Background: Gaucher disease (GD) is an autosomal recessive disorder produced by mutations in the glucocerebrosidase gene (GBA), causing storage of glucosylceramide in reticuloendothelial cells in multiple organs. Traditionally, the prediction of the phenotype based on the genotype has been reported to be limited.Subjects and Methods: We investigated the correlation between the enzymatic residual activity (ERA) and the phenotype at diagnosis of the disease in 45 GD Spanish patients (44 with type I and 1 with type III GD). The genotype involved two of the following previously expressed proteins: c.517A¿>¿C (T134P), 1%; c.721G¿>¿A (G202R), 17%; c.1090G¿>¿T (G325W), 13.9%; c.1208G¿>¿A (S364N), 4.1%; c.1226A¿>¿G (N370S), 17.8%; c.1246G¿>¿A (G377S), 17.6%; c.1289C¿>¿T (P391L), 8.5%; c.1448T¿>¿C (L444P), 3%; and c.1504C¿>¿T (R463C), 24.5%. Recombinant alleles, deletion of 55¿bp in exon 9 and 84GG mutation were considered as mutations with no residual enzymatic activity.Results: The ERA showed a statistically significant correlation with chitotriosidase (P¿<¿0.001), age (P¿<¿0.001), spleen size (P¿<¿0.001), ‘Zimran’s Severity Score Index’ (P¿<¿0.01) and the ‘Gaucher Disease Severity Score Index—Type I’ (P¿<¿ 0.0001) at diagnosis of the disorder. Previous to any medical intervention, a comparison between the ERA and bone involvement, demonstrated a statistically significant relationship (P¿<¿0.01) between the two variables.Conclusions: This study data allowed us to define a new criterion for prognostic assessment of the disease at diagnosis, called Protein Severity Index, which expresses the theoretical severity of the genotype presented by patients, according to the corresponding ERA

Bureo, J. C.

Dalmau, J.

León, P.

Nuñez, R.

Olivera, S.

Torralba, M. A.

Villarrubia, J.

