A molecular survey of hypoxanthine-guanine
phosphoribosyltransferase deficiency in man.
J M Wilson, … , W N Kelley, C T Caskey
J Clin Invest.
1986;
77(1)
:188-195.
https://doi.org/10.1172/JCI112275
.
We characterized 24 unrelated patients with a deficiency of the enzyme
hypoxanthine-guanine phosphoribosyltransferase (HPRT) in an attempt to better understand the nature
and spectrum of mutations that underlie this prototype-inherited disease. Lymphoblast cell
lines derived from each patient were analyzed at multiple molecular levels including the
structure and function of the residual HPRT enzyme, messenger RNA (mRNA), and gene.
Our studies demonstrate the following: (a) at least 16 of the 24 patients represent unique
and independent mutations at the HPRT structural gene; (b) the majority of cell lines have
normal quantities of mRNA but undetectable quantities of enzyme; (c) 33% of patients retain
significant quantities of structurally altered, functionally abnormal, HPRT enzyme variants;
and (d) a minority of patients are void of both enzyme and mRNA, possibly representing
examples of aberrations in gene expression. Our studies provide direct evidence for marked
genetic heterogeneity in this disorder and illustrate the kinds of mutations and mutational
consequences that underlie inherited disease in humans.
Research Article
Find the latest version:
A
Molecular Survey of Hypoxanthine-Guanine Phosphoribosyltransferase
Deficiency
in
Man
JamesM.Wilson, J.Timothy Stout,Thomas D. Palella,BeverlyL.Davidson, WilliamN.Kelley,and C. ThomasCaskey
Departments of Internal MedicineandBiological Chemistry, University ofMichigan, AnnArbor,Michigan 48109,andDepartments of Medicine, Biochemistry, andCellBiology, Baylor College ofMedicine andHowardHughesMedicalInstitute, Houston, Texas77030
Abstract
We
characterized
24 unrelatedpatients
withadeficiency
ofthe enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) inanattempt tobetter understand thenatureand spec-trumof mutations that underlie thisprototype-inheriteddisease. Lymphoblast celllines derived from each patientwereanalyzed atmultiple molecular levels including thestructureand function of the residual HPRTenzyme, messenger RNA(mRNA), and gene.Our studiesdemonstrate thefollowing:
(a)atleast 16of the 24patientsrepresentuniqueandindependent mutationsat the HPRT structural gene; (b) the majority ofcell lines have normal quantities ofmRNA but undetectable quantities of en-zyme;(c) 33% of patients retainsignificant quantities of struc-turallyaltered,functionally abnormal, HPRT enzymevariants;
and(d)aminority of patientsarevoid of bothenzymeandmRNA,
possibly representing examples of aberrations ingeneexpression.
Our studiesprovide direct evidence for markedgenetic
hetero-geneityinthis disorder and illustrate the kinds of mutationsand mutationalconsequencesthatunderlie inherited disease in hu-mans.Introduction
Hypoxanthine-guanine
phosphoribosyltransferase
(HPRT)'
de-ficiency
isan inherited disorder ofpurine
metabolism thathas wide-rangingphenotypic
effects.Avirtual absence ofHPRTac-tivity
is found inpatients
with theLesch-Nyhan syndrome
(1). ThisX-linked recessive disease is characterized by
an overpro-duction of uric acid as well as thedevelopment
of uric acidnephrolithiasis,
choreoathetoid movements,spasticity
andhy-perreflexia,
severemental andgrowth
retardation,
andabizarre patternof patients' self-mutilationcharacterized by compulsivebiting
of theirfingers
andlips (2).Patients
with
a partial deficiency of enzyme activity are spared thedevastatingneurological
andbehavioral abnormalities thatcharacterize the Lesch-Nyhan syndrome. This lattergroup ofpatients usually
presentswith
a severeform ofgoutyarthritis oruric acidnephrolithiasis
in early adulthood (3).Dr.Wilson'scurrentaddressisMedical Services, Massachusetts General
Hospital,Boston, MA 02114.
Receivedfor publication 25 July 1985.
1.Abbreviations used in this paper: CRM', enzyme positive; HPRT, hypoxanthine-guaninephosphoribosyltransferase; PRPP,
5-phosphori-bosyl-l-pyrophosphate; RFLP, restriction fragment length
polymor-phism.
