ISSN 1990-7508, Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 2008, Vol. 2, No. 1, pp. 105–110. © Pleiades Publishing, Ltd., 2008. Original Russian Text © T.M. Boukina, I.V. Tsvetkova, 2008, published in Biomeditsinskaya Khimiya.

CLINICAL STUDIES

Distribution of Mutations of Acid b-D-Glucosidase Gene (GBA) among 68 Russian Patients with Gaucher’s Disease T. M. Boukinaa* and I. V. Tsvetkovab a Research

Centre for Medical Genetics RAMS, ul. Moskvorech’e 1, Moscow, 115478 Russia; tel./fax: (495) 111-8366; e-mail: [email protected] b Institute of Biomedical Chemistry RAMS, Pogodinskaya ul. 10, Moscow, 119121 Russia Received December 21, 2006

Abstract—Gaucher disease (GD) is the most frequent lysosomal storage disease presenting in all populations. Mutations in the acid β-D-glucosidase gene (GBA) cause development of GD, resulting in a decrease or full loss of activity of this enzyme. We report here the results of the molecular-genetic analysis in 68 Russian GD patients from 65 families with the three types of this disease. The GD genotype has been completely elucidated in 58 patients and in all patients we have found at least one mutant allele (92.6%). Besides frequent mutations (p.N370S, c.1263_1317del (del55), p.L444P, p.R463C, Rec NciI) we have identified rare mutations p.R120W, p.R170C, p.R184W, p.G202R, Rec C (p.R120W; p.W184R; p.N188K; p.V191G; p.S196P; p.G202R; p.F213I), presenting in other populations of GD patients. The mutations p.P236T, p.L249Q, p.L288P, p.P319S, p.V352M, p.W381X, p.A384D identified in this study had not been described before. The GBA mutations identified in Russian patients have been compared with those found in patients of other European countries. Genotype-phenotype correlations in GD are discussed. Key words: Gaucher’s disease, acid β-D-glucosidase, acid β-D-glucosidase gene (GBA), molecular-genetic analysis, mutations. DOI: 10.1134/S1990750808010137

INTRODUCTION Gaucher disease (GD; MIM 230800; 230900; 231000) belongs to a group of rare inherited metabolic disorders known as sphingolipidoses (where it is the most frequent disease). GD originally described by Philippe Gaucher in 1882 is inherited as autosomal recessive traits. It is associated with inherited deficit of the lysosomal enzyme, acid β-D-glucosidase (EC 3.2.1.45), resulting in decerased rate of hydrolysis of glucocerebroside followed by its accumulation in GD patients preferentially in macrophage lysosomes. These cells defined as “Gaucher cells” are detected in all tissues and organs. Highest accumulation of these cells found in spleen, liver, and bone marrow determines the development of hepatomegaly, splenomegaly, anemia, pancytemia, and bone damages. Traditionally three types of GD are recognized in dependence of the presence of neurological signs and the rate of their appearance [1]. These include GD type 1 (MIM #230800) known as adult or chronic non-neurological form, GD type 2 (MIM #230900) known as infantile or acute neurological form, and GD type 3 (MIM #231000) juvenile or chronic neurological form. GD is characterized by multiple clinical forms varying *To whom correspondence should be addressed.

from severe one resulting in intrauterine death of a fetus to mild almost symptomless form [2, 3]. Frequency of clinical types of GD also varies in different populations. GD type 1 is the most common form of this disorder. Type 1 occurs more frequently in of Ashkenazi Jewish population (1 : 850); in non Jewish European populations its frequency varies from 1 : 60000 to 1 : 40000). Unlike type 1, types 2 and 3 GD are rarer seen (1 : 100 000 and from 1 : 50 000 to 1 : 100000, respectively). GD is determined by mutations in β-D-glucosidase (GBA) gene. The GBA gene of 7.5 kb in length is located on the long (q) arm of chromosome 1, locus 1q21 and consists of 11 exons (GenBank No. J03059). The pseudogene (psGBA) located at a distance of 16 kb from GBA and shares 97% homology with the functional gene. Primary structure of pseudogene differs from that of GBA by locations of some nucleotides “dissipated” over all length of psGBA, a 55-bp “deletion” from a part of exon 9, and also by four large “deletions” in the introns 2, 4, 6, and 7 (of 313, 626, 320, and 277 bp, respectively), which make this pseudogene shorter than the functional one [4]. About 200 mutations of the GBA gene have been described to date. They have been classified as mild, severe, and lethal, on the basis of the corresponding phenotypic expressions. Mutation is considered as mild

