Increase in interleukin-6 following arterial injury is related to insulin resistance, the -174G-->C polymorphism and complex plaque morphology.

Interleukin-6 (IL-6) is associated with many disease states in humans. We prospectively sought to determine whether IL-6 levels increased following percutaneous coronary intervention (PCI) in the absence of myonecrosis. Additionally, we systematically assessed other clinical and anatomic factors associated with IL-6 levels in a population of patients with coronary atherosclerosis undergoing PCI. Blood samples were collected from 117 patients at baseline, 8 and 16 h following PCI. Samples were assayed for IL-6, creatine kinase-myocardial band (CK-MB), troponin-I (Tn-I), high sensitivity C-reactive protein, glucose, haemoglobin A1c, and a lipid profile. Genotyping of the -174G-->C polymorphism of the IL-6 gene was performed. IL-6 levels increased following PCI among the study group (slope = 0.4 pg/mL/h, P = 0.001). IL-6 levels increased to a similar degree in the absence of myonecrosis. Patients with the XC genotype (either having the GC or the CC allele) had higher IL-6-values at baseline compared to GG genotype patients (4.9 +/- 6.4 vs. 2.6 +/- 1.8 pg/mL, P = 0.02). Multivariable predictors of detectable baseline IL-6 levels included XC genotype (odds ratio [OR]: 4.14, 95% CI 1.58-10.82, P = 0.004), ACC/AHA type C lesion classification (OR: 4.08, 95% CI 1.54-10.84, P = 0.005), elevated baseline Tn-I (OR: 3.31, 95% CI 1.16-9.43, P = 0.025), diabetes (OR: 3.00, 95% CI 1.11-8.09, P = 0.030), and waist circumference (OR: 1.49, 95% CI 1.08-2.06, P = 0.015). Predictors of peak IL-6 following PCI included the XC genotype (estimate 1.4, 95% CI 1.06-1.87, P = 0.019), homeostasis model assessment (estimate 0.99, 95% 0.982-0.999, P = 0.042) and baseline Tn-I > upper limit of normal (estimate 0.7, 95% CI 0.50-0.96, P = 0.039). Lastly, IL-6 increased following PCI even in the absence of myonecrosis as measured by Tn-I elevation. IL-6 levels are also related to the -174G-->C polymorphism, arterial injury, lesion complexity, and insulin resistance.

Quitting smoking in pregnancy.

Smoking doubles the risk of having a low-birthweight baby and significantly increases the rate of perinatal mortality and several other adverse pregnancy outcomes. The mean reduction in birthweight for babies of smoking mothers is 200 g. High quality interventions to help pregnant women quit smoking produce an absolute difference of 8.1% in validated late-pregnancy quit rates. If abstinence is not achievable, it is likely that a 50% reduction in smoking would be the minimum necessary to benefit the health of mother and baby. Healthcare providers perform poorly in antenatal interventions to stop women smoking. Midwives deliver interventions at a higher rate than doctors. The efficacy of nicotine replacement therapy has not been established in pregnancy. Currently, its use should only be considered in women smoking more than 10 cigarettes per day who have made a recent, unsuccessful attempt to quit and who are motivated to quit. Relapse prevention programs have shown little success in the postpartum period.

Airway gene transfer in mouse nasal-airways: importance of identification of epithelial type for assessment of gene transfer.

Mouse nasal airways are often used for the assessment of both reporter and cystic fibrosis transmembrane conductance regulator (CFTR) gene transfer to respiratory epithelia. However, the mouse nasal cavity is lined by both olfactory (OE) and respiratory epithelium (RE). Previous gene transfer studies have suggested that OE may be more efficiently transduced by adenoviral vectors than RE. However, to provide data pertinent to CFTR gene transfer in humans, measurements of CFTR function in mice by transepithelial potential difference (TPD) should be directed towards respiratory rather than olfactory epithelium. We report a new technique to mark the position of the TPD sensing cannula tip in the mouse nasal cavity that permitted us to correlate TPD measurements with epithelial cell type. Using this technique, we found TPD values did not discriminate between respiratory and olfactory epithelia. We next assessed relationships between anatomic regions accessed by the TPD cannula and epithelial type. The frequently used insertion depth of approximately 5 mm from the nose tip predominantly recorded the TPD from anterior dorsal olfactory epithelium. Measurement of the TPD of respiratory epithelium in our study was maximized by insertion of the TPD cannula probe to 2.5 mm depth. Because TPD measurements are not sensitive to epithelial type, adequate control of position and TPD catheter insertion depth are required to ensure accurate estimation of CFTR gene transfer into the target RE in the mouse nasal cavity.