English

Repositorio Universidad de Zaragoza

O R I G I N A L P A P E RResidual enzymatic activity as a prognostic factorin patients with Gaucher disease type 1: correlationwith Zimran and GAUSS-I index and the severityof bone diseaseM.A. Torralba1, S. Olivera1, J.C. Bureo2, J. Dalmau3, R. Nun˜ez4, P. Leon5 andJ. Villarrubia6From the 1Department of Internal Medicine, “Lozano Blesa” University Hospital, Zaragoza, Spain,2Department of Internal Medicine, “Infanta Cristina” University Hospital, Badajoz, Spain, 3Department ofPediatrics, “La Fe´” University Hospital, Valencia, Spain, 4Department of Hematology, “Virgen Del Rocı´o”University Hospital, Sevilla, Spain, 5Department of Hematology, “Dr Peset” University Hospital, Valencia,Spain and 6Department of Hematology, “Ramon Y Cajal” University Hospital, Madrid, SpainAddress correspondence to M.A. Torralba, “Lozano Blesa” University Hospital, Avda. San Juan Bosco, 15; 50009 Zaragoza, Spain. email: mantorralba@gmail.comSummaryBackground: Gaucher disease (GD) is an autosomal recessive disorder produced by mutations in the glucocerebrosidasegene (GBA), causing storage of glucosylceramide in reticuloendothelial cells in multiple organs. Traditionally, the predictionof the phenotype based on the genotype has been reported to be limited.Subjects and Methods: We investigated the correlation between the enzymatic residual activity (ERA) and the phenotype atdiagnosis of the disease in 45 GD Spanish patients (44 with type I and 1 with type III GD). The genotype involved two of thefollowing previously expressed proteins: c.517A>C (T134P), 1%; c.721G>A (G202R), 17%; c.1090G>T (G325W), 13.9%;c.1208G>A (S364N), 4.1%; c.1226A>G (N370S), 17.8%; c.1246G>A (G377S), 17.6%; c.1289C>T (P391L), 8.5%; c.1448T>C (L444P),3%; and c.1504C>T (R463C), 24.5%. Recombinant alleles, deletion of 55 bp in exon 9 and 84GG mutation were considered asmutations with no residual enzymatic activity.Results: The ERA showed a statistically significant correlation with chitotriosidase (P<0.001), age (P<0.001), spleen size(P<0.001), ‘Zimran’s Severity Score Index’ (P<0.01) and the ‘Gaucher Disease Severity Score Index—Type I’ (P< 0.0001) atdiagnosis of the disorder. Previous to any medical intervention, a comparison between the ERA and bone involvement, dem-onstrated a statistically significant relationship (P<0.01) between the two variables.Conclusions: This study data allowed us to define a new criterion for prognostic assessment of the disease at diagnosis,called Protein Severity Index, which expresses the theoretical severity of the genotype presented by patients, according tothe corresponding ERA.Received: 12 November 2015; Revised (in revised form): 5 December 2015VC The Author 2016. Published by Oxford University Press on behalf of the Association of Physicians.All rights reserved. For Permissions, please email: journals.permissions@oup.com449QJM: An International Journal of Medicine, 2016, 449–452doi: 10.1093/qjmed/hcw002Advance Access Publication Date: 19 January 2016Original paperIntroductionGaucher disease (GD) type I (OMIM # 230800) is a lysosomal dis-ease inherited with an autosomal recessive pattern, encom-passing the signs and symptoms occurring in patients with acongenital disorder in the glycolipid metabolism, leading to gly-cosylceramide accumulation. This substance derives from thecellular membrane of senescent leukocytes and it accumulatesin lysosomes of tissue macrophages, due to the deficiency inthe enzyme glycocerebrosidase or b-glycosidase (EC 3.2.1.45).1The gene codifying b-glycosidase is the glucocerebrosidase(GBA) gene, localized in chromosome 1 (q21–q31).2 Mutations inGBA altering the stability of the enzyme or its active site are themain cause of GD. Up to date, more than 300 different muta-tions in GBA have been described and gathered in databases,such as The Human Gene Mutation Database at the Institute ofMedical Genetics in Cardiff (http://www.hgmd.cf.ac.uk/ac/index.php). Out of these mutations, more than 80% are singlenucleotide substitutions and the rest correspond to complexmutant alleles. The four mutant alleles N370S (c.1226 A>G),L444P (c.1448 T>C), 84GG (c.84dupG) and ISV2þ 1 (c.115þ 1G>A)are the most prevalent worldwide.3 Traditionally, it has beenclaimed that it is not possible to foresee the severity of GD basedonly on the genotype; although, patients with a N370S allele areprotected against neuropathic development of the disease,while homozygous patients for L444P probably do developneuropathic pathology.4In 2001, we published the characterization of 10 defective al-leles of GBA with high prevalence in the Spanish population.5Fifteen years later, we plan to use the enzymatic residual activ-ity (ERA) data obtained at the time—by means of expressingeach allele