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Ann.Rev. Genet. 1985.19:127~18 Copyright©1985by AnnualReviewsInc. All rights reserved

HPRT: GENE STRUCTURE, EXPRESSION, AND MUTATION J. Timothy Stout Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030

C. Thomas Caskey Howard Hughes Medical Institute, and Departments of Medicine, Biochemistry, Cell Biology, Baylor College of Medicine, Houston, Texas 77030

and

CONTENTS INTRODUCTION AND PERSPECTIVES ....................................................... NORMAL ENZYME FUNCTION ANDCHARACTERISTICS ............................. Enzymology ........................................................................................ TissueDistribution................................................................................ HPRT GeneStructure............................................................................ HPRT GeneExpression .......................................................................... Pathogenesis of HPRT Deficiency............................................................. MUTATIONS ATTHEHPRTLOCUS ........................................................... Mutations in CulturedCells ..................................................................... Mutations in Man................................................................................. GENE TRANSFER ANDTHERAPEUTIC PROSPECTS .....................................

127 129 129 130 130 132 136 137 137 139 142

INTRODUCTIONAND PERSPECTIVES The biosynthesis of purine and pyrimidine nucleotides is a crucial process for all cells since these molecules are the direct precursors of DNAand RNAand frequently participate as coenzymes in enzymatic reactions. With few exceptions, organisms are able to synthesize purines and pyrimidines via de novo 127 0066-4197/85/1215-0127

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Adenine~.~*

AMP

IMP ~,~

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~ Inosine ’

Hypoxanthine

GMP

Guanosine

Guanine

~ Xanthlne

~

Oxldase

Uric Acid FigureI Pathways of purineinterconversion in mammalian cells. APRT, adeninephosphoribosyltransferase;ADA, adenosinedeaminase; HPRT, hypoxanthine phosphoribosyl transferase; PNP,purinenucleosidephosphorylase. pathways.Since bacteria and humanshave the ability to "salvage" free purine and pyrimidinebases by nucleotide conversion, cellular pools are economically maintained. Ninety percent of free purines in humansare recycled (60). The enzymeshypoxanthine phosphoribosyltransferase (HPRT)and adenine phosphoribosyltransferase(APRT)catalyze these activities in vertebrates (Figure (54, 55). In 1967, Seegmiller et al demonstrated the importance of purine salvage whenthey identified HPRTdeficiency as the defect in the Lesch-Nyhan syndrome (61, 92). The HPRTgene has now been cloned and characterized (14, 46, 47, 69). This permits the molecularanalysis of mutations resulting Lesch-Nyhanand gouty arthritis secondary to HPRTdeficiency (51). The HPRTgene has been used extensively in the study of cultured mammalian cells because of its location on the X chromosome(functional or true + hemizygosity) and because simple selective media allow for growth of HPRT and HPRT-cells. Thusthe HPRT gene has been the most actively studied locus in investigations of mutational agents. Littlefield exploited the HATcounterselective mediumfor selection of somatic cell hybrids used extensively for humangene mapping(62). Similarly KOhler&Milstein used this selection for hybridomafusions (52). Morerecently this selection has been used by Shapiro for the study of X inactivation mechanisms (74). In this review wewill focus the morerecent molecularstudies of mutationand refer the reader to a variety of earlier reviews of HPRTfor cellular behavior (16, 51, 110).

