hprt: gene structure, expression, and mutation · 2017-10-18 · 130 stout & caskey...

Ann. Rev. Genet. 1985. 19:127~18Copyright © 1985 by Annual Reviews Inc. 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, andCell Biology, Baylor College of Medicine, Houston, Texas 77030

CONTENTS

INTRODUCTION AND PERSPECTIVES ....................................................... 127

NORMAL ENZYME FUNCTION AND CHARACTERISTICS ............................. 129Enzymology ........................................................................................ 129Tissue Distribution ................................................................................ 130HPRT Gene Structure ............................................................................ 130HPRT Gene Expression .......................................................................... 132Pathogenesis of HPRT Deficiency ............................................................. 136

MUTATIONS AT THE HPRT LOCUS ........................................................... 137Mutations in Cultured Cells ..................................................................... 137Mutations in Man ................................................................................. 139

GENE TRANSFER AND THERAPEUTIC PROSPECTS ..................................... 142

INTRODUCTION AND PERSPECTIVES

The biosynthesis of purine and pyrimidine nucleotides is a crucial process for

all cells since these molecules are the direct precursors of DNA and RNA and

frequently participate as coenzymes in enzymatic reactions. With few ex-

ceptions, organisms are able to synthesize purines and pyrimidines via de novo

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128 STOUT & CASKEY

Adenine~.~* AMPIMP ~,~ GMP

~ Inosine ’ Guanosine

Hypoxanthine Guanine

~ Xanthlne

~ Oxldase

Uric AcidFigure I Pathways of purine interconversion in mammalian cells. APRT, adenine phosphoribo-syltransferase; ADA, adenosine deaminase; HPRT, hypoxanthine phosphoribosyl transferase;PNP, purine nucleoside phosphorylase.

pathways. Since bacteria and humans have the ability to "salvage" free purineand pyrimidine bases by nucleotide conversion, cellular pools are economicallymaintained. Ninety percent of free purines in humans are recycled (60). Theenzymes hypoxanthine phosphoribosyltransferase (HPRT) and adenine phos-phoribosyltransferase (APRT) catalyze these activities in vertebrates (Figure (54, 55). In 1967, Seegmiller et al demonstrated the importance of purinesalvage when they identified HPRT deficiency as the defect in the Lesch-Nyhansyndrome (61, 92). The HPRT gene has now been cloned and characterized(14, 46, 47, 69). This permits the molecular analysis of mutations resulting Lesch-Nyhan and gouty arthritis secondary to HPRT deficiency (51).

The HPRT gene has been used extensively in the study of cultured mamma-lian cells because of its location on the X chromosome (functional or truehemizygosity) and because simple selective media allow for growth of HPRT+

and HPRT- cells. Thus the HPRT gene has been the most actively studied locusin investigations of mutational agents. Littlefield exploited the HAT coun-terselective medium for selection of somatic cell hybrids used extensively forhuman gene mapping (62). Similarly KOhler & Milstein used this selection forhybridoma fusions (52). More recently this selection has been used by Shapirofor the study of X inactivation mechanisms (74). In this review we will focus the more recent molecular studies of mutation and refer the reader to a variety ofearlier reviews of HPRT for cellular behavior (16, 51, 110).


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HPRT MUTATIONS 129

NORMAL ENZYME FUNCTION ANDCHARACTERISTICS

Enzymology

Hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8) catalyzes thecondensation of 5’-phosphoribosyl-l-pyrophosphate (PRPP) and the purinebases hypoxanthine and guanine in the formation of 5’-IMP and 5’-GMPrespectively (58). Adenine phosphoribosyltransferase (APRT) catalyzes a ilar salvage reaction with adenine (51). These enzymes provide cells with alternative to the energy-expensive de novo synthesis of nucleotides and play acritical role in the maintenance of intracellular purine nucleotide pools in cellsthat have a decreased ability to synthesize new nucleotides [e.g. erythrocytes (24)].

HPRT is found in all cells as a soluble, cytoplasmic enzyme and accounts for0.005-0.04% of total proteins. Wilson et al have determined the amino acidsequence for human erythrocyte HPRT and have shown that each monomercontains 217 amino acids and has a molecular weight of 24,470 (108). Originalestimates of quaternary structure, based on gel filtration chromatography andanalytical sedimentation, predicted dimeric and trimeric structures for func-tional HPRT, while cross-linking studies and isoelectric focusing of human-mouse heteropolymers identified tetrameric structures (5, 41,45, 81, 82, 83).

