Genotyping of apolipoprotein e.

Apolipoprotein E (apo E) is a 299-amino acid plasma protein involved in cholesterol transport and is found in chylomicrons, very low density lipopro-tein, intermediate-density lipoprotein, and high-density lipoprotein (1,1).

Evaluation of chimerism in DNA samples by PCR amplification of D1S80 with detection by capillary electrophoresis.

BACKGROUND: Analysis of the relative amounts of donor and recipient DNA in bone marrow after bone marrow transplantation is frequently used to determine the status of the transplant. We studied the performance of an assay to quantify chimerism based on amplification of the D1S80 variable number tandem repeat marker by PCR with detection of PCR products by capillary electrophoresis (CE). METHODS AND RESULTS: Samples from potential bone marrow donors and recipients were analyzed separately and in mixtures to simulate various degrees of chimerism from 10% to 90% and subjected to PCR/CE analysis. There was excellent agreement between the measured and known relative proportions of DNA components in chimeric samples. The lower limit of sensitivity for detection of chimerism was 1%; between-runs coefficients of variation were <5%. CONCLUSIONS: Amplification of the D1S80 minisatellite by PCR with CE detection is a reliable method for determination of the relative contribution of different DNAs in mixed samples. This method is fast, quantitative, and extremely reproducible.

Association of the prothrombin G20210A mutation with factor V Leiden in a midwestern American population.

The prothrombin G20210A mutation has been identified as a risk factor for thrombosis. We studied the relationship between prothrombin G20210A and factor V Leiden mutations in patients with thrombophilia. The first 264 patients for whom these molecular diagnostic studies were requested at our institution were included in the study. For 116 of the 264 patients, additional coagulation test results were available in the laboratory database. The prothrombin G20210A mutation was found in 16 (6.1%) of the patients and the factor V Leiden mutation in 44 (16.7%). Of the 16 patients with the prothrombin G20210A mutation, 8 also carried factor V Leiden; this association was significant. In contrast, only 2 patients of the 116 with additional coagulation testing harbored more than 1 prothrombotic risk factor. These data support the hypothesis that thrombophilia is a multigenic disorder. Among unselected samples from a Midwestern population evaluated for thrombotic risk factors, the prevalence of factor V Leiden and prothrombin G20210A mutations are similar to those found in other populations in the Western world.

Analysis of CYP21 coding polymorphisms in three ethnic populations: further evidence of nonamplifying CYP21 alleles among whites.

BACKGROUND: Adrenal steroid 21-hydroxylase is essential for the synthesis of both mineralocorticoids and glucocorticoids. The gene for this enzyme, CYP21, contains several frequent coding polymorphisms. Because of its essential function in steroid synthesis, polymorphisms in this enzyme might influence a variety of disease processes. However, before disease-association studies are performed, it is important to understand the frequency of these polymorphisms among normal individuals. METHODS: Using polymerase chain reaction (PCR) with restriction enzyme digestion or size length polymorphism analysis, we measured the frequencies of the +Leu(10), Arg102Lys, and Ser268Thr polymorphisms in CYP21 in healthy whites, blacks, and Indian Americans. The subjects were all young female college students participating in a study of relative risks for cardiovascular disease in these populations. RESULTS: The frequency of each polymorphism among whites, blacks, and Indian Americans were as follows: +Leu(10), 0.55, 0.96, 0.75; Arg102, 0.63, 0.97, 0.82; and Ser268, 0.92, 0.68, 0.79, respectively. With the exception of the frequencies of the Ser268Thr polymorphism among blacks and Indian Americans, there were significantly different frequencies of each polymorphism among all groups (P<.05). Among whites, the distribution of genotypes for the +Leu(10) and Arg102Lys polymorphisms deviated significantly from expected Hardy-Weinberg values because of an excess of homozygotes. CONCLUSIONS: Among the ethnic groups, there are statistically significant differences in the frequencies of these common coding polymorphisms in CYP21 that need to be considered in disease-association studies. Deviation from Hardy-Weinberg distributions might be explained by allelic dropout during PCR, a phenomenon previously reported at this locus.

An overview of genetic factors influencing plasma lipid levels and coronary artery disease risk.

