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Biomedical subjects

J Geisel

Publications and source records attributed to J Geisel.

At least 19 recordsLinked to original sources
PubMed2001

Eight novel LDL receptor gene mutations among patients under LDL apheresis in Dresden and Leipzig.

LDL apheresis is highly efficient in reducing elevated plasma cholesterol. Due to strict indications only patients with severe, refractory hypercholesterolemia are treated with this method. Mutations in the LDL receptor gene are major genetic causes for severe hypercholesterolemia. Screening the entire gene in LDL apheresis patients from Saxony should determine whether an increased frequency of defined mutations is responsible for the atherogenic hypercholesterolemia in this group. 31 unrelated patients (15 male, 16 female, age 33-71 yrs.) were included in the analysis. The LDL-R gene was screened using SSCP and/or automated sequencing. The familial defective apolipoprotein B-100 (FDB) mutation was genotyped using established PCR techniques. Nineteen of 31 patients were carriers of an LDL-R mutation. Ten missense and two nonsense mutations, three insertions and two deletions were detected. The mutations C74S, C74R, T87M, 660delC, 662insCCCCG, 680insGGACAAATCTGA, 1428insC and 2167delG have not been previously described. One patient was compound heterozygous for two missense mutations. Two further patients were heterozygous for FDB. No mutations were found among controls. A genetic background for hypercholesterolemia in the LDL-R could be established in about 61% of the patients examined. Therefore, methods of DNA analysis allow to recognize and adequately treat a large portion of high-risk individuals at an early stage.

Adult↗
PubMed2001

Genetic defects as important factors for moderate hyperhomocysteinemia.

The genes for the enzymes methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MS), methionine synthase reductase (MSR) and cytathionine-beta-synthase (CBS) play an important role in homocysteine metabolism. Rare mutations in these genes cause severe hyperhomocysteinemia and clinical symptoms. Growing interest has focused on common mutations with moderate effects on homocysteine levels. We studied 280 subjects of different age groups for the following mutations: MTHFR677C-->T and 1298A-->C, MS2756A-->G, MSR66A-->G and the 68 bp insertion in the CBS gene. The median value for homocysteine increased significantly with age (median homocysteine levels: 7.5, 12.4 and 16.5 micromol/l in the age groups 20-43, 65-75 and 85-96 years, respectively). The genotypes of the MTHFR677C-->T mutation were associated with differences in plasma homocysteine levels, but without reaching significance. Individuals homozygous for the MTHFR677C-->T mutation had a 2.3 micromol/l higher median homocysteine level compared to individuals with the wild-type allele. This effect was pronounced in combination with low folate levels and abolished with higher folate in plasma. For the other three mutations no association with homocysteine values could be determined. The analysis of homocysteine metabolite cystathionine by backward regression analysis revealed a significant correlation of the MS2756A-->G mutation with cystathionine level. This increase could indicate a disturbed remethylation. In summary, larger and homogeneous study populations are necessary to quantify the small effects of common mutations on homocysteine levels. This may also be the reason that no effects of genetic interactions between two genotypes were observed.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed2001

Homocysteine, cystathionine, methylmalonic acid and B-vitamins in patients with renal disease.