In addition to hypoxanthine and guanine, HPRT can bind and ribosylate awide range of toxic purine analogues, a characteristic first exploited by somaticcell geneticists when HPRT- cells were isolated by their resistance to 6-thioguanine (6-TG) and 8-azaguanine (8-AG) i95). In 1962 Szybalski Szybalska developed a counterselective method for the isolation of HPRT+

cells using media containing hypoxanthine (a purine source), aminopterin (aninhibitor of purine and thymidine synthesis), and thymidine, HAT media (97).The power of these forward and reverse selection procedures has establishedHPRT as an indispensable tool in the development of mutagen testing, hybrido-ma, and somatic cell hybrid techniques.

The phosphoribosyl moiety is provided by 5’-phosphoribosyl-1-pyrophosphate in a transf~rase reaction. Human erythrocyte HPRT Michaelisconstants for guanine and hypoxanthine are 5 x 10-6 M and 1.7 x 10-5 Mrespectively; Km values for PRPP range from 2 x 10-4 tO 5.5 × 10-5 Mdepending upon enzyme source (35, 37, 58, 66). HPRT requires magnesium,and the specific mechanism of catalysis is greatly influenced by the Mg2+ /PRPP ratio (89). Under physiologic conditions, the enzyme first binds PRPPand then the purine base in the establishment of a short-lived ternary complex.The phosphoribosyl moiety is transferred to the base and the enzyme andnucleotide dissociate, presumably with the release of pyrophosphate.

HPRT is one of ten catalytically related phosphoribosyltransferases found inmany organisms. These transferases are involved in the biosynthesis of purines,


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130 STOUT & CASKEY

pyrimidines, and the aromatic amino acids histidine and tryptophan. Whilethese enzymes recognize a wide range of substrates, they all require divalentmetal cations for activity and can use only PRPP as the ribosyl donor (78).Functionally identical HPRT proteins have been purified from hamster, mouse,rat, and human sources (5, 18, 33, 44, 81). Yeast HPRT has also been purifiedand is a single enzyme, capable of recognizing guanine, hypoxanthine, andxanthine (22, 90). The protozoan Leishmania donovani has one enzyme thatrecognizes 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 Salmonella typhimur-ium and Escherichia coli use independent enzymes for conversion of hypoxan-thine and guanine to their respective nucleotides (20, 32, 73). Based presumed substrate and catalytic similarities between these enzymes, Musickpredicted that common structural features may represent shared substrate bind-ing or active sites (78). Argos et al subsequently reported sequence comparisonof three transferases (bacterial and human enzymes) and described a potentialnucleotide binding domain based on secondary and tertiary structural homology(4).

Tissue Distribution

HPRT has been detected in all somatic tissues at low levels (0.005-0.01% oftotal mRNA) (51). Significantly higher levels are found in the central nervoussystem (0.02-0.04%) (51, 70). In normal mice, rats, monkeys, and humans,direct enzyme assay has been used to demonstrate high HPRT-specifi9 activityin brain tissues (43, 49, 57). Furthermore, assay of proteins synthesized in vitrofrom mRNA isolated from Chinese hamster brain, testes, and liver tissuesdemonstrated a sevenfold elevation in brain HPRT activity over other tissues(70). Studies of regional central nervous system (CNS) levels of HPRT activityindicate that in rats and humans, the basal ganglia have the highest level (40,49, 88). The de novo synthesis of purines is lowest in the caudate nucleus (themajor component of the basal ganglia), suggesting this CNS region may bedependent upon salvage of preformed bases for nucleotide pool maintenance(101). This inverse relationship between de novo synthesis and salvage occursin other tissues as well. Erythrocytes, platelets, and bone marrow cells producelittle amidophosphoribosyltransferase (AMPRT, the rate limiting enzyme in denovo purine synthesis) yet are rich in HPRT (24, 42, 59, 65). These tissuedifferences suggest that the role for this enzyme in different tissues is more thansimple "housekeeping" and that it may be important in the pathogenesis of theLesch-Nyhan syndrome.

HPRT Gene Structure

Human, mouse, and hamster HPRT proteins are encoded by a single X-linkedstructural gene. The first evidence that the HPRT gene was X linked came from


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HPRT MUTATIONS 131

pedigree analysis of families that had inborn deficiencies of HPRT (61).Ricciuti & Ruddle demonstrated, via mouse-human somatic cell hybrids,synteny between HPRT and 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 humanHPRT gene to Xq26-27 (84).