HPRT deficiency has recently emerged as a prototype for definingthe nature and functional consequences ofclinically deleterious mutational events and for designing noveltherapeutic modalities that attempt to modulate geneexpression. Several aspects ofthis inherited disorder make it particularly amenable todetailed molecular analyses. Our knowledge of normal HPRT protein(4, 5), mRNA (6, 7), and gene (8, 9) provides a basis for theprecise delineation of the abnormalities present in enzyme-deficient patients. The isolation of mutant genes and their prod-ucts issimplified because the affected malepatients are hemi-zygousforthe HPRTlocus (1, 3), and thegene isexpressed in a wide variety of human tissues, including cultured cells(10). Wedocumentedin anearlierreportthe utility and validityof using viral transformed lymphoblastsintissue cultureas asource for theisolation of mutant HPRT gene products (1 1).
Avarietyofmutationshave nowbeen describedin HPRT-deficient patients. Single unique amino acid substitutions have been identified in four dysfunctional enzyme variants; three mutantenzymes wereisolatedfrom goutypatients (12-14)and one wasisolated from aLesch-Nyhan patient (15). Yangetal. (16)screened aseriesofLesch-Nyhan patientsformajor rear-rangements in the HPRT gene and identified unique abnor-malities in five unrelated patients. These important preliminary investigations have provoked several more fundamental ques-tions.
Acompletedeficiency ofHPRTactivity isnotlethal inutero, but does lead tononreproductiveaffected males(17).Given the genetic lethal characteristics of the disease, whatisthe diversity andprevalence of new mutations in Lesch-Nyhan syndrome? Furthermore, isthereacorrelation betweentypes of mutations or mutational consequences and clinical phenotype? Finally, what is/are the molecular defect(s) in themany Lesch-Nyhan patients who have grossly normal structuralgenesbut lack en-zymeprotein?
In an attempt to answertheseimportantquestionswehave undertaken acomprehensive analysis of the molecular basis of HPRTdeficiency inalargegroupof patients. Lymphoblastoid celllineswereestablished from 24 unrelated enzyme-deficient patientsrepresenting both clinical syndromes. These cell lines were characterized at multiple molecular levels including (a) HPRT enzyme
concentration,
function, andstructure,(b)HPRT mRNAconcentrationandmolecularsize, and (c)gross structure of theHPRT gene.Methods
Subjectsand cell lines. HPRT-deficient patientsweredivided into two clinical subgroups:those who presentedwiththeLesch-Nyhansyndrome andthosewho presented withgoutyarthritisand/oruric acid
nephro-lithiasis.Criteria used in establishingadiagnosisofLesch-Nyhan syn-dromeinclude: (a) virtualabsenceofHPRTactivityinerythrocytes,(b) growthand mental retardation,(c)evidencefor pyramidaland/or
ex-trapyramidaltractdysfunction,and(d)compulsiontoself-inflictpainful
188 Wilson, Stout, Palella, Davidson, Kelley,andCaskey
J.Clin.Invest.
© The American Society for ClinicalInvestigation, Inc. 0021-9738/86/01/0188/08 $1.00
wounds usually manifested by self-mutilation of the hands and lips.The
second clinical subgroup represents a heterogeneous populationof pa-tients who (a) present with gouty arthritis and/or uric acidnephrolithiasis at an early age, (b) demonstrate a broad spectrum of HPRT activity in erythrocytes (from undetectable to -50% of activity), (c) usuallyhave
completely normal neurological function, but may have mild tomoderate
neurological abnormalities (e.g., learning disabilities or hyperreflexia), and (d) never manifest the compulsive self-destructive behavior that is characteristic of the Lesch-Nyhan syndrome.
Lymphoblastoid cell lines were established for each patient by Epstein-Barr virus-mediated transformation of peripheral blood B lymphocytes. Methods used in the establishment and maintenance of lymphoblastoid cell lines have been described(11). Cell lines GM2292, GM 1899, and GM6804 were obtained from The Human Mutant Cell Repository, Camden, NJ.
Enzyme assays. Membrane-free protein extracts were prepared from freshly harvested lymphoblast cultures as described (I 1). Sensitive ra-diochemical and immunochemical techniques were used to quantify HPRT enzyme activity(18) and immunoreactive protein (19), respec-tively.
Measurements of apparent Michaelis constants (Ki) for hypoxanthine
(K.-hypoxanthine)
and5-phosphoribosyl-I-pyrophosphate(K4-PRPP) were performed in the presence of near saturating concentrations of the other substrate for all enzymes exceptHPRTKDiOII andHPRTMU.,h.
