Genetic analysis of case/control data using estimated haplotype frequencies: application to APOE locus variation and Alzheimer's disease.

There is growing debate over the utility of multiple locus association analyses in the identification of genomic regions harboring sequence variants that influence common complex traits such as hypertension and diabetes. Much of this debate concerns the manner in which one can use the genotypic information from individuals gathered in simple sampling frameworks, such as the case/control designs, to actually assess the association between alleles in a particular genomic region and a trait. In this paper we describe methods for testing associations between estimated haplotype frequencies derived from multilocus genotype data and disease endpoints assuming a simple case/control sampling design. These proposed methods overcome the lack of phase information usually associated with samples of unrelated individuals and provide a comprehensive way of assessing the relationship between sequence or multiple-site variation and traits and diseases within populations. We applied the proposed methods in a study of the relationship between polymorphisms within the APOE gene region and Alzheimer's disease. Cases and controls for this study were collected from the United States and France. Our results confirm the known association between the APOE locus and Alzheimer's disease, even when the epsilon 4 polymorphism is not contained in the tested haplotypes. This suggests that, in certain situations, haplotype information and linkage disequilibrium-induced associations between polymorphic loci that neighbor loci harboring functional sequence variants can be exploited to identify disease-predisposing alleles in large, freely mixing populations via estimated haplotype frequency methods.

Large paravaginal pelvic lipoma. A case report.

BACKGROUND: Pelvic lipoma is an extremely rare problem. Current imaging techniques are very helpful in diagnosis and assessment. CASE: A 36-year-old woman, gravida 2, para 3, presented with pelvic pressure and a bulging perineum on the left side. Pelvic examination revealed an 8 x 10-cm, soft mass filling the left hemipelvis. Pelvic ultrasound and computed tomography delineated the mass and suggested a fatty tumor without invasion of surrounding structures. Via laparotomy, a 400-g lipoma was removed from the left paravaginal/ paravesical/pararectal space. The patient had an uneventful recovery. CONCLUSION: Pelvic lipoma should be considered in the differential diagnosis of a soft, solid tumor filling the lateral pelvis. Ultrasonography and computed tomography are very helpful in assessing the nature of the mass. Removal can be done with a transperineal approach.

Efficient synthesis of DNA dumbbells using template-induced chemical ligation in double-stranded polynucleotides closed by minihairpin fragments.

The chemical ligation of 17 50-54-membered nicked DNA dumbbells with different closing fragments, nick positions, and nucleotides facing the nick were investigated. T4, T5, GTA4C, GCGA2GC, and GCGA3GC sequences were chosen as the closing fragments. The nicks were placed in the center of the duplex stem or were adjacent to the closing fragments. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and cyanogen bromide were used as the condensing agents. We showed that the ligation efficiency is 10%-90% depending on the sequence of the closing fragments, nick position, and nucleotides facing the nick. Coupling yields of 80%-90% were observed when the nick was situated in the middle of the molecule between two T residues or was adjacent to GCGA2GC or GCGA3GC minihairpins. In the last case, the reacting 3'-phosphate and 5'-hydroxy groups were brought close together by only two base pair minihairpins. The coupling yields did not depend on the nature of the condensing agent. On the basis of the results obtained, we believe a rational design of nicked DNA dumbbells has been developed for efficient chemical synthesis of closed dumbbells.

Analysis of the distribution of binding sites for a tissue-specific transcription factor in the vertebrate genome.

Hepatocyte nuclear factor 1 (HNF1) is a dimeric homeoprotein expressed in hepatocytes and in a few other epithelial cells where it helps regulate the expression of a specific subset of genes. In an attempt to identify novel target genes for HNF1 and to assess the distribution of its target sites within the vertebrate genome, we performed a computer-assisted search within the available databases using a weighted matrix. Several hundred potential target sequences were identified within the GenBank and EMBL data banks. DNA binding assays demonstrated that more than 95%, of the new sites tested (52 sites among 54) bound HNF1. Surprisingly many HNF1 target sites were found in genes that are transcribed in cell types that do not contain the protein. On the other hand these sites are 2.5 to five times more frequent in hepatic genes than expected. It seems that the presence of HNF1 sites in liver-specific genes was favoured, but that no counter-selection occurred within the rest of the genome. HNF1 binding sites in liver genes are more often associated in clusters with sites for other transcription factors and the enrichment is more pronounced in promoter regions. We identified more than 100 liver specific genes that are potentially regulated by HNF1.