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Enzymology Hypoxanthine phosphoribosyltransferase (HPRT,EC2.4.2.8) catalyzes the condensation of 5’-phosphoribosyl-l-pyrophosphate (PRPP)and the purine bases hypoxanthine and guanine in the formation of 5’-IMP and 5’-GMP respectively (58). Adeninephosphoribosyltransferase (APRT)catalyzes a ilar salvage reaction with adenine (51). Theseenzymesprovide cells with alternative to the energy-expensivede novosynthesis of nucleotides and play a critical role in the maintenance of intracellular purine nucleotide pools in cells that havea decreasedability to synthesizenewnucleotides[e.g. erythrocytes(24)]. HPRT is found in all cells as a soluble, cytoplasmicenzymeand accounts for 0.005-0.04%of total proteins. Wilson et al have determined the aminoacid sequence for humanerythrocyte HPRTand have shown that each monomer contains 217 aminoacids and has a molecular weight of 24,470 (108). Original estimates of quaternary structure, based on gel filtration chromatographyand analytical sedimentation, predicted dimeric and trimeric structures for functional HPRT,while cross-linking studies and isoelectric focusing of humanmouseheteropolymersidentified tetrameric structures (5, 41,45, 81, 82, 83). In addition to hypoxanthine and guanine, HPRTcan bind and ribosylate a wide range of toxic purine analogues,a characteristic first exploited by somatic cell geneticists whenHPRT-cells were isolated by their resistance to 6thioguanine (6-TG) and 8-azaguanine (8-AG) i95). In 1962 Szybalski + Szybalska developed a counterselective methodfor the isolation of HPRT cells using media containing hypoxanthine(a purine source), aminopterin (an inhibitor of purine and thymidine synthesis), and thymidine, HATmedia (97). The powerof these forward and reverse selection procedures has established HPRTas an indispensable tool in the developmentof mutagentesting, hybridoma, and somatic cell hybrid techniques. The phosphoribosyl moiety is provided by 5’-phosphoribosyl-1pyrophosphate in a transf~rase reaction. Humanerythrocyte HPRTMichaelis constants for guanine and hypoxanthine are 5 x 10 -6 Mand 1.7 x 10-5 M respectively; Km values for PRPPrange from 2 x 10 -4 tO 5.5 × 10 -5 M depending upon enzymesource (35, 37, 58, 66). HPRTrequires magnesium, 2+ / and the specific mechanismof catalysis is greatly influenced by the Mg PRPPratio (89). Underphysiologic conditions, the enzymefirst binds PRPP and then the purine base in the establishmentof a short-lived ternary complex. The phosphoribosyl moiety is transferred to the base and the enzymeand nucleotide dissociate, presumablywith the release of pyrophosphate. HPRT is one of ten catalytically related phosphoribosyltransferasesfoundin manyorganisms.Thesetransferases are involvedin the biosynthesis of purines,

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pyrimidines, and the aromatic amino acids histidine and tryptophan. While these enzymesrecognize a wide range of substrates, they all require divalent metal cations for activity and can use only PRPPas the ribosyl donor (78). Functionally identical HPRT proteins have been purified from hamster, mouse, rat, and humansources (5, 18, 33, 44, 81). Yeast HPRThas also been purified and is a single enzyme, capable of recognizing guanine, hypoxanthine, and xanthine (22, 90). The protozoan Leishmania donovani has one enzymethat recognizes hypoxanthine and guanine and another that recognizes xanthine (99). Leishmania lacks de novo purine synthesis and thus is dependent on "salvage" reactions for purine nucleotides. The bacteria Salmonellatyphimurium and Escherichia coli use independent enzymesfor conversion of hypoxanthine and guanine to their respective nucleotides (20, 32, 73). Based presumedsubstrate and catalytic similarities between these enzymes,Musick predicted that common structural features mayrepresent shared substrate binding or active sites (78). Argoset al subsequentlyreported sequencecomparison of three transferases (bacterial and humanenzymes)and described a potential nucleotide binding domainbased on secondaryand tertiary structural homology (4). Tissue Distribution HPRT has been detected in all somatic tissues at low levels (0.005-0.01%of total mRNA) (51). Significantly higher levels are found in the central nervous system (0.02-0.04%) (51, 70). In normal mice, rats, monkeys, and humans, direct enzymeassay has been used to demonstratehigh HPRT-specifi 9 activity in brain tissues (43, 49, 57). Furthermore,assay of proteins synthesizedin vitro from mRNA isolated from Chinese hamster brain, testes, and liver tissues demonstrateda sevenfold elevation in brain HPRT activity over other tissues (70). Studies of regional central nervous system(CNS)levels of HPRT activity indicate that in rats and humans,the basal ganglia have the highest level (40, 49, 88). The de novosynthesis of purines is lowest in the caudate nucleus (the major componentof the basal ganglia), suggesting this CNSregion may be dependent upon salvage of preformed bases for nucleotide pool maintenance (101). This inverse relationship betweende novosynthesis and salvage occurs in other tissues as well. Erythrocytes, platelets, and bonemarrowcells produce little amidophosphoribosyltransferase(AMPRT, the rate limiting enzymein de novo purine synthesis) yet are rich in HPRT(24, 42, 59, 65). These tissue differences suggestthat the role for this enzyme in different tissues is morethan simple "housekeeping"and that it maybe important in the pathogenesis of the Lesch-Nyhan syndrome. HPRT Gene Structure Human,mouse, and hamster HPRTproteins are encoded by a single X-linked structural gene. The first evidence that the HPRT gene was X linked camefrom

