pharmacogenetics; electronic medical record; personalized medicine; cytochrome P450 2D6; thiopurine-methyltransferase
Polymorphisms in CYP2D6 and CYP2C19 affect the efficacy and safety of tricyclics, with some drugs being affected by CYP2D6 only, and others by both polymorphic enzymes. Amitriptyline, clomipramine, doxepin, imipramine, and trimipramine are demethylated by CYP2C19 to pharmacologically active metabolites. These drugs and their metabolites, along with desipramine and nortriptyline, undergo hydroxylation by CYP2D6 to less active metabolites. Evidence from published literature is presented for CYP2D6 and CYP2C19 genotype–directed dosing of tricyclic antidepressants.
Research on genes and medications has advanced our understanding of the genetic basis of individual drug responses. The aim of pharmacogenomics is to develop strategies for individualizing therapy for patients, to optimize outcome through knowledge of human genome variability and its influence on drug response. Pharmacogenomics research is translational in nature and ranges from discovery of genotype-phenotype relationships to clinical trials which provide proof of clinical impact. Advances in pharmacogenomics offer significant potential for subsequent clinical application in individual patients; however, the translation of pharmacogenomics research findings into clinical practice has been slow. Key components to successful clinical implementation of pharmacogenomics will include consistent interpretation of pharmacogenomic test results, availability of clinical guidelines for prescribing based on test results, and knowledge-based decision support systems.
pharmacogenomics; genetic testing; personalized medicine
One approach to circumvent barriers to clinical implementation of pharmacogenetics is to employ pre-prescription genotyping that requires interrogation of multiple pharmacogenetic variants using a high-throughput platform. We compared the performance of the DMET Plus array (1,931 variants in 225 genes) with orthogonal genotyping methods in 220 pediatric patients. A total of 1,692 variants had call rates above 98% and were in Hardy Weinberg equilibrium. Of these, 259 were genotyped by at least one independent method and a total of 19,942 SNP-patient sample pairs were evaluated. The concordance was 99.9% with only 28 genotype discordances observed. For those genes deemed most likely to be clinically relevant (TPMT, CYP2D6, CYP2C19, CYP2C9, VKORC1, DPYD, UGT1A1, and SLCO1B1), a total of 3,799 SNP-patient sample pairs were evaluable and had a concordance of 99.96%. We conclude that the DMET Plus array performs well with primary patient samples when compared to multiple other lower-throughput genotyping methods.
REV1 and DNA Polymerase ζ (REV3 and REV7) play important roles in translesion DNA synthesis (TLS) in which DNA replication bypasses blocking lesions. REV1 and Polζ have also been implicated in promoting repair of DNA double-stranded breaks (DSBs). However, the mechanism by which these two TLS polymerases increase tolerance to DSBs is poorly understood. Here we demonstrate that full-length human REV1, REV3 and REV7 interact in vivo (as determined by co-immunoprecipitation studies) and together, promote homologous recombination repair. Cells lacking REV3 were hypersensitive to agents that cause DSBs including the PARP inhibitor, olaparib. REV1, REV3 or REV7-depleted cells displayed increased chromosomal aberrations, residual DSBs and sites of HR repair following exposure to ionizing radiation. Notably, cells depleted of DNA polymerase η (Polη) or the E3 ubiquitin ligase RAD18 were proficient in DSB repair following exposure to IR indicating that Polη-dependent lesion bypass or RAD18-dependent monoubiquitination of PCNA are not necessary to promote REV1 and Polζ-dependent DNA repair. Thus, the REV1/Polζ complex maintains genomic stability by directly participating in DSB repair in addition to the canonical TLS pathway.
Translesion DNA synthesis (TLS) is a process whereby specialized DNA polymerases are recruited to bypass DNA lesions that would otherwise stall high-fidelity polymerases. We provide evidence that TLS across cisplatin intrastrand cross-links is performed by multiple translesion DNA polymerases. First, we determined that PCNA monoubiquitination by RAD18 is necessary for efficient bypass of cisplatin adducts by the TLS polymerases eta (Polη), REV1, and zeta (Polζ) based on the observations that depletion of these proteins individually leads to decreased cell survival, cell cycle arrest in S phase, and activation of the DNA damage response. Second, we showed that in addition to PCNA monoubiquitination by RAD18, the Fanconi anemia core complex is also important for recruitment of REV1 to stalled replication forks in cisplatin treated cells. Third, we present evidence that REV1 and Polζ are uniquely associated with protection against cisplatin and mitomycin C-induced chromosomal aberrations, and both are necessary for the timely resolution of DNA double-strand breaks associated with repair of DNA interstrand cross-links. Together, our findings indicate that REV1 and Polζ facilitate repair of interstrand cross-links independently of PCNA monoubiquitination and Polη, whereas RAD18 plus Polη, REV1, and Polζ are all necessary for replicative bypass of cisplatin intrastrand DNA cross-links.