Isolation of cloned sequences complementary to HPRT mRNA was original-ly made difficult by the low levels of normal expression of the gene. HPRTmRNA accounts for no more than 0.04% of the total brain message produced(70). In 1981, the isolation of a mouse neuroblastoma cell line (NBR4) expresses high levels of a variant HPRT protein provided the mRNA sourcefrom which murine HPRT cDNA was first cloned (68). Reversion of a 6TGresistant, HPRT- mouse neuroblastoma cell line led to the isolation of HAT-resistant, HPRT+ cells that had elevated levels of an unstable HPRT mutantenzy_me. While immunoprecipitation studies of cellular extracts indicated atenfold elevation of HPRT protein, enzyme activity was only 10-25% ofwild-type levels. In vitro translation of mRNA isolated from this cell lineindicated a 20-50-fold increase ofHPRT mRNA (70). NBR4 HPRT was shownto have a decreased affinity for both hypoxanthine and PRPP relative towild-type HPRT. Sequence comparison of HPRT cDNA from NBR4 andwild-type cDNA has shown that a point mutation is responsible for this unstablecharacter. NBR4 survival in HAT media was achieved by amplification of amutant gene.

HPRT cDNA recombinants were identified by differential hybridizationprocedures using NBR4 cDNA (50 copy amplification) and cDNA from theparental cell line (1 copy) (14). The identity of HPRT cDNA clones confirmed by in vitro translation of mRNA selected by hybridization to candi-date HPRT cDNA recombinants. Probes generated in this fashion were used toisolate a recombinant, pHPT5, that contains an open reading frame of 654nucleotides preceded by 100 nucleotides of 5’ untranslated sequence andfollowed by 550 nucleotides of 3’ untranslated sequence (53). Cross-speciessequence homology facilitated the isolation of human and hamster HPRTcDNA recombinants using pHPT5 as a probe (15). In an alternative approach,Jolly et al used a human HPRT genomic fragment, isolated from transfectedmouse cells, as a probe to isolate a functional HPRT cDNA clone (46, 47).Nucleic acid sequence of these clones was in agreement with amino acidsequence of human erythrocyte HPRT (109).

The human cDNA open reading frame begins with an ATG initiation codon,codes for a protein of 218 amino acids, and ends with a typical TAA terminationcodon (47). Cleavage of the initial methionine from the nascent polypeptide hadpreviously been shown by Wilson et al to be a post-translational event thatresulted in the 217 amino acid monomer (109). Northern analysis of human


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132 STOUT & CASKEY

poly(A)÷ RNA using these cDNA isolates has confirmed a message size of1600 nucleotides (112).

Sequence comparison between mouse, hamster, and human cDNAs in-dicates that homology between these three species is >95% in coding regionsand falls to ~80% in 5’ and 3~ nontranslated regions (19). The differencesbetween the mouse and human sequences result in seven amino acid sub-stitutions, five of which are conserved changes with respect to amino acid class.

The isolation of these cDNA recombinants has facilitated the characteriza-tion of the mouse and human HPRT gene structure. Analysis of overlapping hrecombinants has indicated that human and mouse HPRT genes have nineexons 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 areidentical. The nine exons range in length from 18 to 593 bp in mice and from 18to 637 in humans.

In addition to the functional X-linked HPRT gene, four homologous auto-somal sequences have been detected and localized in man (85). These se-q.uences are processed pseudogenes presumably derived from intronless RNAintermediates. Southern analysis of DNA from a panel of human-Chinesehamster somatic cell hybrids indicated that two of these sequences werelocalized to chromosome 11, one to chromosome 3, and another to the regionbetween p 13 and q 11 on chromosome 5. Single autosomal sequences have beenidentified in mouse and hamster DNA but have not been characterized. Sizeand transcription data suggest they are unexpressed intronless pseudogenes(28).

HPRT Gene Expression

As stated previously, HPRT is expressed in all tissues, albeit at different levels.Melton et al have shown that in the mouse HPRT gene there is no evidence forthe 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 acidsequences 5’ to the start site for both the mouse and human HPRT genes areextremely G-C rich (~80%) (Figure 3). The human HPRT gene also lacksCAAT and TATAA sequences immediately.5 ’ to the initiation site (P. I. Patel,and A. C. Chinault, personal communication). Sequences homologous to theSV40 promoter are found in the 5’ flanking region of the HPRT gene but theirrole in transcription is unresolved. In a description of the hamster HMG CoAreductase gene, Reynolds et al noted a similar lack of CAAT and TATAAsequences and the presence of a G-C rich promoter region containing three 6 bp(CCGCCC) repeats (86). By means of deletion and mutation analysis (11,27),this signal has been shown to have promoter function for the SV40 early geneand the herpes thymidine kinase gene. The human adenosine deaminase gene


