Structures that delineate orphanin FQ and dynorphin A pharmacological selectivities

A genetic polymorphism in the metabolism of the anticonvulsant drug S-mephenytoin has been attributed to defective CYP2C19 alleles. This genetic polymorphism displays large interracial differences with the poor metabolizer (PM) phenotype representing 2-5% of Caucasian and 13-23% of Oriental populations. In the present study, we identified two new mutations in CYP2C19 in a single Swiss Caucasian PM outlier (JOB 1) whose apparent genotype (CYP2C19*1/CYP2C19*2) did not agree with his PM phenotype. These mutations consisted of a single base pair mutation (G395A) in exon 3 resulting in an Arg132-->Gln coding change and a (G276C) mutation in exon 2 resulting in a coding change Glu92-->Asp. However, the G276C mutation and the G395A mutation resided on separate alleles. Genotyping tests of a family study of JOB1 showed that the exon 2 change occurred on the CYP2C19*2 allele, which also contained the known splice mutation in exon 5 (this variant is termed CYP2C19*2B to distinguish it from the original splice variant now termed CYP2C19*2A). The exon 3 mutation resided on a separate allele (termed CYP2C19*6). In all other respects this allele was identical to one of two wild-type alleles, CYP2C19*1B. The incidence of CYP2C19*6 in a European Caucasian population phenotyped for mephenytoin metabolism was 0/344 (99% confidence limits of 0 to 0.9%). Seven of 46 Caucasian CYP2C19*2 alleles were CYP2C19*2B(15%) and 85% were CYP2C19*2A. The Arg132Gln mutation was produced by site-directed mutatgenesis and the recombinant protein expressed in a bacterial cDNA expression system. Recombinant CYP2C19 6 had negligible catalytic activity toward S-mephenytoin compared with CYP2C19 1B, which is consistent with the conclusion that CYP2C19*6 represents a PM allele. Thus, the new CYP2C19*6 allele contributes to the PM phenotype in Caucasians.     

Hypolactasia and metabolic changes in post-menopausal women

The polymorphic cytochrome P450 CYP2D6 is involved in the metabolism of various drugs of wide therapeutic use and is a presumed susceptibility factor for certain environmentally-induced diseases. Our aim was to define the mutations and alleles of the CYP2D6 gene and to evaluate their frequencies in the European population. Using polymerase chain reaction-single strand conformation polymorphism analysis, 672 unrelated subjects were screened for mutations in the 9 exons of the gene and their exon-intron boundaries. A total of 48 point mutations were identified, of which 29 were novel. Mutations 1749 G-->C, 2938 C-->T and 4268 G-->C represented 52.6%, 34.3% and 52.9% of the mutations in the total population, respectively. Of the eight detrimental mutations detected, the 1934 G-->A, the 1795 Tdel and the 2637 Adel accounted for 65.8%, 6.2% and 4.8% respectively, within the poor metabolizer subgroup. Fifty-three different alleles were characterized from the mutation pattern and by allele-specific sequencing. They are derived from three major alleles, namely the wild-type CYP2D6*1A, the functional CYP2D6*2 and the null CYP2D6*4A. Five allelic variants (CYP2D6*1A, *2, *2B, *4A and *5) account for about 87% of all alleles, while the remaining alleles occur with a frequency of 0.1%-2.7%. These data provide a solid basis for future epidemiological, clinical as well as interethnic studies of the CYP2D6 polymorphism and highlight that the described single strand conformation polymorphism method can be successfully used in designing such studies.     

