I Med Genet 1994,31:401-404

Molecular characterisation of type 1 Gaucherdisease families and patients: intrafamilialheterogeneity at the clinical level

0 Amaral, A M Fortuna, L Lacerda, R Pinto, M C Sa Miranda

AbstractType 1 Gaucher disease families were

studied in an attempt to establish aphenotype/genotype correlation in affec-ted persons and also to identify carriersaccurately. In the Portuguese type 1

Gaucher patients, screening for muta-tions N370S, L444P, R463C, and 1066+1G-*A allowed the identification of85% ofthe alleles among unrelated patients. Asubclinical case with genotype N370S/1066 + 1 G-A was identified in one familyin which there were three other symp-tomatic sibs. To our knowledge this isthe first subclinical case with a genotypeother than N370S/N370S. No genotype-phenotype correlation could be estab-lished and considerable clinical hetero-geneity was found even among sibs withthe same genotype. The data collected onthe origins of the Gaucher families indi-cated two areas in northern Portugalwhere a higher frequency of the diseasemay be expected to exist.

(J Med Genet 1994;31:401-404)

Unidade deEnzimologia, Institutode Genetica MedicaJacinto de Magalhaes,Pr Pedro Nunes 74,4000 Porto, Portugal0 AmaralA M FortunaL LacerdaR PintoM C Sa Miranda

Correspondence toDr Sa Miranda.

Received 23 July 1993Revised version acceptedfor publication22 November 1993

Gaucher disease (GD) is the most prevalentlysosomal storage disorder. This autosomalrecessive disease is caused by the defectivecatabolism of glucosylceramide usually owingto a deficiency of glucocerebrosidase (EC3.2.1.45). Clinically, three major types can bedistinguished on the basis of the presence andextent of neurological involvement. The mostcommon form of GD is the heterogeneousnon-neuronopathic type 1,' which is par-ticularly frequent among Ashkenazi Jews.'

In Portugal, type 1 is also the most frequentform of GD. In a previous work we reportedthat in the Portuguese patients mutationN370S was the most common, accounting forabout 60% of the Gaucher alleles.3 Our resultscontrast with reports on other GD patients ofnon-Jewish ancestry; studies on Canadian,4British,5 and Italian6 patients showed that thismutation accounted for approximately 40% ofthe mutant alleles in type 1 patients. The mainaim of the present work was to characteriseGD families, attempting to establish a correla-tion between the patients' genotype and theirclinical phenotype and to identify the carriersaccurately. Information about the origins ofthe families was also collected in an attempt totrace the families as far back as possible anddetermine the geographical origins of the GDalleles in Portugal.

Materials and methodsPATIENTS AND RELATIVESA total of 23 type 1 Gaucher disease patientsand 95 blood relatives from 11 of the 17Gaucher families were studied. All the peoplestudied were white and of Catholic ancestry.No relationship could be detected between thedifferent families.

Patients were subjected to clinical andanalytical examination. They all presented lowglucocerebrosidase activity in leucocytes. Theclinical examinations were performed by thesame physician (AMF), therefore assuringlittle variability in the clinical evaluation, andthe degree of clinical severity established wasbased on previously proposed parameters.7 Allpatients were free of neurological complica-tions.

SAMPLE PREPARATIONGenomic DNA (gDNA) of patients and closerelatives was isolated from white blood cells bystandard methods8 with minor modifications.Buccal wash samples, which proved to be veryconvenient to obtain, were used in the study ofrelatives. The buccal wash consisted of asterile saline solution. PCR amplification wascarried out using either 500 ng of gDNA or10 1l of the cellular extract. The reactions werecarried out in a total volume of 100 gl.

MUTATION ANALYSISThe DNA of most patients and some relativeswas initially studied by dot blot analysis ofPCR products with ASO probes for the mostcommon mutations.3 Patients with unidenti-fied alleles were screened for a total of 19mutations923 by specific restriction endo-nuclease digestion9l0 of PCR amplified pro-ducts.The screening for recombinant alleles in

patients with mutation L444P was also carriedout by specific restriction enzyme digestion,using StyI and HphI, which allow the identifi-cation of pseudogene sequences at genomicnucleotides 5957 and 6306, respectively.Genomic nucleotide numbering is accordingto the sequence published by Horowitz et al.24

In the characterisation of the families, rela-tives were screened for the mutations identi-fied in the index cases.