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if it is detected only in patients with GD type 1 (regardless of the dependence of other alleles). Severe mutations are associated with neurological types of GD and lethal mutations have not been ever found in homozygous or heterozygous states with other lethal mutations [5]. Mutations of structural gene and its pseudogene included nucleotide substitutions, resulting in amino acid substitutions, mutations causing changes in the splicing site, and also insertions, deletions, mutations causing reading frameshift, complex alleles (pooling of several mutations). Since some GBA mutations (e.g., p.L444P, p.R120W, c.1213_1317del, p.G202R) represent normal sequence of the pseudogene it is important to used those methods, which can avoid putative influence of pGBA on results of molecular-genetic analysis of patients [4]. Mutant allele frequency and composition differ in various populations. For example, mutations p.N370S, c.84 insG, p.L444P, IVS2+1G>A represent more than 96% mutations among patients of Jewish population with GD type 1, whereas in non-Jewish population they represent just 75% [1, 6]. Japanese patients with GD have frequent mutations p.L444P and p.F213I, whereas mutations p.N370S and c.84insG have not been found [7, 8]. In Portuguese patients with GD the mutation p.N370S represents 63%, whereas rare mutations (p.G377S and p.N396T) are frequent in this population [9, 10]. Many attempts have been undertaken to explain nature of clinical differences in patients with the same type of GD, however, correlations between genotype and phenotype of this disease have not been determined yet; they mainly involve some features of mild mutation p.N370S and severe mutation p.L444P [11]. Nevertheless, the study of geno-phenotypic correlations in GD is important because it would help to predict the development of this disease and optimization of the beginning of enzyme replacement therapy. We demonstrate here results of molecular genetic analysis of 68 Russian GD patients from 65 families with various types of this disease and results of comparison of the ratio of mutant GD alleles among Russian patients and patients from other populations. METHODS Biochemical Methods GD was diagnosed biochemically by results of assay of acid β-D-glucosidase activity in leukocyte homogenate and activity of the marker enzyme of lysosomal diseases, chitotriosidase in blood plasma using the fluorogenic substrates, 4-methylumbelliferyl-β-Dglucopyranoside (Koch-Light) and 4-methylumbelliferyl-D-N,N',N''-triacetylchitotrioside (Sigma) [12].

Molecular-Genetic Methods Genomic DNA was isolated from peripheral blood of patients and their healthy relatives by means of the standard method [13]. DNA diagnostics was based on polymerase chain reaction (PCR) followed by subsequent single-strand conformation polymorphism analysis (SSCP-analysis) [14] in combination with sequencing and restriction analysis. Taking into consideration a 97%-homology of primary structure of GBA and psGBA we have used the method of nested amplification [15]. Using selected primers special fragments from 1.5 to 2.5 kb corresponding to sequences of GBA gene only were synthesized and then used as templates for synthesis of some exons [16]. Identification of Frequent Mutations in GBA Gene DNA samples obtained from all patients were analyzed for frequent mutations c.84insG, IVS2+1G > A, p.N370S, p.D409H, p.L444P, p.R463C, c.1263_1317del, and Rec Nci I (p.L444P; p.A456P; p.V460V). Screening for mutations IVS2+1G > A, p.N370S, p.D409H, p.L444P, p.R463C was based on the restriction analysis of the amplification products of the functional gene. Screening for the mutation c.84insG employed allele specific amplification and screening for the mutation c.1263_1317del55bp was carried out by analysis of amplification products of 9th exon in 8% polyacryl-amide gel. Samples, containing mutation p.L444P were analyzed by the SSCP method followed by subsequent sequencing (to exclude complex mutant alleles). Identification of Rare and New Mutations in GBA Gene Amplified fragments of GBA exons were analyzed by the SSCP method accompanied by subsequent sequencing. Detected mutations were then validated by allele-specific amplification or restriction analysis. In the case of absence of restriction site for particular substitution modified primers creating corresponding sites were synthesized. Characteristics of the Groups of Patients We have examined 68 patients from 65 families. These included 61 patients with GD type 1, 5 patients with GD type 2, and 2 patients with GD type 3. These include previously reported data [13]. Primary examination of patients was carried out by geneticians at Research Centre for Medical Genetics RAMS and also by physicians of regional clinical hospitals. All patients were from various regions of Russia. RESULTS AND DISCUSSION Biochemical diagnostics. Activities of β-D-glucosidase and chitotriosidase were assayed in the group of patients with GD, in control, and in the group of obli-

