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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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STOUT & CASKEY
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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HPRT MUTATIONS
NORMAL ENZYME
CHARACTERISTICS
FUNCTION
129
AND
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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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130
STOUT & CASKEY
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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HPRT MUTATIONS
131
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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132
STOUT & CASKEY
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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HPRT MUTATIONS
135
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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STOUT & CASKEY
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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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HPRT MUTATIONS
MUTATIONS
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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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138
STOUT & CASKEY
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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HPRT MUTATIONS
139
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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HPRT MUTATIONS
141
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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142
STOUT & CASKEY
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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HPRT MUTATIONS
143
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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144
STOUT & CASKEY
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.
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Annu. Rev. Genet. 1985.19:127-148. Downloaded from arjournals.annualreviews.org
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