Murine Mucopolysaccharidosis Type VIICharacterization of a Mouse with /O-Glucuronidase DeficiencyEdward H. Birkenmeler, Muriel T. Davisson, Wesley G. Beamer, Roger E. Ganschow,* Carole A. Vogler,* Babette Gwynn,Kimberly A. Lyford, Lois M. Maltais, and Cynthia J. Wawrzyniak*The Jackson Laboratory, Bar Harbor, Maine 04609; *Division of Basic Science Research, Children's Hospital Research Foundation,Cincinnati, Ohio 45229; and *Department of Pathology, St. Louis University, St. Louis, Missouri 63104

Abstract

Wehave characterized a new mutant mouse that has virtuallyno /-glucuronidase activity. This biochemical defect causes amurine lysosomal storage disease that has many interestingsimilarities to human mucopolysaccharidosis type VII (MPSVII; Sly syndrome; fl-glucuronidase deficiency). Genetic analy-sis showed that the mutation is inherited as an autosomal re-cessive that maps to the fl-glucuronidase gene complex, [Gus],on the distal end of chromosome 5. Although there is a > 200-fold reduction in the fl-glucuronidase mRNAconcentration inmutant tissues, Southern blot analysis failed to detect any ab-normalities in the structural gene, Gus-s", or in 17 kb of 5'flanking and 4 kb of 3' flanking sequences. Surprisingly, asensitive S1 nuclease assay indicated that the relative level ofkidney gus"P' mRNAresponded normally to androgen induc-tion by increasing 1-fold. Analysis of this mutant mousemay offer valuable information on the pathogenesis of humanMPSVII and provide a useful system in which to study bonemarrow transplantation and gene transfer methods of therapy.

Introduction

f3-Glucuronidase (#-D-glucuronide glucuronohydrolase EC3.2.1.31) is a lysosomal enzyme expressed in most, if not all,mammalian tissues (1). In mouse kidney and liver the enzymeis also found in the microsomes in association with the acces-sory binding protein, egasyn (2). The active enzyme is a tetra-meric glycoprotein that degrades glycosaminoglycans by re-moving fl-glucuronosyl residues at the nonreducing end of oli-gosaccharides (3, 4). Severe deficiency of this enzyme results inthe accumulation of undegraded glycosaminoglycans in thelysosomes and produces the disease mucopolysaccharidosistype VII (5). This disease was first described in humans (6, 7)but there is also a canine model of fl-glucuronidase defi-ciency (8).

Murine #-glucuronidase has been studied extensively be-cause it provides a useful system for understanding mamma-lian gene regulation (1). The structural gene, Gus-s, is locatedon the distal half of chromosome 5 of the mouse. There arethree common alleles designated Gus-sa, Gus-sb, and Gus-s/?which are differentiated by electrophoretic mobility and heat

Address reprint requests to Dr. Birkenmeier, The Jackson Laboratory,600 Main Street, Bar Harbor, ME04609.

Receivedfor publication 13 September 1988.

stability (9-1 1). Three regulatory elements, designated Gus-r,Gus-t, and Gus-u, are closely linked to Gus-s. Gus-r is cis-act-ing and determines the level of Gus-s mRNAin the proximaltubule cells of the kidney in response to androgen induction(12). Gus-t is a trans-acting temporal regulator that determinesthe rate of 3-glucuronidase synthesis in several mouse tissuesduring postnatal development (13, 14). Gus-u is a systemiccis-acting regulator that determines the relative levels of en-zyme activity in all tissues (14, 15). These three regulatoryelements in conjunction with the structural gene define thef3-glucuronidase gene complex, [Gus].

In C3H strains of mice which are of the [GusIH haplotype(16), there are relatively low levels of fl-glucuronidase activityin all tissues but the mice do not have any clinical symptoms.In lieu of a better mouse model, C3Hmice have been utilizedto study the correction of lysosomal enzyme deficiency byallogeneic bone marrow transplantation (17). Several yearsago, three mice with dwarfing characterized by shortness ofnose, limbs, tail, and body length were identified in a colony ofB6.C-H-2bmJ mice at The Jackson Laboratory. The mutantgene was given the provisional name of adipose storage defi-ciency (asd) because, in addition to the obvious skeletal defor-mities, the animals were devoid of visually identifiable whiteadipose tissue (18). While studying this mutation at the geneticand biochemical levels, we found that the mutation is closelylinked to the Gussb allele and causes a severe, if not complete,deficiency of /-glucuronidase activity. Because this mutantmouse has a disease resembling human mucopolysacchari-dosis type VII (MPS VII),' we have renamed the recessivemutation gusmPs. This paper describes our initial characteriza-tion of this mutation at the genetic, cellular, biochemical, andmolecular levels. The results show that the mutation causes asevere lysosomal storage disease that we have named murineMPSVII. In addition, we discuss the importance of this mousein developing methods of therapy for lysosomal storage dis-eases as well as providing information about the regulation ofmammalian gene expression.