Linkage disequilibrium and allele-frequency distributions for 114 single-nucleotide polymorphisms in five populations.

Single-nucleotide polymorphisms (SNPs) may be extremely important for deciphering the impact of genetic variation on complex human diseases. The ultimate value of SNPs for linkage and association mapping studies depends in part on the distribution of SNP allele frequencies and intermarker linkage disequilibrium (LD) across populations. Limited information is available about these distributions on a genomewide scale, particularly for LD. Using 114 SNPs from 33 genes, we compared these distributions in five American populations (727 individuals) of African, European, Chinese, Hispanic, and Japanese descent. The allele frequencies were highly correlated across populations but differed by >20% for at least one pair of populations in 35% of SNPs. The correlation in LD was high for some pairs of populations but not for others (e.g., Chinese American or Japanese American vs. any other population). Regardless of population, average minor-allele frequencies were significantly higher for SNPs in noncoding regions (20%-25%) than for SNPs in coding regions (12%-16%). Interestingly, we found that intermarker LD may be strongest with pairs of SNPs in which both markers are nonconservative substitutions, compared to pairs of SNPs where at least one marker is a conservative substitution. These results suggest that population differences and marker location within the gene may be important factors in the selection of SNPs for use in the study of complex disease with linkage or association mapping methods.

Population distribution and effects on drug metabolism of a genetic variant in the 5' promoter region of CYP3A4.

BACKGROUND: There are large interindividual differences in CYP3A4 expression and in the metabolism of drug substrates for this enzyme. We and others have identified a polymorphism in the 5' promotor region of the CYP3A4 gene; however, its functional significance is not currently known. This study was conducted to determine whether this polymorphism plays a clinically important role in determining CYP3A4 phenotype. METHODS: An adenine (A) to guanine (G) transition was identified in the 5' promotor region of the CYP3A4 gene at position -292 (from the start codon), in a sequence motif known as the nifedipine-specific element. The frequency of this polymorphism was assessed in 802 healthy volunteers from five broadly defined racial groups. The population distribution of the G allele in these groups was as follows: white Americans (3.6%; n = 273), black Americans (54.6%, n = 186), Hispanic Americans (9.3%; n = 188), Japanese Americans (0.0%; n = 77), and Chinese Americans (0.0%; n = 78). In a subsequent study, 90 additional black Americans were genotyped, and a subset of the homozygous subjects (AA, n = 8; GG, n = 23) were given the CYP3A4 probe substrates erythromycin and nifedipine to allow genotype-phenotype comparisons to be made. RESULTS: There was no difference in the rate of CYP3A4-dependent demethylation of erythromycin (erythromycin breath test) or the pharmacokinetics of nifedipine or its CYP3A4-dependent metabolite dehydronifedipine between the two genotype groups (AA or GG). CONCLUSIONS: This promotor region polymorphism does not appear to play a major role in determining constitutive CYP3A4 expression.

A genome-wide search for schizophrenia susceptibility genes.

