J Med Genet 1993 30: 889-894

REVIEW ARTICLE

The glucocerebrosidase locus in Gaucher'sdisease: molecular analysis of a lysosomalenzyme

P K Mistry, T M Cox

Since the concept of lysosomal storage dis-orders was introduced by Hers in 1965' morethan 30 inherited defects affecting this orga-nelle have been recognised.2 Of these the mostprevalent is the autosomal recessive conditionGaucher's disease, which is caused by defi-ciency of glucocerebrosidase (EC 3.2.1.45).Rapid progress has been made in understand-ing the molecular basis of Gaucher's diseaseand in the development of effective enzymereplacement therapy. Analysis of mutationsthat cause Gaucher's disease will shed light onstructure-function relationships of the gluco-cerebrosidase molecule and should define theinfluence of genotype on disease expression.Molecular analysis of the glucocerebrosidasegene can assist the genetic counselling of fami-lies affected by Gaucher's disease and in theprognosis and treatment of individual patientswith this condition.

Department ofMedicine, Universityof Cambridge,Addenbrooke'sHospital, Hills Road,Cambridge CB2 2QQ,UK.P K MistryT M Cox

Correspondence toDr Mistry.

Historical perspectiveGaucher's disease was first described by Phil-lippe Gaucher in 1882, who reported a patientwith hepatosplenomegaly in whom the charac-teristic abnormal macrophages were thoughtto represent a primary neoplasm of the spleen.3Identification of the stored material as gluco-cerebroside in 1934 was followed many yearslater by the finding that glucocerebrosidaseactivity was markedly reduced in the spleen ofpatients with Gaucher's disease by Brady et a14and Patrick5 independently in 1965. Althoughthe enzyme is deficient in most tissues of thebody, it is the cellular specificity of thesubstrate that determines which tissues are

principally affected. The substrate, glucocere-broside, is a constituent of complex glycolipidsand is released in the breakdown of cell mem-branes, particularly leucocytes and erythro-cytes, by mononuclear phagocytes. Thus, inGaucher's disease engorged macrophages ac-

cumulate throughout the body and cause

hepatosplenomegaly, bone marrow infiltrationwith skeletal disease, and, rarely, involvementof the lungs and brain.

Classification of Gaucher's diseaseThere are three main clinical subtypes ofGaucher's disease.6 Most patients suffer from

visceral involvement in the absence of neuro-logical disease: this non-neuronopathic formof the condition is known as type I Gaucher'sdisease. Within this group there is great vari-ability in the rate of disease progression.Patients may present in childhood withhepatosplenomegaly, pancytopenia, bone nec-rosis, and osteoporosis or come to light in theninth decade of life because of the incidentalfinding of splenomegaly. Type I Gaucher'sdisease occurs rarely in all ethnic groups but itis more frequent in the Ashkenazi Jewishpopulation. A wide range of disease activity isseen in .he Jewish population but patients withlate onset or mild disease are usually foundto be of Ashkenazi descent.6 Type II (acuteneuronopathic) disease is a rare disorder withno ethnic predilection. It causes a rapidlyprogressive neurovisceral storage disease anddeath in infancy. Affected infants come to lightas a result of oculomotor abnormalities, cranialand bulbar nerve palsies, spasticity, convul-sions, and opisthotonus. Type III (subacuteneuronopathic) Gaucher's disease progressesless rapidly but is associated with ataxia,myoclonus, and convulsions; there is progres-sive intellectual impairment which is accom-panied by hepatosplenomegaly and skeletaldisease. Death usually occurs in childhood oradolescence. This type of Gaucher's disease isalso rare but it is frequent in a genetic isolatethat represents a single extended pedigree ori-ginating from the Norrbottnian region ofnorthern Sweden.7

DiagnosisTypically the diagnosis of Gaucher's disease isestablished by the finding of characteristicglycolipid laden large macrophages on histolo-gical examination of bone marrow, the liver, orspleen.68 These findings are not absolutelyspecific since pseudo-Gaucher's cells may ap-pear in the marrow of patients suffering from avariety of conditions associated with increasedcell turnover, including chronic myeloid leuk-aemia and thalassaemia.9 Determination ofleucocyte acid 3-glucosidase activity remains areliable and simple means of confirming thediagnosis of Gaucher's disease.610