It was impossible to measure theK,-PRPP
ofHPRTKiPO.
andHPRTMU"ich
in the presence of saturating concentrations of hypoxanthine due to each enzyme's elevated Km-hypoxanthine and the limited solubility of hy-poxanthine in aqueous solution. The following experimental conditions were used for all enzymes except
HPRTKig.,:
apparentKm for hypo-xanthine, varying hypoxanthine (300-3gM)
in the presence of 6mM MgCl2 and 1 mM PRPP; apparentK.
for PRPP, varying PRPP (1-0.01 mM) and MgCl2 (kept in excess of PRPP by 5 mM) in the presence of 300AM
hypoxanthine. The following conditions were used in the analysis ofHPRTKng03:
apparentK.
for hypoxanthine, varying hypoxanthine (300-3 1M) in the presence of 20 mM MgCl2 and 10 mM PRPP; and apparentKm PRPP, varying PRPP (2.5-0.025 mM) and MgCl2 (kept in excess of PRPP by 5mM) in the presence of 300,M
hypoxanthine.Maximal velocities were calculated from measurements of enzyme activity and immunoreactive protein in membrane-free extracts. The maximal velocity is defined as the initial rate (milliunits per milligram protein), measured in the presence of saturating concentrations of hy-poxanthine and PRPP, divided by the level of HPRT immunoreactive protein (nanograms CRM per milligram protein). However, maximal velocities for
HPRTK,..
andHPRTMU..
were extrapolated from assays performed in the presence of subsaturating concentrations of hypoxan-thine.Protein blot analysis. Protein extracts were subjected to polyacryl-amide gel electrophoresis in the presence or absence of sodium dodecyl sulfate (SDS) as described previously. Electrophoretic transfer of proteins from nondenaturing gels to nitrocellulose paper has been well described (1 1). Proteins were transferred from SDS gels to nitrocellulose paper as described by Towbin et al. (20) except that the transfer was carried out for 3 h at 50 V. Detection of HPRT in situ, after transfer from either nondenaturing or SDS gels, was effected immunochemically using pre-viously published methods (I 1).
AnalysisofradiolabeledHPRTproteins. Lymphoblasts from a normal male subject and from patient D.B. were labeled in culture with
[35S]methionine.
"S-labeledHPRT protein was purified to radiochemicalhomogeneity by immunoprecipitation with a highly specific antibody.
Immunoprecipitates were analyzed by SDS-polyacrylamide gel
electro-phoresis. The labeling, purification, and electrophoretic analysis of
[35S]HPRT
has been described (I 1).RNA isolation and Northern blot analysis. Total RNA was isolated in the presence ofguanidine-HCIas described (21). 50
,g
ofRNA was electrophoresed in a 0.8% agarose gel, transferred to nitrocellulose, andhybridized with"P-deoxycytidinetriphosphate (dCTP) nick-translated
HPRT cDNA (21).
DNA isolation and Southern blot analysis. High-molecular-weight
Table I. HPRT Activity and Immunoreactive Protein in
Enzyme-Deficient
LymphoblastsPatients or
cell lines Specific activity Immunoreactiveprotein
mU/mg %control ngCRM/mg %control
Controls 14.5±3.6(3)* 100 332±96(3)* 100 Lesch-Nyhan
D.A. <0.1(2)t <0.7 <1.5(2) <0.5 A.C. <0.1(2) <0.7 <1.5(3) <0.5 E.C. <0.1 (2) <0.7 <1.5(2) <0.5 H.D. <0.1 (4) <0.7 <1.5(2) <0.5 W.E. <0.1 (2) <0.7 <1.5 (2) <0.5 D.G. <0.1(2) <0.7 167±38 (7)§ 50 J.H. <0.1(2) <0.7 <1.5(2) <0.5 D.M. <0.1(2) <0.7 <1.5(3) <0.5
HPRT.,,,,,"Il <0.1(2) <0.7 240±29 (6) 72 B.S. <0.1(2) <0.7 <1.5(3) <0.5
K.T. <0.1(4) <0.7 304±50 (8) 92 GM2292 <0.1(2) <0.7 <1.5(2) <0.5 GM 1899 <0.1(2) <0.7 5.4, 3.6 1.3 GM6804 <0.1(2) <0.7 <1.5(2) <0.5 McA <0.1(2) <0.7 <1.5(2) <0.5 Gout
D.B. 10.0±2.2 (5) 69 172 52 P.C. <0.1 (4) <0.7 15.2, 12.0 4
HPRTA..Ajb.Or 1.5±0.2 (4) 10 37±3 (6) 11 B.D. <0.1 (4) <0.7 5.1, 6.7 2
J.M. 0.2±0.1 (6) 1.4 10.8, 10.0 3
HPRTTo,,ml 4.8±0.3 (4) 33 172±20(6) 52
HPRTLOOd.O 8.6±0.6 (6) 59 116±11 (7) 35
R.T. <0.1 (6) <0.7 2.9,2.7 0.8
HPRTM.,"ch11
0.4±0.1 (6) 3 263±17 (6) 79 *Mean±ISDfor three controllymphoblastcelllines,takenfrom referenceI 1.* Mean; (n)=number of determinations. §Mean± ISD;(n) =numberof determinations.