Methods

Animals. All animals used in these studies were from The JacksonLaboratory. They were obtained from the B6.C-H-2bm`/ByBir-gusmPs/+mutant strain maintained in the research colony of Dr. Birkenmeier,from the MOR/Rkstrain maintained by T. H. Roderick, and from theDBA/2J production colony of The Jackson Laboratory. The mutantstrain was maintained by brother-sister matings of gusPs/+ animalsand each animal was assigned a pedigree number that was recorded inthe breeding records. The mice were fed Wayne Sterilizable Rodent

1. Abbreviations used in this paper: MPSVII, mucopolysaccharidosistype VII; 4-MU, 4-methylumbelliferone; RFLP, restriction fragmentlength polymorphism.

1258 Birkenmeier et al.

J. Clin. Invest.© The American Society for Clinical Investigation, Inc.0021-9738/89/04/1259/09 $2.00Volume 83, April 1989, 1258-1266

Blox (Continental Grain Company, Chicago, IL), which is essential forthe optimal health and reproductive performance of the mutant strain.

Transmission electron microscopy. Tissues were fixed in 2%glutar-aldehyde, 1% paraformaldehyde in 0.1 Mcacodylate buffer, pH 7.4.The tissues were then postfixed in 1% osmium tetroxide, stained enbloc with 0.5% uranyl acetate, dehydrated, and embedded in Epon-ar-aldite. Ultrathin sections were stained again with uranylacetate fol-lowed by lead citrate.

Genetic mapping. To determine the chromosomal location of thegusmPs mutation, heterozygous gusmPs/+ mice were outcrossed to ei-ther DBA/2J or MOR/Rk mice. The F1 progeny were then inter-crossed and the F2 homozygous mutants were screened for the iso-zymes phosphoglucomutase-l (Pgm-J), mitochondrial malate dehy-drogenase (Mor-1), and fl-glucuronidase (Gus) using standard celluloseacetate electrophoretic methods and staining procedures (1 1, 19, 20).DNAwas isolated from the spleens of the F2 mutant animals. TheDNAwas digested with Eco RI or Hind III, Southern blotted, and thenhybridized to cDNAprobes of a-fetoprotein (Afp) or fl-glucuronidase(Gus), respectively. The DNAswere scored using restriction fragmentlength polymorphisms (RFLPs) that were known to exist at these twoloci.

Biochemical analysis. O-Glucuronidase activity was measuredusing a sensitive fluorometric assay (21, 22). Tissues were homoge-nized in 50 mMTris-HCl pH 8.0 (2:1 volume to weight ratio) using amotor-driven pestle designed to fit into a conical 1.5-ml minicentrifugetube. Enzyme assays were in 0.1 Msodium acetate, pH 4.6, in a finalvolume of 0.1 ml. The substrate was 4 mM4-methylumbelliferyl-fl-D-glucuronide and the reaction mixture was incubated at 370C for 1 h.The reaction was stopped by the addition of 1.0 ml of 0.1 Msodiumcarbonate. Protein concentration was determined by the method ofLowry et al. (23). Activity was expressed as nanomoles of 4-methyl-umbelliferone (4-MU) released per milligram protein per hour.

RNA isolation and characterization. Total cellular RNAwas iso-lated from mouse tissues by extraction with guanidine thiocyanatefollowed by centrifugation through CsCl (24). Poly A' RNAwas iso-lated using oligo-dT cellulose chromatography. Northern blots wereprepared after formaldehyde-agarose gel electrophoresis of the RNA(25). The Zetabind (AMF, Meriden, CT) nylon filters were hybridizedto cDNAprobes radiolabeled by the random hexamer method as de-scribed previously (26, 27).