Cytochrome P450 2D6 variants in a Caucasian population: allele frequencies and phenotypic consequences

Cytochrome P450 2D6 (CYP2D6) metabolizes many important drugs. CYP2D6 activity ranges from complete deficiency to ultrafast metabolism, depending on at least 16 different known alleles. Their frequencies were determined in 589 unrelated German volunteers and correlated with enzyme activity measured by phenotyping with dextromethorphan or debrisoquine. For genotyping, nested PCR-RFLP tests from a PCR amplificate of the entire CYP2D6 gene were developed. The frequency of the CYP2D6*1 allele coding for extensive metabolizer (EM) phenotype was .364. The alleles coding for slightly (CYP2D6*2) or moderately (*9 and *10) reduced activity (intermediate metabolizer phenotype [IM]) showed frequencies of .324, .018, and .015, respectively. By use of novel PCR tests for discrimination, CYP2D6 gene duplication alleles were found with frequencies of .005 (*1x2), .013 (*2x2), and .001 (*4x2). Frequencies of alleles with complete deficiency (poor metabolizer phenotype [PM]) were .207 (*4), .020 (*3 and *5), .009 (*6), and .001 (*7, *15, and *16). The defective CYP2D6 alleles *8, *11, *12, *13, and *14 were not found. All 41 PMs (7.0%) in this sample were explained by five mutations detected by four PCR-RFLP tests, which may suffice, together with the gene duplication test, for clinical prediction of CYP2D6 capacity. Three novel variants of known CYP2D6 alleles were discovered: *1C (T1957C), *2B (additional C2558T), and *4E (additional C2938T). Analysis of variance showed significant differences in enzymatic activity measured by the dextromethorphan metabolic ratio (MR) between carriers of EM/PM (mean MR = .006) and IM/PM (mean MR = .014) alleles and between carriers of one (mean MR = .009) and two (mean MR = .003) functional alleles. The results of this study provide a solid basis for prediction of CYP2D6 capacity, as required in drug research and routine drug treatment.     

Oral ulceration as a presenting sign of unknown sarcoidosis mimicking a tumour: report of 2 cases

OBJECTIVE: To test whether some genotypes for CYP2D6 or CYP2C19 could contribute to longevity, we genotyped 241 Danish nonagenarians and centenarians for CYP2D6 and CYP2C19. METHODS: For CYP2D6 we identified the alleles CYP2D6*1, CYP2D6*3 and CYP2D6*4 with allele-specific polymerase chain reaction (PCR). The CYP2D6*5 alleles were identified with a long PCR method. For CYP2C19 we identified the alleles CYP2C19*1, CYP2C19*2 and CYP2C19*3 with an oligonucleotide ligation assay. RESULTS: The four alleles for CYP2D6 did not occur in Hardy-Weinberg proportions. The frequency of poor metabolism was slightly higher (10.2%) than expected [7.7%; odds ratio (OR) = 1.36 (0.75-2.40)]. The genotypes for CYP2C19 occur in Hardy-Weinberg proportions. The frequency of poor metabolism (3.8%) was not significantly different from a young control group [3.1%; OR = 1.21 (0.26-5.75)]. CONCLUSION: CYP2D6 could play a role in human longevity due to the lack of Hardy-Weinberg proportions. If CYP2D6 only plays a role in longevity by protecting the poor metabolizers from cancer, we should expect a rise in the frequency in these genotypes in Denmark from 7.7% among young adults to 10-11% among very old people. We found a frequency of poor metabolism of 10.2% in the very old group. CYP2C19 is - due to the occurrence of Hardy-Weinberg proportions and the expected number of poor metabolizers unlikely to contribute to human longevity.     

Detection of CYP2D6*3 and 2D6*4 allelic variants by PCR-restriction fragment length polymorphism

