Elevated baseline triglyceride levels modulate effects of HMGCoA reductase inhibitors on plasma lipoproteins.

BACKGROUND: The response in levels of very-low-density (VLDL) and low-density (LDL) lipoproteins varies substantially among hyperlipidemic patients during treatment with HMGCoA reductase inhibitors. Apolipoprotein E genotype and gender are known to contribute to the regulation of steady state levels of plasma lipoproteins. This study explores the effect of these and other potential determinants of the response of VLDL and LDL to treatment with reductase inhibitors. METHODS: Using mixed linear statistical models, the response of lipoprotein lipid values was studied in 142 hyperlipidemic individuals who were treated with reductase inhibitors. Patients received one or more of the following drugs individually for a total of 623 treatment observations: lovastatin, pravastatin, simvastatin, or atorvastatin. For evaluation of the effects of treatment in the aggregate, actual doses were expressed as equivalent doses of atorvastatin, using factors based on random assignment comparisons in 16 reported studies. The analysis factors considered were apolipoprotein E genotype, baseline average triglycerides >170 mg/dL (vs less), and gender. RESULTS: Presence of an apo epsilon4 allele was associated with a trend toward greater reduction of triglyceride levels and a diminished ability of the reductase inhibitors to reduce LDL cholesterol levels. Gender had only minimal effect on the response of either LDL cholesterol or triglycerides. However, the effect of elevated baseline triglycerides on the response of both triglycerides and LDL cholesterol was striking and was exerted in opposite directions. The triglyceride-lowering effect of reductase inhibitors was greater in patients with initial triglyceride levels above 170 mg/dL (P=0.0001). The effect was even greater in patients with initial triglyceride levels over 250 mg/dL (P=0.015). Conversely, for LDL cholesterol levels, elevated baseline triglycerides were associated with a significantly decreased response to the drugs (P=0.0015). CONCLUSIONS: These findings indicate that baseline triglyceride levels are an important predictor of response of plasma lipoproteins to HMGCoA reductase inhibitors, perhaps reflecting fundamental differences in mechanism underlying the hyperlipidemic phenotype.

Accurate sequencing by hybridization for DNA diagnostics and individual genomics.

Medical DNA diagnostics will increasingly rely on an accurate and inexpensive identification of mutations that affect the function of a gene. To validate diagnostic sequencing by hybridization (SBH), a number of p53 samples were analyzed with the complete set of 8192 noncomplementary 7-mer oligonucleotides. In four repeated, blind experiments we accurately sequenced 1.1 kb per each of 12 homozygote and heterozygote samples possessing base substitutions, insertions, and deletions. This SBH variant offers a high throughput platform to inexpensively sequence individual gene or pathogen genome samples within the clinical laboratory setting.

Gene-representing cDNA clusters defined by hybridization of 57,419 clones from infant brain libraries with short oligonucleotide probes.

Diverse biochemical and computational procedures and facilities have been developed to hybridize thousands of DNA clones with short oligonucleotide probes and subsequently to extract valuable genetic information. This technology has been applied to 73,536 cDNA clones from infant brain libraries. By a mutual comparison of 57,419 samples that were successfully scored by 200-320 probes, 19,726 genes have been identified and sorted by their expression levels. The data indicate that an additional 20,000 or more genes may be expressed in the infant brain. Representative clones of the found genes create a valuable resource for complete sequencing and functional studies of many novel genes. These results demonstrate the unique capacity of hybridization technology to identify weakly transcribed genes and to study gene networks involved in organismal development, aging, or tumorigenesis by monitoring the expression of every gene in related tissues, whether known or still undiscovered.

Processing of cDNA and genomic kilobase-size clones for massive screening, mapping and sequencing by hybridization.

Efficient procedures for managing a large number of M13 or plasmid clones have been developed. In addition to picking, clones are directly arrayed in multiwell plates by dispensing diluted transformation mixtures. Metal pin arrays are used for fast inoculations of preparative plates filled by medium or by PCR mixture. Growth of M13 clones in multiwell plates is optimized to obtain a consistently high yield, and a PCR protocol is defined for reliable amplification of several thousand M13 or plasmid inserts per day in BioOvens. Over 80,000 cDNA inserts have been amplified. The phages or amplified inserts are spotted on nylon filters using an array of pins having a flat bottom, 0.3 mm in diameter. The procedures are suitable for an automated processing of hundreds of thousands of short clones from representative cDNA and genomic libraries. Hybridization of arrayed clones with oligonucleotide and complex probes can simplify the search for new genes and accelerate large-scale sequencing.

DNA sequence determination by hybridization: a strategy for efficient large-scale sequencing.

The concept of sequencing by hybridization (SBH) makes use of an array of all possible n-nucleotide oligomers (n-mers) to identify n-mers present in an unknown DNA sequence. Computational approaches can then be used to assemble the complete sequence. As a validation of this concept, the sequences of three DNA fragments, 343 base pairs in length, were determined with octamer oligonucleotides. Possible applications of SBH include physical mapping (ordering) of overlapping DNA clones, sequence checking, DNA fingerprinting comparisons of normal and disease-causing genes, and the identification of DNA fragments with particular sequence motifs in complementary DNA and genomic libraries. The SBH techniques may accelerate the mapping and sequencing phases of the human genome project.

Sequencing by hybridization: towards an automated sequencing of one million M13 clones arrayed on membranes.

An immediately applicable variant of the sequencing by hybridization (SBH) method is under development with the capacity to determine up to 100 million base pairs per year. The proposed method comprises six steps: (i) arraying genomic or cDNA M13 clones in 864-well plates (wells of 2 mm); (ii) preparation of DNA samples for spotting by growth of the M13 clones or by polymerase chain reaction (PCR) of the inserts using standard 96-well plates, or plates having as many as 864 correspondingly smaller wells; (iii) robotic spotting of 13,824 samples on an 8 x 12 cm nylon membrane, or correspondingly more, on up to 6 times larger filters, by offset printing with a 96 or 864 0.4 mm pin device; (iv) hybridization of dotted samples with 200-2000 32P-labeled probes comprising 16-256 10-mers having a common 8-mer, 7-mer, or 6-mer in the middle (20 probes per day, each hybridized with 250,000 dots); (v) scoring hybridization signals of 5 million sample-probe pairs per day using storage phosphor plates; and (vi) computing clone order and partial-to-complete DNA sequences using various heuristic algorithms. Genome sequencing based on a combination of this method and gel sequencing techniques may be significantly more economical than gel methods alone.

Reliable hybridization of oligonucleotides as short as six nucleotides.

Although there are many new applications for hybridizing short, synthetic oligonucleotide probes to DNA, such applications have not included determining unknown sequences of DNA. The lack of clear discrimination in hybridization of oligo probes shorter than 11 nucleotides and the lack of a theoretical understanding of factors influencing hybridization of short oligos have hampered the development of their use. We have found conditions for reliable hybridization of oligonucleotides as short as seven nucleotides to cloned DNA or to oligonucleotides attached to filters. Low-temperature hybridization and washing conditions, in contrast to the high stringency conditions currently used in hybridization experiments, have the potential for allowing the simple use of all oligos of six nucleotides or longer in meaningful hybridizations. We also present the hybridization discrimination theory that provides the conceptual framework for understanding these results.