenol blue. Samples were electrophoresed under mild denaturing conditions in a standard 5% polyacrylamide gel [polymerized in 10% ethylene glycol–15% formamide–Tris–taurine buffer (44.5 mM Tris, 28.5 mM taurine, 0.5 mM EDTA, pH 9)] (Ray et al., 1994). The gel was stained with ethidium bromide at 0.5 to 1 mg/ml for 10 min, followed by destaining for 10 min before photography with ultraviolet illumination. Cloning and sequencing of RT-PCR fragments. RT-PCR fragment 2 from MPS VII affected RNA, which formed a heteroduplex with the normal fragment (Fig. 1), was cloned into pCRII vector using the TA cloning kit from Invitrogen (San Diego, CA) and sequenced on both strands to identify the base change. A larger fragment (543 bp) containing residue 599A was subsequently amplified with primers 5*-TCGGCTGGGTGTGGTACGAGC-3* and 5*-CCTGGCTCCCTGTCCCCTGG-3*, cloned into the pCRII vector, and used to introduce the base change at position 559 into the normal canine GUSB cDNA as described below. RT-PCR fragments 1, 3, 4, 5, and 6 were sequenced directly from PCR products.

AID

GENO 5189

/

6r60$$$381

02-11-98 08:43:22

Expression of the normal and mutagenized canine GUSB cDNA. The full-length normal canine GUSB cDNA insert from pcGUSB2 was liberated from the ZAP/pBS vector in two pieces, a 5* 1.8 kb EcoRI/SacI fragment and a 3 * 0.4-kb SacI/BamHI fragment. These fragments were ligated into the plasmid vector pLXSN (Miller and Rosman, 1989), which had been linearized at the cloning sites EcoRI and BamHI, and the desired plasmid clone, pLcgusbSN, was isolated. The 599A mutation was introduced into pLcgusbSN by removing a 255-bp BstBI/SgrAI cDNA fragment that contained the 599G residue. A third restriction enzyme, HindIII, was utilized in the digestion of the pLcgusbSN vector to guarantee the removal of this 255-bp fragment by isolating the desired DNA in two separate fragments from separate digestions (a BstBI/HindIII fragment and a HindIII/ SgrAI fragment). These fragments were ligated to the 255-bp BstBI/ SgrAI fragment isolated from an affected MPS VII dog, thus containing the 599A residue. The desired clone, pLcgusbSNmut, was isolated, and the presence of the 599A residue was confirmed by sequencing across the entire 255-bp region, including the flanking restriction endonuclease sites. The plasmids pLcgusbSN and pLcgusbSN-mutant were introduced into the murine skin fibroblast cell line 3521, a GUSB enzyme activity-deficient cell line established from an MPS VII mouse (Wolfe et al., 1995), by treatment with the cationic lipid, Lipofectamine (Life Technologies, Bethesda, MD). Approximately 4.8 1 105 cells per well were seeded in a 6-well plate 24 h before the addition of 2 mg of plasmid DNA and 16 mg of Lipofectamine per well. Transfected cells were selected in G418 (Geneticin, Life Technologies), and resulting colonies (greater than 100 for each plasmid) were grown to confluence and assayed fluorometrically for GUSB enzymatic activity using 4methylumbelliferyl-b-D-glucuronide substrate (Fischer et al., 1980). Data reported in Fig. 3 are derived from duplicate assays on each cell pellet. Enzyme activity was normalized to total protein in the cell extract. The plasmid pLcgusbSN was electroporated into the amphotropic retrovirus packaging cell line GP/envAm12 (Markowitz et al., 1988), and cells were selected with G418. Supernatants were collected from G418-resistant cells and used to infect 3521 cells. After selection in G418, cells were assayed biochemically (Fischer et al., 1980) for GUSB enzymatic activity.