Deficiency of glucocerebrosidase resultsfrom defects in the enzyme protein itself but a

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few patients have been reported in whom adeficiency of a heat stable activator (SAP2-sphingolipid activator protein 2) of glucocere-brosidase is responsible for a phenocopy ofGaucher's disease." Biochemical studies of theresidual glucocerebrosidase activity in patientswith Gaucher's disease, combined with thewide spectrum of clinical presentation, had ledto the expectation that multiple mutant alleleswould be responsible for the condition. 12Landmarks in the molecular analysis ofGaucher's disease include the purification ofglucocerebrosidase from human placenta in1973,'3 the cloning of glucocerebrosidasecDNA by Sorge et al'4 in 1985, and the com-plete sequence characterisation of the humanglucocerebrosidase gene and its pseudogene byHorowitz et al'5 in 1989.

Glucocerebrosidase gene andpseudogeneThe structural gene for glucocerebrosidaseand its pseudogene map to human chromo-some lq21. ' The structural gene contains 11exons and spans 7 6 kilobases; the pseudogeneis located 16 kb downstream. In the homo-logous regions, 96% nucleotide identity exists.Promoter regions of genes encoding lysosomalenzymes usually conform to the housekeepingmodel. The promoter of the glucocerebrosi-dase functional gene is unusual in this respectsince it contains TATA and CAAT boxes butno SpI binding site.'5 A promoter of the pseu-dogene can direct transcription of reportergenes and pseudogene transcripts have beenisolated from extracts of normal cells main-tained in culture.'617 These transcripts, how-ever, cannot be translated since they containmultiple stop codons; in addition, exons aredeleted during mRNA processing because ofmutations in consensus splice sites. The pseu-dogene is smaller than the structural genebecause of sequence loss in introns 2 (313 bp),4 (626 bp), 6 (320 bp), and 7 (277 bp) as well astwo missing exon sequences in exon 9 (55 bp)and exon 4 (5 bp). The missing intron se-quences appear to be Alu sequences that havebeen inserted into the functional gene afterthe presumed duplication event that gave riseto the pseudogene.'5 In addition, the pseudo-gene contains multiple point substitutionsthroughout its sequence: when transferred tothe structural gene by gene conversion orrecombination events, these mutations resultin defects in glucocerebrosidase that causeGaucher's disease. Such homologies and dif-ferences between the functional and pseudo-gene have important implications for invest-igation of mutations in Gaucher's disease and,as a result of transcription of the pseudogene,particularly affect the molecular analysis ofglucocerebrosidase cDNA.Complementary DNA encoding human glu-

cocerebrosidase spans about 2 kb. The mRNAis unusual in that it has two functional AUGstart codons (-57 and - 118 nucleotides) buttranslation initiated at either site results infunctional glucocerebrosidase. 4 18 Northernanalysis indicates the presence of three gluco-

cerebrosidase mRNA species of approximately5 6, 2 5, and 2 0 kb. The longest mRNA speciesis thought to represent an incompletely splicednuclear transcript whereas the shorter tran-scripts appear to arise from alternative sites fortranscriptional initiation and polyadenylation.'9As expected for a lysosomal enzyme, a 19 aminoacid hydrophobic signal sequence is present.The mRNA is translated into a protein that iscleaved to produce a mature polypeptide of 497amino acids with a molecular weight of55 5 kDa. The polypeptide contains five poten-tial 0 linked glycosylation sites, four of whichappear to be glycosylated. The active site ofglucocerebrosidase resides in the C-terminalhalf of the molecule and investigations withcovalently bound inhibitors suggest a criticalrole for asp443 and asp358 in catalysis.21Numerous polymorphic sites have been

identified in the glucocerebrosidase gene, eightin introns and three in the 5' untranslatedregion.2' These sequences are in linkage dis-equilibrium and constitute two main haplo-types. Various mechanisms have beenproposed to explain the ascendancy of thesehaplotypes. A search for further polymorphicsites might facilitate detection of carriers inrelatives of patients in whom the nature of themutation has not been established. Previousstudies have identified a common polymor-phism with respect to a PvuII restriction site atgenomic nucleotide position 3931 in intron 6which has been linked to many uncharacter-ised alleles in patients with Gaucher's dis-ease.22