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HPRT MUTATIONS 133


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HPRT MUTATIONS 135

also lacks 5’ CAAT and TATAA sequences and contains a G-C rich region withfive GGGCGGG repeats (100). Analogous promoter structures have beenreported for the human DHFR and the X-linked PGK genes (17, 93). It interesting that each of these promoters contains the sequence 5, GGGCGG 3,(or its complement) from 32 to 42 bp upstream from the cap site. Each of thesehousekeeping genes is expressed in all tissues, and each appears to sharecommon nucleic acid sequences, possibly regulatory sequences (67). TwoX-linked genes for clotting factors VIII and IX show no appreciable nucleicacid sequence homology in their promoter regions to the HPRT gene (3, 29).All of these genes contain TATAA-like sequences and are expressed in specifictissues.

Since selective media permit isolation of HPRT+ and HPRT- cells, thislocus provides an excellent opportunity to examine structural differences be-tween active and inactive X chromosomes. Yen and coworkers have suggestedthat patterns of methylation, rather than methylation of specific sites, correlatewith maintenance of X-chromosome inactivation (113). By using methylation-sensitive restriction enzymes to analyze DNA from somatic hybrids containingactive or inactive X chromosomes, they have demonstrated hypomethylation ofactive X chromosomes relative to inactive X chromosomes in the region 5 ’ tothe HPRT structural gene. They found no specific restriction site whosemethylation was directly correlated with gene activity. Wolf et al described a"consensus methylation pattern" for active and inactive HPRT genes using asimilar approach (111). Both studies associated 5’ hypomethylation and hypermethylation with active HPRT genes. While these studies have suggestedthat differences in methylation patterns exist between active and inactive Xchromosomes, no cause and effcct relationship has been established and manyquestions concerning transcriptional repression vs chromosome inactivationremain unanswered.

The molecular basis of tissue variability in the expression of HPRT hasrecently been addressed by studies of transgenic mice that express a humanHPRT minigene. Recombinant molecules containing the human HPRT cDNAflanked by the mouse metallothionein promoter (5’), and the human growthhormone polyadenylation signal (3’), were injected into single cell mouseembryos. These embryos were implanted into pseudopregnant foster mothersand the resulting pups examined for expression of human HPRT. Upon Cd2+

induction, transgenic mice expressed the human enzyme variably in manytissues; however, expression was consistently elevated in brain tissue. Since themetallothionein promoter and the growth hormone polyadenylation signal donot normally influence CNS expression, this study suggests that sequenceswithin the human HPRT transcript (cDNA) influence CNS expression presum-ably via increased mRNA synthesis or stability (94a).


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136 STOUT & CASKEY

Pathogenesis of HPRT Deficiency

Deficiency of HPRT in man results in a spectrum of disease, the severity ofwhich is dependent upon the extent of the deficiency. Complete deficiency ofthe enzyme is associated with the Lesch-Nyhan syndrome (incidence 1 in100,000), while partial deficiency is associated with gout (incidence 1 in 200among males with gout) (51). The Lesch-Nyhan syndrome, first described 1964, is clinically characterized by hyperuricemia, choreoathetoid movements,spasticity, hyperreflexia, mental retardation, and compulsive self-injuriousbehavior (92). Developmental delay is evident at three to six months of agewhile pyramidal and extrapyramidal involvements become apparent within ayear of birth (91). The relationship between HPRT deficiency and CNSdysfunction in Lesch-Nyhan patients remains unclear. Recent biochemical andhistologic studies suggest that inappropriate development of nigrostriataldopaminergic neurons may be important (31). Postmortem analysis of braintissue from three Lesch-Nyhan patients revealed that indexes of dopaminefunction were reduced by 70-90% in the basal ganglia (64). During normaldevelopment, arborization of these neuronal pathways occurs at the same timeas increased HPRT levels in the CNS (2, 63). Moreover, behavioral changesincluding self-mutilation occur when drugs are used to arrest development ofthese terminal dopaminergic neurons (7, 13). Since it has been shown thatdopamine receptor binding is regulated by guanine triphosphate and di-phosphate nucleotides, perhaps purine imbalance during development results ininappropriate synaptogenesis in the basal ganglia (21).