Moderate hyperhomocysteinemia is very frequent in renal patients. Aside from homocysteine (HCY) itself, the metabolites methylmalonic acid (MMA) and cystathionine (CYS) supply further information about disturbances in HCY metabolism. In two groups of renal patients, transplant and hemodialysis patients, we measured HCY, MMA and CYS and evaluated their diagnostic value for impaired HCY metabolism due to vitamin deficiency and renal insufficiency. We investigated serum samples from 63 transplant patients and 38 patients undergoing hemodialysis. HCY, MMA and CYS were assayed by gas chromatography-mass spectrometry, vitamin B6 by HPLC, B12 and folate by chemiluminescence immunoassay. The determination of HCY, MMA, and CYS in renal patients provides specific information about intracellular disturbances of HCY metabolism. The frequency of increased metabolite levels in renal patients was much higher than the frequency of lowered vitamin concentrations in serum. Furthermore, the metabolite levels in transplant patients were only moderately increased, whereas they were strongly increased in patients on hemodialysis (HCY 19.2 vs. 28.8 micromol/l, MMA 292 vs. 1025 nmol/l, CYS 733 vs. 2711 nmol/l). Our findings may support the use of MMA determination in the diagnosis of vitamin B12 deficiency in renal patients. Compared to vitamin B12 deficiency, renal dysfunction itself appears to cause only a modest elevation in serum MMA. Regression analysis revealed that the moderate elevation of HCY and CYS in transplant patients is mainly a consequence of impaired remethylation of HCY to methionine with activated transsulfuration, whereas the mildly elevated MMA level is attributable to renal dysfunction. In patients on hemodialysis, all three metabolites were markedly elevated, indicating a strongly disturbed HCY metabolism. Based on a backward regression, we discovered that the HCY metabolism was strongly disturbed by renal insufficiency and vitamin deficiency. The markedly elevated HCY level was mainly attributable to functional vitamin B12 deficiency indicated by high MMA, and the strong CYS elevation was due to renal dysfunction and inhibition of this pathway by low levels of vitamin B6. In conclusion, besides HCY, the determination of MMA and CYS levels supports an early diagnosis of B-vitamin deficiency in renal patients. MMA is a more sensitive indicator of intracellular vitamin B12 deficiency than vitamin B12 in serum.

Adolescent↗
PubMed2000

Fluorescence-based single-strand conformation polymorphism analysis of mutations by capillary electrophoresis.

Capillary electrophoresis in combination with fluorescence-based single-strand conformation polymorphism (SSCP) analysis was used to screen for known mutations as well as for unknown mutations. The mutations causing hemochromatosis and thrombogenetic diseases (factor V Leiden mutation and prothrombin mutation) are well defined. Familial hypercholesterolemia is caused by mutations in the low density lipoprotein (LDL) receptor gene. Because the mutations are heterogeneously localized in all 18 exons of the LDL receptor gene, effective screening procedures are necessary. The three well known mutations and 59 of 61 previously characterized mutations in the LDL receptor gene were detected by a distinct abnormal fragment pattern in capillary electrophoresis. The remaining two mutations in the LDL receptor gene showed only slight abnormalities under standard electrophoresis conditions (13 kV, 30 degrees C, 30 min). However, the abnormal pattern could be amplified by increasing the electrophoresis temperature. In all cases, heterozygous and homozygous mutations could clearly be differentiated from wild-type alleles. Because of the high efficiency of mutation detection, capillary electrophoresis in combination with fluorescence-based SSCP analysis would be attractive for the detection of well-defined mutations as well as for the screening of unknown mutations. The accuracy and the degree of automation make this technique well suited for routine genetic diagnosis.

Animals↗
PubMed2000

Genetic Polymorphism of Methylenetetrahydrofolate Reductase (MTHFR) and Coronary Artery Disease.

A high plasma homocysteine concentration is a risk factor for atherosclerotic disease and venous thrombosis. Homocysteine levels are influenced by folic acid, vitamin B 6 and vitamin B 12, as well as by hereditary factors. A common genetic variant of the methylenetetrahydrofolate reductase (MTHFR) gene CC 677 T) is associated with thermolability of the MTHFR enzyme and elevated plasma homocysteine concentration, especially in those with low folic acid concentration. The prevalence of point mutation (nucleotide 677 C --> T) in MTHFR was measured in patients with coronary artery disease (CAD) who all underwent coronary artery bypass surgery (62 cases; age 64.0 +/- 9.5 years), and was compared with, age-matched control subjects. In patients with coronary artery disease (CAD), we investigated the prevalence of point mutation (nucleotide 677 C --> T) in MTHFR in comparison with control subjects. Heterozygous (C/T) prevalence for the 677 C --> T mutation in the MTHFR was higher in patients with CAD than in control subjects (P < 0.05). The prevalence of homozygosity (C/C) for wild-type MTHFR was lower in patients with CAD in comparison with control subjects (P < 0.05).