Mutational analysis in Gaucher'sdiseaseHETEROGENEITY OF MUTATIONSThe multiplicity of mutations responsible forGaucher's disease had long been suspectedfrom the clinical heterogeneity of the conditionand the different kinetic properties of the resi-dual enzyme activity.'2 Marked differences inthe synthesis and processing of enzyme proteinhad also been shown,'8 but the extent of theheterogeneity of lesions at the glucocerebrosi-dase locus has only recently become evident asa result of detailed molecular analysis. Excel-lent reviews, to which the reader is referred,catalogue these mutations.'82324 In AshkenaziJews with type I disease, four mutations ac-count for over 98% of disease alleles. Thesesame four mutations account for approxim-ately 60% of disease alleles in patients notknown to have Jewish ancestry; rare or privatemutations account for the remainder.2526 Manypatients with Gaucher's disease exhibit com-pound heterozygosity for mutations at the glu-cocerebrosidase locus.

COMMON MUTATIONSMolecular lesions in the glucocerebrosidasegene described hitherto include missense mu-tations, a splice site mutation, a nucleotideinsertion, deletions, crossovers between thestructural gene and pseudogene, and geneconversion events (non-homologous recombi-

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The glucocerebrosidase locus in Gaucher's disease: molecular analysis of a lysosomal enzyme

nation). The most prevalent mutation in non-neuronopathic Gaucher's disease is asn370_ ser(N370S) which is caused by replacement of anA by G at cDNA nucleotide position 1226.27This mutation accounts for 75% of diseasealleles among Ashkenazi patients and approx-imately 25% among patients not known to beJewish.2528 In vitro expression of this mutantenzyme in insect cells indicates that, comparedwith the wild type enzyme, it has reducedspecific activity and other abnormal catalyticproperties in relation to activation by negat-ively charged phospholipids and SAP2.29 InAshkenazi Jews the N370S mutation is linkedto a PvuII polymorphism; it is invariablyfound in the context of the Pvl.1- genotype.22The observation that most unknown alleles inAshkenazi patients occurred in the context ofthe Pvl.1+ genotype prompted Beutler et aP1to search for other widespread mutations inthis ethnic group. Recently they reported theinsertion of a single nucleotide, G, at cDNAposition 84, that results in a frameshift thatabolishes translation of glucocerebrosidase.30This mutation has so far only been found as asingle copy in compound heterozygotes ofAshkenazi descent and accounts for 13% ofdisease alleles. A less common missense muta-tion, leu4"-4pro (L444P) is caused by replace-ment of a T for a C at cDNA nucleotide 1448.3'This mutation occurs normally in the pseudo-gene and, thus, has the potential to complicatemolecular analysis of the structural gene inGaucher's disease.'5 The L444P mutation ac-counts for only about 2% of disease alleles inAshkenazi patients but for approximately 40%of the alleles in non-Jewish patients.2532 Themutation is associated with all three subtypesof disease but tends to cause severe or neuro-nopathic disease when present in the homo-zygous form. Leucine 444 is located in thecatalytic domain previously identified in theenzyme by reaction with the substrate analo-gue f-conduritol epoxide.20 Disruption of theprotein structure in this region produces anunstable protein that possesses little residualactivity.33