individually—to study the putative correlation be-tween the genotype and phenotype in a series of patients withpredominately GD type I, in order to use this variable to guidegenetic advice.Materials and methodsWe conducted a non-interventionist study, in which six phys-icians from five different University hospitals in Spain, locatedin the cities of Badajoz, Madrid, Seville, Valencia and Zaragoza,voluntarily participated and were submitted the clinical data.Every participating physician provided the first data registeredin the clinical history of their patients with GD at the momentof diagnosis. The data provided included age, gender, genotype,hemoglobin and platelets values, liver and spleen size weremeasured in cm below costal rib margin, presence or absence ofsplenectomy, the activity of plasmatic chitotriosidase, and theZimran’s severity score index (SSI).6 Since every patient (exceptone type III) had GD type I, the ‘Gaucher Disease SSI—Type I’(GauSSI-I) was calculated.7 Special attention was placed on theassessment of bone pathology, which was classified as follows:(i) no bone pathology, (ii) mild bone pathology (osteopenia/osteoporosis or bone pain with no other alteration) and (iii) se-vere bone pathology (any bone lesion other than osteopenia/osteoporosis or bone pain with no other alteration).The residual enzyme activity in each patient was estimatedby adding the activity obtained after individually expressingeach of the two alleles responsible for the disease in COS-1cells.5 The residual activities corresponding to the mutant al-leles have been previously published and are shown in Table 1.After excluding those patients with a genotype containingan allele not previously expressed in COS-1 cells, the final num-ber of patients was 45, and all of them had been diagnosed withGD years earlier by a reduced activity of acid b-glycosidase inleukocytes. Mutant recombinant alleles, the 55 bp deletion inexon 9 and the 84GG mutation were considered as mutationswith no residual enzymatic activity.StatisticsThe statistical analyses were conducted using the SPSS 20.0 soft-ware. Qualitative variables were expressed as percentages andquantitative variables by measures of central tendency and dis-persion (mean, standard deviation and range). A 95% confidenceinterval was calculated for the means. The normality of the vari-ables was assessed by the Kolmogorov–Smirnov test. The correl-ation between quantitative variables was performed using thePearson correlation or Spearman rho, according to the normalityof the variable. Both, the Zimran’s SSI and the GauSSI-I were nor-malized for the maximum score of both indexes in our popula-tion (26 in the case of the Zimran’s SSI, and 17 in the case of theGauSSI-I index) to perform correlation analyses with other vari-ables. Significance was considered for correlations with P< 0.05.A comparison between the ERA and bone pathology was per-formed by means of the analysis of variance (ANOVA) test, andlater on the post hoc analysis to assess the correlation betweenthe ERA and bone pathology of any degree. Last, receiver operat-ing characteristic (ROC) curves were performed to obtain the cor-relation of bone pathology with the different quantitativevariables; when the correlation was significant, the optimal cut-off diagnostic point of such variable was obtained.Table 1. Residual activities corresponding to expressed wild typeand mutant allelesPlasmid Activity (nM/h/mg) Corrected expression %GBA wild type 69.0 233.5 100T134P 2.4 2.4 1G202R 28.1 39.9 17G325W 24.2 32.5 14S364N 9.4 9.6 4N370S 35.6 41.6 18G377S 31.4 40.8 18P391L 6.9 19.7 9L444P 6.9 6.9 3R463C 28.6 57.2 24Figure 1. Genotypes of the studied population and ERA.450 | QJM: An International Journal of Medicine, 2016, Vol. 109, No. 7ResultsOut of the 45 patients with GD, 51% were males. Mean age atdiagnosis was 23 years (range 1–68). The most frequent geno-type was the N370S/L444P (28%), followed by N370S/N370S (21%)and N370S/c.1263del55 (13%) (Figure 1). Clinical and biologicalparameters are shown in Table 2. At the moment of diagnosis,the mean residual activity was 21 (range 0–41). Thirty-ninepoint five percent of patients had no bone disease; 30% hadmild disease and 30% severe disease (Figure 2).When assessing the correlation of the residual enzymatic ac-tivity with the chitotriosidase, age at diagnosis and spleen size, amoderate correlation statistically significant (P< 0.001) wasobserved, in a way such that the lower residual activity, thegreater were the chitotriosidase activity and the spleen size, andthe earlier the age at diagnosis. And this is exactly the same thinghappens with the Zimran´s SSI and the GAUSS-I score (P< 0.001).No significant correlation was observed between the residual en-zymatic activity and