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gate heterozygotes (patient’s parents) (Tables 1, 2). Mean values of β-D-glucosidase and chitotriosidase activities calculated for these three groups are consistent with literature data [17, 18]. Analysis of chitotriosidase activity in the groups of genotyped patients with GD, obligate heterozygotes and in control has shown that in the case of border values of activity of acid β-D-glucosidase GD may be diagnosed by significantly increased activity of chitotriosidase. Some authors believe that GD severity depends on the level of remaining activity of acid β-D-glucosidase and lower values of the enzyme activity determine neurological forms of this disease [19]. In the examined patients there was not correlation between acid β-Dglucosidase activity with clinical phenotype. In our group the patients with neurological type of GD had activity of acid β-D-glucosidase of 3 nmol/mg/h, whereas patients with non-neurological type of GD had the activity of acid β-D-glucosidase of 0.6 nmol/mg/h. Thus, the decrease of activity of β-D-glucosidase may be used for diagnostics of GD, but not for determination of type of this disease. Molecular-genetic diagnostics. Study of DNA from 68 patients with diagnosed GD (confirmed by biochemical methods) 126 mutant alleles (92.6%) have been identified. Initial screening of all samples for frequent mutations c.84insG, IVS2+1G > A, p.N370S, c.1263_1317del55bp, p.D409H, p.L444P, p.R463C, and Rec Nci I (p.L444P, p.A456P, p.V460V) revealed 98 mutant alleles (72.1%). The substitution p.N370S in exon 9 was the most frequent one (detected in 61 of 136 GD; 44.9%); it was found in five patients in homozygous state. There was substitution p.L444P in exon 10 (detected in 35 of 136 GD alleles); it was also found in five patients in homozygous state. However, only in 22 of 30 patients (27 of 136 GD alleles; 19.9%) the substitution p.L444P was an independent mutation, whereas in 8 patients (8 of 136 GD alleles; 5.9%) it represented a part of complex mutation RecNciI, where it was associated with two substitutions p.A456P and p.V460V of the same exon 10. Lack of mutations p.D409H and del 55 bp in combination with p.L444P allowed to exclude the presence of other complex mutations involving p.L444P. In one patient deletion of 55 bp (c.1263_1317del) was found in heterozygous state in exon 9 as an independent mutations (0.7%). Another patient had substitution p.R463C (0.7%) in heterozygous state. We did not find mutations p.D409H, c.84insG, and IVS2+1G > A in our group of patients. During the second step of this study we found two other mutations p.W184R and p.A384D, which were quite frequent for Russian patients. The substitution p.W184R in exon 6 was found in eight heterozygote patients (5.9%); (in one patient it was detected in the complex Rec C mutation (p.R120W; p.W184R; p.N188K; p.V191G; p.S196P; p.G202R; p.F213I),

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Table 1. Activity of acid β-D-glucosidase in leukocytes Range of valActivity Range ues ( X ± SD) Examined group ( X ± SD) of values (nmol/mg/h) (nmol/mg/h) (nmol/mg/h) [17] Control (n = 219) 10.8 ± 6.3 Heterozygotes 6.8 ± 2.7 (n = 50) Patients (n = 86) 1.9 ± 0.9

2.4–52.7 2.8–12.9

8.2 ± 3.1 –

0–4.2

0.3–3

Table 2. Values of catalytic activity of plasma chitotriosidase in GD Examined group

Activity ( X ± SD) (nmol/ml/h)

Range of valRange of values ues ( X ± SD) (nmol/ml/h) (nmol/ml/h) [18]