Lately, Beutler et aP4 have described a splic-ing mutation (IVS2 + 1) that results in thedeletion of exon 2 from the mature transcript.This accounts for approximately 3% of diseasemutations in unrelated Jewish patients.32 TheIVS2 + 1 mutation (replacement of G by A atgenomic position 1067) is also normally pre-sent in the glucocerebrosidase pseudogene.Several mutant alleles of glucocerebrosidasediffer from the wild type sequence in as manyas four codons and correspond to sequencevariations found in the pseudogene. Thesecomplex alleles represent chimeric molecules,part functional gene and part pseudogene, thatmay result from gene conversion events orunequal crossing over.3537The most widespread missense mutations

responsible for Gaucher's disease N370S,L444P, and R463C (cDNA 1226G, 1448C and1504T respectively) occur in residues at exons9 and 10 which have been considered criticalfor the formation of the active site. However,other missense mutations occur throughout

the gene (four in exon 5, two in exon 6, one inexon 7, five in exon 8, two in exon 11)182432 andour understanding of the part played by theseresidues in the functional integrity of humanglucocerebrosidase will depend in the firstinstance on the determination of the threedimensional structure of the enzyme at atomicresolution.

Mutational analysis in clinical practiceThe human glucocerebrosidase gene has beensubject to intensive study in relation toGaucher's disease. Given the prevalence ofGaucher's disease and the morbidity associ-ated with its more severe forms, there is a needfor methods to facilitate prenatal screening anddiagnosis. The emergence of an effective al-ternative to marrow transplantation in thetreatment for non-neuronopathic Gaucher'sdisease also has a bearing on diagnosis andprognosis. Although enzymatic assay of acid P-glucosidase activity may serve to confirm thediagnosis of Gaucher's disease when sus-pected, activities obtained in up to 20% ofobligate heterozygotes are within the normalrange of enzymatic activity. Under these cir-cumstances, molecular analysis of the gluco-cerebrosidase gene should aid the definitivedetection of carriers in families at risk forGaucher's disease. In addition, proceduresbased on the polymerase chain reaction for theanalysis of genomic DNA should improve thepotential for early prenatal diagnosis by chor-ionic villus sampling where there has beenexperience of neuronopathic Gaucher's disease(figure).Molecular analysis of the glucocerebrosidase

gene is complicated by the presence of thetranscribed pseudogene and thus methods todetect mutations must either amplify thestructural gene sequences selectively or allowseparation of the amplified structural genefrom the amplified pseudogene sequences.2538Screening for known mutations has relied onhybridisation to allele specific oligonucleotideprobes or, more simply, when the mutationalters a restriction site, by digestion with ap-propriate restriction endonucleases. Examplesof the latter include the NciI site associatedwith the L444P mutation,3' the HhaI site asso-ciated with the P415N mutation,39 and deletionof a CfrlO site in association with N463C.25However, most of the mutations that causeGaucher's disease do not alter restriction sitesand screening for these has been facilitated bythe application of the 'mismatch PCR' tech-nique.' The search for known mutations,regardless of whether or not a restriction site ispresent, has been further simplified by the useof the amplification refractory mutation sys-tem (ARMS) which permits rapid genotypingbased on allele specific amplification in thePCR in the single step25 (figure). These pro-cedures allow genetic diagnosis of Gaucher'sdisease to be carried out in diagnostic labora-tories outside major research centres. In ourlaboratory, among seven Ashkenazi Jewishpatients with Gaucher's disease, 11 diseasealleles were identified as N370S, and two as

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K-Prenatal diagnosis of the L444P mutation in the human glucocerebrosidase gene by ARMS analysis. The PCR wascarried out simultaneously with a primer complementary to the wild type sequence (a) and the mutant sequence (b).The pseudogene, which normally carries the L444P sequence, gives rise to a 55 bp smaller product. I and 2 parents(first cousins); 3 affected child previously treated by bone marrow transplantation at the age of 2 years; 4 CVS:chorionic villous DNA obtainedfrom a further pregnancy at 9 weeks' gestation. 5 DNA obtained from healthydaughter. The assignment of genotypes, including heterozygosity in the fetus, was later confirmed by digestion withthe restriction endonuclease NciI.

84GG, leaving only one unknown allele.Among our other 17 patients from other ethnicbackgrounds, ARMS analysis for commonmutations showed six N370S alleles, nineL444P alleles, two N463C alleles, and oneIVS2 + 1 leaving less than half the diseasealleles in this diverse group of patients uniden-tified.