Objective. To describe the distribution and severity of muscle weakness using manual muscle testing (MMT) in 172 patients with PM, DM and juvenile DM (JDM). The secondary objectives included characterizing individual muscle group weakness and determining associations of weakness with functional status and myositis characteristics in this large cohort of patients with myositis.
Methods. Strength was assessed for 13 muscle groups using the 10-point MMT and expressed as a total score, subscores based on functional and anatomical regions, and grades for individual muscle groups. Patient characteristics and secondary outcomes, such as clinical course, muscle enzymes, corticosteroid dosage and functional status were evaluated for association with strength using univariate and multivariate analyses.
Results. A gradient of proximal weakness was seen, with PM weakest, DM intermediate and JDM strongest among the three myositis clinical groups (P ≤ 0.05). Hip flexors, hip extensors, hip abductors, neck flexors and shoulder abductors were the muscle groups with the greatest weakness among all three clinical groups. Muscle groups were affected symmetrically.
Conclusions. Axial and proximal muscle impairment was reflected in the five weakest muscles shared by our cohort of myositis patients. However, differences in the pattern of weakness were observed among all three clinical groups. Our findings suggest a greater severity of proximal weakness in PM in comparison with DM.
Myositis; Manual muscle test; Strength; Rehabilitation
To describe the distribution and severity of muscle weakness using manual muscle testing (MMT) in 172 patients with polymyositis (PM), dermatomyositis (DM) and juvenile dermatomyositis (JDM). The secondary objectives included characterizing individual muscle group weakness and determining associations of weakness with functional status and myositis characteristics in this large cohort of patients with myositis.
Strength was assessed for 13 muscle groups using the 10-point MMT and expressed as a total score, subscores based on functional and anatomical regions, and grades for individual muscle groups. Patient characteristics and secondary outcomes such as clinical course, muscle enzymes, corticosteroid dosage, and functional status were evaluated for association with strength using univariate and multivariate analyses.
A gradient of proximal weakness was seen, with PM weakest, DM intermediate and JDM strongest among the three myositis clinical groups (p ≤ 0.05). Hip flexors, hip extensors, hip abductors, neck flexors, and shoulder abductors were the muscle groups with the greatest weakness among all three clinical groups. Muscle groups were affected symmetrically.
Axial and proximal muscle impairment was reflected in the five weakest muscles shared by our cohort of myositis patients. However, differences in the pattern of weakness were observed among all three clinical groups. Our findings suggest a greater severity of proximal weakness in PM in comparison to DM.
Myositis; manual muscle test; strength; rehabilitation
Oxidative stress plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). To investigate the correlation between the progression of COPD and plasma biomarkers of chronic inflammation and oxidative injury, blood samples were obtained from healthy volunteers (HV, n = 14) and stabilized COPD patients. The patients were divided into three groups according to their GOLD stage (II, n = 34; III, n = 18; IV, n = 20). C-reactive protein (CRP), protein carbonyls (PC), malondialdehyde (MDA), susceptible lipoperoxidation of plasma substrates (SLPS), and myeloperoxidase activity (MPO) were measured. The plasma concentration of SLPS was measured as the amount of MDA generated by a metal ion-catalyzed reaction in vitro. PC, SLPS, and CPR were increased significantly (p < 0.001) in COPD patients when compared to HV. MDA concentrations and MPO activities were not significantly different from those of the HV group. In conclusion, increased oxidation of lipids and proteins resulting in a progressive increase in the amount of total plasma carbonyls and oxidative stress the presence of oxidative stress during COPD progression, concomitant with an increased oxidation of lipids and proteins resulting in a progressive and significant increase in the amount of total carbonyls formed from lipid-derived aldehydes and direct amino acid side chain oxidation in plasma, may serve as a biomarker and independent monitor of COPD progression and oxidative stress injury.
Chronic obstructive pulmonary disease (COPD); oxidative stress; protein carbonyls; reactive oxygen species (ROS); lipoperoxidation of plasma substrates (SLPS).