Journal Article↗
PubMed2000

Role of homocysteine, cystathionine and methylmalonic acid measurement for diagnosis of vitamin deficiency in high-aged subjects.

BACKGROUND: Intracellular B-vitamin and folate deficiency indicated by hyperhomocysteinemia is very frequent in the elderly population. Hyperhomocysteinemia increases the risk of atherothrombotic diseases and neuropsychiatric complications. Our aim was to evaluate the prevalence of increased serum metabolite concentrations in subjects of a higher age, and whether the measurement of metabolite concentrations is more effective in diagnosing B-vitamin deficiency than mere homocysteine. MATERIALS AND METHODS: Homocysteine (HCY), cystathionine (CYS) and methylmalonic acid (MMA) were investigated in serum together with vitamin B-12, B-6 and folate in 90 high-aged subjects (85-102 years), 92 seniors (65-75 years), and in 50 younger subjects (19-50 years). RESULTS: Elderly subjects (high-aged and senior) had elevated serum concentrations of metabolites. High-aged subjects had a higher frequency of pathological increases than seniors: HCY 62% vs. 24%; MMA 62% vs. 23%; CYS 81% vs. 36%. Folate and vitamin B-6 concentrations were significantly decreased in both elderly groups; vitamin B-12 was only decreased in high-aged subjects. Utilising vitamin B-6, B-12 and folate for diagnosis of intracellular vitamin deficiency, the rate was 30% in seniors and 55% in high aged subjects. However, utilising the metabolites (HCY, MMA and CYS) for the diagnosis of intracellular vitamin deficiency, there was a distinctly increased rate of 55% in seniors respective to 90% in high-aged subjects. Backward multiple regression analysis revealed that only folate, MMA, creatinine and age were independent variables influencing the HCY concentration. Furthermore, the MMA concentration was significantly and independently influenced by folate, vitamin B-12, HCY and creatinine, and the serum concentration of CYS by vitamin B-12, creatinine and age. CONCLUSION: The metabolites HCY, MMA and CYS are sensitive indicators diagnosing impaired remethylation of homocysteine to methionine with parallel activation of catabolic pathway. Compared to mere HCY or B-vitamins in serum, the efficiency of diagnosing a disturbed HCY metabolism increases very much in utilising the metabolites HCY, MMA and CYS. For differential diagnosis, parallel measurement of folate and creatinine is recommended. The early and correct diagnosis of B-vitamin deficiency in elderly subjects is of high clinical relevance.

Adult↗
PubMed2000

Plasma homocysteine and lipoprotein profile in patients with peripheral arterial occlusive disease.

Several studies have identified moderate hyperhomocysteinemia (HCy) as an independent risk factor for atherosclerosis. The purpose of this case control study was to determine lipoprotein profile and homocysteine concentration in serum of 85 male patients with peripheral arterial occlusive disease (PAOD) and in 51 normolipidemic age-matched male controls. Cholesterol, triglycerides, and high-density lipoprotein (HDL) cholesterol as well as subfractions HDL2 and HDL3 cholesterol, low-density lipoprotein (LDL) cholesterol, apo B, apo A-I, and lipoprotein particles LpA-I and LpA-I:A-II were measured in serum. Homocysteine, folic acid, and vitamins B6 and B12 were determined with the help of high-pressure liquid chromatography. The 677 C --> T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene was analyzed in PAOD patients. Patients with peripheral arterial occlusive disease showed a significantly higher mean concentration of homocysteine than control subjects (p<0.001). There was a negative correlation between the levels of homocysteine and vitamin B12 as well as folic acid (for vitamin B12: r=-0.40 and for folic acid: r=-0.38). The prevalence of hyperhomocysteinemia (Hcy >16 micromol/L) in the patients was 45% in contrast to 8% in controls. HDL cholesterol, HDL3 cholesterol, Apo A-I, and Lp A-I were significantly reduced in patients and triglycerides were elevated. The elevated plasma homocysteine concentration is frequently seen in homozygous carriers of a point mutation (677 C --> T) in the methylenetetrahydrofolate reductase gene, as the product of this gene is an enzyme, participating in homocysteine remethylation. The homozygous state for the 677 C --> T mutation was found in 13.3% of PAOD patients.