Population geneticsGaucher's disease appears to be most commonin Ashkenazi Jews in whom a recent survey hassuggested that the frequency of homozygosityfor mutations may be as high as 1 in 976.4' Thisis largely because of the prevalence of theN370S mutation which accounts for 75% ofdisease alleles.28 Other common mutations inthis population are 84GG (13%), IVS2+ 1(3%), leaving less than 5% of allelesunknown.3034 As a result of studies in morethan 2000 Ashkenazi subjects, Beutler et a14'estimated the gene frequency for N370S andthe 84GG mutation to be 0 032 and 0-00217,respectively; thus 1 in 980 offspring of unse-lected marriages between Ashkenazi Jews arepredicted to be N370S homozygotes. In theirsurvey, Beutler et al discovered four asympto-matic persons harbouring two copies of theN370S mutation when they examined a ran-dom Ashkenazi sample. The N370S mutationwas found to be more prevalent than expectedin the healthy Ashkenazi population in relationto a large sample of Ashkenazi patients withGaucher's disease. Beutler et al estimated thatapproximately two-thirds of the alleles weremissing from the patient population withGaucher's disease, once again indicating vari-able expressivity of the homozygous form ofthis mutation and its association with relativelymild disease. This mutation exists at polymor-

phic frequency among Ashkenazi Jews andraises the possibility that it confers a selectiveadvantage in the heterozygous state. TheN370S and 84GG mutations are found consis-tently in the context of their respective haplo-type associations, an observation that suggeststhat they arose on a single ancestral chromo-some and were selected for or spread by diffu-sion from a single founding population. TheSwedish isolate found in Norrbotten isstrongly associated with type III Gaucher'sdisease.7 All 10 patients studied were found tobe homozygous for L444P and careful pedi-gree analysis later showed that these subjectswere descended from a common ancestor innorthern Sweden.742 Among non-Jewishpatients, L444P is the most common diseaseallele and accounts for approximately 40% ofdisease alleles. In the general population,L444P occurs in the context of different haplo-types, a finding that suggests that this patholo-gical variant has arisen repeatedly as a result ofdistinct mutational events.23

Genotype-phenotype correlations inGaucher's diseaseMany inherited diseases show variable clinicalexpressivity and Gaucher's disease is no ex-ception: type II (neuronopathic disease) is acondition of infancy that progresses rapidly todeath whereas other persons homozygous forother mutations in the glucocerebrosidase genemay remain asymptomatic in their ninthdecade of life. Striking heterogeneity of clini-cal expression may also be observed in full sibswithin a given pedigree affected by Gaucher'sdisease and provides clear evidence that factorsoutside the glucocerebrosidase locus may alsoinfluence the degree to which the enzymaticdeficiency is responsible for clinical manifesta-

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The glucocerebrosidase locus in Gaucher's disease: molecular analysis of a lysosomal enzyme

tions. In general, it may be said that the moresevere the disease the lower the level of resi-dual enzymatic activity that can be expected.The level of activity needed to protect

against the more severe manifestations ofGaucher's disease may be a small fraction ofthe normal. Conzelman and Sandhoffl" pro-posed that there exists a critical threshold inthe activity of lysosomal enzymes below whichaccumulation of undegraded substrate occurs.This critical threshold and the rate of accumu-lation of undegraded substrate may differbetween cells, that is, between Kupffer cellsand splenic macrophages or between the samecells at different developmental stages in dif-ferent tissues, depending on the rate of influxof substrate. Thus a very small difference ofresidual activity could markedly affect the ageof onset of the disease, the rate of its progres-sion, and its clinical manifestations. An addi-tional factor in relation to Gaucher's disease isthat, during its course, when the spleenreaches a critical size, hypersplenism may re-sult in the dramatic increase in glycolipidturnover from blood cell membranes. Thiswould be expected to accelerate the depositionof undegraded substrate. Thus secondarypathophysiological disturbances may influencethe apparent severity of Gaucher's diseasedisproportionately and lead to bias in the cor-relation of given genotypes with clinical mani-festations. Other mechanisms may influencethe activity of glucocerebrosidase in Gaucher'scells: there may be enhanced transcription,resulting in a 2 to 3 fold increase in steady statemRNA that would partially compensate fordecreased enzymatic activity'9 and partialamelioration of the disease.