The mutant of CYP2D6*3 allele with A2637 deletion in exon 5 and the mutant of CYP2D6*4 allele G1934-->A, splice site defect are among the most common polymorphic alleles of CYP2D6 gene, resulting in a decreased or no activity of CYP isoenzyme. In this study, a reliable polymerase chain reaction-restriction fragment length polymorphism method for identification of CYP2D6*3 and CYP2D6*4 alleles was used to investigate the genotype and phenotype prevalence in the groups of normal controls, and of cirrhosis and cancer patients. The results showed none of 36 controls genotyped for 2D6*3 and 2D6*4 allele to have the 2D6*3 allele with frameshift mutation in exon 5, while 33% (n=12) were found to bear the 2D6*4 allele with G to A mutation at the intron 3-exon 4 junction. In breast cancer patients (n=35) genotyped for 2D6*3 and 2D6*4 alleles, none with 2D6*3 allele was found either, but 60% (n=18) were found to bear the 2D6*4 allele. In patients with head and neck squamous cell cancer, there was only one subject with 2D6*3 allele and he was heterozygous. Among them, as many as ten (40%) patients were found to bear 2D6*4 allele. In the cirrhosis group, none of the patients was found to have the 2D6*3 allele, while the CYP2D6*4 allele was found in 23% (n=6) patients. The phenotype predicted according to the genotype was as follows: in the control group, 3% of individuals were identified as poor metabolizers, 70% as extensive metabolizers, and 27% as heterozygote extensive metabolizers. In the group of breast cancer, 7% of the patients were identified as poor metabolizer, 57% as extensive metabolizer and 36% as phenotype. In squamous cell cancer and cirrhosis patients, the incidence of poor metabolizer was zero, and of heterozygotes extensive metabolizer 42% and 31%, respectively.     

CYP2C19 genotype and phenotype determined by omeprazole in a Korean population

Omeprazole (20 mg orally) was given to 103 healthy Korean subjects and blood was taken 3 h after administration. The plasma concentration ratio of omeprazole and hydroxyomeprazole, used as an index of CYP2C19 activity, was bimodally distributed. Thirteen subjects (12.6%) were identified as poor metabolizers (PMs) with an omeprazole hydroxylation ratio of 6.95 or higher. Among the 206 CYP2C19 alleles, CYP2C19*2 and CYP2C19*3 were found in 43 alleles (21%) and 24 alleles (12%), respectively. Twelve subjects (12%) carried two defect alleles (*2/*2, *2/*3 or *3/*3), 43 subjects (42%) were heterozygous for a mutated (*2 or *3) and a wild type (*1) allele, and the remaining 48 subjects (47%) were homozygous for the wild type allele. The distributions of the metabolic ratio between these three genotype groups were significantly different (Kruskal-Wallis test: p < 0.0001). The genotypes of 19 additional Korean PMs has been identified in a previous mephenytoin study. From a total of 32 PMs, 31 were genotypically PMs by analysis of the CYP2C19*2 and *3 alleles and only one PM subject was found to be heterozygous for the *1 and *2 alleles. At present it cannot be judged whether this subject has a defective allele with a so-far unidentified mutation or a true wild type allele. We thus confirm a high incidence (12.6%) of PMs of omeprazole in Koreans and of the 32 Korean PMs 97% could be identified by the genotype analysis.     

CYP2D6 phenotype-genotype relationships in African-Americans and Caucasians in Los Angeles

CYP2D6 genotyping (CYP2D6*3, CYP2D6*4, CYP2D6*5, CYP2D6*13, CYP2D6*16 alleles and gene duplications) was previously performed on 1053 Caucasian and African-American lung cancer cases and control individuals and no significant difference in allele frequencies between cases and control individuals detected. We have carried out additional genotyping (CYP2D6*6, CYP2D6*7, CYP2D6*8, CYP2D6*9, CYP2D6*10, CYP2D6*17 alleles) and debrisoquine phenotyping on subgroups from this study to assess phenotype-genotype relationships. African-Americans showed significant differences from Caucasians with respect to frequency of defective CYP2D6 alleles, particularly CYP2D6*4 and CYP2D6*5. The CYP2D6*17 allele occurred at a frequency of 0.26 among 87 African-Americans and appeared to explain higher average metabolic ratios among African-Americans compared with Caucasians. CYP2D6*6, CYP2D6*8, CYP2D6*9 and CYP2D6*10 were rare in both ethnic groups but explained approximately 40% of higher than expected metabolic ratios among extensive metabolizers. Among individuals phenotyped with debrisoquine, 32 out of 359 were in the poor metabolizer range with 24 of these (75%) also showing two defective CYP2D6 alleles. Additional single strand conformational polymorphism analysis screening of samples showing large phenotype-genotype discrepancies resulted in the detection of three novel polymorphisms. If subjects taking potentially interfering drugs were excluded, this additional screening enabled the positive identification of 88% of phenotypic poor metabolizers by genotyping. This sensitivity was comparable with that of phenotyping, which identified 90% of those with two defective alleles as poor metabolizers.     