Aged↗
PubMed1999

Fluorescence-based single-strand conformation polymorphism analysis of the low density lipoprotein receptor gene by capillary electrophoresis.

We describe here a new method to screen for unknown mutations in the low density lipoprotein (LDL) receptor gene by the use of capillary electrophoresis in single-strand conformation polymorphism (SSCP) analysis. To analyze the promoter and all 18 exons, 20 different amplification reactions were necessary. For each polymerase chain reaction (PCR), the forward and reverse primers were 5' fluorescent-labelled with FAM and HEX, respectively. To test the accuracy of the newly developed method, 61 genetic variants distributed in 16 exons were analyzed. Under identical electrophoresis conditions (13 kV, 30 degrees C, 30 min), 59 mutations were detected by a distinct abnormal SSCP pattern. The two remaining mutations showed only slight abnormalities, which could be amplified by increasing the electrophoresis temperature. The high accuracy, the degree of automation and the speed of analysis make fluorescence-based SSCP analysis with capillary electrophoresis ideal for rapid mutation screening and the technique is well-suited for clinical applications.

Base Sequence↗
PubMed1999

Hyperhomocysteinemia in high-aged subjects: relation of B-vitamins, folic acid, renal function and the methylenetetrahydrofolate reductase mutation.

Moderate hyperhomocysteinemia is an atherogenic risk factor and plays an important role in geriatrics. Here, we have investigated the role of hyperhomocysteinemia in two elderly groups: 104 longeval subjects of 85-102 years, 100 seniors aged 65-75 years, and 75 controls of 19-60 years. Elevated homocysteine levels were found in 58% of longeval subjects in comparison with 32% in seniors. The homocysteine level in serum correlated positively with age as well as serum creatinine, and inversely with serum folate, but there was no correlation with serum B-vitamins. The frequency of vitamin B deficiency in serum of longeval subjects compared to seniors was as follows: vitamin B6 43% vs. 22%, vitamin B12 20% vs. 8%, and folic acid 1% in both groups. Increased serum creatinine levels (> 1.1 mg/dl) were found in 63% of the longeval subjects and in 32% of seniors. The 677-missense mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, responsible for moderate homocysteine elevation, was found in 35, 37 and 27% of alleles in longeval persons, senior subjects and younger controls, respectively, showing no significant difference in frequency distributions of the MTHFR gene mutation. It can be concluded that hyperhomocysteinemia is very common with increased age. Its importance as an atherogenic risk factor with advanced age has to be clarified.

Adult↗
PubMed1998

Lipid-free apolipoprotein (apo) A-I is converted into alpha-migrating high density lipoproteins by lipoprotein-depleted plasma of normolipidemic donors and apo A-I-deficient patients but not of Tangier disease patients.

Plasma of patients with Tangier disease (TD) is devoid of alpha-LpA-I (apolipoprotein A-I-containing lipoprotein), which in normolipidemic plasma constitutes the majority of high density lipoprotein (HDL). The residual amounts of apolipoprotein A-I (apo A-I) in TD plasma have electrophoretic prebeta1-LpA-I mobility. We have previously demonstrated that TD plasma does not convert prebeta1-LpA-I into alpha-LpA-I. In this study we found that plasmas of normolipidemic controls, apo A-I-deficient patients and patients with fish-eye disease, but not plasmas of six TD patients, convert biotinylated lipid-free apo A-I into alpha-LpA-I. Supplementation of plasma with free oleic acid or fatty acid free albumin neither inhibited conversion activity in normal plasmas nor reconstituted it in TD plasma. In normal plasma the conversion activity was assessed in HDL and in the lipoprotein-free fraction. The latter fraction, however, generated larger particles only in the presence of exogenous phospholipid vesicles. To obtain particles with alpha-mobility, these vesicles had to contain phosphatidylinositol and/or cholesterol. Lipoprotein-depleted TD plasma did not convert lipid-free apo A-I into alpha-LpA-I even in the presence of exogenous vesicles with phospholipids or cholesterol. Taken together we conclude that disturbed transfer of glycerophospholipds onto apo A-I or prebeta1-LpA-I prevents maturation of HDL and thereby possibly causes deficiency of HDL cholesterol in patients with TD. Moreover, the lack of alpha-LpA-I in TD plasma together with its failure to convert exogenous apo A-I into an alpha-migrating particle provide specific tests for the diagnosis of TD.