Crosslinking of double-stranded oligonucleotides containing O-methyl-substituted pyrophosphate groups to the HNF1 transcription factor in nuclear cell extract.

Probing of the HNF1 (hepatocyte nuclear factor I) DNA-binding region using a set of DNA duplexes containing pyrophosphate or O-methyl-substituted pyrophosphate internucleotide groups at different positions of the HNF1 recognition sequence was performed. The histidine-tagged HNF1/1-281 DNA binding domain and nuclear extract from rat liver were used. We showed that HNF1 from these species specifically binds to modified DNA duplexes. A correlation in binding affinity of both types of duplexes was detected. Crosslinking of the HNF1 DNA-binding domain and HNF1 in nuclear liver extract to DNA duplexes carrying O-methyl-substituted pyrophosphate groups was observed. The crosslinking efficiency of HNF1 in liver extract to substituted pyrophosphate-modified DNA duplex, containing a reactive internucleotide group between nucleotides G and T of the GT dinucleotide immediately 5' to the TAAT recognition sequence, amounts to 40% of the efficiency of non-covalent association. Nonspecific crosslinking of the reactive DNA duplexes to other components of nuclear extract was not observed. These results indicate that DNA duplexes carrying substituted pyrophosphate internucleotide groups can specifically bind and crosslink with DNA-binding proteins, especially transcription factors in crude preparations and could constitute a potential tool to control the expression of disease-causing genes.

Mechanisms of inhibition of in vitro dimerization of HIV type I RNA by sense and antisense oligonucleotides.

Retroviruses display a strong selective pressure to maintain the dimeric nature of their genomic RNAs, suggesting that dimerization is essential for viral replication. Recently, we identified the cis-element required for initiation of human immunodeficiency virus type I (HIV-I) RNA dimerization in vitro. The dimerization initiation site (DIS) is a hairpin structure containing a self-complementary sequence in the loop. We proposed that dimerization is initiated by a loop-loop kissing interaction involving the self-complementary sequence present in each monomer. We tested the ability of sense and antisense oligonucleotides targeted against the DIS to interfere with a preformed viral RNA dimer. Self-dimerization and inhibition properties of the tested oligonucleotides are dictated by the nature of the loop. An RNA loop is absolutely required in the case of sense oligonucleotides, whereas the nature and the sequence of the stem is not important. They form reversible loop-loop interactions and act as competitive inhibitors. Antisense oligonucleotides are less efficient in self-dimerization and are more potent inhibitors than sense oligonucleotides. They are less sensitive to the nature of the loop than the antisense oligonucleotides. Antisense hairpins with either RNA or DNA stems are able to form highly stable and irreversible complexes with viral RNA, resulting from complete extension of base pairing initiated by loop-loop interaction.

Hairpin, dumbbell, and single-stranded phosphodiester oligonucleotides exhibit identical uptake in T lymphocyte cell lines.

The cellular binding, uptake, and intracellular distribution of structured double-stranded phosphodiester oligonucleotides (decoys) have been examined in T lymphocytes using fluorescein-labeled molecules. Intracellular localization of hairpin and dumbbell decoys was similar to that of single-stranded oligonucleotides. At short incubation times, oligonucleotides were localized only in cytoplasmic vesicles, whereas at longer times, they were also found in the nucleus. Cellular uptake was dependent on temperature, time, and extracellular concentration. Oligonucleotide efflux was similar for all types of molecules and was very rapid (t1/2 = 10-15 minutes). These results imply that phosphodiester double-stranded oligonucleotides can enter cellular compartments of interest for their potential biologic function and, thus, provide a potential tool to control gene transcription.

BCR-ABL antisense oligodeoxyribonucleotides suppress the growth of leukemic and normal hematopoietic cells by a sequence-specific but nonantisense mechanism.

We have examined the effect of BCR/ABL junctional antisense phosphodiester oligodeoxyribonucleotides (ODNs) on BV173 and other chronic myeloid leukemia (CML) cell lines. Various control ODNs were used to understand the mechanism of the observed antiproliferative effect. Not only the antisense ODNs but also several control ODNs inhibit the proliferation of the leukemic cell lines. All the ODNs that inhibit the cell proliferation share a TAT consensus sequence at their 3' end. A 1-base mismatch within this consensus sequence abolishes the antiproliferative effect. Mismatches of several bases at any other position within the sequence of the active ODNs do not suppress the observed effect. Similar experiments on normal or CML CD34+ cell fraction led to the same observations. We conclude that the antiproliferative effect of the phosphodiester BCR/ABL antisense ODNs cannot be attributed to an antisense mechanism but rather to a nonelucidated effect of a 3' terminal TAT sequence. This effect is not CML specific.