For the SI nuclease protection assays, part of the pGUS-l cDNAclone representing exons 2 through 12 was subcloned into a BlueScribe vector (Stratagene, LaJolla, CA). T7 RNApolymerase was usedwith [32P]UTP to make labeled anti-sense RNA (cRNA) that wasseparated from the template by agarose gel electrophoresis. For eachassay 100 ,g of total RNAwas suspended in a formamide hybridiza-tion buffer containing 1-5 X I05 cpm of the cRNAprobe. The samplewas denatured at 85°C and then allowed to hybridize at 65°C from 3 hto overnight. After hybridization, the samples were treated with 400 US1 nuclease. To assay for the SI -resistant hybrid molecules, the sam-ples were electrophoresed in nondenaturing polyacrylamide gels. Thegels were dried and autoradiography performed at -70°C with anintensifying screen. To confirm that equal amounts of RNAwerepresent in each assay, parallel assays were performed using a y-actincRNA probe. An additional control assay utilized Escherichia coliRNArather than mouse tissue RNAin the hybridization reaction inorder to verify the specificity of the bands detected.

Southern blot analysis. Genomic DNAswere digested with restric-tion enzymes, electrophoresed in 0.8% agarose gels, and transferred toZetabind nylon filters by the Southern method (28). Probes were madefrom the various cloned DNAs using the random hexamer labelingmethod and were hybridized to the nylon filters as described pre-viously (26).

Testosterone induction. In our initial experiments and for theNorthern blots shown in this paper, female gusmPs/gusmPs mice weretreated with testosterone by implanting capsules made from Silastictubing (Dow Corning Corp., Midland, MI) in the nape of the neck asdescribed previously (29). Each capsule contained either 0 or 5 mgof

testosterone. Normal female mice of unknown genotype (+/+ orgusmPs/+) were included as controls to determine whether f3-glucuroni-dase enzyme activity and mRNAconcentration had been induced inthe kidney in response to this treatment. For the S I nuclease protectiondata that are presented in this paper, the mice received a 30-mg pelletof testosterone in the nape of the neck. The mutant animals respondedin a similar fashion to each of these two methods of testosteronedelivery.

Molecular clones. The cloned DNAsused to make probes for theNorthern and Southern blots were obtained from several sources. ThepGUS-l cDNA clone of murine fl-glucuronidase (12) was obtainedfrom Dr. Ganschow. The subclones from the cosmid clone D23 (orPGY-1) of the Gus-sb allele from the YBRmouse strain (30) wereprovided by K. Paigen (University of California, Berkeley, CA). Thea-fetoprotein cDNAclone, pHcII 440, was obtained from S. M. Tilgh-man (Princeton University, Princeton, NJ).

Results

History and clinical description of the gus1P1mgusPS mouse.The mutation occurred on the congenic mouse strain B6.C-H-2b'/By, which was originally called H(z 1). This strain car-ries a mutant H-2Kb allele named H-2Kbml. The strain origi-nated from a C57BL/6By female mated to an irradiatedBALB/cBy male (31). The H-2Kbml mutation was backcrossedfor 10 generations to C57BL/6By mice and then inbred inorder to establish the congenic strain mentioned above. Ap-proximately 10 yr later in 1976, the first gusmPs/gusmPs animalswere identified in the B6.C-H-2bm'/By colony at generationF34. The parents and their offspring were used to develop aseparate inbred colony of mice maintained by strict brother-sister matings of pedigreed animals. Mutant males are repro-ductively sterile for reasons independent of gonadal and re-productive tract morphology or of sperm number, morphol-ogy, and motility. Mutant females are capable of producinglitters but, because of insufficient lactation, the offspring mustbe raised by foster mothers. Therefore, the strain, namedB6.C-H-2bmI/ByBir-gusmPsI+, is maintained by mating hetero-zygous gusrPs/+ siblings identified by their ability to producemutant offspring.