Adult↗
PubMed1998

Assignment of Tangier disease to chromosome 9q31 by a graphical linkage exclusion strategy.

A low level of high density lipoprotein (HDL) cholesterol is a strong predictor of ischaemic heart disease (IHD) and myocardial infarction. One cause of low HDL-cholesterol is Tangier disease (TD), an autosomal codominant inherited condition first described in 1961 in two siblings on Tangier Island in the United States of America. Apart from low HDL-cholesterol levels and an increased incidence of atherosclerosis, TD is characterized by reduced total cholesterol, raised triglycerides, peripheral neuropathy and accumulation of cholesteryl esters in macrophages, which causes enlargement of the liver, spleen and tonsils. In contrast to two other monogenic HDL deficiencies in which defects in the plasma proteins apoA-I and LCAT interfere primarily with the formation of HDL (refs 7-10), TD shows a defect in cell signalling and the mobilization of cellular lipids. The genetic defect in TD is unknown, and identification of the Tangier gene will contribute to the understanding of this intracellular pathway and of HDL metabolism and its link with IHD. We report here the localization of the genetic defect in TD to chromosome 9q31, using a genome-wide graphical linkage exclusion strategy in one pedigree, complemented by classical lod score calculations at this region in a total of three pedigrees (combined lod 10.05 at D9S1784). We also provide evidence that TD may be due to a loss-of-function defect.

Chromosome Mapping↗
PubMed1998

Mutation analysis of exon 3 of the LDL receptor gene in patients with severe hypercholesterolemia.

Single-strand conformation polymorphism analysis was used to screen for mutations in exon 3 of the low density lipoprotein receptor gene in a group of 218 unrelated patients with severe hypercholesterolemia (low density lipoprotein cholesterol > 6.7 mmol/l) living in the Cologne area of Germany. Including the complementary primers the fragment studied had a length of 176 bp. An abnormal single-strand conformation polymorphism pattern was observed in eight patients, four of whom had an identical abnormal fragment pattern indicating that five different mutations were present. By direct DNA sequencing, the underlying mutations could be confirmed (Cys54-->Tyr, Trp66-->Gly, Glu80-->Lys, 2 bp insertion (AT between codon 44 and 45, 9 bp deletion (codons 65 to 67)). The analysis of the pathogenicity indicates that all mutations were causative for the low density lipoprotein cholesterol elevation. The Trp66-->Gly and Glu80-->Lys mutations were previously described in a French-Canadian population and in an English population, respectively. The 2 bp insertion was detected in four unrelated patients and is one of the most frequent mutations detected up to now in the German population.

Base Sequence↗
PubMed1998

Plasma and fibroblasts of Tangier disease patients are disturbed in transferring phospholipids onto apolipoprotein A-I.