"IObtained from reference 11.
DNA wasisolated from culturedlymphoblastsasdescribed (21).DNA
wasdigested to completion with one of several restriction endonucleases underconditions recommended by the manufacturer (Bethesda Research Laboratories, Gaithersburg, MD, and NewEngland Biolabs, Beverly, MA).RestrictedDNA wasfractionated in 0.8% agarosegels,transferred
tonitrocellulosepaper,andprobed with 32P-labeledDNA asdescribed(16).
Results
HPRTenzymeactivity
and
immunoreactiveprotein. Lympho-blast cell lines were established from 24 unrelated HPRT-defi-cientpatients. 15 patients presented with the Lesch-Nyhan syn-dromeand 9patients presented with gout and/or nephrolithiasis. Previous studies have demonstrated unique mutant forms of HPRT in five of these patients (11-15,19).2
These variants have been namedHPRTItinswon,
HPRTAnn
Arbor, HPRTToronto,
HPRTLondon,
andHPRTMunich-Levelsof HPRT enzyme activity and immunoreactive pro-teinaresummarized in TableI.
Activity
assayswerecarriedout in the presence of hypoxanthine andPRPPconcentrationsthat exceedthenormalMichaelisconstantsby 60-foldand120-fold,
respectively.
HPRTenzymeactivity
wasundetectable (<0.7% ofcontrol) in all Lesch-Nyhan patients, while HPRTimmu-2.TwopatientsdescribedbyYangetal.(16)werealso included inour
study;lymphoblastcelllinesMcA andGM6804
correspond
toYang
etnoreactive protein was undetectable (<0.5% of control) in 11 of 15 Lesch-Nyhan patients; a low level of enzyme protein was measuredin cellline GM 1899 (1% of control) while substantially higher levels were measured in patients D.G. (50% of control),
HPRTKIcSSOfl
(72%of control), and K.T. (92% of control). Patients whopresented with gout exhibited a broad range of activity from undetectable (patients R.T., P.C., and B.D.) to 69% of control (patient D.B.). HPRT immunoreactive protein was detectedin each patient with gout; however, the range of enzyme concen-tration was wide (from 0.8 to 79% of control).Kineticproperties of
enzyme variants.
Culturedlymphoblasts
with >10% residual HPRT protein werefurther
characterized with respect to the kinetic properties of the residualHPRT pro-tein. Included in this groupwere cell lines expressing thefive known HPRT variants and threepreviously
undescribed celllines,
D.B.,D.G., and K.T. Table IIsummarizes
the measured Michaelis constants forhypoxanthine
andPRPP, and the cal-culated maximal velocity of each variant.Significant
kinetic ab-normalities weredetected
in all variants exceptHPRTTOnlODO.
These data
confirm earlier
kinetic
analyses
of
HPRTKin.Son
(22),
HPRTMUnich
(23),HPRTTOTOfltO
(24), and enzymefrom
D.B.(25),isolated
from either
erythrocytes orfibroblasts.
HPRTfrom
pa-tient D.B.wasindistinguishable
fromHPRTLfndOn
intermsof kineticdysfunction;
both enzymesexhibited
aslight increase
inV.,
and asixfoldincreaseinKm-hypoxanthine.
Enzymeactivity was undetectable in extractsfrom
D.G. and K.T., precluding kineticanalysis
of the mutant enzymes.Electrophoretic analysis of
enzyme variants. Thepreviously
undescribed
enzyme-positive
patients
werefurther
characterized with respect to the electrophoreticproperties
of theresidual
HPRTprotein. Membrane-free
extractsweresubjected
topoly-acrylamide gel electrophoresis,
in the presence or absence of SDS, and HPRT enzymesweredetectedimmunochemically
us-ingtheprotein blottechnique.
TableII.KineticPropertiesofHPRT Variants
Michaelisconstants
Maximal Mutant Hypoxanthine PRPP velocity*
MM MM mU14gCRM
Controls 3.5±2.4(3)t 16.0±7.0
(3)t
44(3)t
HPRTToronto 4.6±2.1 (4)§ 14.5±7.7(3) 28
HPRTLofdon
20.2±4.3 (12) 10.2±5.0 (6) 74HPRTAnn
Ae0or
33(31,34)"
38±13 (6) 41HPRTMuniCh 261±53 (4) 5.9±2.4 (3) 2.6¶
HPRTKintlon
555±107 (3) 2,025(2,100, 1,950)50¶
D.B. 20.2±3.2 (4) 11.9±4.0 (3) 58D.G. NM** NM NM
K.T. NM NM NM
*Definedasthemaximumenzymaticactivity divided by the total
quantity ofHPRT enzyme as measured byradioimmunoassay(see
Methods).
f Mean± 1
SD; (n)
=numberofnormallines studied.§ Mean±1SD;(n)=numberof determinations.