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pedigree analysis of families that had inborn deficiencies of HPRT(61). Ricciuti & Ruddle demonstrated, via mouse-humansomatic cell hybrids, synteny between HPRTand two genes on the long arm of the X chromosome-glucose 6-phosphate dehydrogenase (G6PD) and phosphoglycerate kinase (PGK)(87). Subsequent mapping by Pal and Sprenkle localized the human HPRTgene to Xq26-27 (84). Isolation of cloned sequences complementaryto HPRTmRNA was originally madedifficult by the low levels of normal expression of the gene. HPRT mRNA accounts for no more than 0.04%of the total brain message produced (70). In 1981, the isolation of a mouseneuroblastomacell line (NBR4) expresses high levels of a variant HPRTprotein provided the mRNA source from which murine HPRTcDNAwas first cloned (68). Reversion of a 6TG resistant, HPRT-mouseneuroblastomacell line led to the isolation of HAT+ cells that had elevated levels of an unstable HPRTmutant resistant, HPRT enzy_me.While immunoprecipitationstudies of cellular extracts indicated a tenfold elevation of HPRTprotein, enzymeactivity was only 10-25%of wild-type levels. In vitro translation of mRNA isolated from this cell line indicated a 20-50-fold increase ofHPRTmRNA (70). NBR4HPRTwas shown to have a decreased affinity for both hypoxanthine and PRPPrelative to wild-type HPRT. Sequence comparison of HPRTcDNAfrom NBR4and wild-type cDNA has shownthat a point mutationis responsible for this unstable character. NBR4survival in HATmedia was achieved by amplification of a mutant gene. HPRTcDNArecombinants were identified by differential hybridization procedures using NBR4cDNA(50 copy amplification) and cDNAfrom the parental cell line (1 copy) (14). The identity of HPRTcDNAclones confirmedby in vitro translation of mRNA selected by hybridization to candidate HPRTcDNA recombinants. Probes generated in this fashion were used to isolate a recombinant, pHPT5,that contains an open reading frame of 654 nucleotides preceded by 100 nucleotides of 5’ untranslated sequence and followed by 550 nucleotides of 3’ untranslated sequence (53). Cross-species sequence homology facilitated the isolation of human and hamster HPRT cDNA recombinants using pHPT5as a probe (15). In an alternative approach, Jolly et al used a humanHPRTgenomicfragment, isolated from transfected mousecells, as a probe to isolate a functional HPRTcDNA clone (46, 47). Nucleic acid sequence of these clones was in agreement with amino acid sequence of humanerythrocyte HPRT(109). The humancDNAopen reading frame begins with an ATGinitiation codon, codes for a protein of 218 aminoacids, and ends with a typical TAAtermination codon(47). Cleavageof the initial methioninefrom the nascent polypeptidehad previously been shownby Wilsonet al to be a post-translational event that resulted in the 217 amino acid monomer(109). Northern analysis of human

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poly(A)÷ RNAusing these cDNAisolates has confirmed a message size of 1600 nucleotides (112). Sequence comparison between mouse, hamster, and human cDNAsindicates that homologybetweenthese three species is >95%in coding regions and falls to ~80%in 5’ and 3~ nontranslated regions (19). The differences between the mouse and humansequences result in seven amino acid substitutions, five of whichare conservedchangeswith respect to aminoacid class. The isolation of these cDNA recombinantshas facilitated the characterization of the mouseand humanHPRTgene structure. Analysis of overlapping h recombinants has indicated that humanand mouse HPRTgenes have nine exons within a 44 kb expanse of genomic DNA(Figure 2; see also 69, 85). (Patel et al, paper submitted). The intron/exon junctions for both species are identical. The nine exonsrange in length from18 to 593 bp in miceand from 18 to 637 in humans. In addition to the functional X-linked HPRTgene, four homologousautosomal sequences have been detected and localized in man (85). These seq.uences are processed pseudogenespresumably derived from intronless RNA intermediates. Southern analysis of DNAfrom a panel of human-Chinese hamster somatic cell hybrids indicated that two of these sequences were localized to chromosome 11, one to chromosome3, and another to the region betweenp 13 and q 11 on chromosome 5. Single autosomal sequences have been identified in mouseand hamster DNAbut have not been characterized. Size and transcription data suggest they are unexpressed intronless pseudogenes (28). HPRT Gene Expression Asstated previously,HPRT is expressedin all tissues, albeit at different levels. Meltonet al have shownthat in the mouseHPRT gene there is no evidence for the presence of a CAAT box and that the nearest sequence corresponding to a "TATAA" box (normally located 20-30 bp upstream from structural genes) occurs >700 bp 5’ to the cap site (Figure 2; see also 64). The nucleic acid sequences 5’ to the start site for both the mouseand humanHPRTgenes are extremely G-C rich (~80%) (Figure 3). The humanHPRTgene also lacks CAAT and TATAA sequencesimmediately.5’ to the initiation site (P. I. Patel, and A. C. Chinault, personal communication). Sequences homologousto the SV40promoter are found in the 5’ flanking region of the HPRTgene but their role in transcription is unresolved. In a description of the hamster HMG CoA reductase gene, Reynolds et al noted a similar lack of CAATand TATAA sequencesand the presence of a G-Crich promoter region containing three 6 bp (CCGCCC) repeats (86). By meansof deletion and mutation analysis (11,27), this signal has been shownto have promoter function for the SV40early gene and the herpes thymidine kinase gene. The humanadenosine deaminase gene