[Synthesis of modified oligonucleotides and use of a duplex from them with covalently bound chains]. / Sintez modifitsirovannykh oligonukleotidov i poluchenie iz nikh dupleksa s kovalentno sviazannymi tsepiami.

A method for the synthesis of DNA duplex with covalently linked strands was elaborated, and the thermal and hydrolytic stability of the duplex was studied. The strands were connected via an amide bond between carboxyl and aliphatic amino groups in the presence of water-soluble carbodiimide. For this purpose, a series of modified 5- to 26-mer oligonucleotides with primary amino or carboxyl group were prepared, and their properties were investigated.

Inhibition of HSV-1 proliferation by decoy phosphodiester oligonucleotides containing ICP4 recognition sequences.

Transcriptional control in eukaryotes results from the interplay between DNA sequences in promoters, enhancers, or silencers and transcription factors. Selective control of gene expression can thus be achieved by inhibiting specific transcription factor/DNA interactions. Transcriptional activity of DNA binding transcription factors can be inhibited by competition with double-stranded oligonucleotides (decoys) that contain their specific recognition sequences. The immediate early protein ICP4 of herpes simplex virus type 1 (HSV-1) is a sequence-specific DNA binding protein that is essential for viral replication. We synthesized double-stranded hairpin phosphodiester oligonucleotides carrying ICP4 sites and demonstrated their ability to specifically titrate ICP4. Upon addition to Vero cells, ICP4 hairpin decoys significantly reduced HSV-1 titers (IC50 = 0.3 microM), whereas a control hairpin oligonucleotide had no activity. Antiviral activity of ICP4 hairpin decoys was correlated to their relative binding affinities. These results show that phosphodiester oligonucleotides can compete for binding of specific transcription factors within cells, thus providing a potential therapeutic tool to control disease-causing genes.

Image-guided automated core biopsies of the breast, chest, abdomen, and pelvis.

PURPOSE: A prospective study was undertaken to measure success at obtaining definitive histologic diagnoses with an automated biopsy gun technique. MATERIALS AND METHODS: Image-directed biopsies (n = 300) were performed between November 1989 and June 1992 in 288 patients. Biopsy specimens of breast lesions were obtained with ultrasound (US) or stereotaxic mammographic guidance. Biopsy samples of lesions from other areas of the body were obtained with computed tomographic or US guidance. Clinical follow-up and surgical biopsy were used to measure diagnostic accuracy. RESULTS: Overall, a definitive diagnosis was reached with core specimen biopsy techniques in 95.3% of cases (286 of 300), and all 286 definitive findings were accurate. CONCLUSION: Because of the success of this approach, the adoption of an automated biopsy gun technique that includes histologic examination of a core specimen should be considered by all radiologists who perform image-guided biopsies.

[Synthesis of cyclic oligodeoxyribonucleotides with non-nucleotide inserts]. / Sintez tsiklicheskikh oligodezoksiribonukleotidov s nenukleotidnymi vstavkami.

An effective method for the oligonucleotide cyclization using BrCN-induced chemical ligation was developed. The novel idea to incorporate non-nucleotide inserts makes the process of cyclizations independent of the inner secondary structure of the linear precursor. An set of assays were developed to confirm the cyclic structure of the compounds obtained.

Improved anti-herpes simplex virus type 1 activity of a phosphodiester antisense oligonucleotide containing a 3'-terminal hairpin-like structure.

We synthesized a series of 20-mer antisense phosphodiester oligonucleotides constituting of a 5'-dodecameric sequence, complementary to the acceptor splice junction of herpes simplex virus type 1 (HSV-1) pre-mRNAs IE4 and IE5, flanked in 3' by octameric sequences adopting hairpin-like structures of different stabilities. The presence of the minihairpins in 3' protected the 20-mer phosphodiester oligonucleotides against serum nuclease degradation, this protection being well correlated to the reported melting temperatures of the minihairpins, and to the gel mobilities of the 20-mer oligonucleotides. While no protection was observed using a linear 8-mer, the addition in 3' of the most stable minihairpin--H8--increased more than eightfold the nuclease resistance of the linear antisense dodecamer. We analyzed the effect of such a protection on the anti-HSV-1 antisense activities of the oligonucleotides. When bearing H8 in 3', the antisense dodecamer was 10 times more active than in the absence of 3'-flanking sequence, while a linear 20-mer control containing the antisense sequence was only 3 times more active. This work provides the basis for a further rational design of phosphodiester antisense oligonucleotides, taking advantage of the specific properties conferred by their conformations.