RESULTS

Normal Canine GUSB cDNA Cloning Screening of two canine cDNA libraries, constructed from normal dog testis and cultured kidney epithelial cells, resulted in the isolation of two partial cDNA clones and one full-length cDNA clone. One partial clone (pcGUSB23) covered 1235 bp of the GUSB sequence, including the entire 3* untranslated region and the poly(A) tail, and the other (pcGUSB3) contained 1866 bp that included most of the coding sequence and overlapped with the pcGUSB23 sequence except for the last 6 bp before the poly(A) tail. The full-length clone (pcGUSB2) was 2199 nucleotides in length, with an open reading frame extending from nucleotide position 63 through 2018 and a 14-nucleotide poly(A) tail. The deduced polypeptide was 651 amino acids long. Sequence differences between the cDNA clones include an A to C substitution unique to clone pcGUSB23 at position 1108 of the full-length cDNA, causing a predicted lysine to threonine substitution at amino acid position 349, and a C to G substitution unique to clone pcGUSB3 at position 1730 of the full-length cDNA, causing a predicted phenylalanine to leucine substitution at amino acid position 556. Since both nucleotide

gnma

250

RAY ET AL.

TABLE 1 Canine b-Glucuronidase cDNA Primers Used for Amplification of Normal and MPS VII Affected RPE RNA by RT-PCR Fragment

Size (bp)

Primer sequence (5*–3*)

Primer name

Location

1

335

2

377

3

481

4

360

5

435

6

413

CAC GCG CGG CCG CGA CGG AA CCT CCC GCT CGT ACC ACA CC TTG TCG GCT GGG TGT GGT AC GTA GAG GAG CAC AGG GCG AT CAA GTA CCC CAA GGG TTA CT ATC CTC ATG TTT GTT GAC CC CCC GCC TAC CTG TAC TCG TT CTG GAC CAG CAT GAT GCC CA GAC CGC TAT GGG ATC GTG GTC GTA CTC GCT CTG ATT ATT GG CAC ATG GAG GTG ATT CAG CT AAC GGG CTG TTT TCC AAA CAC

BG-16 BG-19 BG-1 BG-2 BG-3 BG-4 BG-7 BG-10 BG-5 BG-8 BG-11 BG-12

41–60 375–356 346–365 722–703 638–657 1118–1099 948–967 1307–1288 1248–1268 1682–1662 1596–1615 2008–1988

substitutions caused amino acid substitutions in residues of the polypeptide that are conserved in all GUSB sequences reported, even in Escherichia coli, it is possible that these substitutions represent reverse-transcriptase artifacts. Neither substitution is seen in the functional (see below) full-length pcGUSB2 clone. Similar to its human counterpart, the canine GUSB cDNA contained two potential poly(A) addition sites, with the second site having been used in all three cDNA clones analyzed. The region surrounding these two sites was conserved between human and canine GUSB cDNAs, but no conservation of sequences could be detected between the 3* untranslated regions of rodents and that of other mammalian species. Comparison of the full-length dog cDNA and deduced amino acid sequences with those from human, rat, and mouse (see Fig. 2 for GenBank accession numbers and references) show that, as expected, dog GUSB cDNA is more closely related to the human GUSB cDNA, with 81% identical amino acid residues, and more distantly related to the rodent species (76 and 78% identity with mouse and rat, respectively). All four mammalian species have similar alignments with E. coli GUSB, and the amino acids that do align show approximately 50% identical residues. Similar to the rodent GUSB proteins, canine GUSB is lacking leucine residue 166 seen in the human GUSB sequence (Fig. 2). However, canine GUSB does contain residues corresponding to human amino acid positions 313 through 315, which are missing in the rodent GUSB sequences. The three asparagine-linked glycosylation sites in the canine GUSB protein (positions 174, 421, and 632) are conserved among all mammalian species whereas human, rat, and mouse GUSB proteins all contain an additional N-linked glycosylation site that is not conserved among all mammalian GUSB proteins (Shipley et al., 1993b). Canine GUSB has an additional amino acid at its carboxyl terminus compared to human GUSB. Sequence of GUSB cDNA in MPS VII Affected Dogs The GUSB cDNA sequence from affected dogs was examined by RT-PCR of mRNA isolated from retinal

