Biomedical subjects
Publications and source records attributed to E Beutler.
A polymorphic site has been found in the human glucose phosphate isomerase (GPI) gene. This site is produced by a A-->G substitution at nt 489 of GPI cDNA, resulting in a silent mutation. To our knowledge, it is the first defined polymorphism in the gene. It is present with similar gene frequencies in Asian, American White, African American, and Jewish populations.
Five unrelated patients with hereditary glucosephosphate isomerase (GPI) deficiency resulting in nonspherocytic hemolytic anemia were studied. Three new mutations were found in the coding region of the GPI gene: two patients were heterozygous for 223 A-->G (R75G) and 898 G-->C(R300P), respectively and one was homozygous for 1415G-->A(R472H). Surprisingly, 2 previously reported mutations, 286 C-->T and 1039 C-->T, were found in 2 and 3 patients respectively. Until now only 4 of 18 GPI mutations had been found more than once in unrelated patients and these 4 in only 2 patients each. Eleven of the 20 known point mutations have occurred at CpG "hot spots" and the 286 C-->T and 1039 C-->T are among these. The 489 G/A polymorphism in the GPI coding region was used to demonstrate unequivocally that the 1039 C-->T mutation occurred in both haplotypes and therefore probably originated more than once. Because no common GPI mutation has been found we suggest that heterozygosity for GPI confers little if any selective advantage.
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.
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked enzyme defect. We report a new variant, G6PD Durham713G, that is associated with chronic nonspherocytic hemolytic anemia. The G6PD Durham713G variant has a unique biochemical and enzymatic profile and a novel A-->G substitution mutation at nucleotide 713, changing lysine to arginine at amino acid 238. This mutation was not found in the mother of our patient, indicating that G6PD Durham713G resulted from a de novo mutation.
Modified placental human glucocerebrosidase (alglucerase) and recombinant glucocerebrosidase (imiglucerase) are effective means of treating Type 1 Gaucher's disease. Amelioration of hepatosplenomegaly and of haematological manifestations is usually apparent within 6 months. Bone disease responds more slowly but within several years improvement is evident in most patients. Analysis of a large body of data demonstrates that the rate of response of all manifestations of Gaucher's disease is independent of dose over the range of 30 to 260 U/kg body weight per month. Even the response to 15 U/kg per month appears to be equivalent under most circumstances; treatment failures are the same in patients treated with 15, 30 and 130 U/kg per month. Patients with severe manifestations respond more rapidly than those with mild disease, and this, too, is true at all but the 15 U/kg per month dosage level. All available data thus support the administration of no more than 15 to 30 U of alglucerase or imiglucerase per kg/month. Frequent dosing, i.e. three times weekly, appears to be the most effective means of administration.
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Many mutations affecting the glucocerebrosidase gene have been defined as causes of the glycolipid storage disorder, Gaucher disease. These correlate to a certain extent with the clinical course of the disease. Studies of the natural history of Gaucher disease show that progression is usually very slow. Pulmonary involvement, usually regarded as a very rare manifestation, has received more attention recently, and minor abnormalities have been documented in many patients. Replacement of the deficient enzyme with alglucerase or the recombinant from of glucocerebrosidase imiglucerase is effective in treating the disease. All published data confirm that the response to treatment is entirely independent of dose, and this has now been shown to be true of skeletal manifestations of Gaucher disease as well. Consequently, there is no justification for the administration of the large doses of this expensive medication. A number of efforts are being made to develop gene therapy for Gaucher disease, but formidable obstacles must be overcome before such an approach can become feasible.
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BACKGROUND: The 845A(C282Y) mutation in the HLA-H gene accounts for most cases of hereditary hemochromatosis in patients who are of European origin. Some lack this mutation, however, and it is not present in Asian patients. Thus, other mutations either in HLA-H or associated proteins may be present in such patients. HLA-H associates with beta-2-microglobulin. Calreticulin associates with class 1 HLA proteins and appears to be identical with mobilferrin, a putative iron transport protein. These two proteins are therefore candidates for mutations in patients with hemochromatosis. MATERIALS AND METHODS: We have sequenced the coding region and parts of introns of the HLA-H gene, the beta-2-microglobulin gene, and the calreticulin (mobilferrin) gene of 10, 7, and 5 hemochromatosis patients, respectively, selecting those who were not homozygous for the 845A(C282Y) mutation. The number of chromosomes at risk studied were 18 for HLA-H, 14 for beta-2-microglobulin and 10 for calreticulin. RESULTS: We detected 3 new intronic polymorphisms in the HLA-H gene, each a point mutation. Some differences from published sequences of beta-2-microglobulin and calreticulin were documented, but these were uniformly present in all samples. CONCLUSIONS: The lack of additional mutations in the HLA-H gene is remarkable, and we speculate that the C282Y mutation may be a gain-of-function change.