Control 20.5 ± 1.3 0.0–200.0 5–199.4 (n = 760) Heterozy23.1 ± 5.5 0.0–94.0 – gotes (n = 50) Patients 11328 ± 1850.5 1175–35550 5580–51800 (n = 85)

formed by 7 point substitutions 6 of which were “originated” from psGBA pseudogene (marked in bold) [20]. Substitution p.A384D in exon 9 was found in five heterozygote patients (3.7%). Three patients had substitution p.R120W (2.1%), two patients had substitution p.G202R (1.4%), two patients had substitution p.L288P (1.4%); one patient in homozygous state had substitution p.V352M (1.4%) and two patients had substitution p.W381X (1.4%). Mutations p.R170C, Rec C (p.R120W, p.W184R, p.N188K, p.V191G, p.S196P, p.G202R, p.F213I), p.P236T, p.L249Q, p.P319S were found only once (by 0.7%). Seven mutations (p.P236T, p.L288P, p.L249Q, p.P319S, p.V352M, p.A384D, p.W381X) have not been described before. Restriction analysis of the amplified fragments of 100 alleles of GBA gene of healthy individual did not reveal any of newly described mutations. This suggests that these new mutations cannot be attributed to manifestations of DNA polymorphisms. In patients of our group mutations were found in exons 5–10 (Table 3). These exons are responsible for control of stability and catalytic activity of acid β-Dglucosidase. Most of previously detected mutations were located in exons 6, 9 and 10. Most of mutations detected in our patients are also located in these exons. Among the 12 mutations identified in this study p.W381X is a nonsense mutation, the 55-bp deletion in exon 9 of GBA gene caused a reading frameshift and formation of premature termination codon in the mid-

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Table 3. Distribution of mutations among exons of GBA gene Changes in nucleotide sequence

No. 1 2 3 4

c.475 C > T c.625 C > T c.667 T > C

5 6 7 8 9 10 11 12 13 14 15 16

c.721 G > A c.823 C > A c.980 T > C c.863 T > A c.1072 C > T c.1171 G > A c.1226 A > G c.1263_1317de155bp c.1259 G > A c.1268 C > A c.1448 T > C c.1448 T > C; c.1483 G > C; c.1497 G > C c.1504 C > T

17

Exon number

Changes in amino acid sequence

5 6

p.R120W p.R170C p.W184R Rec C (p.R120W, p.W184R, p.N188K, p. V191G, p.S196P, p.G202R, p.F213I) p.G202R p.P236T p.L288P p.L249Q p.P319S p.V352M p.N370S p.N382 FS, stop386 p.W381X p.A384D p.L444P Rec NciI (p.L444P; p.A456P; p.V460V) p.R463C

7 8 9

10

Number of mutant alleles in the set

Reference

3 1 7 1

27 6 20 20

2 1 2 1 1 2 61 1 2 5 27 8

5 New mutation New mutation New mutation New mutation New mutation 30 34 New mutation New mutation 30 21

1

31

Note: For designation of mutations traditional amino acid nomenclature has been used; it does not take into consideration first 39 amino acid residues, constituting the signal peptide sequence.

Table 4. Mutation profile of populations of GD patients [8, 9, 28, 29, 32, 33] Mutations p.N370S p.L444P Rec Nci I Others

Russia (n = 68)

Czech Republic (n = 29)

England (n = 46)

Spain (n = 35)

Germany (n = 21)

Portugal (n = 16)

Japan (n = 32)

61/136 27/136 8/136 40/136

28/58 11/58 5/58 14/58

36/92 31/92 4/92 21/92

31/70 18/70 1/70 20/70

17/42 8/42

20/32 7/32 2/32 3/32

0 26/64

dle of exon 9. Complex mutations RecNciI (p.L444P, p.A456P, p.V460V) and Rec C (p.R120W, p.W184R, p.N188K, p.V191G, p.S196P, p.G202R, p.F213I) are formed as recombinations between gene and pseudogene in exones 10 and 5–6, respectively [20, 21]. As in most European populations three mutations were also the most frequent ones in the Russian population of GD patients studied. These included p.N370S, p.L444P, and RecNciI, representing 44.9, 19.9, and 5.9% of total number of alleles, respectively. Quantitative distribution of mutations p.N370S, p.L444P, and recNciI was similar in Russian, Czech, and German populations (Table 4). In contrast to other European populations the Russian population of patients also carried two other frequent mutations: p.W184R, found in

17/42

38/64

eight heterozygote patients from seven families, and p.A384D, found in six unrelated patients. Substitution of Trp-184 for Arg was earlier described as severe one determining GD type 2. In seven our patients with GD type 1 this mutations was found in combination with p.N370S and/or Rec C. In one patient with GD type 2 this substitution was inherited in combination with unidentified mutation (Table 5). A new (previously unknown) substitution of Ala-384 for Asp was found (in combination with p.N370S) in five patients with GD type 1. In one patient this substitution was combined with p.L444P and this determined the development of GD type 2. Functional analysis of mutations p.N382K, p.L383R, p.L385P, which (in combination with p.L444P) determine the development of GD type 2 in