Average oftwo separatedeterminations. Individual values enclosed in parentheses.
¶Extrapolatedtomaximumvaluesfromassaysin whichPRPP was
saturatingbuthypoxanthinewas not.
**NM,notmeasured because the enzymevarianthad nodetectable
catalytic activity.
Differences in thenet
charge
of HPRT variantswereassessedby nondenaturing polyacrylamide gel
electrophoresis. The known HPRT variants have been classified previously into three groups basedonthiselectrophoretic
parameter(I 1):
normalmi-gration,
HPRTLod.f
andHPRTAnf Arbor;
cathodalmigration,
HPRTKinstOf
andHPRTMUnsch;
and anodal migration,HPRTTX,tO.
Membrane-free extractsofthe previously uncharacterized pa-tients, D.B., K.T., and D.G.,weresubjectedtosimilar electro-phoretic analysis, asshown in Fig. 1. Enzyme from D.B. was indistinguishable from normal HPRT (lane N), while HPRT from D.G.was moreanodal than normal(but
indistinguishable
fromHPRTTorono),
and HPRT from K.T. wasmore cathodal than normal (but similartothe other known cathodal variantsHPRTKjnston
andHPRTMunich).
Differences in theapparentsubunit molecular weight of the variantenzymes wereidentified by comparing their relative
mi-gration during SDS-polyacrylamide gel
electrophoresis. Previous experiments demonstratedanessentially normalapparent sub-unit molecular weight for all of the known variants exceptHPRTLondon,
which exhibitedaslightlylowerapparent subunit molecular weight (11, 19). The protein blot techniquewas em-ployedtoquicklyscreenfor marked alterationsinapparent sub-unit molecular weight.Membrane-free extracts from a normal cell line,thethree previously uncharacterized enzyme-positive cell lines, and se-lected known HPRTvariants, wereelectrophoresed in the
pres-enceofSDS, and theHPRTenzymesweredetected immuno-chemicallyasdescribed (Fig. 2). A single predominate molecular species with an apparent subunit molecular weight equal to 25,000, aswell asmultiple additional nonpredominate bands, weredetectedin each cell linestudied.Severalobservations in-dicate that the multiple nonpredominate bandsaredue to non-specific binding of antibody, and that the 25,000-mol-wt species representsbonafide HPRT. All bands exceptthe25,000-mol-wt band are present when (a) control serum is used in place of HPRTantiserum (datanotshown),or(b)extractsfrom enzyme-deficient cell lines lackingdetectable HPRT immunoreactive
pG
N
DBI
-S°
N
KT
(/'N
_.
....~~~~~~
SPIqI,1
0
tS ..~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Figure 1.Proteinblotanalysis oflymphoblastHPRTafter nondena-turing polyacrylamidegelelectrophoresis.Lymphoblastextracts were
electrophoresed innondenaturingpolyacrylamidegelsandanalyzed forHPRTimmunoreactive proteinasdescribed in theMethods
sec-tion. Similarquantitiesof cellularextract(500-250,gprotein)were
electrophoresed ineachlane.LaneNrepresentsextractfroma lym-phoblastcellline derivedfromanormalcontrol.
N
DB
NDB
N
--M4*_
_ _o
Figure2.Proteinblotanalysis oflymphoblastHPRTafter SDS-poly-acrylamide gel electrophoresis. Lymphoblast extracts were electropho-resed under denaturing conditions inpolyacrylamidegelscontaining
SDS,and HPRTimmunoreactive proteinwasdetected in situ. Each lanecontained similar quantities ofcellular extract (500-250
,g
totalprotein). Lane N represents extractfromalymphoblastcell line
de-rived froma normal control. The arrow indicates the location of HPRT.
protein were analyzed (data not shown). The apparent subunit molecular weight of HPRT from K.T. and D.G. is
indistin-guishable
from normalHPRT(lane N). However, the migration ofHPRTfrom
D.B. appears to be slightly faster than normal HPRT and, therefore, the apparent subunit molecular weight somewhatlower.Weattempted to confirm the subtle decrease in apparent subunit molecular weight of HPRT from D.B. using an alter-native method. HPRT from D.B. and fromanormal
lympho-blastoid
cell line was labeled in culture with[35S]methionine
andpurified
tohomogeneity
byimmunoprecipitation.
Thera-diochemically
homogeneous enzymes were subjected toSDS-polyacrylamide
gelelectrophoresis
anddirectly visualized
by autoradiography(Fig.