BACKGROUND: Coronary artery disease (CAD) is a major cause of morbidity and mortality in most Western countries and its origin involves a significant genetic component. METHODS: Genetic and epidemiologic studies have been performed to identify factors that influence the CAD risk in the population. RESULTS: The primary loci that have been demonstrated to be associated with increased CAD risk owing to genetic mutations include the low-density lipoprotein receptor, apolipoprotein B-100, and lipoprotein(a). Additional implicated loci include lipoprotein lipase, apolipoprotein CII, cholesteryl ester transfer protein, apolipoprotein AI, and lecithin-cholesterol acyl transferase. CONCLUSIONS: Numerous mutations in known genes exert a major effect on CAD risk in some patients. However, in most patients with CAD, the genetic component is believed to be attributable to the aggregate effect of loci that, individually, exert only a minor influence on lipoprotein levels.

Chromosomal aneuploidy in leukemic blast crisis: a potential source of error in interpretation of bone marrow engraftment analysis by VNTR amplification.

BACKGROUND: Polymerase chain reaction (PCR) amplification of polymorphic microsatellite or minisatellite DNA markers has proven to be a fast, sensitive, and specific technique in post-transplantation monitoring of bone marrow engraftment, as well as early detection of residual disease and relapse. Deletion or amplification of chromosomal segments carrying marker loci, as can occur in leukemia and other hematologic malignancies, may result in loss or increased dosage of marker alleles. Examination of these marker alleles by PCR therefore may give aberrant results, which might lead to misinterpretation of bone marrow transplantation (BMT) engraftment studies. METHODS AND RESULTS: We report a case of chronic myelogenous leukemia treated by BMT. PCR amplification of the minisatellite at the apoB locus on chromosome 2 was used to monitor the donor bone marrow engraftment. The patient experienced relapse in blast crisis with a near-haploid karyotype with loss of recipient-specific apoB allele causing an aberrant PCR result for bone marrow engraftment that mimicked full donor engraftment. CONCLUSIONS: Loss or gain of polymorphic DNA markers because of chromosomal losses or gains in some hematologic malignancies may affect the interpretation of bone marrow engraftment studies by PCR. When choosing polymorphic markers for such studies, it is important to avoid those that will be affected by expected chromosomal alteration, if possible. In addition, any abberant post-transplantation typing should prompt further investigation to rule out the possibility of chromosomal aberration. Review of all pertinent laboratory studies is important to avoid misinterpretation of results from a single test for engraftment analysis.

Single-nucleotide polymorphisms in intron 2 of CYP21P: evidence for a higher rate of mutation at CpG dinucleotides in the functional steroid 21-hydroxylase gene and application to segregation analysis in congenital adrenal hyperplasia.

BACKGROUND: Intron 2 of CYP21, the functional steroid 21-hydroxylase gene contains several single-nucleotide polymorphisms (SNPs). We tested the hypothesis that intron 2 of the pseudogene, CYP21P, might also be polymorphic and provide markers for segregation analysis of this region of the genome, including observable markers for segregation analysis of CYP21 gene deletions. A comparison of SNPs in both genes might provide insights into the rates of mutation in these duplicated genes. METHODS: After amplification with PCR, we examined restriction site polymorphisms in intron 2 of CYP21P in 24 members of the parental generation of the Centre d'Etude du Polymorphisme Humain families and selected offspring. RESULTS: Intron 2 of CYP21P contains frequent SNPs around nucleotide 398 and nucleotide 509, which can be typed by PCR/restriction enzyme digestion with HaeIII. Of the 48 CYP21P alleles examined, 44 could be characterized unambiguously. Of these 44 alleles, 4 were deleted, and the frequencies of restriction at the polymorphic HaeIII sites were 20 of 40 at nucleotide 398 and 30 of 40 at nucleotide 509. Both polymorphisms result from C-->T transitions that occur at CpG dinucleotides. The frequencies of C at these nucleotides in CYP21P are significantly higher than at the corresponding nucleotides in CYP21 of the same individuals (P <0.01). CONCLUSION: These data suggest that these CpG dinucleotides are more frequently mutated in CYP21 than in CYP21P, and that several mutations at CpG dinucleotides in the coding regions of CYP21 might result from CpG instability rather than the more usually proposed mechanism of gene conversion. These frequent SNPs provide useful markers for studying both allelic segregation of CYP21, particularly for chromosomes with known CYP21 deletions, and for investigating the origin of these polymorphisms.