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ACID β-D-GLUCOSIDASE MUTATIONS IN GAUCHER’S DISEASE Table 5. Genotype distribution in Russian patients with various types of GD Type

Genotype

Number of patients

1 type

p.N370S/p.N370S 5/68 (6.7%) p.N370S/p.L444P 14/68 (20.6%) p.N370S/Rec Nci I 8/68 (11.8%) p.N370S/p.W184R 6/68 (8.8%) p.N370S/p.A384D 5/68 (7.4%) p.N370S/p.R120W 2/68 (2.9%) p.N370S/p.G202R 2/68 (2.9%) p.N370S/p.W381X 2/68 (2.9%) p.N370S/p.P236T 1/68 (1.5%) p.N370S/p.L249Q 1/68 (1.5%) p.N370S/p.L288P 1/68 (1.5%) p.N370S/Rec C 1/68 (1.5%) p.N370S/c.1263_1317del55bp 1/68 (1.5%) p.L444P/p.R463C 1/68 (1.5%) p.R120W/p.R170C 1/68 (1.5%) p.L288P/p.P319S 1/68 (1.5%) p.V352M/p.V352M 1/68 (1.5%) p.N370S/? 8/68 (11.8%) p.L444P/? 1/68 (1.5%) 2, 3 types p.L444P/p.L444P 5/68 (7.4%) p.A384D/p.L444P 1/68 (1.5%) p.W184R/? 1/68 (1.5%)

Chinese patients has shown that they should be referred to the group of severe mutations [22]. Taking into consideration these facts we suggest that p.A384D should be also referred to the group of severe mutations. The most frequent genotypes in the group of investigated Russian patients were p.N370S/p.L444P, 14/68 (20.6%), p.N370S/Rec Nci I, 8/68 (11.8%), p.N370S/p.W184R, 6/68 (8.8%), p.N370S/p.A384D, 5/68 (7.4%), p.N370S/p.N370S, 5/68 (7.4%), p.L444P/p.L444P, 5/68 (7.4%). Other 12 genotypes were found in 1–2 cases (Table 5). Geno-phenotypic correlations. Study of genotypephenotype correlations in GD is rather complex and slow process; such complexity is determined by variability of all three types of GD. Differences in clinical manifestations of this disease are observed in patients with the same genotype and also in relatives and even in monozygote twins [23]. Reasons for such variability remain unknown and may be induced by both certain mutations (influencing catalytic function of the protein product) and also by epigenetic factors. Enzymatic activity of acid β-D-glucosidase requires interaction of the enzyme, phospholipids and/or receptor on the inner lysosomal membrane, sphingolipid

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activator protein, saposin C (SAP C) and insoluble substrate, glucosylceramide [24]. In vitro millimolar concentrations of SAP C activate acid β-D-glucosidase in the presence of negatively charged phospholipids. Grabowski et al. proposed a kinetic model of active site of acid β-D-glucosidase; this model suggests the existence of three sites: hydrophilic glycon binding site, aglycon site interacting with acyl chain of glucosylceramide and a site interacting with sphingosine chain of glucosylceramide. Putative region of SAP C binding is located at C-terminal part of this enzyme [25]. Liou et al. studied and characterized 42 amino acid substitutions resulting in GD. These authors indicate that most variants of mutant molecules of acid β-D-glucosidase are characterized by impairments in activation of this enzyme by negatively charged phospholipid molecules (catalytic activity, proteolytic stability and kinetics have been investigated). The most characteristic changes were typical for mutations p.N370S and p.V394L. Mutant variant of acid β-D-glucosidase (substitution p.L444P) demonstrated low effectiveness of SAP C binding and decreased glucosidase activity [26]. Although there are difficulties in determination of geno-phenotypic correlations relationship between some mutations and certain types of GD has been elucidated. For example, the homozygote mutations p.W184R, p.G202R, and p.L444P cause neurological forms of GD, whereas p.N370S/p.N370S has been found in patients with GD type 1. The presence of at least one p.N370S allele usually prevents the development of neurological form of GD. Severity of GD in heterozygote p.N370S carriers is probably determined by the second mutant allele. Since most mutations determining the development of GD are rare ones the evaluation of possible role of newly described mutations in the development of GD may be elucidated either in patient in homozygous state either during analysis of clinical signs of representative patients with the same genotypes. CONCLUSIONS GD is one of the common sphigolipidoses. It is characterized by a wide range of clinical forms varying from mild symptomless forms to severe forms with central nervous system involvement. Determination of β-D-glucosidase activity in leukocytes validates GD diagnosis without histological studies. Study of plasma chitotriosidase activity is also important in GD diagnostics because in some cases remaining activity of β-D-glucosidase of patients may overlap with the enzyme activity in heterozygotes. In this case the level of chitotriosidase activity becomes an additional marker during validation of GD diagnosis. Molecular-genetic analysis of 68 patients verified 92.6% mutant alleles. As in most European populations the most frequent mutations were p.N370S, p.L444P, and RecNciI; they represented 44.8, 19.8, and 5.9% of total number of alleles, respectively. Population of Rus-