ResultsOf all the mutations tested in the patients, onlyfour were encountered, namely N370S, L444P

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Amaral, Fortuna, Lacerda, Pinto, Sa Miranda

25 -

20 -

15 -

10 -

5-

Fl F2 F3 F4 F5 F6 F7 F8 F9 F10Families

F1l F12 F13 F14 F15 F16 F17

::N370S/N370S; *:N370S/R463C; * :N370S/L444P;

* N370S/1066+1G->A; +:N370S/?; 0 :L444P/?

Genotype and clinical severity scores of type 1 GD patients. Patients within families are all sibs. Severity scorescalculated according to Zimran et al.7

(including pseudogene recombinant alleles),R463C, and 1066 + 1 G--A, allowing the iden-tification of 85% of the alleles among unrelatedpatients.

Clinically, the patients' phenotypes rangedfrom subclinical to severe, with no apparentcorrelation with the genotype at the gluco-cerebrosidase locus, as shown in the figure.Intrafamilial variability in the severity ofsymptoms, shown in the table, was particularlyevident in two families (Fl and F12). Family 1was one of N370S homozygotes where, at thetime of the study of two affected sisters, a

subclinical adult male was identified. In family12, there were two moderately affected sisters(P15 and P16), both with early onset, and an

additional patient was diagnosed during herthird pregnancy when haematological prob-lems arose. In this same family, a subclinicalcase (aged 28) was picked up at the time of this

study. All four patients presented the same

genotype (N370S/1066 + 1 G-+A). Additional

clinical heterogeneity was verified in family 6,where P8 and P9 (both N370S/L444P) pre-

sented diverse clinical histories. Patient 8, fouryears younger than her affected brother, hadearlier onset than him followed by massivehepatosplenomegaly and lung involvement.The older brother, P9, who was only identifiedwhile his sister was being studied, was foundto present milder hepatosplenomegaly, radio-graphic signs of bone involvement, anddelayed growth.Of the six patients in the N370S/L444P

group, two (P11 and P12) were found to havepseudogene recombinant alleles. The presenceof recombinant alleles could not be related to

the degree of severity of the illness, althoughone patient died of liver disease (P11) and theother one is a moderately affected woman in

Clinical and analytical characteristics of patients from three unrelated families

Clinical and analytical characteristics*

Family Patient Onset (y) Cytopenia Splenomegaly Hepatomegaly Bone involvement Other organ involvementt

Fl P1 7 + + + ++ -Fl P2 20 - + +

Fl P3+ 29 -F6 P8 3 + + + + + + + - +

F6 P9 6 - + + + + ++ -F12 P15 3 + s + + + ++-F12 P16 12 + s + + + + +

F12 P17 32 + -F12 P18* 28 -

*-not detectable, + mild, + + moderate, + + + massive, s splenectomised.t Other organ involvement refers to kidneys and lungs.+ Subclinical cases presenting glucocerebrosidase deficiency in leucocytes.

x

CD

-0ci

a)

L.._

0)

co

E

Nil

m Ml~~~1 15,16

mm

m r-ii~~~~o M21 M2

in17*K

m M18~~~~~~~

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Molecular characterisation of type 1 Gaucher disease families and patients

her forties (P12). It is worth noting that bothpatients had been splenectomised. In the caseof patients 19 to 23 only one of the alleles wasidentified by testing all the 19 previously men-tioned mutations.Among the 95 blood relatives examined

(including 37 obligate carriers) two subclinicalcases and 72 carriers were identified; 55 carriedmutation N370S, six carried mutation 1066 + 1G--A, five carried mutation L444P, and sixwere obligate carriers for undefined mutations.In two of the families examined (F6 and FIO)two persons were found to be N370S carrierseven though they had not contributed to thepatients' genotype. In one case the carrier wasa non-consanguineous sister in law and in theother case it was a grandmother who hadtransmitted the normal allele to the patient'sfather.The establishment of parental origins

showed that 62-5% of 32 parents originatedfrom two geographical areas in northern Por-tugal, which comprise a population of 0 5million. The remaining 37 5% were scatteredthroughout the country (including the Azoresislands). No interfamilial relationships weredetected; however, detailed genealogy wasunavailable earlier than the third generation.