Fig. 1 shows a 269-d-old mutant animal compared with anormal littermate. The mutant phenotype is easily discerniblesince the affected animals are smaller and have shorter, stubbylimbs as well as a shorter, thicker tail. The most unusual fea-ture is the peculiar facial dysmorphism shown in Fig. 1 B. Thenasal bones are reduced markedly in size, resulting in a pug-nosed appearance. Although the mutant animals look grosslynormal at birth, the abnormal appearance of the body and facedescribed above is usually evident by the time the animals areweaned at 21 d of age. The syndrome is characterized furtherby early sudden death from unknown causes. Fig. 2 shows theages at which adult male and female mice died in the colonyduring the previous 2 yr. Although normal C57BL/6J micelive to 850 or more d of age, the mutant male animals lived anaverage of 170±62 d (n = 38) while the females had a slightlyshorter mean lifespan (P < 0.025, Student t test) of 141±67 d(n = 53). Of the 91 animals included in these statistics, only 10lived longer than 241 d, the oldest a female who lived 340 d.

The mutant animals have several other unusual features.In addition to the peculiar facies, they are dwarfs with severeskeletal deformities. Smears of the peripheral blood showedabundant granulocytic inclusions in nucleated cells. Electronand light microscopy revealed evidence of vacuolar storage inmany tissues. Fig. 3 shows the pronounced cytoplasmic vacu-

,8-Glucuronidase Deficiency in Mice 1259

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Figure 1. Appearance of gusmPs/gusmPs mice. Two photographs of amale gusmPs/gusmPs mouse standing next to a normal littermate male+/? mouse for comparison. The mice are 269 d of age. (A) The mu-tant is the smaller of the two animals. (B) The mutant is standing be-hind the normal mouse and has the shortened nose characteristic ofthe face in murine MPSVII.

olization present in endothelial cells of spleen and liver. Thesefindings are remarkably similar to human patients with fl-gluc-uronidase deficiency (6, 22). However, the specific diagnosis ofmurine MPSVII required the genetic and biochemical datapresented below.

Genetic analysis of the gusrPs mutation. Initial geneticstudies confirmed that the mutation was autosomal recessive.Of 1,179 animals that were born to heterozygous parents overa 2 yr period and survived to weaning, 233 (19.8%) weregusmPS/gusmPS and 946 (80.2%) were +/?. The female to maleratios for normal and mutant animals were 0.86 and 0.74,respectively.

Genetic analysis to determine the chromosomal location ofthe mutant gene led to the identification of the biochemicaldefect as fl-glucuronidase deficiency. In the first 36 mutantanimals obtained from a DBA/2J intercross, the recombina-tion frequency with phosphoglucomutase-l (Pgm-J) was 31%(23/72 chromosomes). Because this was less than the 50%levelexpected for random segregation, these results suggested thatthe mutation was on chromosome 5. Linkage to chromosome5 was confirmed using an a-fetoprotein (Afp) cDNA clone,pHcII 440, that detects an Eco RI RFLP between C57BL/6Jand DBA/2J. Of the 27 F2 mutant animals (54 chromosomes)typed for both Pgm-J and Afp, we found 37.03±6.75% and

20.37±5.48% recombination, respectively, between these lociand gusmPs.

These results suggested that guSr'Ps was located on the distalhalf of chromosome 5. This was confirmed using an intercrosswith MOR/Rkmice. MORmice carry the Mor-lb and Gus-saalleles, while the mutant strain carries the Mor-Ja and GUSSballeles (20). The gene order on chromosome 5 is Pgm-J, Afp,f3-glucuronidase (Gus), and mitochondrial malate dehydroge-nase (Mor-J). Among 74 mutant animals typed for Mor-J,there was 6.08±1.96% recombination between gusmPs andMor-J. To complete the genetic studies, the mutation wasmapped to the [Gus] complex using a f3-glucuronidase cDNAclone, pGUS-1, that detects a Hind III RFLP between thestructural genes Gus-sa and GUSSb (16). Out of 40 DNAstested (80 chromosomes), no recombination occurred betweengusmPs and GUSSb. Thus, at the 95% upper confidence limit,these two loci are located within 3.7 centimorgans of one an-other. In addition, these results showed that the congenicstrain carrying the gusmPs mutation had the C57BL/6By Gus-5b allele rather than the BALB/cBy Gus-sa allele. Therefore,the radiation treatment given the original BALB/cBy father ofthe H(zl) mouse could not have been the direct cause of thegusmPs mutation. In all probability, gusPs is a spontaneousmutation of the [GUS]B haplotype that occurred in the B6.C-H-2bm'/By colony.