3). The apparent subunit molecular weight ofHPRTfrom D.B. was decreased as compared with the normal enzyme (lane N).This
subtledifference
in subunit molecular weight was better illustrated by demonstrating partial resolution of the normal and variant enzyme when co-electrophoresed (lane N+D.B.).Analysis
of HPRT mRNA. Northern analysis revealedes-sentially
normal concentrations of HPRT mRNA in 12 of 15No-Figure3.SDS-polyacrylamidegelelectrophoresis oflabeled lympho-blastHPRT. Lymphoblast HPRTlabeledin culture with
[35S]methioninewasdenatured andsubjectedtoSDS-polyacrylamide gel electrophoresis. RadiolabeledHPRT wasidentifiedby autoradiog-raphy.Lane N,purifiedenzymefroma normallymphoblastcellline;
lane DB,purifiedenzymefromD.B.; and laneN+DB, a mixtureof purifiedenzymefrom normal andD.B.
Lesch-Nyhan patients and 8 of 9 gouty patients (Fig. 4). Cell lines
from
McA(Lesch-Nyhan), W.E. (Lesch-Nyhan), GM2292 (Lesch-Nyhan), and B.D. (gout) had no detectable HPRT mRNA, evenafter
longerautoradiographic
exposures (Fig. 4). Weestimate the sensitivity of this technique to be >5% normal HPRT mRNAconcentration.
GM6804wasthe
only
cell line in which the HPRTmRNAexhibited
alteredelectrophoretic properties;
this abnormal mRNAmigrated
at anabnormally slow rate, consistent withan increase in molecularsize
from 1.6 to 1.8 kb. Similarfindings
havebeenreported in
fibroblasts derived
from thispatient
(16).
Southern blot
analysis of the
HPRT gene. DNA from eachcell
linewasexamined by
themethod of Southern foralterations
RT EC BD DA
*O
"to
0
o -o
c O
O
C:
0
MCA1899 1- 6804 -J
...I
Figure 4.Northernblot
analysis
of lymphoblastHPRT mRNA.TotalRNA(50
,g/lane)
waselectropho-resedin agarose,transferredto
ni-trocellulose,andprobedwith
32p_
labeled HPRT cDNA. Results fromD.B., P.C.,and
HPRTAflf
Aboare notshown.
DB
N KT
DG
0
,n
in HPRT gene structure using restriction endonucleases. The presence of linked Bam H I and Taq I restriction fragment length polymorphisms (RFLPs) were determined with HPRT and DSX- I0 probes, respectively.
Thehuman HPRT gene is composed of nine exons distrib-utedover >40 kb of DNA (8; 9; T. Patel, personal communi-cation). The integrity of the HPRT structural gene in mutant cell lines was assessed as recently described by Yang et al. (16); DNA was analyzed according to the method of Southern (26) usingfull-length HPRT cDNA as a probe. Using this approach, we demonstrated that the majority of these patients (13 out of
15 Lesch-Nyhan patients and 9 out of 9 patients with gouty arthritis) have
mutations
that do not involve major gene rear-rangements (data not shown). An abnormal fragment, of low molecularweight, is consistently seen in Southern blots of Bam H 1 digested DNA from GM 1899 (data not shown). Abnormalfragments
are not detected with other enzymes; hence, this rep-resentseither a rare Bam HI RFLP or the detection of the mu-tation. Three mutations, previously reported, which are detect-able by Southern analysis include GM6804, McA, andHPRTTOOntO.
Analysis of DNA from GM6804 (lymphoblast culture from pt. GM 1662) suggests aninternal duplication
of the HPRTgeneinvolving exons 2 and 3(16).
DNAfrom
McA (lymphoblast culture from pt. RJK853) demonstrates no X-spe-cificfragments thathybridize
toHPRT cDNA,indicative
of a total gene deletion (16). Detection ofthepoint mutation
in exon 3 ofHPRTTOrOnt,,
bySouthern analysis
of DNA has been de-scribed (27).Two
HPRT-linked
RFLPs have beendescribed.
Nussbaum reported a three-allele Bam H1 RFLP that occurs within the HPRT gene (28). These alleles are expressed phenotypically on Southern blots as threedistinct pairs
offragments:
a 22-kb, 25-kb pair; a 12-kb, 25-kb pair; and a 22-kb, 18-kb pair. An additional two-allele TaqI RFLPhas been reported foran anon-ymous sequence (DXS-10)
separatefrom,
but closely linked to (95%confidence limits
0 <15cM), the HPRT locus (29). These alleles are represented as 5 or7kbfragments
onSouthernanal-AC BS
44
\.I.-ysis.
These RFLP patternscanbe combinedtoestablish sixhap-lotypes,
four of which arerepresented
in thisgroupofpatients
(Fig. 5).
Themostfrequent haplotype (68%)
wasthatcombining
the
22-kb,
25-kb Bam HI allele and the 5-kbTaq
I allele(22,
25/5).