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also lacks 5’ CAAT and TATAA sequences and contains a G-Crich region with five GGGCGGG repeats (100). Analogous promoter structures have been reported for the humanDHFRand the X-linked PGKgenes (17, 93). It interesting that each of these promoters contains the sequence 5, GGGCGG 3, (or its complement)from32 to 42 bp upstreamfrom the cap site. Eachof these housekeepinggenes is expressed in all tissues, and each appears to share commonnucleic acid sequences, possibly regulatory sequences (67). Two X-linked genes for clotting factors VIII and IX showno appreciable nucleic acid sequence homologyin their promoter regions to the HPRT gene (3, 29). All of these genes contain TATAA-like sequences and are expressed in specific tissues. + and HPRT-cells, this Since selective media permit isolation of HPRT locus provides an excellent opportunity to examinestructural differences between active and inactive X chromosomes.Yen and coworkers have suggested that patterns of methylation,rather than methylationof specific sites, correlate with maintenanceof X-chromosome inactivation (113). By using methylationsensitive restriction enzymesto analyze DNA from somatic hybrids containing active or inactive X chromosomes,they have demonstratedhypomethylationof active X chromosomes relative to inactive X chromosomes in the region 5 ’ to the HPRTstructural gene. They found no specific restriction site whose methylationwas directly correlated with gene activity. Wolfet al described a "consensus methylation pattern" for active and inactive HPRTgenes using a similar approach (111). Both studies associated 5’ hypomethylationand hypermethylationwith active HPRTgenes. While these studies have suggested that differences in methylation patterns exist betweenactive and inactive X chromosomes,no cause and effcct relationship has been established and many questions concerning transcriptional repression vs chromosome inactivation remain unanswered. The molecular basis of tissue variability in the expression of HPRThas recently been addressed by studies of transgenic mice that express a human HPRTminigene. Recombinant molecules containing the human HPRTcDNA flanked by the mousemetallothionein promoter (5’), and the humangrowth hormonepolyadenylation signal (3’), were injected into single cell mouse embryos. These embryoswere implanted into pseudopregnant foster mothers 2+ and the resulting pups examinedfor expression of humanHPRT.UponCd induction, transgenic mice expressed the humanenzymevariably in many tissues; however,expressionwasconsistently elevated in brain tissue. Since the metallothionein promoter and the growth hormonepolyadenylation signal do not normally influence CNSexpression, this study suggests that sequences within the humanHPRTtranscript (cDNA)influence CNSexpression presumably via increased mRNA synthesis or stability (94a).