Biochemical characterization ofgusmPs/gusmPs mice. Whilethe chromosome mapping studies were in progress, we at-tempted to biochemically type the mutant mice for the twof3-glucuronidase alleles present in the second intercross. How-ever, when the cellulose acetate plates were stained for ,B-gluc-uronidase activity by a simultaneous dye coupling methodusing naphthol-AS-BI-f3-D-glucuronide as substrate (l1), noactivity was observed. This finding indicated that the biochem-ical defect was probably f3-glucuronidase deficiency. To mea-sure more accurately the specific activity ofB-glucuronidase invarious tissues, we used a sensitive fluorometric assay with4-methylumbelliferyl-,3-D-glucuronide as the substrate (21,22). This method detected extremely low levels of what waspresumably fl-glucuronidase activity. Assays of liver, kidney,

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Figure 2. Lifespan of adult male and female gusmPs/gusm'Ps mice. Thishistogram shows the age in days at- which 38 male (solid bars) and 53female (striped bars) mutant mice died of natural causes in the col-ony over a 2-yr period. The histogram is a plot of the percentage ofthe animals for each sex that died within each consecutive 30-d pe-riod versus the minimum age in days represented in each time pe-riod. These data were obtained from animals that lived long enoughto be weaned and had reached a minimum age of 30 d.

1260 Birkenmeier et al.

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brain, thymus, and spleen from mutant animals gave < 1%normal levels, and the specific activity was always < 1.0 nmolof 4-MU released/mg of protein per h. The levels of activity inkidneys and liver of mutant mice (male and female) at fourmonths of age were 0.08±0.02 and 0.18±0.03 nmol of 4-MU/mgof protein per h, respectively. This is in striking contrast tothe kidneys and liver of a control C57BL/6J male mousewhich had ,B-glucuronidase levels of 79 and 201 nmol of4-MU/mg of protein per h, respectively.

Next, we measured the levels of ,-glucuronidase activitywithin the mutant strain. If the effects of the normal andgUSmPsalleles on enzyme activity are additive in heterozygousanimals, then the gusmPs allele is semidominant. Fig. 4 showsthat the gusrPs/+ parents producing mutant offspring hadlevels of activity in liver that were approximately one-half ofthat observed in C57BL/6J mice. In contrast, parents thatfailed to produce mutant offspring after having at least 14 pupsclearly showed that one parent appeared normal (+/+) whilethe other had reduced activity similar to the heterozygous par-ents. Since all of the obligate heterozygous animals had inter-mediate levels of fl-glucuronidase activity, we concluded thatthe guSmPs allele is semidominant at the biochemical level eventhough gusmPs/+ mice otherwise appeared normal.

j3-Glucuronidase mRNAlevels. To determine the levels offl-glucuronidase mRNAin mutant mice, we isolated total cel-lular RNAfrom the kidneys of two sibling male mice that were87 d of age. One was phenotypically normal (+/?) and theother was a mutant (gusmPs/gusmPs). A Northern blot contain-ing 15 jg of each RNAwas hybridized to a cDNA clone,pGUS- 1, that represents - 1.7 kb of the 2.9-kb murine fl-gluc-uronidase mRNA(12). Although the expected mRNAwasdetected in the normal animal, there was no hybridizing RNAof any size in the mutant RNA (data not shown). Since amRNAat 10% of the normal level would have been detected,it appears that the deficiency of,3-glucuronidase activity in themutant animals is caused by a low concentration of ,B-gluc-uronidase mRNA.

Fig. 5 shows the results of another attempt to detectmRNAin mutant mice by Northern blot analysis. Weisolated

poly A' RNAfrom the kidneys and liver of a testosterone-treated mutant female mouse and prepared a Northern blotcontaining 20 ,ug of each mRNA.Control lanes contained 1.0and 5.0 ,g of kidney and liver poly A' RNAs, respectively, thatwere obtained from a testosterone-treated +/? normal femalemouse. After hybridization to the pGUS- 1 probe, severalbands were detected in the mutant kidney. A dominant smallband represented a mRNAthat is about 1.0 kb in size. Theorigin and nature of this mRNAis unknown. Another largerband comigrated with the f3-glucuronidase mRNAthat waspresent in the lanes containing mRNAfrom the normalmouse. The intensity of these bands on the film indicated thatif the comigrating band present in the mutant kidney wasfl-glucuronidase mRNA, then its concentration was at least200-fold less than that found in normal kidney. Interestingly,the mutant liver poly A+ RNAlane had only a very faint bandat this position and it was apparent only on the original autora-diogram after an 88-h exposure. This large relative differencebetween the f3-glucuronidase mRNAlevels in mutant kidneyand liver from testosterone treated mice is similar to that seenin normal mice where testosterone causes a > 10-fold induc-tion of renal f3-glucuronidase mRNAlevels (32).