We completed a systematic genome-wide search for evidence of loci linked to schizophrenia using a collection of 70 pedigrees containing multiple affected individuals according to three phenotype classifications: schizophrenia only (48 pedigrees; 70 sib-pairs); schizophrenia plus schizoaffective disorder (70 pedigrees; 101 sib-pairs); and a broad category consisting of schizophrenia, schizoaffective disorder, paranoid or schizotypal personality disorder, psychosis not otherwise specified (NOS), delusional disorder, and brief reactive psychosis (70 pedigrees; 111 sib-pairs). All 70 families contained at least one individual affected with chronic schizophrenia according to DSM-III-R criteria. Three hundred and thirty-eight markers spanning the genome were typed in all pedigrees for an average resolution of 10.5 cM (range, 0-31 cM) and an average heterozygosity of 74.3% per marker. The data were analyzed using multipoint nonparametric allele-sharing and traditional two-point lod score analyses using dominant and recessive, affecteds-only models. Twelve chromosomes (1, 2, 4, 5, 8, 10, 11, 12, 13, 14, 16, and 22) had at least one region with a nominal P value <0.05, and two of these chromosomes had a nominal P value <0.01 (chromosomes 13 and 16), using allele-sharing tests in GENEHUNTER. Five chromosomes (1, 2, 4, 11, and 13) had at least one marker with a lod score >2.0, allowing for heterogeneity. These regions will be saturated with additional markers and investigated in a new, larger set of families to test for replication.

X-Linked chronic granulomatous disease: mutations in the CYBB gene encoding the gp91-phox component of respiratory-burst oxidase.

Chronic granulomatous disease (CGD) is a hereditary disorder of host defense due to absent or decreased activity of phagocyte NADPH oxidase. The X-linked form of the disease derives from defects in the CYBB gene, which encodes the 91-kD glycoprotein component (termed "gp91-phox") of the oxidase. We have identified the mutations in the CYBB gene responsible for X-linked CGD in 131 consecutive independent kindreds. Screening by SSCP analysis identified mutations in 124 of the kindreds, and sequencing of all exons and intron boundary regions revealed the other seven mutations. We detected 103 different specific mutations; no single mutation appeared in more than seven independent kindreds. The types of mutations included large and small deletions (11%), frameshifts (24%), nonsense mutations (23%), missense mutations (23%), splice-region mutations (17%), and regulatory-region mutations (2%). The distribution of mutations within the CYBB gene exhibited great heterogeneity, with no apparent mutational hot spots. Evaluation of 87 available mothers revealed X-linked carrier status in all but 10. The heterogeneity of mutations and the lack of any predominant genotype indicate that the disease represents many different mutational events, without a founder effect, as is expected for a disorder with a previously lethal phenotype.

Linkage of bipolar affective disorder to chromosome 18 markers in a new pedigree series.

Several groups have reported evidence suggesting linkage of bipolar affective disorder (BPAD) to chromosome 18. We have reported data from 28 pedigrees that showed linkage to marker loci on 18p and to loci 40 cM distant on 18q. Most of the linkage evidence derived from families with affected phenotypes in only the paternal lineage and from marker alleles transmitted on the paternal chromosome. We now report results from a series of 30 new pedigrees (259 individuals) genotyped for 13 polymorphic markers spanning chromosome 18. Subjects were interviewed by a psychiatrist and were diagnosed by highly reliable methods. Genotypes were generated with automated technology and were scored blind to phenotype. Affected sib pairs showed excess allele sharing at the 18q markers D18S541 and D18S38. A parent-of-origin effect was observed, but it was not consistently paternal. No robust evidence of linkage was detected for markers elsewhere on chromosome 18. Multipoint nonparametric linkage analysis in the new sample combined with the original sample of families supports linkage on chromosome 18q, but the susceptibility gene is not well localized.

A p47-phox pseudogene carries the most common mutation causing p47-phox- deficient chronic granulomatous disease.

The predominant genetic defect causing p47-phox-deficient chronic granulomatous disease (A47 degrees CGD) is a GT deletion (DeltaGT) at the beginning of exon 2. No explanation exists to account for the high incidence of this single mutation causing a rare disease in an unrelated, racially diverse population. In each of 34 consecutive unrelated normal individuals, both the normal and mutant DeltaGT sequences were present in genomic DNA, suggesting that a p47-phox related sequence carrying DeltaGT exists in the normal population. Screening of genomic bacteriophage and YAC libraries identified 13 p47-phox bacteriophage and 19 YAC clones. The GT deletion was found in 11 bacteriophage and 15 YAC clones. Only 5 exonic and 33 intronic differences distinguished all DeltaGT clones from all wild-type clones. The most striking differences were a 30-bp deletion in intron 1 and a 20-bp duplication in intron 2. These results provide good evidence for the existence of at least one highly homologous p47-phox pseudogene containing the DeltaGT mutation. The p47-phox gene and pseudogene(s) colocalize to chromosome 7q11.23. This close linkage, together with the presence within each gene of multiple recombination hot spots, suggests that the predominance of the DeltaGT mutation in A47 degrees CGD is caused by recombination events between the wild-type gene and the pseudogene(s).