Microarray Comparative Genomic Hybridization (array CGH) provides a means to examine DNA copy number aberrations. Various platforms, brands and underlying technologies are available, facing the user with many choices regarding platform sensitivity and number, localization, and density distribution of probes.
We evaluate three different platforms presenting different nature and arrangement of the probes: The Agilent Human Genome CGH Microarray 44 k, the ROMA/NimbleGen Representational Oligonucleotide Microarray 82 k, and the Illumina Human-1 Genotyping 109 k BeadChip, with Agilent being gene oriented, ROMA/NimbleGen being genome oriented, and Illumina being genotyping oriented. We investigated copy number changes in 20 human breast tumor samples representing different gene expression subclasses, using a suite of graphical and statistical methods designed to work across platforms. Despite substantial differences in the composition and spatial distribution of probes, the comparison revealed high overall concordance. Notably however, some short amplifications and deletions of potential biological importance were not detected by all platforms. Both correlation and cluster analysis indicate a somewhat higher similarity between ROMA/NimbleGen and Illumina than between Agilent and the other two platforms. The programs developed for the analysis are available from .
We conclude that platforms based on different technology principles reveal similar aberration patterns, although we observed some unique amplification or deletion peaks at various locations, only detected by one of the platforms. The correct platform choice for a particular study is dependent on whether the appointed research intention is gene, genome, or genotype oriented.
It is unclear whether it is best to use high-viscosity or low-viscosity cement for fixation of total hip replacement (THR) femoral components. This study examines the influence of cement viscosity on the migration of the Exeter femoral component using roentgen stereophotogrammetric analysis (RSA). Simplex, CMW1 and CMW3 G cements were examined in a total of 46 patients over a 12-month period. The overall pattern of migration for all cohorts was one of subsidence and rotation into valgus. There was no significant difference in any aspect of migration between the groups. In vitro studies demonstrate that low-viscosity cement forms a more stable bone-cement interface. Several groups have examined the in vivo effect of cement viscosity on stem longevity with conflicting results. For a polished, tapered implant that is designed to subside, cement viscosity does not influence the 1-year migration, and it is therefore unlikely to affect long-term outcome.
This report describes the case of a 44-year-old woman presenting to a Sleep and Alertness clinic with symptoms of narcolepsy. The patient had clinical and polysomnographic features of narcolepsy, which disappeared after disclosure of severe psychological stress. Following a discussion of the differential diagnosis of narcolepsy, alternative diagnoses are considered. The authors suggest that the patient had a hysterical conversion disorder, or "pseudo-narcolepsy." Careful inquiry into psychological factors in unusual cases of narcolepsy may be warranted.
Little information is available on the relationship between occupational exposure to inorganic arsenic in coal fly ash and urinary excretion of arsenic metabolites. This study ws undertaken in a coal-fired power plant in Slovakia during a routine maintenance outage. Arsenic was measured in the breathing zone of workers during 5 consecutive workdays, and urine samples were obtained for analysis of arsenic metabolites--inorganic arsenic (Asi), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA)--prior to the start of each shift. Results from a small number of cascade impactor air samples indicated that approximately 90% of total particle mass and arsenic was present in particle size fractions >/= 3.5 micron. The 8-hr time-weighted average (TWA) mean arsenic air concentration was 48.3 microg/m3 (range 0.17-375.2) and the mean sum of urinary arsenic (SigmaAs) metabolites was 16.9 microg As/g creatinine (range 2.6-50.8). For an 8-hr TWA of 10 microg/m3 arsenic from coal fly ash, the predicted mean concentration of the SigmaAs urinary metabolites was 13.2 microg As/G creatinine [95% confidence interval (CI), 10.1-16.3). Comparisons with previously published studies of exposure to arsenic trioxide vapors and dusts in copper smelters suggest that bioavailability of arsenic from airborne coal fly ash (as indicated by urinary excretion) is about one-third that seen in smelters and similar settings. Arsenic compound characteristics, matrix composition, and particle size distribution probably play major roles in determining actual uptake of airborne arsenic.