Characterization of frequent polymorphisms in intron 2 of CYP21: application to analysis of segregation of CYP21 alleles.

The gene encoding adrenal steroid 21-hydroxylase, CYP21, is located in the MHC class III region. Most cases of congenital adrenal hyperplasia (CAH) are caused by mutations in this gene, and most mutations appear to arise from gene conversion-like events involving the transfer of deleterious sequences from the pseudogene, CYP21P, which is located within 30 kb of CYP21. Approximately 20-30% of mutations are caused by deletions of CYP21. The second intron of CYP21 is polymorphic, and several base substitutions that include nt395, nt453, and nt601 have been reported; however, the frequencies of these polymorphisms are unknown. Using a combination of cleavase fragment length polymorphism analysis and direct sequencing, we examined the sequence of intron 2 in seven wild-type CYP21 genes and determined the frequency of polymorphisms at nt395, nt453, and nt601 in 48 chromosomes from the parental generation of Centre d'Etude du Polymorphisme Humain families. The observed frequencies of bases at these positions were as follows: 395C, 0.17; 395T, 0.83; 453C, 0.71; 453T, 0.29; 601A, 0.1; and 601C, 0.9. Using a PCR/restriction digestion approach to examine these intragenic markers, we could follow the segregation of alleles in informative families with 21-hydroxylase deficiency and identify deletions of CYP21. We emphasize that this method should be used in conjunction with other molecular genetic techniques for diagnosis of CAH. In addition to their potential use in families with CAH, these markers may be of use in genetic studies of the MHC in humans.

Genotyping of apolipoprotein E by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The genotyping of the various isoforms of Apolipoprotein E (apo E) has been performed using matrix-assisted laser desorption/ionization (MALDI-MS). The polymerase chain reaction was used to amplify the specific apo E gene sequence followed by digestion with Cfo I (Clostridium formicoaceticum), for generating restriction fragments for rapid and accurate mass analysis. An exonuclease I digestion step was introduced to remove the unused primers after PCR, which can otherwise interfere in the mass spectral analysis. By replacing the gel electrophoresis detection step with MALDI-MS, restriction isotyping of the apo E gene was achieved. Genotyping of an unknown sample and obtained from an independent diagnostic laboratory demonstrated the validity of the MALDI-MS method for the routine analysis of apo E.

Quantification of nucleic acids.

Quantification of nucleic acids is an area of growing importance in the clinical laboratory. In addition to routine measurements of total nucleic acid concentrations, quantitative techniques are used to determine the number of microorganisms in samples, the level of expression of genes, or the presence of alterations in gene dosage. This article provides an overview of current methodologies for measurement of total and specific nucleic acid concentrations.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as a rapid screening method to detect mutations causing Tay-Sachs disease.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used as a rapid method for the detection of human genetic polymorphisms. In particular, the mutations in the human HEXA gene that cause the infantile Tay-Sachs disease have been studied using MALDI-MS to demonstrate the feasibility of this technique for use in clinical and diagnostic analysis. The protocols involved in this approach include, polymerase chain reaction for the amplification of the mutation site from buccal cell DNA, followed by restriction enzyme digestion of the amplified regions of the template cells. The products of amplification and digestion were studied using MALDI-MS. MALDI-MS experiments are shown to provide essentially the same information as obtained from gel electrophoresis but orders of magnitude faster.

Sequence analysis of the hepatitis C virus (HCV) core gene suggests the core protein as an appropriate target for HCV vaccine strategies.