Use of wild-type p53 to achieve complete treatment sensitization of tumor cells expressing endogenous mutant p53.

It is known that transfer of the wild-type p53 gene into p53-negative cells from transgenic mice increases their sensitivity to drug and radiation-induced apoptosis. However, unlike many human tumors, these transgenic cells do not express mutant p53, and it is not known from these earlier studies whether wild-type p53 dominates the effects of mutant p53 with respect to drug and radiation sensitivity. We addressed this question in glioblastoma, a disease characterized by an unusually high level of intrinsic resistance to therapy and poor prognosis: mean survival time from diagnosis is only about 1 yr. We introduced the gene for wild-type p53 into human T98G glioblastoma cells, which express endogenous mutant p53 but not wild-type p53. Stable transfectants that co-expressed mutant and wild-type p53 had enhanced sensitivity to cisplatin and gamma radiation, compared with parental cells, control vector-transduced cells, and transduced cells that had lost expression of wild-type p53. Transient wild-type p53 expression after high-efficiency gene transfer by a p53 adenovirus also sensitized the cells to cisplatin and correlated with the induction of apoptosis. The sensitization effect was also observed in p53 adenovirus-infected H23 small cell lung carcinoma cells, which express endogenous mutant p53. Therefore, wild-type p53 gene transfer has dominant effects over mutant p53 in sensitizing tumor cells to therapy, which supports the potential of p53 gene therapy to enhance the efficacy of traditional therapy.

Characterization of the gene encoding carbonic anhydrase I from the pigtail macaque.

The structure of the gene encoding carbonic anhydrase I (CA I) was determined for the pigtail macaque Macaca nemestrina. When the deduced amino-acid sequence was compared with those of five other primates, four non-primate mammals and a turtle, seven residues were found to be unique and invariant to all of the CA I sequences. A scheme is presented for the probable evolutionary order of the six polymorphic nucleotide changes found in the coding regions of the CA I locus of pigtail macaques.

Mutations in the promoter region of the gene for gp91-phox in X-linked chronic granulomatous disease with decreased expression of cytochrome b558.

We examined the molecular defect in two kindreds with "variant" X-linked chronic granulomatous disease (CGD). Western blots of neutrophil extracts showed decreased immunoreactive cytochrome b558 components gp91-phox and p22-phox. Analysis of mRNA demonstrated reduced gp91-phox transcripts, with relative preservation of an alternative mRNA species created by transcription initiation in the third exon of the gene. Single strand conformation polymorphism analysis of the 5' flanking region of the patients' gp91-phox genes revealed an electrophoretic abnormality not detected in 40 other gp91-phox genes. Genomic sequencing demonstrated a single base change associated with CGD in each kindred: in one, adenine to cytosine at base pair-57 and in the other, thymidine to cytosine at -55. These mutations are located between the "CCAAT" and "TATA" box consensus sequences involved in eukaryotic gene transcription. Gel shift assays revealed two specific DNA-protein complexes formed between phagocyte nuclear extracts and an oligonucleotide probe representing bases -31 to -68 of the gp91-phox promoter region; the faster-migrating complex could not be formed with oligonucleotides containing either of the promoter mutations. Thus, these promoter region mutations appear to be causally related to the loss of association of a DNA-binding protein and lead to diminished gp91-phox expression, abnormal transcription initiation, and the development of CGD.

Mutation creates an open reading frame within the 5' untranslated region of macaque erythrocyte carbonic anhydrase (CA) I mRNA that suppresses CA I expression and supports the scanning model for translation.