hemoglobin, platelets values or liver size.The Zimran’s SSI and GauSSI-I were determined at diagnosiswith a mean of 7.9 (range 1–26) and 7.44 (range 1–17), respect-ively. By means of the Spearman test, it was confirmed a greatcorrelation between both severity indexes (r ¼ 0.691, P< 0.0001).Both indexes were normalized as stated in methods for the fol-lowing correlation analyses. The correlation between theZimran’s SSI and the residual enzymatic activity was moderateand statistically significant (r ¼ 0.548, P< 0.01) and that be-tween the GauSSI-I index and the residual enzymatic activitywas strong (r ¼ 0.665, P< 0.0001), in such a way that a lesser re-sidual activity involves a more severe phenotype presented bypatients at diagnosis. A subanalysis in non-pediatric patients(age 14 years or older) showed a similar moderate correlationbetween the Zimran´s SSI and the residual activity (r ¼ 0.478,P< 0.01), and a stronger correlation between the GauSSI-I indexand the residual activity (r ¼ 0.787, P< 0.0001).The ANOVA test showed a statistically significant (P< 0.01)association between the residual enzymatic activity and bonepathology (Levene statistic: 0.104, F: 9.152, P¼ 0.001). In the posthoc analysis (Scheffe test), a statistically significant difference ofgreater magnitude was observed in the correlation between theresidual activity and the group with bone disease, whether itwas mild or severe (Mild bone disease F:19.23, Severe bone dis-ease F: 20.33, P< 0.05).The correlation between bone disease and the differentquantitative variables was obtained by means of ROC curves andTable2.ClinicalandbiologicalparametersofstudypatientsGenotypeNResidualactivity(%)TypeofGaucherdiseaseAgeatdiagnosisyearsChitotriosidase(mM/mlh)CCL18/PARC(ng/ml)Bonedisease(%ofpatients)Liver(cm)Averageand(statisticalrange)Spleen(cm)Averageand(statisticalrange)SSIAverageand(statisticalrange)GauSSI-IAverageand(statisticalrange)N370S/L444P1321119.6(SD9.5;10–41)10309(7632;1640–28420)489(338;176–859)No27.3%Mild45.5%aSevere27.3%2.6(2.3;0–8)12.5(7.6;0–25)7.1(2.4;4–10)7.55(2.6;2–12)N370S/N370S1036139.9(SD16.3;21–68)6687(3934;1060–12503)1285No80%Mild10%Severe10%1.3(1.6;0–3)1.4(1.4;0–4)4.7(2.3;1–10)3.9(2;1–7)N370S/Del55718122(SD16.4;5–53)21187(11412;1123–33582)209(223;51–367)No14.3%Mild71.4%Severe14.3%5.4(9;0–24)15(10.5;0–30)8.5(2.3;5–11)9.29(3.6;5–16)N370S/84GG41819(SD4.2;4–14)20406(1363;7226–32758)573(131;381–676)No50%Severe50%2.8(0.9;2–4)9.8(3.9;6–14)9(3.2;6–13)11.25(5.1;6–17)N370S/G325W232139.5(SD3.5;37–42)15535(49.5;15500–15570)Mild2.5(0.7;2–3)11.3(10.3;4–19)9(5.7;5–13)7N370S/T134P219112.5(SD0.7;12–13)19275(1539;18187–20364)371Mild50%Severe50%2.5(0.5;2–3)10(4.2;7–13)9.5(0.7;9–10)7.5(0.7;7–8)L444P/L444P16339142Severe100%0726–N370S/Del2pbEx1011811410860Severe100%016910N370S/G202R13517011NotavailableN370S/P391L1261255706Severe100%1711128N370S/S364N1221517085Severe100%57116R463C/G377S1411117461Severe100%5865RecNcil/c225-227delTAC10112371323763Severe100%41410aMildbonedisease(osteopeniaorosteoporosis);severebonedisease(fracturesoravascularnecrosisorbonecrisisordeformitiesorprosthesis).Figure 2. Patients percentage with different degree of bone disease.M.A. Torralba et al. | 451were significant: Zimran’s SSI (Area Under the Curve(AUC):0.872; 95% CI¼ 0.766–0.979); GauSSI-I (AUC:0.799; 95%CI¼ 0.666–0.931); Splenomegaly (AUC:0.721; 95% CI¼ 0.562–0.879)and Hepatomegaly (AUC:0.695; 95% CI¼ 0.523–0.867).DiscussionOur study demonstrates that the residual activity obtained fromthese rare expressed mutant proteins justify a percentage of theclinical profile observed in these Spanish patients, before anyintervention. However the clinical experience with GD and theresults of genotype/phenotype correlation studies suggest thatGBA deficiency is necessary but not sufficient to explain the ul-timate clinical outcome.8 In general, the great variability in sys-temic involvement in GD patients requires considering thatthere are other underlying mechanisms playing a very import-ant role such as promoter mutations, environment, and otherepigenetics and non-genetic causes.9Despite the limitations of this study we consider it is import-ant to communicate our findings to the Gaucher´s scientific com-munity. In the 1990s, the expression studies performed byGrabowski et al.10 and Grace et al.11 showed that the mutationsinvolving exons 5, 8, 9 and 10 are the ones causing the greatestimpact in the catalytic activity of the enzyme. Yet, there aremany mutations described in the literature which have not beenstudied in vitro. Thus, it has not been possible to establish a com-plete classification of the alleles able to determine the severity oftheir phenotype according to their expression. In the studied pa-tients sample, it is clear the statistical correlation between theresidual