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Pathogenesis

of HPRT Deficiency

Deficiency of HPRTin manresults in a spectrum of disease, the severity of which is dependentupon the extent of the deficiency. Completedeficiency of the enzymeis associated with the Lesch-Nyhansyndrome (incidence 1 in 100,000), while partial deficiency is associated with gout (incidence 1 in 200 amongmales with gout) (51). The Lesch-Nyhansyndrome, first described 1964, is clinically characterized by hyperuricemia, choreoathetoid movements, spasticity, hyperreflexia, mental retardation, and compulsiveself-injurious behavior (92). Developmentaldelay is evident at three to six monthsof age while pyramidal and extrapyramidal involvements becomeapparent within a year of birth (91). The relationship between HPRTdeficiency and CNS dysfunction in Lesch-Nyhanpatients remains unclear. Recent biochemical and histologic studies suggest that inappropriate developmentof nigrostriatal dopaminergic neurons maybe important (31). Postmortemanalysis of brain tissue from three Lesch-Nyhanpatients revealed that indexes of dopamine function were reduced by 70-90%in the basal ganglia (64). During normal development,arborization of these neuronal pathwaysoccurs at the same time as increased HPRTlevels in the CNS(2, 63). Moreover, behavioral changes including self-mutilation occur whendrugs are used to arrest developmentof these terminal dopaminergic neurons (7, 13). Since it has been shownthat dopamine receptor binding is regulated by guanine triphosphate and diphosphatenucleotides, perhaps purine imbalanceduring developmentresults in inappropriate synaptogenesisin the basal ganglia (21). Hyperuricemia in these patients is associated with an increase in the rate of de novo purine synthesis (51). This increase is probably due to the combined effects of debreasedfeedbackinhibition by nucleotide end products and cellular loss of hypoxanthine. Lymphoblasts from both normal and Lesch-Nyhan patients are capable of accelerated de novosynthesis whencultured in hypoxanthine-depleted media, but only normal cells reduce de novo synthesis upon addition of exogenoushypoxanthine(39). The inability of Lesch-Nyhan cells convert intracellular hypoxanthineto IMPleads to cellular loss and subsequent conversion of hypoxanthine to uric acid by hepatocyte xanthine oxidase. In patients with partial HPRT deficiency, excessive production of uric acid leads to hyperuricemia,nephrolithiasis, and a severe form of gout. Evidenceof spasticity, cerebellar ataxia, and mild mentalretardation can be foundin 20%of patients with partial HPRTdeficiency, but these individuals do not become self-mutilating. Allopurin~l, an inhibitor of xanthine oxidase, is helpful in reducing the uric acid production in these patients but has no effect on the neurological manifestations of disease. Fluphenazine, an antagonist of ’postsynaptic dopaminereceptors, is purportedly effective in reducing selfmutilating behavior in these patients, presumably via interaction with D~ nigrostriatal receptors.

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Mutations

AT THE HPRT

in Cultured

137

LOCUS

Cells

Three factors have contributed to the development of the HPRTgene as an important test systemin the study of mammalian cell mutagenesis.First, as an X-linked gene, HPRTis hemizygousin male cells and functionally hemizygous in female cells, makingthis locus particularly useful in the examination of recessive mutations. Secondly, HPRT is a nonessential enzymefor cells growing in culture since purines can be provided by de novo synthesis. Finally, + powerful selection procedures exist for the isolation of HPRT-or HPRT cells, makingmeasurementof mutation and reversion rate possible (95, 97). Purine analogue resistance in mammalian cells results from an inability to Convert these analogues to toxic nucleotides, an activity dependent upon functional HPRTo Thusquantitation of 6-thioguanine or 8-azaguanineresistant cells provides a simple measureof mutation frequency (76). HPRTantibodies and cDNA probes permit the detailed characterization of individual mutations and mechanismsof mutation (8). Suchstrategies were utilized by Fuscoe et al in their analysis of 19 HPRT Chinese hamster mutants (28). Southern blots of DNAfrom ten spontaneous and nine UV-inducedmutants detected two subclones (one spontaneous, one UVirradiated) with major deletions at the HPRT locus. Cytogeneticanalysis of these cell lines showed the deletions to be associated with chromosomal translocations. DNAfrom the remaining 17 subclones appeared normal in Southernblots. Noneof these mutantlines showedHPRTactivity, and only one producedmaterial that could cross-react with antibodies raised against HPRT. Subsequent analysis of mRNAsynthesized in 20 HPRT-, CRM-Chinese hamster lines, in whichno genomicalterations were detected by Southernblots, indicated that 18 of these mutants produced normal levels of HPRTmRNA (79). Cumulativelythese data suggest that point mutations in the HPRT locus account for the majority of spontaneous or UV-inducedmutants. Recent work by Simonshas shownthat 12 of 19 mutantChinese hamster cell lines generated by exposureto ionizing radiation (600 rads) contained deletions detectable Southern analysis (J. Simons, personal communication).Crawfordfound four of six a-irradiated humanfibroblast lines selected for 6-TGresistance to have HPRTgene deletions (B. Crawford, personal communication). These studies demonstratethe usefulness of recombinantDNA techniques in the characterization of mutational mechanisms. Anapproachfor differentiating betweenbase pair substitution and frameshift mutants of the HPRTgene has recently been described by Stone-Wolff & Rossman(94a). This methodutilizes a reversion systemdependentupon single base pair substitution reversion following N-mettiyl-N’-nitro-nitrosoguanidine (MNNG) treatment, or frameshift reversion following ICR-191treatment.