Hyperuricemia in these patients is associated with an increase in the rate of denovo purine synthesis (51). This increase is probably due to the combinedeffects of debreased feedback inhibition by nucleotide end products and cellularloss of hypoxanthine. Lymphoblasts from both normal and Lesch-Nyhanpatients are capable of accelerated de novo synthesis when cultured in hypoxan-thine-depleted media, but only normal cells reduce de novo synthesis uponaddition of exogenous hypoxanthine (39). The inability of Lesch-Nyhan cells convert intracellular hypoxanthine to IMP leads to cellular loss and subsequentconversion of hypoxanthine to uric acid by hepatocyte xanthine oxidase.

In patients with partial HPRT deficiency, excessive production of uric acidleads to hyperuricemia, nephrolithiasis, and a severe form of gout. Evidence ofspasticity, cerebellar ataxia, and mild mental retardation can be found in 20% ofpatients with partial HPRT deficiency, but these individuals do not becomeself-mutilating. Allopurin~l, an inhibitor of xanthine oxidase, is helpful inreducing the uric acid production in these patients but has no effect on theneurological manifestations of disease. Fluphenazine, an antagonist of’postsynaptic dopamine receptors, is purportedly effective in reducing self-mutilating behavior in these patients, presumably via interaction with D~nigrostriatal receptors.


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HPRT MUTATIONS 137

MUTATIONS AT THE HPRT LOCUS

Mutations in Cultured Cells

Three factors have contributed to the development of the HPRT gene as animportant test system in the study of mammalian cell mutagenesis. First, as anX-linked gene, HPRT is hemizygous in male cells and functionally hemizygousin female cells, making this locus particularly useful in the examination ofrecessive mutations. Secondly, HPRT is a nonessential enzyme for cells grow-ing in culture since purines can be provided by de novo synthesis. Finally,powerful selection procedures exist for the isolation of HPRT- or HPRT+

cells, making measurement of 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 uponfunctional HPRTo Thus quantitation of 6-thioguanine or 8-azaguanine resistantcells provides a simple measure of mutation frequency (76). HPRT antibodiesand cDNA probes permit the detailed characterization of individual mutationsand mechanisms of mutation (8).

Such strategies were utilized by Fuscoe et al in their analysis of 19 HPRTChinese hamster mutants (28). Southern blots of DNA from ten spontaneousand nine UV-induced mutants detected two subclones (one spontaneous, oneUV irradiated) with major deletions at the HPRT locus. Cytogenetic analysis ofthese cell lines showed the deletions to be associated with chromosomaltranslocations. DNA from the remaining 17 subclones appeared normal inSouthern blots. None of these mutant lines showed HPRT activity, and only oneproduced material that could cross-react with antibodies raised against HPRT.Subsequent analysis of mRNA synthesized in 20 HPRT-, CRM- Chinesehamster lines, in which no genomic alterations were detected by Southern blots,indicated that 18 of these mutants produced normal levels of HPRT mRNA(79). Cumulatively these data suggest that point mutations in the HPRT locusaccount for the majority of spontaneous or UV-induced mutants. Recent workby Simons has shown that 12 of 19 mutant Chinese hamster cell lines generatedby exposure to ionizing radiation (600 rads) contained deletions detectable Southern analysis (J. Simons, personal communication). Crawford found fourof six a-irradiated human fibroblast lines selected for 6-TG resistance to haveHPRT gene deletions (B. Crawford, personal communication). These studiesdemonstrate the usefulness of recombinant DNA techniques in the characteriza-tion of mutational mechanisms.

An approach for differentiating between base pair substitution and frameshiftmutants of the HPRT gene has recently been described by Stone-Wolff &Rossman (94a). This method utilizes a reversion system dependent upon singlebase pair substitution reversion following N-mettiyl-N’-nitro-nitrosoguanidine(MNNG) treatment, or frameshift reversion following ICR-191 treatment.


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138 STOUT & CASKEY

Distinction between these two types of mutations is predicated on the idea thatframeshift mutations most frequently revert by a second frameshift mutationand that base substitution mutations most frequently revert by another substitu-tion mutation. Thus frameshift mutations conferring 6-TG resistance will morefrequently revert to the 6-TG sensitive phenotype following treatment withICR-191 (a frameshift mutagen) than with MNNG (a substitution mutagen), converse being true for base substitution mutations. Different reversionfrequencies have been observed for different HPRT- cell lines treated withthese compounds (94). DNA sequence analysis will be needed to specify thenature of these mutants and revertants, although such analysis might be difficultgiven the large size of the HPRT gene and the difficulties inherent in cloningand sequencing HPRT mutants.