AID

GENO 5189

/

6r60$$$381

02-11-98 08:43:22

pigment epithelial cells, followed by conformation-sensitive gel electrophoresis (CSGE) and sequencing. The cDNA was amplified on six overlapping fragments that cover 1915 of the 1956 nucleotides in the open reading frame (Table 1). An initial screen for nucleotide differences between normal and MPS VII affected dogs was performed using CSGE (Ganguly et al., 1993). In this system, RT-PCR-amplified DNA fragments derived from normal and affected dogs were mixed, denatured, allowed to reanneal, and electrophoresed in a partially denaturing gel to produce bends that retard the migration of heteroduplex molecules. Of the six fragments, only fragment 2 produced a heteroduplex band (Fig. 1). Direct sequencing of all six RT-PCR fragments isolated from an affected dog found only a single nucleotide difference, a guanosine (G) to adenine (A) base change at nucleotide position 559, when compared to the normal canine GUSB cDNA sequence. The sequence of the 41 nucleotides not present in the RT-PCR fragments was determined from direct sequencing of PCR fragments amplified directly from genomic DNA of an MPS

FIG. 1. Conformation sensitive gel electrophoresis (CSGE) scanning of canine GUSB mRNA for mutations in MPS VII. RT-PCR was performed on RNA isolated from known normal (N), MPS VIIaffected (A), and carriers (C) dogs. (N / A) represents normal sample mixed with affected sample. Numbers 1 through 6 represent DNA fragments amplified from regions of cDNA; sequences of the primers and sizes of PCR products are given in Table 1. PCR products were screened for heteroduplex formation by electrophoresis in the CSGE gel system as described in the text. The fragment 2 PCR product of the carrier sample shows a heteroduplex band, as does the mixed N / A sample.

gnma

251

CANINE MPS VII GENE DEFECT

FIG. 2. Alignment of the predicted GUSB polypeptides from various species. Sequence sources and GenBank accession numbers are as follows: human (Oshima et al., 1987) M15182; rat (Nishimura et al., 1986) M13962; mouse (Gallagher et al., 1988) J03047; and E. coli (Jefferson et al., 1986) M14641. The region surrounding amino acid residue 166 of the canine GUSB polypeptide is shown. Identical residues are boxed, and gaps introduced to optimize the alignment are indicated by dashes. The location of the missense mutation in MPS VII affected dogs is shown by an arrow. Amino acid residue positions are shown to the right, and amino acid residue positions below the alignment refer to the human polypeptide sequence. Residues that are mutated in human MPS VII patients are shown in italics (P148S, Yamada et al., 1995; E150K, Vervoort et al., 1996; D152N and L176F, Vervoort et al., 1995).

VII affected dog and agreed with the normal sequence. Fragment 2 was cloned and sequenced, confirming the G to A change at position 559. This base change caused an arginine (R) to histidine (H) substitution at position 166 in the predicted polypeptide and is referred to as canine R166H (cR166H). This residue corresponds to Arg167 in the human GUSB polypeptide and is conserved in the other mammalian and in E. coli GUSBs, as illustrated in Fig. 2. As expected, the cR166H mutation segregates appropriately in dog MPS VII pedigrees in over 30 dogs tested (data not shown). Expression of Normal and Mutated Canine GUSB cDNAs The normal canine GUSB cDNA (pcGUSB2) was cloned into the retroviral vector pLXSN to produce the plasmid pLcgusbSN, for use as a mammalian expression vector. The 559 G to A missense mutation was introduced into this vector, and the resulting plasmid, pLcgusbSNmut, was shown by sequencing to differ from the parent plasmid by only the 559 G to A substitution. These plasmids were introduced independently into the murine fibroblast cell line 3521 (Wolfe et al., 1995), which was derived from a MPS VII mouse and has no GUSB enzyme activity due to a deletion mutation in the GUSB gene (Sands and Birkenmeier, 1993). G418-resistant colonies were grown from each transfection and assayed for GUSB enzymatic activity. The GUSB activities of the transfected 3521 cells containing the mutant and normal expression plasmids are shown in Fig. 3. While the cells expressing the mutant cDNA had GUSB activities that were 4- to 5fold higher than those of untransfected 3521 cells, this activity was 100-fold less than in cells expressing the normal canine GUSB cDNA. Therefore, introduction of the cR166H mutation into a normal canine GUSB cDNA reduced the enzymatic activity to approximately 1% of normal. This is in close agreement with the measured levels of residual GUSB enzymatic activity (0.2– 1.7% of normal) in tissues from MPS VII dogs (Schuchman et al., 1989). Correction of the GUSB Deficiency in MPS VII Cells by Retroviral Gene Transfer The recombinant retrovirus vector pLcgusbSN was introduced into an amphotropic packaging cell line to