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sian patients was characterized by seven p.W184R alleles and five p.A384D alleles in unrelated patients. The rare or single mutations in this group included p.R120W, p.G202R, p.W381X, p.R170C, p.P236T, recC, L288P, p.L288Q, p.P319S, p.R463C, and del55. 7.4% of mutant alleles remained unidentified. The population of Russian patients was characterized by significant molecular heterogeneity (16 various mutant alleles and 17 genotypes). In this population the most frequent genotypes were: p.N370S/p.L444P—10/55 (18%), p.N370S/Rec Nci I—8/55 (15%), p.N370S/p.N370S—5/55 (9%), p.L444P/p.L444P— 4/55 (7%). (1) The molecular-genetic analysis was carried out in 68 Russian GD patients. Screening of frequent mutations revealed five dominating alleles in exons 6, 9, and 10 of GBA gene: p.W184R, p.N370S, p.A384D, p.L444P, and RecNciI. Studies have identified 16 mutations; 7 mutations (p.P236T, p.L288P, p.L288Q, p.P319S, p.V352M, p.W381X, p.A384D) have been described for the first time. (2) Clear correlations have been recognized between severity of GD and detected mutations. In patients in homozygous state p.N370S mutation determines mild or intermediate form of GD type 1. Clinical manifestation of GD in heterozygote carriers of p.N370S is determined by the second mutant allele. In homozygotes mutation p.L444P determined the development of the neurological form of GD type 2 or type 3 and in combination with mutation p.A384D it determines GD type 2. REFERENCES 1. Beutler, E. and Grabowski, G.A., in The Metabolic and Molecular Bases of Inherited Disease, Scriver, C.R., Beaudet, A.L., Valle, D., Sly, W.S., Eds., New York: McGraw-Hill, 2001, pp. 3635–3668. 2. Sidransky, E., Sherer, D.M., and Ginns, E.I., Pediatr. Res., 1992, vol. 32, pp. 494–498. 3. Berrebi, A., Wishnitzer, R., and Von-der-Walde, U., Nouv. Rev. Fr. Hematol., 1984, vol. 26, pp. 201–203. 4. Horowitz, M., Wilder, S., Horowitz, Z., Reiner, O., Gelbart, T., and Beutler, E., Genomics, 1989, vol. 4, p. 87. 5. Beutler, E., Demina, A., and Gelbart, T., Mol. Med., 1994, vol. 1, pp. 82–92. 6. Koprivica, V., Stone, D.L., Park, J.K., Callahan, M., Frish, A., Cohen, I.J., Tavebi, N., Sidransky, E., et al., J. Hum. Genet., 2000, vol. 66, pp. 1777–1786. 7. Kawame, H., Maekawa, K., and Eto, Y., Hum. Mut., 1993, vol. 2, pp. 362–367. 8. Eto, Y. and Ida, H., Neurochem. Res., 1999, vol. 24, pp. 207–211. 9. Amaral, O., Lacedra, L., Santos, R., Pinto, R.A., Aerts, J., SaMirada, M.C., et al., Biochem. Med. Metab. Biol., 1993, vol. 49, pp. 97–107. 10. Amaral, O., Pinto, F., Fortuno, M., Lacerda, L., SaMirada, M.C., et al., Hum. Mut., 1996, vol. 8, pp. 280– 281.