A variant allele at the CA I locus that produces a deficiency of erythrocyte-specific CA I occurs as a widespread polymorphism in pigtail macaques from southeast Asia. Sequence analyses revealed a C----G substitution 12 nucleotides downstream of the cap site in the variant erythrocyte CA I mRNA. This mutation forms a new AUG start site and an open reading frame coding for 26 amino acids that terminates 6 nucleotides before the normal AUG initiation codon for CA I. It appears that the presence of this upstream open reading frame greatly diminishes reinitiation of translation from the normal start site, resulting in trace levels of CA I in erythrocytes. Preferential use of the first AUG codon supports the scanning model for translation initiation in eukaryotes.

Variation in coding exons of two electrophoretic alleles at the pigtail macaque carbonic anhydrase I locus as determined by direct, double-stranded sequencing of polymerase chain reaction (PCR) products.

Two, electrophoretically distinct, forms of carbonic anhydrase I (CA Ia and CA Ib) are found at high polymorphic frequencies in red cells of natural populations of pigtail macaques, Macaca nemestrina, from southeast Asia. By use of the polymerase chain reaction, exons of the CA I gene were amplified from homozygous (a/a, b/b) and heterozygous (a/b) animals. Direct sequencing of the amplified DNA from four animals revealed differences between the a and the b electrophoretic alleles ranging from three to six nucleotides, and from one to three differences within each allele. These results indicate a greater genetic variability at the CA I locus in this macaque species than previously realized.

Chronic granulomatous disease: diagnosis and classification at the molecular level.

Chronic granulomatous disease (CGD) is caused by the failure of phagocytes to produce microbicidal derivatives of molecular oxygen, such as hydrogen peroxide. It is one of the best characterized of the phagocyte disorders and represents an important consideration in the differential diagnosis of recurrent infections. The clinical, biochemical, and molecular genetic aspects of CGD are reviewed in this context in this article.

Three case reports of the metabolic and electroencephalographic changes during advanced Buddhist meditation techniques.

To examine the extent to which advanced meditative practices might alter body metabolism and the electroencephalogram (EEG), we investigated three Tibetan Buddhist monks living in the Rumtek monastery in Sikkim, India. In a study carried out in February 1988, we found that during the practice of several different meditative practices, resting metabolism (VO2) could be both raised (up to 61%) and lowered (down to 64%). The reduction from rest is the largest ever reported. On the EEG, marked asymmetry in alpha and beta activity between the hemispheres and increased beta activity were present. From these three case reports, we conclude that advanced meditative practices may yield different alterations in metabolism (there are also forms of meditation that increase metabolism) and that the decreases in metabolism can be striking.

Origins and molecular evolution of the carbonic anhydrase isozymes.

Work on membrane-bound and subcellular forms of CA at the protein level, and the possibility of multiple forms of the mouse CA II gene at the DNA level, indicate that CA may represent an extensive multigene family. A method for classifying newly sequenced CA molecules, or genes encoding them, is discussed. Phylogenetic trees based on the existing sequence data are presented and discussed in terms of gene evolution. The active-site residues of CA II have been more conserved in evolution than those of CA I or CA III. After the gene duplications, CA III and CA I initially evolved more rapidly than CA II. Since the mammalian radiation, the CA II molecule as a whole has been accepting substitutions more frequently than CA I, which in turn is evolving more rapidly than CA III. These findings can be explained if external regions of CA I and CA III have been conserved in evolution owing to interactions with other molecules. Two such regions appear to be residues 18-37 in CA I and 231-250 in CA III. Spinach CA was purified and a small amount of sequence data collected. The difficulty in aligning it with animal CAs suggests that a plant CA may not be suitable to shed light on the active site and character of the ancestral eukaryote CA.

Differential fatness gain of low income boys and girls.

As shown in 564 girls and 553 boys followed for a period of 18 yr, long-term gain in both subscapular and triceps skinfold thickness was higher in children of lower family income level than those of higher family incomes. This differential fatness gain accounts for the socioeconomic "reversal" of fatness in the female shown in cross-sectional studies and newly extends the phenomenon to both sexes. The finding that low-income children show a greater long-term increase in fatness bears on the prevention and control of obesity.