Conventional molecular diagnosis of steroid 21-hydroxylase deficiency using mismatched primers and polymerase chain reaction

We tested a conventional method based on polymerase chain reaction (PCR) and specific primers with one mismatched base at the 3' end to introduce restriction enzyme sites in order to detect mutations of the CYP21 gene without radioisotope. Using this method, the intron 2 mutation causing aberrant splicing of mRNA (In2G) and the exon 4 mutation (Ile->Asn, Ex4) in the CYP21 gene were analyzed. The nonsense mutation in exon 8 (Ex8NON) of the CYP21 gene was also investigated by PCR and subsequent restriction enzyme digestion. The mismatched primers successfully amplified the CYP21 gene containing the In2G and the Ex4 mutation sites, and the presence of these two mutations could be determined by restriction enzyme digestion after PCR. We used this new method to study 33 patients. Twenty-five of these patients were found to have at least one mutation (In2G and/or Ex4 mutation). By enzyme digestion after PCR, the Ex8NON mutation was also identified (7 out of 33 patients). In conclusion, we have developed a new method to detect point mutations in the CYP21 gene. This method was proved to be sensitive and rapid for the detection of the mutations studied. Therefore, this method is suitable for clinical genetic diagnosis.     

MR imaging of rabbit hip cartilage with a clinical imager and specifically designed surface coils

To study whether an in-vitro model for three different genotypes of human CYP2C9*3 polymorphism would be useful for predicting the in-vivo kinetics of (S)-warfarin in patients with the corresponding genotypes, the intrinsic clearance (Cl(int) or Vmax/Km) for (S)-warfarin 7-hydroxylation obtained from recombinant human CYP2C9*1 [wild-type (wt)] and CYP2C9*3 (Leu359/Leu) expressed in yeast and the mixture of equal amounts of these were compared with the in-vivo unbound oral CI (CI(po,u)) of (S)-warfarin obtained from 47 Japanese cardiac patients with the corresponding CYP2C9 genotypes. The in-vitro study revealed that the recombinant CYP2C9*1 (wt/wt), 2C9*3 (Leu359/Leu) and their mixture (Ile359/Leu) possessed a mean Km of 2.6, 10.4 and 6.6 microM and Vmax of 280, 67 and 246 pmol/min/nmol P450, respectively. Thus, the mean in-vitro Cl(int) obtained from recombinant CYP2C9*3 (Leu359/Leu) and the mixture (Ile359/Leu) of 2C9*3 and 2C9*1 were 94% and 65% lower than that obtained from CYP2C9*1 (wt/wt) (6.7 versus 38 versus 108 ml/min/micromol P450, respectively). The in-vivo study showed that the median Cl(po,u) for (S)-warfarin obtained from patients with homozygous (Leu359/Leu, n = 1) and heterozygous (Ile359/Leu, n = 4) CYP2C9*3 mutations were reduced by 90% (62 ml/min) and 66% (212 ml/min, P < 0.05) compared with that obtained from those with homozygous 2C9*1 (625 ml/min, n = 42). Consequently, there was a significant correlation (r = 0.99, P < 0.05) between the in-vitro Cl(int) for (S)-warfarin 7-hydroxylation and the in-vivo Cl(po,u) for (S)-warfarin in relation to the CYP2C9*3 polymorphism. In conclusion, the in-vitro model for human CYP2C9*3 polymorphism using recombinant cytochrome P450 proteins would serve as a useful means for predicting changes in in-vivo kinetics for (S)-warfarin and possibly other CYP2C9 substrates in relation to CYP2C9*3 polymorphism.     