enzymatic activity—calculated as the addition of the ex-pression percentage of both alleles with respect to the normalprotein expressed in mammalian cells—and determined biolo-gical and clinical parameters before any intervention.Particularly noticeable is the correlation at disease diagnosis,such as age, activity of plasmatic chitotriosidase, spleen size andZimran´s SSI. Furthermore it is mainly illustrative the high correl-ation between the residual enzymatic activity and the GauSSI-Icoefficient. The GauSSI-I index is the most valuable tool from aclinical and phenotypical point of view and for the first time, itscorrelation with the presence or absence of bone involvementhas been shown. In addition, the current study shows that, atleast in this series of patients the GauSSI-I and the Zimran´s SSIshow a high correlation; thus, a more severe genotype—thatwith lower residual activity from the alleles responsible for thedisease—causes a more severe phenotype and vice-versa.The main limitation of the study is the individual assessmentof the alleles; thus, we still need to find out what happens with theresidual activity when both alleles are expressed at the same time.In view of the report data, we can define a new criterion forprognostic evaluation at disease diagnosis, which we havecalled Protein Severity Index, which expresses the theoreticalseverity of the patient genotype, according to the correspondingresidual enzymatic activity. Further studies with a high numberof patients should be performed to establish the utility of thisnew prognosis index.ConclusionsThe study shows that the mutations found in this group of pa-tients cause a decrease of the enzymatic activity, constitutingthe first link in the chain of events participating in the patho-genesis of GD, and explaining a significant amount of the finalphenotype. In addition, this finding allows for the classificationof the studied mutations according to their severity, with thecorresponding implications from the point of view of geneticadvice and disease prognosis.Conflict of interest: Dr Torralba, Bureo, Dalmau, Nu´n˜ez, Leon andVillarrubia are consultants for Genzyme Corporation and ShireCompany and participate in advisory panels and conferenceson lysosomal storage diseases. They have received researchsupport from both companies, but the preparation of this casereport was carried out entirely independently.References1. Brady RO, Kanfer JN, Bradley RM, Shapiro D. Demonstrationof a deficiency of glucocerebroside-cleaving enzyme inGaucher’s disease. J Clin Invest 1966; 45:1112–5.2. Devine EA, Smith M, Arredondo-Vega FX, Shafit-Zagardo B,Desnick RJ. Regional assignment of the structural gene forhuman acid beta-glucosidase to q42 leads to qter on chromo-some 1. Cytogenet Cell Genet 1982; 33:340–4.3. Horowitz M, Tzuri G, Eyal N, Berebi A, Kolodny EH, Brady RO,et al. Prevalence of nine mutations among Jewish and non-Jewish Gaucher disease patients. Am J Hum Genet 1993;53:921–30.4. Dahl N, Lagerstrom M, Erikson A, Pettersson U. Gaucherdisease type III (Norrbottnian type) is caused by a single mu-tation in exon 10 of the glucocerebrosidase gene. Am J HumGenet 1990; 47:275–8.5. Torralba MA, Perez-Calvo JI, Pastores GM, Cenarro A, GiraldoP, Pocovi M. Identification and characterization of a novelmutation c.1090G>T (G325W) and nine common mutant al-leles leading to Gaucher disease in Spanish patients. BloodCells Mol Dis 2001; 27:489–95.6. Zimran A, Kay A, Gelbart T, Garver P, Thurston D, Saven A,et al. Gaucher disease. Clinical, laboratory, radiologic, andgenetic features of 53 patients. Medicine 1992; 71:337–53.7. Di Rocco M, Giona F, Carubbi F, Linari S, Minichilli F, BradyRO, et al. A new severity score index for phenotypic classifica-tion and evaluation of responses to treatment in type IGaucher disease. Haematologica 2008; 93:1211–8.8. Koprivica V, Stone DL, Park JK, Callahan M, Frisch A, Cohen IJ,et al. Analysis and classification of 304 mutant alleles in pa-tients with type 1 and type 3 Gaucher disease. Am J Hum Genet2000; 66:1777–86.9. Goker-Alpan O, Hruska KS, Orvisky E, Kishnani PS,Stubblefield BK, Schiffmann R, et al. Divergent phenotypes inGaucher disease implicate the role of modifiers. J Med Genet2005; 42:e37.10.Grabowski GA, Gatt S, Horowitz M. Acid beta-glucosidase: en-zymology and molecular biology of Gaucher disease. Crit RevBiochemMol Biol 1990; 25:385–414.11.Grace ME, Graves PN, Smith FI, Grabowski GA. Analyses ofcatalytic activity and inhibitor binding of human acid beta-glucosidase by site-directed mutagenesis. Identification ofresidues critical to catalysis and evidence for causality of twoAshkenazi Jewish Gaucher disease type 1 mutations. J BiolChem 1990; 265:6827–35.452 | QJM: An International Journal of Medicine, 2016, Vol. 109, No. 7