DiscussionIn the Portuguese patients, the proportions ofGaucher alleles were found to differ from thosepreviously described in non-Ashkenazipatients.2 Identification of85% of the Gaucheralleles among unrelated patients was possibleby screening for mutations N370S (59%),L444P including recombinant alleles (17 5%),R463C (5-9%), and 1066+1 G-.A (2-9%).The severity score applied, although useful

because it allows the standard comparison ofthe degree of disease severity, has a majordraw-back in that patients with differentsymptoms may present the same severityscore, as happened with P13, P15, and P16 (allwith a score of 17).The results obtained clearly indicate that it

is not possible to predict the evolution of theclinical symptoms even among affected sibs.The finding of subclinical cases with alleleN370S in the homozygous state or associatedwith a null allele, such as the 1066 + 1G--Aallele (in P18), contradicts the hypothesis ofthis latter genotype being associated with aparticularly severe clinical phenotype" andseems to indicate that a correlation betweenthe amount of functional residual glucocere-brosidase and the clinical phenotype cannot beestablished in Gaucher disease. The remark-able capacities of activation of the N370Smutated glucocerebrosidase when in the pres-ence of saposin C and phosphatidylserine25could partially account for the considerableheterogeneity verified among patients withallele N370S.On the basis of the N370S carrier frequency

in Portugal,26 a large number of subclinicalhomozygotes is expected to exist. The presentidentification of a subclinical compoundheterozygote (N370S/1066 + 1G-.A) leads us

to believe that the number ofundiagnosed type1 patients may be larger than previouslythought.The lack of correlation between the geno-

type at the glucocerebrosidase locus and thepatient's phenotype suggests that additionalfactors have to be involved in the clinicalexpression of this disease. The identification ofthese factors seems to be essential for thebetter understanding of the pathophysiologyof GD.

We wish to thank the patients and their relatives for theircooperation as well as the clinicians who referred the patients tous and provided their medical records. This work was sup-ported by grants BD/2299/92-ID and BD/1042/90-ID fromJNICT (Portugal).

1 Barranger J, Ginns E. Glucosylceramide lipidoses: Gaucherdisease. In: Scriver CS, et al, eds. The metabolic basis ofinherited disease. New York: McGraw-Hill, 1989:1677-98.

2 Beutler E. Gaucher disease: new molecular approaches todiagnosis and treatment. Science 1992;256:794-9.

3 Amaral 0, Lacerda L, Santos R, Pinto RA, Aerts H, SaMiranda MC. Type 1 Gaucher disease: molecular, bio-chemical, and clinical characterization of patients fromnorthern Portugal. Biochem Med Metab Biol 1993;49:97-107.

4 Choy F, Woo M, Der Kaloustian V. Molecular analysis ofGaucher disease: screening of patients in the Montreal/Quebec region. Am J Med Genet 1991;41:469-74.

5 Walley A, Barth L, Ellis I, Fensom A, Harris A. Gaucher'sdisease in the United Kingdom: screening non-Jewishpatients for the two common mutations. J Med Genet1993;30:280-3.

6 Tuteja R, Bembi B, Agosti E, Baralle F. 1448C mutationlinked to the Pvl .1-genotype in Italian patients withGaucher disease. Hum Molec Genet 1993;2:781-4.

7 Zimran A, Gross E, West C, Sorge J, Kubitz M, Beutler E.Prediction of severity of Gaucher's disease by identifica-tion of mutations at the DNA level. Lancet 1989;ii:349-52.