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BACKGROUND: Gene transfer is a potential treatment modality of genetic disease. Efficient, practical methods of DNA transfection are currently under investigation. MATERIALS AND METHODS: A beta-galactosidase reporter plasmid interacted electrostatically with histones, poly-L-Lys, poly-L-Arg, and a combination of poly-L-Lys and poly-L-Arg. This complex was then used to transfect COS-7 cells. beta-galactosidase activity was quantified and used to compare the efficiency of gene transfection in vitro. A comparison was also made of DNA transfection with the most active histone subclass, i.e., histone H2A, in the absence and presence of an anionic liposome. RESULTS: There was a marked increase in DNA transfection in the presence of histone H2A when compared with the control, whereas each of the other histones and polycations showed little, if any, effect. The extent of activation depends strongly on the DNA/histone ratio and is also a function of the molarity of the final Tris-acetate, pH 8, solution. The anionic liposomes used demonstrated an inhibitory effect. CONCLUSIONS: Histone H2A significantly enhances in vitro DNA transfection whereas other histones and anionic liposomes do not. A study of the difference between histone H2A and other histone subclasses may serve to clarify some of the mechanisms and the essential components of efficient gene delivery.
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We have studied four unrelated Italian patients with chronic hemolytic anemia associated with glucose phosphate isomerase (GPI) deficiency. Using intronic primers, we were able to detect the gene alterations on the genomic DNA of the patients. Five different mutations were identified among the eight mutated alleles found: three missense mutations (301A,584T,1028G), one nonsense mutation (286T), and a four nucleotides deletion [Del 1473-IVS16(+2)]. All of these were new except for mutation 1028G, which was previously identified in a Japanese variant (GPI Narita). Two patients were homozygotes (301A/301A and 1028G/1028G), whereas the other two were compound heterozygotes sharing a common mutation [286T/584T and Del 1473-IVS16(+2)/584T]. The missense mutations were found to involve highly conserved amino acids, suggesting that these residues are crucial for the maintenance of the enzyme function. The mutation 286T results in a truncated protein of 95 amino acids in comparison with the 558 of the normal one. The four nucleotides deletion located at the junction of exon/intron 16(5'-TTGGTCGgtgagt-3') is the first GPI mutation affecting a splice site. Moreover one difference from the published sequence (473T-->G) was found in exon five in all of the eight alleles studied and in 30 normal subjects. Correlation was made between mutations, biochemical characteristics of the enzyme, and clinical course of the disease.
A 2-year, placebo-controlled, double-blind, crossover study was started in 1992 to evaluate cladribine, an immunosuppressive drug, in the treatment of chronic progressive multiple sclerosis. In the first year patients were given cladribine 0.10 mg/kg per day for 7 days as four monthly courses for a total of 2.8 mg/kg or placebo. During the second year patients treated with placebo during the first year were given i.v. infusions of 0.10 mg, 0.05 mg, and 0.05 mg of cladribine per kg of body weight per day for 7 consecutive days in three successive monthly courses, for a total dose of 1.4 mg/kg. Patients who had been treated previously with cladribine were crossed over to placebo. Analysis of the results revealed a favorable influence on the neurological performance scores, both in the Kurtze extended disability status and the Scripps neurological rating scale, and on MRI findings in patients treated with cladribine. In the first year the most striking finding was that while clinical deterioration continued in the placebo-treated patients, the condition of patients who received cladribine stabilized or even improved slightly. Toxicity and therapeutic response were dose-related.
A new mutation of the G-6PD gene at position 910 is described. Biochemical analysis suggests that a conformational change results in the enzymatic deficiency associated with G-6PD(West Virginia).
Pyruvate kinase (PK) deficiency due to mutations of the PKLR gene is a common cause of hereditary nonspherocytic hemolytic anemia. Thus far, 55 different mutations have been described in patients with PK-deficient hemolytic anemia. Polymorphisms within the PKLR gene and in the tightly linked glucocerebrosidase (GBA) gene suggest that PK deficiency may represent a balanced polymorphism.
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