AID

GENO 5189

/

6r60$$$381

02-11-98 08:43:22

produce retroviral particles that were used to infect the murine MPS VII fibroblast cell line 3521 (Wolfe et al., 1995). After selection in G418, 3521 cells infected with the pLcgusbSN retrovirus expressed high levels of GUSB activity, as seen in Fig. 3, demonstrating correction of the GUSB enzyme deficiency by retroviral gene transfer of the canine GUSB cDNA. The level of enzyme activity in retrovirus-infected cells corresponded to 5to 10-fold of the activity in normal canine fibroblasts (not shown) and, as expected for retrovirally expressed GUSB cDNAs (Wolfe et al., 1990; 1995; Taylor and Wolfe, 1994), the level of enzyme activity was higher in cells infected with a retroviral vector than in those transfected with plasmid DNA. DISCUSSION

We report here the sequence of the canine GUSB cDNA, the identification of a missense mutation in the

FIG. 3. GUSB enzymatic activity in MPS VII 3521 cells containing canine GUSB cDNA expression vectors. GUSB enzymatic activity in cell lysates of MPS VII 3521 cells are expressed as nmol/ h/mg protein. Transfections were performed with the canine GUSB cDNA expression vectors pLcgusbSN, containing the normal cDNA, and pLcgusbSN-mutant, containing the cDNA with the single 599 G to A mutation. The label RV-infected under the right bar indicates that the cells were infected with retrovirus particles produced from packaging cells that contained the pLcgusbSN vector.

gnma

252

RAY ET AL.

GUSB gene in MPS VII dogs that drastically reduces normal GUSB enzymatic activity, and the expression of the canine GUSB cDNA. The canine GUSB cDNA sequence shows the expected relative relationships to GUSB cDNAs from other species; that is, it is more closely related to human GUSB than to rodent GUSB sequences, at both the nucleotide and the amino acid levels. The missense mutation found in the GUSB cDNA of MPS VII dogs changes an amino acid residue that is conserved in all known GUSB genes, including E. coli. While the Arg to His substitution does not appear to disrupt any important structure in the polypeptide, as assessed by analysis of predicted secondary structural features (not shown), it does occur in a region that is conserved in E. coli (see Fig. 2). The missense mutation does not correspond to any of the mutations found in human MPS VII cases, with the closest mutations occurring either 15 residues toward the amino terminus or 9 residues toward the carboxyl terminus of the polypeptide (Vervoort et al., 1995; see Fig. 2). Mutagenesis demonstrates that the cR166H mutation reduces the normal canine GUSB enzymatic activity by more than 100-fold. Biochemical studies of the defective enzyme in canine MPS VII demonstrate residual activity as high as 1.7% in tissues (Schuchman et al., 1989). No significant physical differences, such as thermostability or pH stability, were detected in the enzyme analyzed from MPS VII dogs. However, the residual activity showed an altered Km toward the artificial substrate 4-methylumbelliferyl-b-D-glucuronide. These findings are consistent with a missense mutation in the GUSB gene, as demonstrated here. The presence of a mutant enzyme protein in the MPS VII affected animals will reduce the likelihood that an animal treated with the normal canine enzyme will develop an immune response against the transferred gene product, as has been observed in gene transfer studies in canine MPS I, which has a nonsense mutation and no protein expression (Menon et al., 1992; Shull et al., 1996). Similarly, mouse knockout models and the MPS VII mouse strain are null mutations that do not express the mutant protein and can respond immunologically to therapeutic normal gene products. Thus the MPS VII dog is an important model for treatment of human lysosomal storage diseases because many patients have missense mutations expressing enzymatically inactive proteins. The molecular characterization of a missense mutation and the cloning of the normal canine GUSB cDNA are important steps in advancing our ability to utilize this homologue of a human genetic disease effectively.

supported by Consolidated Research Grant, College of Veterinary Medicine, Cornell University (J.R.), Donnelley Development Award (J.R.), University of Pennsylvania Research Foundation (P.H.), the Lucille P. Markey Charitable Trust (D.F.P.), and grants NS33526 (M.E.H.), DK42707 (J.H.W.), DK46637 (J.H.W.), DK45347 (J.C.F.), EY11142 (G.D.A., J.R.), and RR02512 (D.F.P.) from the National Institutes of Health. A.B. was supported by the Kleberg Foundation.