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Distinction betweenthese twotypes of mutationsis predicated on the idea that frameshift mutations most frequently revert by a second frameshift mutation and that base substitution mutationsmostfrequently revert by another substitution mutation. Thusframeshift mutations conferring 6-TGresistance will more frequently revert to the 6-TGsensitive phenotype following treatment with ICR-191(a frameshift mutagen)than with MNNG (a substitution mutagen), converse being true for base substitution mutations. Different reversion frequencies have been observed for different HPRT-cell lines treated with these compounds(94). DNAsequence analysis will be needed to specify the nature of these mutantsand revertants, althoughsuch analysis mightbe difficult given the large size of the HPRT gene and the difficulties inherent in cloning and sequencing HPRTmutants. Two developments may make the HPRTlocus more amenable to rapid molecular analysis. First, somatic cell lines have been developedin which a single intronless HPRTminigene functions in place of a deleted endogenous gene, thus effectively reducingthe target of mutationfrom44 kb to 1 kb in size (S. M. W. Chang, personal communication). Second, Myers et al have developed a simple methodof identifying and locating point mutations based on novel denaturation properties of wild-type:mutant heteroduplexes during gradient electrophoresis (71). These developments may overcome the disadvantageof the large size of the HPRT gene in the analysis of point mutants. To examinethe in vivo mutagenicity of toxic compounds in mice, Jones et al have developed a clonogenic assay to quantify 6-TGresistant spleen lymphocytes isolated from mice that have been treated with various drugs (48). These r coloinvestigators have demonstrateda linear dose-related increase of 6-TG nies from mice treated with ethylnitrosourea (ENU).Fifteen days following intraperitoneal injections of ENU,isolated lymphocyteswere plated at high density (4-8 × 105 cells/250 t~1 well) in the presence of 6-TGand concanavalin A. 6-TGresistant colonies were scored after 8 days in selective media. This system offers the advantages of a direct method of measuring in vivo mutagenicity of manycompoundsand the ability to expand cloned cells for mutantcharacterization at the genetic level. Albertini (1) and Morley(75) have reported the ability to measurethe rate 6-TGresistance from peripheral T cells grownin culture. Turner et al reports that a relatively high proportion (57%) of spontaneous mutations in human lymphocytesinvolve substantial gone alterations (deletions, exon amplification. s, etc) that are not evident cytogenetically(98). Twelveof 21 6-TGresistant clones had altered HPRT Southernpatterns, while none of the unselected clones from the same individual showedalteration. Since stringent 6-TGselection conditions were used, however, mutations resulting in undetectable HPRT activity were favored. Geneswith deletions and/or other major gene rearrangements mayhave appeared at an artificially high frequency. These results are

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interesting in light of observations that only 18%of patients with the LeschNyhansyndrome and 10%of 6-TG resistant hamster cells have major gene rearrangements(79, 112). The data suggest that in vivo mutations of somatic cells mayoccur more commonly by gene deletion. This maybe relevant to the deletion mutationsassociated with the neoplastic transformationof retinoblastoma and Wilms’ tumor (9, 23, 56).

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Mutations

in Man

Since the description of the familial nature of the Lesch-Nyhansyndromeand the biochemicalelucidation of its etiology, HPRTdeficiency has been a model for the study of X-linked disease. Advances in our knowledge of HPRT genomicand protein structure have allowed characterization of a numberof heterogeneousmutant alleles. First reports characterizing HPRT deficiency in humancell lysates described enzymaticdata suggestive of mutational heterogeneity (66, 92). Thesechanges included altered sensitivity to product inhibition, thcrmolability, Kmvalue alterations, and changesin electrophoretic mobility (6, 10, 25, 34, 38, 50, 66, 92, 96). Withthe developmentof effective purification and protein sequencing protocols, Wilsonet al have been able to compareaminoacid sequence data from three HPRTdeficient patients with gouty arthritis and one Lesch-Nyhan patient (Figure 4; see also 104, 107, 108, 110). Oneof the patients with gout, HPRTT .... to, has a mutationresulting in the replacementof an arginine residue with glycine at position 50 (107). A single to G base changein the arginine codoncan explain this substitution and would result in the loss of a TaqIrestriction site, a prediction confirmedby Southern analysis (105). HPRTLondon and HPRTMunich have serine to leucine (position 109) and serine to arginine (position 103) changes respectively (108, 110). Each of these mutationsresults in gouty arthritis. HPRTKinston occurs in association with the Lesch-Nyhansyndrome and has a mutation that substitutes asparagine for arginine at position 193 (106). Whilethis methodof mutantanalysis has proved valuable in the description of pathogenicmutations, it is limited to those that result in the productionof a protein product. A recent survey of cell extracts from HPRT-deficientpatients indicates that 11 of 15 Lesch-Nyhanpatients producedno material recognized by HPRTantibodies (J. T. Stout, and J. M. Wilson,in preparation). Mutationsthat inhibit transcription, RNA processing, or involve major rearrangements of the HPRTgene generally fail to produce detectable protein and are not amenableto this type of examination. Studies by Yanget al examinedthe organization of the HPRTgene in 28 unrelated Lesch-Nyhanpatients (112). Five (18%) of the patients screened showedunique Southern patterns suggestive of major gene alterations. These alterations include a total gene deletion (RJK853), two3’ deletions (RJK849,