While 13% of allpatients
had the22,
25/7
pattern, the22, 18/5
and12, 25/5
patternswereeachseeninsingle patients.
Therefore,
9% ofpatients
have gene defects identifiableby
Southern
analysis.
Discussion
The inbornerrorsof
metabolism
areexamplesof
disorders thatareoften caused
by
aberrations
in thefunction
orexpressionof
single
macromolecules. We have undertaken acomprehensive
molecularanalysis
ofalarge
populationof
patients withade-ficiency
of the enzyme HPRT, inan attempt toillustrate the spectrum andprevalence
ofgenetic
aberrations
that underlie thisprototype-inherited
disease.Westudied 24 unrelated HPRT-deficient
patients;
15patients
presented
with theLesch-Nyhan syndrome,
while 9patients
presented
with eithergoutornephrolithiasis.
Acomprehensive
analysis
of the HPRTenzyme,mRNA,
andgeneprovided the basis for the molecular classification summarized in Table III andFig.
6.Approximately
33% of all HPRT-deficient patients retained >5% residual HPRT immunoreactive protein. Thesepatients,
referredto asthe enzyme-positive(CRM+)
group,providemanyexamples
ofanenzymedeficiency
statecausedby
anabnormality
in enzyme function. Five of these
eight
patients have been de-scribedpreviously,
four of which have been showntocontainunique
enzymevariants that differ from the normalenzymeby
a
single
amino acid substitution. Patients D.B., D.G., and K.T. havenotbeen describedpreviously.
HPRTenzymesfrom D.G. and K.T. exhibitunique
constellations of electrophoretic andcatalytic
properties and therefore have been assigned thefollow-ing
namesbasedonthe geographical residence of thepatients:
HPRTNew
Haven(D.G.)
andHPRTy8IC (K.T.).
In both cases,unique
-O c
~ ~
~~~~~~~o
DG DM WE HD JH : KT 2292 JM RT EC BD DA F CS PC 1899 6804
l t
t wW>.9 .. ... w ~^8M|
*
:WS
tEV
~~~~~~~~~~~~~~~~~t*~~~1
C
C2
WE HD JH KT
Figure
5.Southern blotanalysis
of selectedlymphoblastHPRT DNA. (A) (top)DNAwasdigestedtocompletion
with BamHI, electro-phoresed through0.8% agarose, transferredtonitrocellulose, and g PC 6804 0probed with full-length 32P-labeled HPRT cDNA. Theportionofthe
autoradiograph
demonstrating
the Et;. :polymorphisms
is shown.(B)
(bot-B4 -tom)
DNAwasdigested
to comple-3k tion withTaq
I,electrophoresedthrough 0.8% agarose, transferred '[
m zto nitrocellulose, and probed with 32P-labeled DXS-1 DNA.
Table III. Molecular Heterogeneity ofHPRT Mutations
Protein analysis
Nucleic acid analysis Electrophoretic
Patients CRM Kinetic properties properties mRNA DNA haplotype* Type
%control kb
Lesch-Nyhan D.A.,A.C., E.C.,
H.D., J.H., D.M. <0.6 NMt ND§ + 22, 25/5 I
B.S. <0.6 NM ND + 12,25/5 II
GM 1899 1.3 NM ND + 22,
25/511
IIIGM6804 <0.6 NM ND Variant¶ Duplication/5** IV
McA <0.6 NM ND - Deletion/7tt V
W.E. <0.6 NM ND - 22, 25/7 VI
GM2292 <0.6 NM ND - 22,25/5 VII
K.T.
(HPRTY¢e)
92 NM Cathodal + 22, 25/7 VIIIHPRT1cinaon
72 TKmHx and PRPP Cathodal + 22, 18/5 IXD.G.(HPRTNeWHaven) 50 NM Anodal + 22, 25/7 X
Gout
P.C., R.T., J.M. 4-0.8 NM ND + 22, 25/5 XI
B.D. 2 NM ND - 22,25/5 XII
HPRTAnn
Arbor 11 TKmHxand PRPP Neutral +22,
25/5
XIIIHPRTToronto 52 Normal Anodal + 22,25/5 XIV
HPRTMUniCh
79 TKmHx and IV..
Cathodal + 22, 25/5 XVHPRTLflndin
and D.B. 35-52 TKmHx Neutral and + 22, 25/5 XVIlapparentsize
* DNA haplotype: Bam
HI
polymorphisms/DXS-10
polymorphisms. tNM, not measured because of limitedcatalytic activity oftheresidualenzyme. §Notdeterminedduetolimited residualenzymeprotein. "ArareBam H1 polymorphism linkedtothe HPRT gene resultsin the production oflow molecularweight restriction fragment. ¶This mRNAis slightly larger than normal. ** Internalduplication ofexons 2and 3. j4 The entire structuralgenehas been deleted.
structural genemutations have rendered theenzymes
completely
incompetent
and haveresulted, clinically,
in the Lesch-Nyhansyndrome.