Two developments may make the HPRT locus more amenable to rapidmolecular analysis. First, somatic cell lines have been developed in which asingle intronless HPRT minigene functions in place of a deleted endogenousgene, thus effectively reducing the target of mutation from 44 kb to 1 kb in size(S. M. W. Chang, personal communication). Second, Myers et al have de-veloped a simple method of identifying and locating point mutations based onnovel denaturation properties of wild-type:mutant heteroduplexes during gra-dient electrophoresis (71). These developments may overcome the dis-advantage of the large size of the HPRT gene in the analysis of point mutants.

To examine the in vivo mutagenicity of toxic compounds in mice, Jones et alhave developed a clonogenic assay to quantify 6-TG resistant spleen lympho-cytes isolated from mice that have been treated with various drugs (48). Theseinvestigators have demonstrated a linear dose-related increase of 6-TGr colo-nies from mice treated with ethylnitrosourea (ENU). Fifteen days followingintraperitoneal injections of ENU, isolated lymphocytes were plated at highdensity (4-8 × 105 cells/250 t~1 well) in the presence of 6-TG and concanavalinA. 6-TG resistant colonies were scored after 8 days in selective media. Thissystem offers the advantages of a direct method of measuring in vivomutagenicity of many compounds and the ability to expand cloned cells formutant characterization at the genetic level.

Albertini (1) and Morley (75) have reported the ability to measure the rate 6-TG resistance from peripheral T cells grown in culture. Turner et al reportsthat a relatively high proportion (57%) of spontaneous mutations in humanlymphocytes involve substantial gone alterations (deletions, exon amplifica-tion. s, etc) that are not evident cytogenetically (98). Twelve of 21 6-TG resistantclones had altered HPRT Southern patterns, while none of the unselected clonesfrom the same individual showed alteration. Since stringent 6-TG selectionconditions were used, however, mutations resulting in undetectable HPRTactivity were favored. Genes with deletions and/or other major gene rearrange-ments may have appeared at an artificially high frequency. These results are


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HPRT MUTATIONS 139

interesting in light of observations that only 18% of patients with the Lesch-Nyhan syndrome and 10% of 6-TG resistant hamster cells have major generearrangements (79, 112). The data suggest that in vivo mutations of somaticcells may occur more commonly by gene deletion. This may be relevant to thedeletion mutations associated with the neoplastic transformation of retinoblas-toma and Wilms’ tumor (9, 23, 56).

Mutations in Man

Since the description of the familial nature of the Lesch-Nyhan syndrome andthe biochemical elucidation of its etiology, HPRT deficiency has been a modelfor the study of X-linked disease. Advances in our knowledge of HPRTgenomic and protein structure have allowed characterization of a number ofheterogeneous mutant alleles.

First reports characterizing HPRT deficiency in human cell lysates describedenzymatic data suggestive of mutational heterogeneity (66, 92). These changesincluded altered sensitivity to product inhibition, thcrmolability, Km valuealterations, and changes in electrophoretic mobility (6, 10, 25, 34, 38, 50, 66,92, 96). With the development of effective purification and protein sequencingprotocols, Wilson et al have been able to compare amino acid sequence datafrom three HPRT deficient patients with gouty arthritis and one Lesch-Nyhanpatient (Figure 4; see also 104, 107, 108, 110).

One of the patients with gout, HPRTT .... to, has a mutation resulting in thereplacement of an arginine residue with glycine at position 50 (107). A single to G base change in the arginine codon can explain this substitution and wouldresult in the loss of a TaqI restriction site, a prediction confirmed by Southernanalysis (105).

HPRTLondon and HPRTMunich have serine to leucine (position 109) and serineto arginine (position 103) changes respectively (108, 110). Each of thesemutations results in gouty arthritis. HPRTKinston occurs in association with theLesch-Nyhan syndrome and has a mutation that substitutes asparagine forarginine at position 193 (106). While this method of mutant analysis has provedvaluable in the description of pathogenic mutations, it is limited to those thatresult in the production of a protein product. A recent survey of cell extractsfrom HPRT-deficient patients indicates that 11 of 15 Lesch-Nyhan patientsproduced no material recognized by HPRT antibodies (J. T. Stout, and J. M.Wilson, in preparation). Mutations that inhibit transcription, RNA processing,or involve major rearrangements of the HPRT gene generally fail to producedetectable protein and are not amenable to this type of examination.