Comparative analysis of histology, DNA content, p53 and Ki-ras mutations in colectomy specimens with long-standing ulcerative colitis

CYP2C19 (S-mephenytoin hydroxylase) is a polymorphically expressed enzyme. Currently, two defective alleles are known--CYP2C19*2 and CYP2C19*3. The authors have developed an oligonucleotide ligation assay to detect these two alleles. This assay combines the hybridization of one common, biotinylated capture probe and two allele-specific probes to the target DNA, with the ability of a DNA ligase to distinguish mismatched nucleotides. The probes are only ligated if they are base paired correctly to the target strand. The biotin is bound to streptavidin, and all DNA not covalently bound to the biotin-labeled capture probe, is removed in a washing procedure. The allele-specific probes are labeled with either europium or samarium, and their emission can be measured simultaneously. The ratio between the emission separates the genotypes. This method was applied on DNA from 19 whites and 21 Vietnamese living in Denmark. All genotypes determined by the assay were consistent with the results from restriction enzyme cleavage. There were 12 poor metabolizers; 10 homozygous CYP2C19*2/CYP2C19*2, one heterozygous CYP2C19*2/CYP2C19*3, and one heterozygous CYP2C19*1/CYP2C19*2. The authors conclude that this assay is well-suited for a high throughput of samples in a routine laboratory. The finding of an apparently heterozygous CYP2C19*1/CYP2C19*2 poor metabolizer, confirms that there are still unknown mutations in CYP2C19.     

Updated sequence information for TEM beta-lactamase genes

Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs such as 6-mercapto-purine, 6-thioguanine and azathioprine. TPMT activity is inherited as an autosomal co-dominant trait, and several mutations in the TPMT gene have been identified which correlate with a low activity phenotype. Although ethnic differences in TPMT activity have been described, population frequency analysis of TPMT alleles has not been well defined in different ethnic groups. The frequency of four allelic variants of the TPMT gene, TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C were compared in British Caucasian (n = 199) and Ghanaian (n = 217) populations using PCR-RFLP and allele-specific PCR-based assays. TPMT*3C was found in 14.8% of Ghanaians (31 heterozygotes, one homozygote). The TPMT*2, TPMT*3A and TPMT*3B alleles were not detected in any of the Ghanaian samples analysed. In contrast, 10.1% of British subjects had variant alleles, consisting of TPMT*2 (n = 2), TPMT*3A (n = 17) and TPMT*3C (n = 1) alleles. The frequencies of mutant alleles in this study were 5.3 and 7.6% in British Caucasians and Ghanaians, respectively. Among Ghanaian tribes, Ewe subjects had a lower frequency of mutant alleles (5.9%) than Ga (13.2%) or Fanti (11.6%), although this did not reach statistical significance. This study provides the first analysis of TPMT mutant allele frequency in an African population and indicates that, unlike Caucasians, TPMT*3C is the most common allele in African subjects.     

An efficient strategy for detection of known and new mutations of the CYP2D6 gene using single strand conformation polymorphism analysis

To detect mutations in the cytochrome P450 CYP2D6 gene (CYP2D6), we developed a strategy based on single-strand conformation polymorphism (SSCP) analysis of the gene amplified by polymerase chain reaction (PCR). The efficiency of the method was evaluated by analysing DNA samples from extensive metabolizers (EM) and poor metabolizers (PM) of debrisoquine. Haplotypes, alleles and mutations of CYP2D6 had previously been characterized in each individual using PCR assays, Xba I restriction fragment length polymorphism (RFLP) and sequencing. PCR-SSCP results were in complete agreement with those obtained using established methods. All previously characterized mutations were associated with particular shifts in the electrophoretic mobility of DNA fragments allowing their identification. We further tested the efficiency of PCR-SSCP for detecting new CYP2D6 mutations. DNA from a PM subject presumed to carry an unknown non-functional mutant allele of CYP2D6 was amplified and bands with aberrant migration patterns were observed on SSCP gels. Sequence analysis of the corresponding DNA fragments revealed the causative mutations. In this way, a novel non-functional allele of the gene, carrying three previously reported mutations and a new mutation in the third exon which results in a premature termination codon, was characterized. Finally, CYP2D6 SSCP analysis was performed on DNA amplified with fluorescent primers and an automated DNA sequencer was used for SSCP analysis of products. We conclude that the PCR-SSCP approach is a powerful method of identifying simultaneously known and new mutations of the CYP2D6 gene.     