Residual enzymatic activity as a prognostic factor in patients with Gaucher disease type 1: Correlation with Zimran and GAUSS-I index and the severity of bone disease

Gaucher disease (GD) is an autosomal recessive disorder produced by mutations in the glucocerebrosidase gene (GBA), causing storage of glucosylceramide in reticuloendothelial cells in multiple organs. Traditionally, the prediction of the phenotype based on the genotype has been reported to be limited. We investigated the correlation between the enzymatic residual activity (ERA) and the phenotype at diagnosis of the disease in 45 GD Spanish patients (44 with type I and 1 with type III GD). The genotype involved two of the following previously expressed proteins: c.517A > C (T134P), 1%; c.721G > A (G202R), 17%; c.1090G > T (G325W), 13.9%; c.1208G > A (S364N), 4.1%; c.1226A > G (N370S), 17.8%; c.1246G > A (G377S), 17.6%; c.1289C > T (P391L), 8.5%; c.1448T > C (L444P), 3%; and c.1504C > T (R463C), 24.5%. Recombinant alleles, deletion of 55 bp in exon 9 and 84GG mutation were considered as mutations with no residual enzymatic activity. The ERA showed a statistically significant correlation with chitotriosidase (P  This study data allowed us to define a new criterion for prognostic assessment of the disease at diagnosis, called Protein Severity Index, which expresses the theoretical severity of the genotype presented by patients, according to the corresponding ERA

Torralba, M A

Olivera, S

Bureo, J C

Dalmau, J

Nuñez, R

León, P

Villarrubia, J

Fondo Bibliográfico Digital Institucional

Residual enzymatic activity as a prognostic factor in patients with Gaucher disease type 1: correlation with Zimran and GAUSS-I index and the severity of bone disease.

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Residual enzymatic activity as a prognostic factor in patients with Gaucher disease type 1: Correlation with Zimran and GAUSS-I index and the severity of bone disease

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