Studies by Yang et al examined the organization of the HPRT gene in 28unrelated Lesch-Nyhan patients (112). Five (18%) of the patients screenedshowed unique Southern patterns suggestive of major gene alterations. Thesealterations include a total gene deletion (RJK 853), two 3’ deletions (RJK 849,


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GM 3467), a potential internal insertion (GM 2227), and an exon duplication(GM 1662). All of these patients are cytogenetically normal and fail to producea stable protein product. None of the deletion patients produce a stable mRNA.GM 1662 produces an HPRT mRNA approximately 200 nt larger than wildtype. This is consistent with Southern and sequence data that have confirmed aperfect duplication of exons 2 and 3 (D. S. Konecki, in preparation). While thispatient produces a stable mRNA, enzyme activity is undetectable and nocross-reactive material can be detected in Western blots.

Family study of the Lesch-Nyhan patient GM 1662 permitted the identifica-tion of the origin of this mutational event. An abnormal 4.1 kb Bgl II band,characteristic of the duplication mutation, was identified in the propositus, hismother, and two sisters but was absent in his maternal grandmother. These dataindicate that this mutation originated in the germ line of a maternal grandparentand gave rise to an asymptomatic carrier female.

Two additional family studies provide data on the gametic origin of muta-tions (J. T. Stout, in preparation). The Lesch-Nyhan patient RJK 853 has X-specific HPRT sequences while his mother has two normal copies of theHPRT gene, indicating that the deletion event occurred in a maternal gamete. Ina second example, the patient (RJK 983) has a partial 3’ deletion with thebreakpoint 3’ to the fourth exon. The mother of this patient has two normalcopies of the HPRT gene, again identifying a maternal gametic deletion eventas the origin of the new mutation.

These findings support Haldane’s prediction that lethal, X-linked mutationsthat confer no selective advantage to heterozygotes will frequently arise asspontaneous new mutations (36). HPRT deficiency syndromes show no ethnicpredilection. Males with the Lesch-Nyhan syndrome fail to reproduce, andfemale heterozygotes have no selective advantage. The maintenance of theLesch-Nyhan syndrome in the population is thus dependent upon the sporadicoccurrence of these heterogenous mutations.

In earlier studies, Francke et a! compiled the results of carrier detection testson mothers and maternal grandmothers from 54 families with a single affectedchild (26). These tests suggest that 11 of 54 affected males were the result new mutations. In addition, 10 of the heter0zygous mothers were shown to becarriers of new mutations. Molecular analysis of the origins of mutations insuch families will permit identification of paternal and maternal gametic muta-tions as well as determine mechanisms of mutation for each.

To address the question of genetic heterogeneity among mutations of theHPRT gene, Southern, Northern, Western, kinetic, and immunoquantitativedata have been compiled for 24 unrelated patients with HPRT deficiency. Usingthese molecular parameters to create a composite picture for each mutant, it wasfound that 14 of 24 patients have unique genetic backgrounds, suggestive ofextensive mutant heterogeneity.


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142 STOUT & CASKEY

Indirect evidence for genomic heterogeneity in this population came fromlimited haplotype analysis. Two HPRT-linked restriction fragment length poly-morphisms (RFLPs) have been described (12, 80). In 1983, Nussbaum scribed a three-allele BamHI RFLP that occurs within the HPRT gene. Thesealleles are expressed phenotypically on Southern blots as three distinct pairs offragments: (a) a 22 kb/25 kb pair, (b) a 12 kb/25 kb pair, and (c) a 22 kb/18 pair. An additional two-allele TaqI RFLP was reported for an anonymoussequence (DXS-10) separate from, but closely linked to (95% confidencelimits, 0< 15cM), the HPRT gcne. These alleles are represented as 5 kb or 7 kbbands on Southern blots. These RFLP patterns can be combined to establish sixhaplotypes, four of which are represented in this group of 24 patients. The mostfrequent haplotype pattern (68%) was that combining the 22 kb/25 kb BamHIallele and the 5 kb Taql allele (22/25/5 pattern). The 22/25/7 pattern repre-sented 9% of this group, and the 12/25/5 and 22/18/5 patterns were each seen ina single patient. Patients having unique, readily identifiable major gene alter-ations accounted for 9% surveyed (112; J. T. Stout, and J. M. Wilson, inpreparation).

This study reports that a minority of HPRT deficient patients are able toproduce a protein product detectable by immunologic methods. The majority ofthose that are CRM+ show the less severe, gouty phenotype. Only 15% ofpatients surveyed failed to produce a detectable HPRT mRNA and, as ex-pected, those patients exhibited the severe Lesch-Nyhan phenotype. In general,the more severe the molecular defect (i.e. major gene alteration, HPRTmRNA-, etc) the more severe the clinical phenotype.