To measure the testosterone induction of f3-glucuronidasemRNAin gusmPs/gusmPs mice, we used a S1 nuclease protec-tion assay with anti-sense RNAtranscribed from the pGUS- IcDNA clone. The 1.4-kb subclone of pGUS-I used to makethe cRNA probe contains exons 2 through 12 of the f3-gluc-uronidase gene and thus represents most of the coding se-quences. The results of the S1 protection analysis are shown in

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1262 Birkenmeier et al.

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Figure 6. Testosterone induction of fl-glucuronidase mRNAin mu-tant mice. (A) This autoradiogram shows the relative levels of ,B-gluc-uronidase mRNAin the livers of female mutant [Gus]mP1 mice andnormal [Gus]a mice that either were or were not treated with testos-terone. For each lane, 100 ,g of total cellular RNAwas hybridizedwith an antisense fl-glucuronidase riboprobe followed by SI nucleasetreatment and gel electrophoresis. The source of RNAin each lane isas follows: lane 1, three untreated [Gus]a mice (U); lane 2, one[Gus]'mP mouse treated with testosterone (T); lanes 3 and 4, eachlane has one untreated [Gus]m'P mouse (U); lane 5, E. coli; and lane6, size markers of 1448, 517, 396, 239, and 147 base pairs. (B) Thisautoradiogram is similar to A but shows the relative levels of fl-gluc-uronidase mRNAin the kidneys of mutant and normal mice. Thesource of RNAin each lane is as follows: lane 1, three [Gus]' micetreated with testosterone (T); lane 2, three untreated [Gus]' mice (U);lane 3, one [Gus]mP' mouse treated with testosterone (T); lane 4, oneuntreated [Gus]m1P mouse (U); and lane 5, E. coli.

Fig. 6. The liver RNAfrom mutant animals generated a 1.4-kbdsRNA fragment identical in size to that found in a normalliver. However, in the mutant mice, the f3-glucuronidasemRNAwas present at a much lower concentration. Testoster-one treatment did not change the concentration of liver

mRNAin either the mutant mouse or the normal mouse. Asimilar relatively faint 1.4-kb band was observed in the mutantkidney. However, the intensity of the bands indicated that therenal f3-glucuronidase mRNAin mutant mice is present at an11-fold higher concentration after testosterone treatment. De-spite this response to testosterone, the f3-glucuronidase mRNAconcentration in the kidneys of mutant animals remains muchlower than that seen in normal uninduced animals. Althoughit may not be possible to correlate the Northern blot resultsdirectly with these data, both experiments suggest that the f-glucuronidase gene is being transcribed in gusmPs/gusmPs miceand that a polyadenylated mRNAof normal or nearly normalsize is produced. This mRNAis present at a concentration atleast 200-fold less than that found in normal mice. The relativeconcentration of mutant kidney mRNAincreases in responseto testosterone treatment in a manner that appears to be simi-lar to that seen in normal mice.

Structure of the Gus-s allele in mutant mice. To investigatethe gusPS mutation at the DNAlevel, DNAfrom +/+ andgus PS/+ normal animals was compared with DNAfrom ho-mozygous gusmPs/gusmPS mutant animals by Southern blotanalysis. These DNAswere digested with BamHI, Eco RI, andHind III and then hybridized to a 3.2-kb Bam HI fragmentobtained from the 5' flanking region of a genomic clone ofGus-sb from a YBRmouse (30). With all three enzymes, themutant allele looked identical to the normal allele (data notshown). The pGUS-l cDNA clone also failed to detect anydifferences between the normal and mutant DNAs. Therefore,Southern blot analysis did not reveal any large deletions, du-plications, insertions, or rearrangements within a 35-kb seg-ment of the mutant genome that contains the Gus structuralgene. Either the mutation has not generated a RFLP that isdetectable with the three restriction enzymes we tested or themutation is located outside of the region covered by ourprobes.