ACKNOWLEDGMENTS

Markowitz, D., Goff, S., and Bank, A. (1988). Construction and use of a safe and efficient amphotropic packaging cell line. Virology 167: 400–406.

The authors thank Drs. E. Neufeld and B. Dlott for the canine testis cDNA library, Drs. Kunal Ray, Mark Sands, and the late E. Birkenmeier for helpful discussions, and V. Rininger, T. Gidalevitz, and M. Parente for excellent technical assistance. This work was

AID

GENO 5189

/

6r60$$$381

02-11-98 08:43:22

REFERENCES Birkenmeier, E. H., Davisson, M. T., Beamer, W. G., Ganschow, R. E., Vogler, C., Gwynn, B., Lyford, K. A., Maltais, L. M., and Wawrzyniak, C. J. (1989). Murine mucopolysaccharidosis type VII: Characterization of a mouse with b-glucuronidase deficiency. J. Clin. Invest. 83: 1258–1266. Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem. 162: 156–159. Fischer, H. D., Nataowicz, M., Sly, W. S., and Bretthauer, R. K. (1980). Fibroblast receptor for lysosomal enzymes mediates pinocytosis of multivalent phosphomannan fragment. J. Cell Biol. 84: 77–86. Fyfe, J. C., Kurzhals, R. L., Giger, U., Haskins, M. E., Patterson, D. F., Wang, P., Wolfe, J. H., Yuhki, N., and Henthorn, P. S. (1996). A missense mutation causes beta-glucuronidase deficiency in feline MPS VII. Am. J. Hum. Genet. 59S: A197. Gallagher, P. M., D’Amore, M. A., Lund, S. D., and Ganschow, R. E. (1988). The complete nucleotide sequence of murine b-glucuronidase mRNA and its deduced polypeptide. Genomics 2: 215–219. Ganguly, A., Rock, M. J., and Prockop, D. J. (1993). Conformationsensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: Evidence for solvent-induced bends in DNA heteroduplexes. Proc. Natl. Acad. Sci. USA. 90: 10325–10329. Gitzelmann, R., Bosshard, N. U., Superti-Furga, A., Spycher, M. A., Briner, J. Wiesmann, U., Lutz, H., and Litschi, B. (1994). Feline mucopolysaccharidosis VII due to beta-glucuronidase deficiency. Vet. Pathol. 31: 435–443. Haskins, M. E., Desnick, R. J., DiFerrante, N., Jezyk, P. F., and Patterson, D. F. (1984). b-Glucuronidase deficiency in a dog: A model of human mucopolysaccharidosis VII. Pediatr. Res. 18: 980–984. Haskins, M. E., Aguirre, G. D., Jezyk, P. F., Schuchman, E. H., Desnick, R. J., and Patterson D. F. (1991). Animal models of human disease. Mucopolysaccharidosis type VII (Sly syndrome). Betaglucuronidase-deficient mucopolysaccharidosis in the dog. Am. J. Pathol. 138: 1553–1555. Haskins, M., Abkowitz, J., Aguirre, G., S., Casal, M., Evans, S., Hasson, C., Just, C., Lexa, F., Miranda, S., Schuchman, E., Simonaro, C., Thrall, M. A., Wang, P., Weil, M., Weimelt, S., Wolfe, J., and Patterson, D. (1997). Bone marrow transplantation in animal models of lysosomal storage diseases. In Correction of Genetic Diseases by Transplantation IV. (O. Ringden, J. R. Hobbs, and C. G. Steward (Eds.), pp. 1–11, COGENT Press, Middlesex G.B. Islam, M. R., Vervoort, R., Lissens, W., Hoo, J. J., Valentino, L. A., and Sly, W. S. (1996). Beta-Glucuronidase P408S, P415L mutations: Evidence that both mutations combine to produce an MPS VII allele in certain Mexican patients. Hum. Genet. 98: 281–284. Jefferson, R. A., Burgess, S. M., and Hirsh, D. (1986). b-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc. Natl. Acad. Sci. USA 83: 8447–8451.