HPRT from D.B. appears identical toHPRTL,,d00
in terms of itselectrophoretic
andcatalytic properties, despite
thefact
thatpatients
D.B. andG.S. sharenoapparentcommonheritage.
Molecularstudies
areneededtodetermine ifD.B.has theidentical missence mutation ofHPRTLOJdOO
(Ser
109- Leu). Themajority
of
HPRT-deficientpatients
demonstrate no15
c)
z
10
Ua.
0
zr
G
z
H
rIi
CRM+
CRM-CRM-mRNA+
mRNA-Figure6.Molecularclassification of HPRT-deficientpatients.o, gout; o,Lesch-Nyhan.
detectable HPRTprotein.Quantification of HPRT mRNA fur-ther subdividedthis large group of patients into those with HPRT mRNA (CRM- mRNA+) and those without detectable HPRT mRNA(CRM- mRNA-). The former group, comprising 50% of all patients,represents those for which the genetic lesion has affected eitherthe function of mRNA (i.e., efficiency of trans-lation)or thestability of the translation product. Mutations that have a selective effect on the efficiency of mRNA translation without affecting the stability of either the mRNA or its trans-lation product are, to the best of our knowledge, without prec-edent in mammalian systems. Thealternative and more likely explanation isthatthe native enzyme is relatively intolerant to slight perturbations so that minor structural changes, such as amino acid substitutions,mayfrequently affectenzymestability andtherebylead to an accelerated intracellulardegradation of enzymeprotein. Afirststepin understandingthemolecular pa-thology of thislarge groupof patients is to definethe specific mutations involved.
The final category of patients, comprising only 17% of all patients studied, includesthosewithundetectableHPRTprotein and mRNA (CRM- mRNA-). This molecular form of HPRT deficiency is analogoustothethalassemia syndromes,whichare
who were void of detectable HPRT mRNA (1 6).3 Identical anal-yses failed to identify abnormalitiesin the HPRT geneof the three new CRM- mRNA- patients described in this study. However, smalldeletionsorinsertions may have inperceptible changesinrestriction fragment mobilityandtherefore maybe missed when analyzed accordingtothemethod ofSouthern.
The molecular and clinical characterization of 24 unrelated HPRT-deficient patients summarized above
provides
direct ev-idence for theexistence of 12 uniquemutations
attheHPRT locus. Indirect evidence for even greater genetic heterogeneity in this population was provided through the study of linked RFLPs.Neutral haplotype analysis permits the identification of genetic backgrounds on which HPRT mutational events oc-curred,thusexpandingthe number of documentedindependent mutations. Several HPRT-deficient patientswho werepreviouslyindistinguishable
nowbecomeidentifiableasunique
mutational events.Characterizationof the clinical biochemical and molec-ularproperties of the24unrelatedHPRT-deficientpatients,
in concertwith haplotype analysis, providesindirect evidence for the existence of 16 unique aberrant genesin this population. Pertinent data that define theseunique
mutations
are summa-rized in Table III. This marked molecularheterogeneity
provides furthersupportforthe conceptoriginally proposed by
Haldane, thatX-linkedrecessivedisordersaresustainedinthepopulation bythe constantintroduction ofnewmutations. Furthermore, the present study demonstrates that each category ofmutation,
asoutlinedinFig. 6, isrepresentedinbothclinical syndromes. However, mutations with
less deleterious
effects on enzyme functioninvivo(e.g.,mostaminoacidsubstitutions thataffect enzymefunction butnotenzymeconcentration)
are moreoftenassociated with patients
whoarespared
thedevastating
neuro-logic
andbehavioral abnormalities
of Lesch-Nyhan syndrome.
Inconclusion, we analyzed a large population of unrelated HPRT-deficient patients with respect to abnormalities in the HPRT enzyme, mRNA, and gene. This study provides insight into the spectrum and prevalence of mutations that underlie
this heterogeneous disorder
andillustrates
thekind of mutations that can lead toinherited
disease in man.Acknowledgments
We wouldliketothank L. Landa and K. Holmes for their excellent
researchassistance.
Thiswork wassupportedby grantsAM-19045and AM-3 1428 from
theNational Institutes of Healthand also by theHowardHughes
Foun-dation.Mr.Stoutwassupported bythePhilipM. Berlozheimer
Fellow-ship. Dr. Wilsonwas supported by theMedicalScientistTrainingProgram
attheUniversity of Michigan.
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