Because methylation of specific DNAsequences some-times correlates with levels of expression of certain genes, weused the methylation-sensitive enzyme, Hpa II, to determinethe methylation status of some of the cytosine residues withinthe mutant Gus locus. Liver DNAfrom the mice used in theSouthern blots described above, was digested with either HpaII or the methylation-insensitive isoschizomer, Msp I. TheDNAs were Southern blotted and hybridized to the pGUS-1cDNAprobe and the results are shown in Fig. 7. The mutantand normal DNAslooked identical when digested with Msp Iand three major bands were resolved in the gel. The Hpa IIdigests were different from the Msp I digests, and at least sixeasily discernible bands were present in each lane. However,the Hpa II digests of all three DNAswere essentially identicalto one another. These results indicate that methylation of cy-tosine residues has occurred at some of the Msp I sites in liverDNA, and that the sites which are methylated are identical innormal and mutant animals.

Discussion

This article describes a recently discovered mutant mouse atthe genetic, cellular, biochemical, and molecular levels. Thedata show that the phenotype and clinical presentation of thedisease in this mouse can be attributed to the lack of ,-gluc-uronidase activity in the various tissues tested. Weconcluded

13-Glucuronidase Deficiency in Mice 1263

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Figure 7. Methylation pattern of the fl-glucuronidase gene in mutantmice. This is an autoradiogram of a Southern blot hybridized to thepGUS-I cDNA probe. It contains liver DNAfrom (1) gus PS/gusmPsmice, (2) gusrPs/+ mice, and (3) +/+ mice. Each DNAwas digestedwith Msp I (M) and Hpa II (H).

that the mouse has a lysosomal storage disease that is themurine counterpart of a human disease, mucopolysacchari-dosis type VII (MPS VII), described originally in a patient byWilliam S. Sly and colleagues (6). Since this original article,there have been 19 patients reported in the literature (33).There is also a single report of f3-glucuronidase deficiency in adog (8). Although there are inbred strains of mice with highand low levels of f-glucuronidase activity, none have a severeenough deficiency to cause a disease with similarities tohuman MPSVII (15, 17). Therefore, as a null mutation, thegusmPs/gusmPs mouse is a unique resource that will have abroad application in answering many important scientificquestions about (a) fl-glucuronidase-deficient mucopolysac-charidosis and (b) regulation of Gus gene expression.

The [Gus] complex is one of the most thoroughly charac-terized genetic loci in the mouse (1). Some of its interestinggenetic characteristics and various regulatory elements havebeen referenced previously in this paper. The gus"Ps mutationhas been mapped to the [Gus] complex on the distal half ofchromosome 5. Based upon the physical appearance of the

mice, it is a recessive mutation. However, at the biochemicallevel, it appears as a semidominant mutation because gusmPs/+animals have one half the 13-glucuronidase activity present in+/+ animals. Even though there is a large reduction in thef3-glucuronidase mRNAconcentration in the mutant tissues,the proximal tubule cells of the kidney appear to respond totestosterone induction since the relative concentration of f3-glucuronidase mRNAin the kidney increased > 10-fold. It isnot known whether the small amount of mRNApresent in themutant mice is translated to produce a protein with j3-gluc-uronidase activity. Our assays indicated that activity, if pres-ent, was only slightly above background. To fully understandthe cause of this severe enzyme deficiency will require that themutation be defined at the DNAsequence level. One likelypossibility is that the DNAsequence of the promoter or anassociated regulatory element such as an enhancer has beenaltered in the mutant. Alternatively, the promoter may be ca-pable of functioning normally as suggested by the testosteroneinduction experiment. The mRNAspecies observed on theNorthern blots are consistent with the possibility that the de-fect is related to mRNAstability or processing. Whatever thecause of this mutation, further study of the genetic defect at themolecular level will lead to a better understanding of the regu-lation of f3-glucuronidase gene expression.