Menon, K. P., Tieu, P. T., and Neufeld, E. F. (1992). Architecture of the canine IDUA gene and mutation underlying canine mucopolysaccharidosis I. Genomics 14: 763–768.

gnma

CANINE MPS VII GENE DEFECT Miller, A. D., and Rosman, G. J. (1989). Improved retroviral vectors for gene transfer and expression. BioTechniques 7: 980–990. Moullier, P., Bohl, D., Heard, J. M., and Danos, O. (1995). Correction of lysosomal storage in the liver and spleen of MPS VII mice by implantation of genetically modified skin fibroblasts. Nat. Genet. 4: 154–159. Nishimura ,Y., Rosenfeld, M. G., Kreibich, G., Gubler, U., Sabatini, D. D., Adesnik, M., and Andy, R. (1986). Nucleotide sequence of rat preputial gland b-glucuronidase cDNA and in vitro insertion of its encoded polypeptide into microsomal membranes. Proc. Natl. Acad. Sci. USA 83: 7292–7296. Oshima, A., Kyle, J. W., Miller, R. D., Hoffmann, J. W., Powell, P. P., Grubb, J. H., Sly, W. S., Tropak, M., Guise, K. S., and Gravel, R. A. (1987). Cloning, sequencing and expression of cDNA for human bglucuronidase. Proc. Natl. Acad. Sci. USA 84: 685–689. Paigen, K. (1989). Mammalian b-glucuronidase: Genetics, molecular biology and cell biology. Prog. Nucleic Acid Res. Mol. Biol. 37: 155– 205. Ray, K., Baldwin, V., Acland, G., Blanton, S., and Aguirre, G. (1994). Co-segregation of codon 807 mutation of the rod cGMP phosphodiesterase bgene (PEDB) in rcd 1. Invest. Ophthalmol. Vis. Sci. 35: 4291–4299. Sambrook, J., Fritsch, E. F., and Maniatis, J. (1989). ‘‘Molecular Cloning: A Laboratory Manual,’’ 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.. Sands, M. S,. and Birkenmeier, E. H. (1993). A single-base-pair deletion in the b-glucuronidase gene accounts for the phenotype of murine mucopolysaccharidosis type VII. Proc. Natl. Acad. Sci. USA 90: 6567–6571. Schuchman, E. H., Toroyan, T. K., Haskins, M. E., and Desnick, R. J. (1989). Characterization of the defective b-glucuronidase activity in canine mucopolysaccharidosis type VII. Enzyme 42: 174–180. Shipley, J. M., Klinkenberg, M., Wu , B. M., Bachinksy, D. R., Grubb, J. H., and Sly, W. S. (1993a). Mutational analysis of a patient with Mucopolysaccharidosis type VII and identification of pseudogenes. Am. J. Hum. Genet. 52: 517–526. Shipley, J. M., Grubb, J. H., and Sly, W. S. (1993b). The role of glycosylation and phosphorylation in the expression of active human bglucuronidase. J. Biol. Chem. 268: 12193–12198. Shull, R., Lu, X., Dube, I., Lutzko, C., Kruth, S., Abrams-Ogg, A., Kiem H.-P., Goehle, S., Schuening, F., Millan, C., and Carter, R. (1996). Humoral immune response limits gene therapy in canine MPS I. Blood 88: 377–379. Sly, W. S., Quinton, B. A., McAlister ,W. H., and Rimoin, D. L. (1973). b- Glucuronidase deficiency: report of clinical, radiologic, and biochemical features of a new mucopolysaccharidosis. J. Pediatr. 82: 249–257. States, B., Reynolds, R., Lee, J., and Segal, S. (1990). Cystine uptake by cultured cells originating from dog proximal tubule segments. In Vitro Cell. Dev. Biol. 26: 105–112. Stolzfus, L. J., Sosa-Pineda , B., Moskowitz, S. M., Menon, K. P., Dlott, B., Hoopert, L., Teplow, D. B., Shull, R. M., and Neufeld, E. F. (1992). Cloning and characterization of cDNA encoding ca-