Distribution of epithelial-specific antigen in uterine cervix with endocervical glandular dysplasia

The human orosomucoid (ORM) is controlled by two closely linked loci, ORM1 and ORM2, and two tandem genes, AGP1 and AGP2, encoding the proteins produced by the two loci, have been cloned. In this study the molecular basis of ORM1 polymorphism was investigated. For the detection of mutations the products of the six exons of each gene, amplified by the polymerase chain reaction (PCR), were screened by single-strand conformation polymorphism analysis. Subsequently, the exons with an altered migration pattern were gene-specifically amplified by nested PCR. Sequencing of the gene-specific PCR products showed that the three common ORM1 alleles result from A-->G transitions at the codons for amino acid positions 20 in exon 1 and 156 in exon 5 of the AGP1 gene: ORM1*F1 was characterized by CAG (Gln) and GTG (Val), ORM1*F2, by CAG (Gln) and ATG (Met), and ORM1*S, by CGG (Arg) and GTG (Val). The phylogenesis of the genes encoding these three ORM1 alleles is discussed.     

Simple detection of a point mutation in LDL receptor gene causing familial hypercholesterolemia in southern Italy by allele-specific polymerase chain reaction

Polymerase chain reaction (PCR) amplification of specific alleles allowed the rapid detection of a point mutation (missense Gly528 --> Asp) in exon 11 of the low density lipoprotein receptor gene which was otherwise not detectable by exon amplification and enzymatic digestion as it does not modify the normal restriction pattern. The mutant allele, designated as FH-Palermo-1 from the origin of the first carrier family identified, gave a specific PCR product of 109 bp clearly distinct from the product of 168 bp obtained from other alleles with a nonspecific couple of primers. This method allowed us to distinguish one positive sample mixed with up to 11 parts of normal DNA. Furthermore, the specific amplification product was characterized by a Bsm I restriction site not present in nonspecific products.     

Glucokinase gene variants in the common form of NIDDM

To determine whether a structural defect in glucokinase could be a primary cause of glucose intolerance in the common form of NIDDM, the prevalence of mutations in the gene in 60 American black NIDDM patients was investigated. First, by Southern blot analysis of DNA from a subset of randomly selected subjects (n = 20), no gross deletions, insertions, or rearrangements of the gene were detected. Next, the 5'-untranslated and coding regions of the gene were amplified directly from genomic DNA by the polymerase chain reaction. PCR products were screened for mutations by using single-strand conformational polymorphism analysis. A total of nine variants were identified, with two in the 5'-UT regions of islet exon 1, two in the 5'-UT region of liver exon 1, and five in the coding regions. For islet exon 1, 5 of 60 NIDDM patients had both variants in the 5'-UT region; and for liver exon 1, two variants each occurred in 1 of 60 NIDDM patients. The coding region variants included a missense mutation in islet exon 1, substitution of Ala11 (GCC) with Thr11 (ACC), found in 2 patients. The biological consequences of this mutation and the mutations in the 5'-UT portion of the gene have yet to be determined. The rest of the variants were third base pair changes of codons, i.e., silent. A common polymorphism, which was in linkage equilibrium with microsatellite repeats GCK1 and GCK2, was found in intron 9, and a variant in intron 2 in both alleles of 1 patient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of omeprazole as a probe drug for CYP2C19 phenotype in Swedish Caucasians: comparison with S-mephenytoin hydroxylation phenotype and CYP2C19 genotype