Oligonucleotide circularization by template-directed chemical ligation.

An efficient method for producing the covalent closure of oligonucleotides on complementary templates by the action of BrCN was developed. A rational design of linear precursor oligonucleotides was studied, and the effect of factors such as oligonucleotide concentration and oligomer-template length ratio was evaluated. The efficiency of circularization was shown to correlate well with the secondary structure of the precursor oligomer (as predicted by a simple computer analysis), hairpin-like structures bearing free termini clearly favouring the circularization reaction. A novel idea, consisting of the incorporation of non-nucleotide insertions in the precursor oligomer (namely, 1,2-dideoxy-D-ribofuranose residues), may render this method universal and highly effective. An original set of assays was developed to confirm the circular structure of the covalently closed oligonucleotides.

Ex vivo regulation of specific gene expression by nanomolar concentration of double-stranded dumbbell oligonucleotides.

Inhibition of specific transcriptional regulatory proteins is a new approach to control gene expression. Transcriptional activity of DNA-binding proteins can be inhibited by the use of double-stranded (ds) oligodeoxynucleotides that compete for the binding to their specific target sequences in promoters and enhancers. As a model, we used phosphodiester dumbbell oligonucleotides containing a binding site for the liver-enriched transcription factor HNF-1 (Hepatocyte Nuclear Factor 1). Binding affinity of HNF-1 to dumbbell oligonucleotides was the same as that to ds oligonucleotides, as determined by gel retardation assays. HNF-1 dumbbells specifically inhibited in vitro transcription driven by the albumin promoter by more than 90%. HNF-1-dependent activation of a CAT reporter plasmid was specifically inhibited when the HNF-1 dumbbell oligonucleotide was added at nM concentration to transiently transfected C33 cells. On the contrary, HNF-1 ds oligonucleotides, which displayed the same activity as the dumbbell oligonucleotides in the in vitro assays, were no more effective in the ex vivo experiments. These results might reflect the increased stability of the circular dumbbell oligonucleotides towards cellular nuclease degradation, as shown in vitro with nucleolytic enzymes. Dumbbell oligonucleotides containing unmodified phosphodiester bonds may efficiently compete for binding of specific transcription factors within cells, then providing a potential therapeutic tool to control disease-causing genes.

Further studies on the primer formation for glycogen biosynthesis in rat heart.

Using selected incubation conditions we have identified intermediate steps, between the first glucose transferred to protein and the appropriate substrate for glycogen synthase. Mn2+ stimulates the addition of the first, and probably, the second glucose molecule to the acceptor protein but inhibits further elongation. In the presence of Mn2+ only one radioglucosylated protein band of M(r) 42 kDa was evident. In the absence of Mn2+, two bands of 60.7 and 64.6 kDa were obtained indicating elongation of the glucan chains. After Glc6P addition a family of glucosylated proteins with higher M(r) was obtained, as reported previously. Mn2+ inhibition of the second step, is reversed by PMSF+Glc6P addition. Under these conditions a family of radioglucosylated protein bands with M(r) far in excess of 42 kDa, similar to that obtained without Mn2+, was obtained. Therefore, two different transglucosylating activities were necessary, at least, to prepare the appropriate substrate for glycogen synthase. Based on these observations the model we proposed earlier for glycogen biogenesis is modified. The original "Glycogen Initiator" implies at present two enzymatic activities, Glycogen Initiator 1 (activated by Mn2+) and Glycogen Initiator 2 (inhibited by Mn2+).

Electronic properties and free radical production by nitrofuran compounds.

Substitution of nifurtimox tetrahydrothiazine moiety by triazol-4-yl, benzimidazol-l-yl, pyrazol-l-yl or related aromatic nitrogen heterocycles determines changes in the quantum chemistry descriptors of the molecule, namely, (a) greater negative LUMO energy; (b) lesser electron density on specific atoms, especially on the nitro group atoms, and (c) modification of individual net atomic charges at relevant atoms. These variations correlate with the greater capability of nifurtimox analogues for redox-cycling and oxygen radical production, after one-electron reduction by ascorbate or reduced flavoenzymes. Variation of the nitrofurans electronic structure can also explain the greater activity of nifurtimox analogues as inhibitors of glutathione reductase and Trypanosoma cruzi growth, although other factors, such as molecular hydrophobicity and connectivity may contribute to the latter inhibition.