8 Miller S, Dykes D, Polensky H. A simple salting outprocedure for extracting DNA from human nucleatedcells. Nucleic Acids Res 1988;16:1215.

9 Beutler E, Gelbart T, Kuhl W, Zimran A, West C. Muta-tions in Jewish patients with Gaucher disease. Blood1992;79: 1662-6.

10 Beutler E, Gelbart T, West C. Identification of six newGaucher disease mutations. Genomics 1993;15:203-5.

11 He GS, Grabowski GA. Gaucher disease: a G+ I--A+ 1IVS2 splice donor site mutation causing exon 2 skippingin the acid i-glucosidase mRNA. Am J Hum Genet1992;51:810-20.

12 Beutler E, Gelbart T, Kuhl W, Sorge J, West C. Identifica-tion of the second common Jewish Gaucher disease muta-tion makes possible population based screening for theheterozygote state. Proc Natl Acad Sci USA1991;88: 10544-7.

13 Graves PN, Grabowski GA, Eisner R, Palese P, Smith FI.Gaucher disease type 1: cloning and characterization of acDNA encoding acid ,B-glucosidase from an AshkenaziJewish patient. DNA 1988;7:521-7.

14 Eyal N, Firon N, Wilder S, Kolodny EH, Horowitz M.Three unique base pair changes in a family with Gaucherdisease. Hum Genet 1991;87:328-32.

15 Latham TE, Theophilus BDM, Grabowski GA, Smith FI.Heterogeneity of mutations in the acid ,3-glucosidase geneof Gaucher disease patients. DNA Cell Biol 1991;10: 15-21.

16 Beutler E, Gelbart T. Gaucher disease associated with aunique KpnI restriction site: identification of the amino-acid substitution. Ann Hum Genet 1990;54:149-53.

17 Eyal N, Wilder S, Horowitz M. Prevalent and rare muta-tions among Gaucher patients. Gene 1990;96:277-83.

18 Tsuji S, Martin BM, Barranger JA, Stubblefield BK,LaMarca ME, Ginns EI. Genetic heterogeneity in type 1Gaucher disease: multiple genotypes in Ashkenazic andnon-Ashkenazic individuals. Proc Natl Acad Sci USA1988;85:2349-52.

19 Beutler E, Gelbart T, West C. The facile detection of the nt1226 mutation of glucocerebrosidase by 'mismatched'PCR. Clin Chim Acta 1990;194:161-6.

20 Theophilus BDM, Latham T, Grabowski GA, Smith FI.Comparison of RNase A, chemical cleavage, and GC-clamped denaturing gradient gel electrophoresis for thedetection of mutations in exon 9 of the human acid -glucosidase gene. Nucleic Acids Res 1989;17:7707-22.

21 Widgerson M, Firon N, Horowitz Z, etal. Characterizationof mutations in Gaucher patients by cDNA cloning. AmJHum Genet 1989;44:365-77.

22 Tsuji S, Choudary PV, Martin GM, et al. A mutation in thehuman glucocerebrosidase gene in neuronopathicGaucher disease. N EnglI Med 1987;316:570-5.

403

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404

23 Hong CM, Ohashi T, Yu XJ, Weiler S, Barranger JA.Sequence of two alleles responsible for Gaucher disease.DNA Cell Biol 1990;9:233-41.

24 Horowitz M, Wilder S, Horowitz Z, Reiner 0, Gelbart T,Beutler E. The human glucocerebrosidase gene and pseu-dogene: structure and evolution. Genomics 1989;4:87-96.

25 Sa Miranda MC, Aerts J, Pinto R, et al. Activity of

Amaral, Fortuna, Lacerda, Pinto, Sa Miranda

glucocerebrosidase in extracts of different cell types fromtype 1 Gaucher disease patients. Clin Genet 1990;38:218-27.

26 Lacerda L, Amaral 0, Pinto R, Oliveira P, Aerts J, SaMiranda MC. Gaucher disease: 5841G glucocerebrosi-dase gene frequency in the Portuguese, a non AshkenaziJewish population. Clin Genet (submitted).

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