Plasmas of patients with Tangier disease (TD) lack lipid-rich alpha-HDL which, in normal plasma, constitutes the majority of high density lipoprotein (HDL). Residual amounts of apolipoprotein (apo)A-I in TD plasma occur as lipid-poor or even lipid-free prebeta-HDL. By contrast to normal plasma, TD plasma does not convert prebeta-HDL into alpha-HDL. Moreover, fibroblasts of TD patients were found to be defective in secreting cholesterol or phospholipids in the presence of lipid-free apoA-I. We have therefore hypothesized that both defective conversion of prebeta-HDL into alpha-HDL and defective lipid efflux from TD cells onto lipid-free apoA-I result from a disturbance in phospholipid transfer occurring in both cellular and extracellular compartments. To test this hypothesis we established an assay that measures the activity of plasma, cells, and cell culture media to transfer radiolabeled phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) from vesicles onto apoA-I, apoA-II, albumin, or reconstituted HDL. Plasmas, HDL, and lipoprotein-depleted plasma of normolipidemic probands as well as cell homogenates and culture media of normal fibroblasts were active at 37 degrees C but not at 4 degrees C in transferring radiolabeled PC, PI, and PE dose- and time-dependently onto either lipid-free apoA-I or reconstituted HDL. Transfer of glycerophospholipids onto apoA-II was much lower than onto apoA-I; transfer onto albumin was close to background. Compared to ten normolipidemic plasmas and four apoA-I-deficient plasmas, plasmas of six TD patients were significantly reduced by 40-50% in their glycerophospholipid transfer activities. Compared to eight normal fibroblast cell lines, homogenates and culture media of four TD fibroblast cell lines were reduced by 40-50% and 30-35%, respectively, in their activity to transfer PC, PI, or PE onto apoA-I. Our data suggest that in TD the same mechanism underlies both defective conversion of prebeta-HDL into alpha-HDL and impaired efflux of cellular lipids, namely a defective phospholipid transfer.

Adult↗
PubMed1998

Detection of mutations in the apolipoprotein CII gene by denaturing gradient gel electrophoresis. Identification of the splice site variant apolipoprotein CII-Hamburg in a patient with severe hypertriglyceridemia.

Familial apolipoprotein (apo) CII deficiency is a rare autosomal recessive inborn error of metabolism clinically resembling lipoprotein lipase deficiency. A number of mutations of the apo CII gene are known to date; they are located in the promoter region, the coding exons, or in the splice junctions. We present a simple assay based on PCR and denaturing gradient gel electrophoresis, which allows scanning of the promoter, the entire coding sequence, and the splice junctions of the apo CII gene for sequence variants. All gene fragments are amplified using a common PCR protocol and are examined for mutations on a single gradient gel. Using this method and direct sequencing, we identified homozygosity for a donor splice-site mutation in the second intron, previously designated apo CII-Hamburg, as the genetic cause of apo CII deficiency in a 9-year-old boy presenting with chylomicronemia, eruptive xanthoma, and pancreatitis. In addition, the method allowed us to detect all of six different other known mutations of the apo CII gene. We conclude, therefore, that our assay is highly sensitive; in addition, it is easy to perform and may facilitate the differential diagnosis of disorders of lipoprotein metabolism at the genetic level.

Alternative Splicing↗
PubMed1996

Screening for mutations in the LDL receptor gene and apolipoprotein B-100 gene in 218 patients with severe hypercholesterolemia.

A group of 218 patients with severe hypercholesterolemia (LDL cholesterol > 260 mg/dl) living in the Cologne area were screened for mutations in the LDL receptor gene and apolipoprotein B-100 gene. In the LDL receptor gene Southern blotting was used for detection of major DNA rearrangements and the single-strand conformation polymorphism (SSCP) method was used to screen for micro-deletions and insertions and single base alterations. The Arg3500-->Glu mutation, which is the only relevant mutation in the apolipoprotein B-100 gene causing hypercholesterolemia, was detected by a modified PCR and restriction enzyme digestion. Three different major rearrangements, all of which were deletion, were found in the LDL receptor gene. The SSCP screening was started with exon 4. In 20 cases an abnormal fragment pattern was observed. The apolipoprotein B-100 mutation was detected in 15 patients. In summary, by the combined analysis of major rearrangements, micro-deletions, insertions and single base alterations in the LDL receptor gene and the Arg3500-->Glu mutation in the apolipoprotein B-100 gene, mutations causing or probably causing hypercholesterolemia could be detected in 38 of the 218 studied patients. The expansion of SSCP screening to other exons of the LDL receptor gene will greatly increase the identification of mutations causing hypercholesterolemia.

Apolipoprotein B-100↗