AID

GENO 5189

/

6r60$$$381

02-11-98 08:43:22

253

nine a-L-iduronidase. mRNA deficiency in mucopolysaccharidosis I dog. J. Biol. Chem. 267: 6570–6575. Stramm, L. E., Wolfe, J. H., Schuchman, E. H., Haskins, M. E., Patterson, D. F., and Aguirre, G. D. (1990). b-Glucuronidase mediated pathway essential for retinal pigment epithelial degradation of glycosaminoglycans. Disease expression and in vitro disease correction using retroviral mediated cDNA transfer. Exp. Eye Res. 50: 521–532. Taylor, R. M., and Wolfe, J. H. (1994). Cross-correction of b-glucuronidase deficiency by retroviral vector-mediated gene transfer. Exp. Cell Res. 214: 606–613. Tomatsu, S., Fukuda, S., Sukegawa , K., Ikedo, Y., Yamada, S., Yamada, Y., Sasaki, T., Okamoto, H., Kuwahara, T., Yamaguchi , S., Kimn, T., Shintaku , H., Isshiki, G., and Orii, T. (1991). Mucopolysaccharidosis type VII: Characterization of mutations and molecular heterogeneity. Am. J. Hum. Genet. 48: 89–96. Vervoort, R., Lissens, W., and Liebaers, I. (1993). Molecular analysis of a patient with hydrops fetalis caused by b-glucuronidase deficiency, and evidence for additional pseudogenes. Hum. Mutat. 2: 443–445. Vervoort, R., Islam, M. R., Sly, W., Chabas, A., Wevers, R., de Jong, J., Liebaers, I., and Lissens, W. (1995). A pseudodeficiency allele (D152N) of the human beta-glucuronidase gene. Am. J. Hum. Genet. 57: 798–804. Vervoort, R., Islam, M. R., Sly, W. S., Zabot, M. T., Kleijer, W. J., Chabas, A., Fensom, A., Young, E. P., Liebaers, I., and Lissens, W. (1996). Molecular analysis of patients with beta-glucuronidase deficiency presenting as hydrops fetalis or as early mucopolysaccharidosis VII. Am. J. Hum. Genet. 58: 457–471. Vervoort, R., Buist, N. R. M., Kleijer, W. J., Wevers, R., Fryns, J. P., Liebaers, I., and Lissens, W. (1997). Mutations in mucopolysaccharisosis type-VII and heterogeneity of the polyadenylation region. Hum. Genet. 99: 462–468. Wolfe, J. H., Schuchman, E. H., Stramm, L. E., Concaugh, E. A., Haskins, M. E., Aguirre, G. D., Patterson, D. F., Desnick, R. J., and Gilboa, E. (1990). Restoration of normal lysosomal function in mucopolysaccharidosis type VII cells by retroviral vector-mediated gene transfer. Proc. Natl. Acad. Sci. USA 87: 2877–2881. Wolfe, J. H., Sands, M. S., Barker, J. E., Gwynn, B., Rowe, L. B., Vogler, C. A., and Birkenmeier, E. H. (1992). Reversal of pathology in murine mucopolysaccharidosis type VII by somatic cell gene transfer. Nature 360: 749–753. Wolfe, J. H., Kyle, J. W., Sands, M. S., Sly, W. S., Markowitz, D. G., and Parente, M. K. (1995) High level expression and export of bglucuronidase from murine mucopolysaccharidosis VII cells corrected by a double-copy retrovirus vector. Gene Ther. 2: 70–78. Wu, B. M., and Sly, W. S. (1993). Mutational studies in a patient with the hydrops fetalis form of mucopolysaccharidosis type VII. Hum. Mutat. 2: 446–457. Yamada, S., Tomatsu, S., Sly, W. S., Islam, M. R., Wenger, D. A., Fukuda, S., Sukegawa, K., and Orii, T. (1995). Four novel mutations in mucopolysaccharidosis type VII including a unique base substitution in exon 10 of the b-glucuronidase gene that creates a novel 5* splice site. Hum. Mol. Genet. 4: 651–655.

gnma