GENE TRANSFER AND THERAPEUTIC PROSPECTS

The transfer of an expressible HPRT cDNA molecule into HPRT-deficientmouse cells was first described by Jolly et al (47). Transfection of mouse LA cells with a plasmid containing the human HPRT cDNA resulted in the appear-ance of HAT-resistant colonies at a frequency of approximately 1 x 10-3 .

Brennand and coworkers subsequently reported the expression of human andChinese hamster cDNA recombinants in fibroblasts from Lesch-Nyhan patientsand HPRT-deficient Chinese hamster cells (15). These recombinants wereintroduced into cells by calcium phosphate coprecipitation, and HAT-resistantcolonies were recovered at frequencies of 1.9 x 10-4 (hamster cDNA in humancells) and 5 x 10-4 (human eDNA into human cells). These frequencies arecomparable to those obtained by Mulligan and Berg, who introduced into asimilar Lesch-Nyhan cell line a chimeric plasmid containing the bacterialguanine phosphoribosyltransferase (gpt) gene and SV40 promoter elements(77).


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Microinjection techniques have substantially enhanced gene transferefficiencies. When HPRT eDNA molecules, under the control of a variety ofpromoters, are microinjected into HPRT- cells, HAT-resistant colonies appearat frequencies 10~--103 fold greater than frequencies obtained by calciumphosphate precipitation (S. M. W. Chang, personal communication). Studiesinvolving the introduction of human HPRT eDNA sequences into mouseembryos via microinjection suggest that human minigenes can become stablecomponents of the mouse genome. These genes can be expressed in a variety oftissues and can be transmitted to progeny in a Mendelian fashion. Alternativesto these relatively inefficient and labor-intensive methods of gene transfer havebeen found in the form of naturally occurring RNA and DNA viruses.

The unique structure and capabilities of viruses may make them the idealgene transfer system in mammalian cells. Retroviruses are vectors whose lifecycle depends upon the integration, replication, and expression of their geneticmaterial in host cells (30, 102). The retroviral system is very efficient (100% cells infected carry an integrated copy of the gene), and many cell typesrefractory to CaPO4 transfection can be infected with retroviruses.

Infection of cultured Lesch-Nyhan cells with amphotrophic retrovirusescontaining human HPRT cDNA sequences was shown by Willis et al to result inthe production of enzymatically active human HPRT (103). Concomitant withexpression of HPRT in these cells was the partial correction of other aberrantmetabolic parameters, i.e. elevated purine excretion and increased intracellularhypoxanthine. A. Miller et al have reported using similar viruses to infectmouse bone marrow cells that were subsequently transplanted into lethallyirradiated mice (72). In these studies, HPRT-virus production was detected mouse spleen and bone marrow cells as long as 133 days after transplantation.Human HPRT protein was detected in spleen cells for two months. Thesestudies suggest that retroviral vectors may serve as an efficient mechanism forsomatic gene transfer.

The ultimate goal of medical geneticists involved with gene regulation andtransfer is the management of inborn errors of metabolism at. the genetic ratherthan symptomatic level. Gene therapy will depend upon the development ofeffective delivery systems, capable of safely introducing new genetic materialthat can be expressed at the appropriate times and in the appropriate tissues.Lesch-Nyhan syndrome may be amenable to gene therapy but available data donot assure success.

It is not clear if gene introduction into bone marrow cells will influence CNSdysfunction in these patients since therapeutic bone marrow transplantation forthe Lesch-Nyhan syndrome has not been reported. Many aspects concerningthe delivery of genes via retroviral vectors remain unclear. Questions concern-ing tissue requirements and temporal restraints on HPRT expression need to beanswered. The devastating nature of this disease, combined with the lack of


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144 STOUT & CASKEY

therapeutic altematives, warrants the investigation of gene transfer as a poten-

tial treatment for Lesch-Nyhan patients.

ACKNOWLEDGMENTS

The authors wish to thank Lillian Tanagho for her help in the preparation of thismanuscript. JTS gratefully recognizes support from the Philip Michael Be-

rolzheimer Medical Scientists Fellowship. CTC is an Investigator for the

Howard Hughes Medical Institute.

This work was supported by NIH Grant # AM31428.

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hprt: gene structure, expression, and mutation · 2017-10-18 · 130 stout & caskey...

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