11. McGabe, E.R.B., Fine, B.A., Globus, M.S., et al., JAMA, 1996, vol. 275, pp. 548–553. 12. Beier, E.M., Bukina, T.M., and Tsvetkova, I.I., Vopr. Med. Khim., 2000 vol. 46, pp. 451–454. 13. Bukina, T.M., Vestnik RGMU, 2002, vol. 25, no. 4, pp. 39–42. 14. Condie, A., Borresen, A-L.,Eeles, R., Eeles, R., Borresen, A.L., Coles, C., Cooper, C., Prosser, J., et al., Hum. Mut., 1993, vol. 2, pp. 58–66. 15. Stone, D.L, Tayebi, N., Orvisky, E., Stubblefield, B., Madike, V., and Sidransky, E., Hum. Mut., 2000, vol. 15, pp. 181–188. 16. Bukina, T.M., Biochemical and Molecular Biological Characteristics of Gaucher’s Disease in Russian Patients, Candidate’s Dissertation, GU MGNTS RAMN, Moscow, 2005 17. Galjaard, H., Genetic Metabolic Diseases. Early Diagnosis and Prenatal Diagnosis, Amsterdam-New YorkOxford: Elsevier/North-Holland Biomedical Press, 1980. 18. Guo, Y., He, W., Boer, A.M., Wevers, R.A., de Bruun, A.M., Groener, J.E.M., Hollak, C.E.M., Aerts, J.M., Galiaard, H., van Diggelen, O.P., et al., J. Inher. Metab. Dis., 1995, vol. 18, pp. 717–722. 19. Sandhoff, K., van Echten, G., Schroder, M., Schnabel, D., and Suzuki K., Biochem. Soc. Trans., 1992, vol. 20, p. 695. 20. Latham, T.E., Theophilus, B.D., Grabowsky, G.A., and Smith, F.I., DNA Cell Biol., 1991, vol. 10, pp. 15–21. 21. Latham, T., Grabowski, G.A., Theophilus, B.D., and Smith, F.I., Am. J. Hum. Genet., 1990, vol. 47, pp. 79–86. 22. Tang, N.L.S., Zhang, W., Grabowski, G.A., To, K.F., Choy, F.Y.M., Ma, S.L., and Shi, H.P., Hum. Mut., 2005, vol. 26, pp. 59–60. 23. Lachmann, R.H., Grant, I.R., Halsall, D., and Cox, T.M., QJM, 2004, vol. 97, pp. 199–204. 24. Berent, S.L. and Radin, N.S., Biochim. Biophys. Acta, 1981, vol. 664, pp. 572–582. 25. Grabowski, G.A. and Horowitz, M., Baillieres Clin. Haematol., 1997, vol. 10, pp. 635–656. 26. Liou, B., Kazimierczuk, A., Zhang, M., Scott, C.R., Hegde, R.S., and Grabowski, G., J. Biol. Chem., 2005, vol. 281, pp. 4242–4253. 27. Beutler, E. and Gelbart, T., Hum. Mut., 1996, vol. 8, pp. 207–213. 28. Countre, P., Demina, A., Beutler, T., Beck, M., and Petrides, P.E., Hum. Genet., 1997, vol. 99, pp. 816–820. 29. Cormand, B., Grinbeg, D., Gort, L., Chabas, A., Vilageliu, L., et al., Hum. Mut., 1998, vol. 11, pp. 295–305. 30. Tsuji, S., Choudary, P.V., Martin, B.M., Stubblefield, B.K., Mayor, J.A., Borrangez, J.A., Ginns, E.L., et al., N. Engl. J. Med., 1987, vol. 316, pp. 570–574. 31. Hong, C.M., Ohashi, T., Yu, X.J, Weiler, S., and Barranger, J.A., DNA Cell Biol., 1990, vol. 9, pp. 233. 32. Hatton, C.E., Cooper, A., Whitehouse, C., and Wraith, J.E., Arch. Dis. Chieldhood, 1997, vol. 77, pp. 17–22. 33. Hodanova, K., Hrebicek, M., Cervenkova, M., Mzazova, L., Veprekova, L., Zemen, J. et al., Blood Cell Mol. Dis., 1999, vol. 25, n. 18, pp. 287–298. 34. Beutler, E., Gelbart, T., and West, C., Genomics, 1993, vol. 15, pp. 203–205.

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