Hepatitis C virus (HCV) is a major healthproblem with a prevalence of 1% in the United States population, and a significant percentage of infected patients progress to chronic liver disease and cirrhosis. Interferon therapy has demonstrated that the immune system can be modulated to alter the acute course of the disease, but long-term treatments remain elusive. Prevention of hepatitis C infection is therefore an important strategy to mitigate the impact of this disease. Initial attempts at vaccination have focused on recombinant envelope vaccines, which have shown an ability to protect against very low titre challenges of HCV in chimps. The need for vaccines capable of protecting against higher titre challenges has led to the search for alternative vaccine strategies. The most highly conserved structural protein in the HCV genome is the core protein, and vaccine strategies targeting the core protein have been proposed to increase vaccine efficacy. The variability of HCV core sequences and genotypes in the Ann Arbor patient population are not known, and the present study was undertaken to assess the theoretical feasibility of developing a HCV core vaccine by excluding promiscuous core (C) gene variability as a mechanism of vaccine failure. Results of nucleotide and deduced amino acid sequence analysis from 13 of 14 patients studied reveal a 93% nucleotide and 96.4% amino acid core sequence homology in the C gene regions studied. Genotype analysis revealed four of 14 to be type 1a and nine of 14 to be type 1b with one infection not being sufficiently characterized to determine genotype. These results demonstrate a sufficiently high degree of conservation of HCV core sequences in our patient population to permit design of a vaccine directed against core protein.

Carbamazepine measurement in samples from the emergency room.

The measurement of carbamazepine (CBZ) in samples from the emergency room (ER) raises several issues for drug monitoring. First, the ER frequently requires rapid turnaround time for clinical samples; this need may be more conveniently met by automated immunoassays than by high-performance liquid chromatography (HPLC), the other major analytical technique for measurement of carbamazepine. On the other hand, immunoassays often do not completely measure the pharmacologically active carbamazepine epoxide metabolite and therefore may not indicate the full extent of serum anticonvulsant activity. Second, patients may be admitted to the ER specifically because of seizure activity, which may be an indication of under- or overmedication with carbamazepine and which, if due in large part to high levels of the epoxide metabolite, may not be fully assessed by immunoassay. We examined the results of carbamazepine determination in 102 consecutive samples sent from an ER. Each sample was analyzed by a fluorescence polarization immunoassay (FPIA) and by HPLC. There were good correlations between the FPIA and the HPLC for the parent drug and for the sum of the parent drug plus metabolite (carbamazepine-10,11-epoxide, CBZ-E) with these regression equations: FPIA = 1.13 (HPLC CBZ) + 0.09 (r2 = 0.93) and FPIA = 0.93 (HPLC CBZ+CBZ-E)-0.55 (r2 = 0.89), respectively. There were weak correlations between the FPIA and the epoxide and between the parent drug and the epoxide. Based on the FPIA and HPLC results, we classified each value relative to the therapeutic range, i.e., supratherapeutic, subtherapeutic, or therapeutic.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative analysis in molecular diagnostics.

Quantitative analysis of DNA products derived from polymerase chain reaction (PCR)-based assays depends on the careful optimization of each of the reaction parameters to achieve highly efficient amplification of target sequences. In practice, however, measurement of the accumulated PCR product is reliable only when analyses are performed at points in the exponential phase of the PCR amplification curve and before the onset of the plateau phase. The recent development of more sensitive DNA product detection systems has permitted the analysis of PCR assays after fewer amplification cycles, where the accumulation of product approaches linearity, while at the same time maintaining superior assay specificity. These methods include the use of high performance liquid chromatography, automated fluorescence detection, electrochemiluminescence, and the ligase chain reaction. Clinical applications of these methods are numerous and include diagnostic testing as well as therapeutic monitoring for neoplastic, infectious, and inherited genetic disease.

Catalase, superoxide dismutase and glutathione peroxidase activity in neutrophils of sham-operated and olfactory-bulbectomised rats following chronic treatment with desipramine and lithium chloride.

In the olfactory-bulbectomised rat model of depression, neutrophil phagocytosis was significantly decreased and phagocytosis started later in comparison to sham-operated animals. Both desipramine and lithium chloride treatment significantly reversed the depressed neutrophil phagocytosis and shortened the time to commencement of phagocytosis in drug-treated bulbectomised rats. The catalase and glutathione peroxidase (GSH-PX) activities in bulbectomised rats were decreased, while superoxide dismutase (SOD) was significantly increased. Chronic desipramine and lithium chloride treatment slightly improved catalase activity in the bulbectomised rats. Desipramine significantly reversed the reduction in activity of GSH-PX, but failed to reverse the increased activity of SOD. In contrast, lithium chloride significantly reversed SOD activity to normal values, without affecting GSH-PX activity in the bulbectomised rats.