It is also important to recognize the usefulness of this nullmutation in providing a genetic background in which to studycertain cellular processes. Lysosomes are involved in manycellular processes including killing infectious agents, degrada-tion of hormones and transport proteins, turnover of intracel-lular proteins, and remodeling of tissues and bones. Crucial toappropriate lysosomal function is the trafficking of lysosomalenzymes (34). Thus, in addition to understanding the biologi-cal consequences of lysosomal enzyme deficiencies, it is neces-sary to understand the mechanisms responsible for normallysosomal enzyme synthesis, sorting, and transport. It is nowtechnically possible to make transgenic mice carrying eithervarious naturally occurring Gus structural alleles or in vitromutated f3-glucuronidase coding sequences. If these transgenesare then placed on the gusmPs/gusmPS genetic background byappropriate genetic crosses, it should provide a valuable bio-logical system in which to identify and characterize the molec-ular signals required for correct cellular processing and target-ing of lysosomal enzymes.

In the area of clinical investigation, research on murineMPSVII may have important implications. HumanMPSVIIhas been reported to have considerable phenotypic variation(33, 35). It is unclear which of the various symptoms and theirseverity relate directly to enzyme deficiency and which are alsoaffected by genetic background and other disease processes.The murine gusmPs mutation is on a well-defined homoge-neous genetic background. All of the mice are pedigreed andthe colony has been maintained by strict brother-sister mat-ings. Thus, the gusmP11gusmPs mouse offers a model system inwhich to study the pathogenesis of a lysosomal storage diseasein a large number of animals with a uniform genetic back-ground. Although MPSVII is a rare disease, all of the humanlysosomal storage diseases taken together have a significantclinical incidence. Studies using animal models of the humandiseases may provide important information about the clinicalcourse of the disorder and suggest ways to treat humans withsimilar lysosomal enzyme defects (17, 36). Two interestingpossibilities for treatment of murine MPSVII are transplanta-

1264 Birkenmeier et al.

tion of normal syngeneic bone marrow cells and infection ofmutant bone marrow stem cells with defective retrovirusesthat encode murine or human 3-glucuronidase. Our prelimi-nary results with bone marrow transplantation into sublethallyirradiated (200-400 rads) mutant mice have shown remark-able reversal of storage disease pathology in many tissues. Todate, 78% (14/18) of the animals that received transplants arestill alive and are between 400 and 500 d of age (Birkenmeieret al., unpublished results). We predict that some may ap-proach a normal lifespan and reach an age of at least 2 yr.

In summary, we have identified a murine lysosomal stor-age disease that has many similarities to human MPSVII.There is little if any fl-glucuronidase activity present in thesemice because of a very low level of f3-glucuronidase mRNA.The exact nature of the defect at the DNAlevel has yet to bedefined but the gus nPs mutation maps to the [Gus] complex onchromosome 5. The mice may be useful in further basic re-search involving regulation of gene expression and traffickingof lysosomal enzymes as well as clinical research involvinglysosomal storage diseases and their treatment.

Acknowledgments

There are many individuals who contributed intellectual and technicalassistance to make this paper possible. Wegive special recognition toJeanette Reed of The Jackson Laboratory who first discovered thegusmPs/gusmPs mice in 1976 and to Carole Spencer of The JacksonLaboratory for excellent technical assistance. Wethank Patricia Gal-lagher (Children's Hospital Research Foundation, Cincinnati, OH)and Ken Paigen (University of California, Berkeley, CA) for providingunpublished information about the molecular structure of the normalf-glucuronidase gene. Benjamin Taylor and Thomas Roderick of TheJackson Laboratory made helpful suggestions in the chromosomalmapping studies. William S. Sly (St. Louis University, St. Louis, MO)and Jane E. Barker of The Jackson Laboratory, who are collaboratingon additional studies involving the gusmPs mutation, made many help-ful suggestions on the initial work presented in this paper. Wealsothank Raymond Negrel from the University of Nice, France, whospent a summer at The Jackson Laboratory characterizing the white fatdeficiency present in this mutant mouse and whose data will be pub-lished elsewhere. The genetic mapping was supported partially by theNational Science Foundation grant BSR 84-18828 to Dr. Davissonand by the National Cancer Institute Cancer CORECenter grant 5 P30CA 34196 to The Jackson Laboratory. The SI mapping studies werefunded by the U. S. Public Health Service grant AM 14770 to Dr.Ganschow. Otherwise, the reported research and the guSmPs mousecolony were supported by the Public Health Service grant DK-34384to Dr. Birkenmeier. The Public Health Service is not responsible forthe paper's contents nor do the contents necessarily represent the viewsof the Public Health Service.

The Jackson Laboratory is fully accredited by the American Asso-ciation for Accreditation of Laboratory Animal Care.

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