Because of the implications for prognosisand counselling of carriers, an important ob-jective in the molecular analysis of the gluco-cerebrosidase gene has been to correlate thenature of the mutation with the clinicalfeatures of the disease. As indicated above,precise correlation has been difficult to achievein Gaucher's disease. Nevertheless, it is clearthat genotype does influence phenotypestrongly. Indices of disease severity, that is,age of presentation or a composite score basedon other clinical criteria, have been used toestimate rates of disease progression." Ingeneral, homozygosity for the N370S mutationconfers the mildest form of disease and, al-though the presence of another allele is usuallyassociated with more aggressive features,expression of one copy of this mutated genemay indeed provide sufficient enzymatic ac-tivity to preclude development of neurologicaldisease. Some homozygotes for N370S remainasymptomatic and have very few manifesta-tions of the disease even on careful clinicalstudy and, as indicated above, up to two-thirdsof persons with this genotype may escapemedical attention for Gaucher's disease.4'Among those who present with symptoms, themedian age of detection is 30 years but a fewsubjects have a disease with onset in adoles-cence or early adult life. In one such case thepresence of a deleted allele resulting in incor-rect assignment of a homozygous genotype was

later shown.24 Despite these exceptions thereare clearly genetic and environmental factorsthat influence the severity of the disease butwhich are not yet understood. The role ofvariation in the expression or quality of SAP 2in relation to disease expression has yet to besystematically investigated.Homozygosity for the L444P mutation

causes severe Gaucher's disease and correlateswith neurological complications. Here again,however, striking variability in phenotype hasbeen observed ranging from fulminant neuro-logical involvement characteristic of type IIdisease to the more subacute neuronopathicform in type III. A few patients with the type Iphenotype have been found to be homozygousfor the L444P mutation.45 In some type IIpatients who were originally designated L444Phomozygotes, numerous additional mutationsin the glucocerebrosidase gene resulting fromcrossover events with the pseudogene havebeen found.3-37 The correlation with neuro-logical complications of Gaucher's diseasedoes not appear to apply in all populationgroups, however. In Japanese patients homo-zygosity for L444P has been found among thenon-neuronopathic forms of Gaucher's diseaseand a different mutation has primarily beencorrelated with type III disease (phe2'3-8ile)(F2131).4647 Recent investigations of the effectof the R463C mutation illustrate another as-pect of genotype-phenotype correlations inGaucher's disease. In two studies R463C hasbeen found to be associated with severe diseaseand neurological involvement,25 45 but inanother investigation it is associated with milddisease,23 yet when this mutant enzyme isexpressed in insect cells, its specific activityhas been found to be little impaired. However,the enzyme is also much less susceptible toactivation by the sphingolipid activator pro-tein in vitro.48Thus, in any individual patient it is difficult

to predict accurately the outcome of diseasesolely from knowledge of the genotype. Todetermine the origin of phenotypic variation isa scientific question of importance which mayeventually lead to therapeutic approaches todisease modification, for example, in relationto the sphingolipid activator protein. The in-vestigation of the source of phenotypic varia-tion will be greatly helped by the developmentof methods to measure residual glucocerebro-sidase activity in situ and this might beachieved by loading lysosomes in macrophagesobtained from patients with Gaucher's diseasewith radiolabelled glucocerebroside or a suit-able fluorescent artificial substrate.49 Thiswould in the first place allow the hypothesis ofConzelman and SandhoffV3 to be examined. Inthe same way, the production of models ofGaucher's disease in experimental animals50may also assist in the investigation of thecellular pathophysiology of glucocerebrosidasedeficiency in man.

We wish to thank Drs Ian Ellis and AnthonyFensom of the Paediatric Research Unit,UMDS-Guy's Campus, London SEI 9RTfor referring the family depicted in the figure

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Mistry, Cox

for prenatal diagnosis and the Wellcome Trustfor support. Mrs Joan Grantham kindly ren-

dered the manuscript suitable for publication.

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enzyme.disease: molecular analysis of a lysosomal The glucocerebrosidase locus in Gaucher's

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