A single oral dose of omeprazole (20 mg) was given orally to 160 healthy Caucasian Swedish subjects and tested as a probe for CYP2C19. The study was nonrandomized and included seven subjects previously classified as poor metabolizers (PM) of S-mephenytoin. The ratio between the plasma concentrations of omeprazole and hydroxyomeprazole (metabolic ratio; MR) was determined by HPLC in a blood sample drawn 3 h after drug intake. In 17 subjects the test was repeated and the MRs of omeprazole on the two occasions were correlated (rs = 0.85; p < 0.0001). There was a significant correlation between the MR of omeprazole and the S/R mephenytoin ratio among 141 subjects, in whom both ratios were determined (rs = 0.63, p < 0.001). All seven PMs of S-mephenytoin had higher MRs of omeprazole (7.1-23.8) than extensive metabolizers (EM) (0.1-4.9). All 160 subjects and another 15 Caucasian Swedish PMs previously phenotyped with mephenytoin were analysed with respect to the presence of the CYP2C19m1 allele by PCR amplification of the intron 4/exon 5 junction followed by Sma I digestion. EMs heterozygous for the CYP2C19m1 gene had MRs of omeprazole and S/R ratios of mephenytoin that were higher than those of subjects who were homozygous for the wild-type allele (p = 0.0001). Nineteen of the 22 PMs were homozygous for the CYP2C19m1 gene. Three were heterozygous for this allele. Thus, 41 of the 44 alleles (93%) of PMs were defective CYP2C19m1. One of the remaining three PM alleles was subsequently found to contain the CYP2C19m2 mutation, which has earlier been shown to be associated with the PM phenotype in Oriental populations. In conclusion, the phenotype determined by omeprazole correlated with that of mephenytoin, and was in good agreement with the genotype.     

Response to ICRF-159 in cell lines resistant to cleavable complex-forming topoisomerase II inhibitors

Gaucher disease is the most common lysosomal storage disease with a high prevalence in the Ashkenazi Jewish population but it is also present in other populations. The presence of eight mutations (1226G, 1448C, IVS2+1. 84GG, 1504T, 1604T, 1342C and 1297T) and the complete deletion of the beta-glucocerebrosidase gene was investigated in 25 unrelated non-Jewish patients with Gaucher's disease in Germany. In the Jewish population, three of these mutations account for more than 90% of all mutated alleles. In addition, relatives of two patients were included in our study. Restriction fragment length polymorphism analysis and sequencing of PCR products obtained from DNA of peripheral blood leukocytes was performed for mutation analysis. Gene deletion was detected by comparison of radioactively labelled PCR fragments of both the functional beta-glucocerebrosidase gene and the pseudogene. Among the unrelated patients, 50 alleles were investigated and the mutations identified in 35 alleles (70%), whereas 15 alleles (30%) remained unidentified. The most prevalent mutation in our group of patients was the 1226G (370Asn-->Ser) mutation, accounting for 18 alleles (36%), followed by the 1448C (444Lcu-->Pro) mutation, that was found in 12 alleles (24%). A complete gene deletion was present in two alleles (4%). The IVS1+2 (splicing mutation), the 1504T (463Arg-->Cys) as well as the 1342C (409Asp-->His) mutations were each present in one allele (2%). None of the alleles carried the 84GG (frame-shift), 1604A (496Arg-->His) or the 1297T (394Val-->Leu) mutation. This distribution is different from the Ashkenazi Jewish population but is similar to other Caucasian groups like the Spanish and Portuguese populations. Our results confirm the variability of mutation patterns in Gaucher patients of different ethnic origin. All patients were divided into nine groups according to their genotype and their clinical status was related to the individual genotype. Genotype/phenotype characteristics of the 1226G, 1448C, and 1342C mutations of previous studies were confirmed by our results.