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GM3467), a potential internal insertion (GM2227), and an exon duplication (GM1662). All of these patients are cytogenetically normaland fail to produce a stable protein product. Noneof the deletion patients producea stable mRNA. GM1662 produces an HPRTmRNA approximately 200 nt larger than wild type. This is consistent with Southernand sequencedata that have confirmeda perfect duplication of exons 2 and 3 (D. S. Konecki,in preparation). Whilethis patient produces a stable mRNA, enzymeactivity is undetectable and no cross-reactive material can be detected in Westernblots. Family study of the Lesch-Nyhanpatient GM1662permitted the identification of the origin of this mutational event. Anabnormal4.1 kb Bgl II band, characteristic of the duplication mutation,was identified in the propositus, his mother, and two sisters but was absent in his maternal grandmother.Thesedata indicate that this mutationoriginated in the germline of a maternalgrandparent and gave rise to an asymptomaticcarrier female. Twoadditional family studies provide data on the gametic origin of mutations (J. T. Stout, in preparation). The Lesch-Nyhanpatient RJK853 has X-specific HPRTsequences while his mother has two normal copies of the HPRT gene, indicating that the deletion event occurredin a maternal gamete.In a second example, the patient (RJK983) has a partial 3’ deletion with the breakpoint 3’ to the fourth exon. The mother of this patient has two normal copies of the HPRTgene, again identifying a maternal gametic deletion event as the origin of the newmutation. Thesefindings support Haldane’sprediction that lethal, X-linked mutations that confer no selective advantage to heterozygotes will frequently arise as spontaneous new mutations (36). HPRTdeficiency syndromesshowno ethnic predilection. Males with the Lesch-Nyhansyndromefail to reproduce, and female heterozygotes have no selective advantage. The maintenance of the Lesch-Nyhansyndromein the population is thus dependent upon the sporadic occurrence of these heterogenous mutations. In earlier studies, Franckeet a! compiledthe results of carrier detection tests on mothers and maternal grandmothersfrom 54 families with a single affected child (26). Thesetests suggest that 11 of 54 affected maleswere the result newmutations. In addition, 10 of the heter0zygous mothers were shownto be carriers of newmutations. Molecularanalysis of the origins of mutations in such families will permit identification of paternal and maternalgametic mutations as well as determine mechanismsof mutation for each. To address the question of genetic heterogeneity amongmutations of the HPRTgene, Southern, Northern, Western, kinetic, and immunoquantitative data have been compiledfor 24 unrelated patients with HPRT deficiency. Using these molecularparametersto create a compositepicture for each mutant, it was found that 14 of 24 patients have unique genetic backgrounds, suggestive of extensive mutant heterogeneity.

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Indirect evidence for genomicheterogeneity in this population camefrom limited haplotype analysis. TwoHPRT-linkedrestriction fragment length polymorphisms (RFLPs) have been described (12, 80). In 1983, Nussbaum scribed a three-allele BamHIRFLPthat occurs within the HPRTgene. These alleles are expressedphenotypicallyon Southernblots as three distinct pairs of fragments:(a) a 22 kb/25 kb pair, (b) a 12 kb/25kb pair, and (c) a 22 kb/18 pair. An additional two-allele TaqI RFLPwas reported for an anonymous sequence (DXS-10)separate from, but closely linked to (95% confidence limits, 0< 15cM),the HPRT gcne. Thesealleles are represented as 5 kb or 7 kb bands on Southernblots. TheseRFLPpatterns can be combinedto establish six haplotypes,four of whichare represented in this groupof 24 patients. Themost frequent haplotype pattern (68%) was that combiningthe 22 kb/25 kb BamHI allele and the 5 kb Taql allele (22/25/5 pattern). The 22/25/7 pattern represented 9%of this group, and the 12/25/5 and 22/18/5 patterns wereeach seen in a single patient. Patients havingunique, readily identifiable major genealterations accounted for 9%surveyed (112; J. T. Stout, and J. M. Wilson, in preparation). This study reports that a minority of HPRT deficient patients are able to producea protein product detectable by immunologicmethods. The majority of + show the less severe, gouty phenotype. Only 15%of those that are CRM patients surveyed failed to produce a detectable HPRTmRNA and, as expected, those patients exhibited the severe Lesch-Nyhan phenotype.In general, the more severe the molecular defect (i.e. major gene alteration, HPRT mRNA-,etc) the more severe the clinical phenotype.

GENE TRANSFER

AND THERAPEUTIC

PROSPECTS

The transfer of an expressible HPRTcDNAmolecule into HPRT-deficient mousecells was first described by Jolly et al (47). Transfection of mouseLA cells with a plasmid containing the humanHPRTcDNA resulted in the appearance of HAT-resistant colonies at a frequency of approximately 1 -3 x .10 Brennandand coworkers subsequently reported the expression of humanand Chinese hamster cDNArecombinantsin fibroblasts from Lesch-Nyhanpatients and HPRT-deficient Chinese hamster cells (15). These recombinants were introduced into cells by calcium phosphatecoprecipitation, and HAT-resistant colonies were recovered at frequencies of 1.9 x 10-4 (hamster cDNA in human cells) and 5 x 10-4 (humaneDNAinto humancells). These frequencies are comparable to those obtained by Mulligan and Berg, who introduced into a similar Lesch-Nyhancell line a chimeric plasmid containing the bacterial guanine phosphoribosyltransferase (gpt) gene and SV40promoter elements (77).

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Microinjection techniques have substantially enhanced gene transfer efficiencies. WhenHPRTeDNA molecules, under the control of a variety of promoters, are microinjected into HPRT-cells, HAT-resistantcolonies appear at frequencies 10~--103 fold greater than frequencies obtained by calcium phosphate precipitation (S. M. W.Chang, personal communication).Studies involving the introduction of human HPRTeDNAsequences into mouse embryosvia microinjection suggest that humanminigenes can becomestable componentsof the mousegenome.These genes can be expressed in a variety of tissues and can be transmitted to progenyin a Mendelianfashion. Alternatives to these relatively inefficient and labor-intensive methodsof genetransfer have been found in the form of naturally occurring RNAand DNAviruses. The unique structure and capabilities of viruses maymakethem the ideal gene transfer systemin mammalian cells. Retroviruses are vectors whoselife cycle dependsuponthe integration, replication, and expressionof their genetic material in host cells (30, 102). Theretroviral systemis very efficient (100% cells infected carry an integrated copy of the gene), and manycell types refractory to CaPO4transfection can be infected with retroviruses. Infection of cultured Lesch-Nyhancells with amphotrophic retroviruses containing humanHPRTcDNA sequences was shownby Willis et al to result in the production of enzymatically active humanHPRT(103). Concomitantwith expression of HPRT in these cells was the partial correction of other aberrant metabolicparameters,i.e. elevated purine excretion and increased intracellular hypoxanthine. A. Miller et al have reported using similar viruses to infect mousebone marrowcells that were subsequently transplanted into lethally irradiated mice (72). In these studies, HPRT-virusproduction was detected mousespleen and bone marrowcells as long as 133 days after transplantation. HumanHPRTprotein was detected in spleen cells for two months. These studies suggest that retroviral vectors mayserve as an efficient mechanism for somatic gene transfer. The ultimate goal of medical geneticists involved with gene regulation and transfer is the management of inborn errors of metabolismat. the genetic rather than symptomatic level. Gene therapy will depend upon the development of effective delivery systems, capable of safely introducing newgenetic material that can be expressed at the appropriate times and in the appropriate tissues. Lesch-Nyhansyndromemaybe amenableto gene therapy but available data do not assure success. It is not clear if geneintroduction into bone marrowcells will influence CNS dysfunction in these patients since therapeutic bonemarrowtransplantation for the Lesch-Nyhansyndromehas not been reported. Manyaspects concerning the delivery of genes via retroviral vectors remainunclear. Questionsconcerning tissue requirements and temporal restraints on HPRT expression need to be answered. The devastating nature of this disease, combinedwith the lack of

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therapeutic altematives, warrants the investigation tial treatment for Lesch-Nyhan patients.

of gene transfer

as a poten-

ACKNOWLEDGMENTS

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The authors wish to thank Lillian Tanagho for her help in the preparation of this manuscript. JTS gratefully recognizes support from the Philip Michael Berolzheimer Medical Scientists Fellowship. CTC is an Investigator for the Howard Hughes Medical Institute. This work was supported by NIH Grant # AM31428. Literature

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Annu. Rev. Genet. 1985.19:127-148. Downloaded from arjournals.annualreviews.org by Auburn University on 01/06/07. For personal use only.

Annu. Rev. Genet. 1985.19:127-148. Downloaded from arjournals.annualreviews.org by Auburn University on 01/06/07. For personal use only.