DNA polymerase II (Pol II) is regulated as part of the SOS response to DNA damage in Escherichia coli. We examined the participation of Pol II in the response to oxidative damage, adaptive mutation, and recombination. Cells lacking Pol II activity (polB delta 1 mutants) exhibited 5- to 10-fold-greater sensitivity to mode 1 killing by H2O2 compared with isogenic polB+ cells. Survival decreased by about 15-fold when polB mutants containing defective superoxide dismutase genes, sodA and sodB, were compared with polB+ sodA sodB mutants. Resistance to peroxide killing was restored following P1 transduction of polB cells to polB+ or by conjugation of polB cells with an F' plasmid carrying a copy of polB+. The rate at which Lac+ mutations arose in Lac- cells subjected to selection for lactose utilization, a phenomenon known as adaptive mutation, was increased threefold in polB backgrounds and returned to wild-type rates when polB cells were transduced to polB+. Following multiple passages of polB cells or prolonged starvation, a progressive loss of sensitivity to killing by peroxide was observed, suggesting that second-site suppressor mutations may be occurring with relatively high frequencies. The presence of suppressor mutations may account for the apparent lack of a mutant phenotype in earlier studies. A well-established polB strain, a dinA Mu d(Apr lac) fusion (GW1010), exhibited wild-type (Pol II+) sensitivity to killing by peroxide, consistent with the accumulation of second-site suppressor mutations. A high titer anti-Pol II polyclonal antibody was used to screen for the presence of Pol II in other bacteria and in the yeast Saccharomyces cerevisiae. Cross-reacting material was found in all gram-negative strains tested but was not detected in gram-positive strains or in S. cerevisiae. Induction of Pol II by nalidixic acid was observed in E. coli K-12, B, and C, in Shigella flexneri, and in Salmonella typhimurium.
We have identified sequences at the telomeres of the yeast Candida albicans and have found that they are composed of tandem copies of a 23-bp sequence. Through the cloning of native telomeric ends and the characterization and cloning of a "healed" end, we demonstrate that these repeated sequences are sufficient to function as a telomere. All copies of the 23-bp repeat that have been sequenced from a number of C. albicans strains are identical. In contrast, adjacent subtelomeric sequences are variable both between strains and within the WO-1 strain. In the WO-1 strain, the lengths of the telomeres are dependent upon growth temperature and are substantially longer at higher temperatures. Telomere growth is accompanied by increases in the number of the 23-bp repeats present on the telomeric fragments. These results suggest that either telomerase-maintained telomeres can be more complex in structure than was previously imagined or that Candida telomeres are maintained via a telomerase-independent mechanism.
We have isolated a gene, designated CAG1, from Candida albicans by using the G-protein alpha-subunit clone SCG1 of Saccharomyces cerevisiae as a probe. Amino acid sequence comparison revealed that CAG1 is more homologous to SCG1 than to any other G protein reported so far. Homology between CAG1 and SCG1 not only includes the conserved guanine nucleotide binding domains but also spans the normally variable regions which are thought to be involved in interaction with the components of the specific signal transduction pathway. Furthermore, CAG1 contains a central domain, previously found only in SCG1. cag1 null mutants of C. albicans created by gene disruption produced no readily detectable phenotype. The C. albicans CAG1 gene complemented both the growth and mating defects of S. cerevisiae scg1 null mutants when carried on either a low- or high-copy-number plasmid. In diploid C. albicans, the CAG1 transcript was readily detectable in mycelial and yeast cells of both the white and opaque forms. However, the CAG1-specific transcript in S. cerevisiae transformants containing the C. albicans CAG1 gene was observed only in haploid cells. This transcription pattern matches that of SCG1 in S. cerevisiae and is caused by a1-alpha 2 mediated repression in diploid cells. That is, CAG1 behaves as a haploid-specific gene in S. cerevisiae, subject to control by the a1-alpha 2 mating-type regulation pathway. We infer from these results that C. albicans may have a signal transduction system analogous to that controlling mating type in S. cerevisiae or possibly even a sexual pathway that has so far remained undetected.
We report the isolation of middle-repetitive DNA sequences from Cryptococcus neoformans that are species and variety specific. These probes were used for assessing strain relatedness among cryptococcal isolates from patients with and without AIDS who were from Zaire and the United States. Five distinct hybridization patterns were observed for the 60 isolates examined, regardless of the restriction enzyme used for digestion. The most common pattern among the isolates from the patients without AIDS was also the most common among the isolates from the patients with AIDS who were from the United States and was the only pattern observed for all isolates tested from patients with AIDS who were from Zaire. On the basis of the high specificity and sensitivity of the signals observed by hybridization, we suggest that these sequences provide a means for both biotyping and early diagnosis of C. neoformans.
The WO-1 strain of Candida albicans is capable of alternating between two highly distinct yeast cell types termed white and opaque (E. H. A. Rikkerrink, B. B. Magee, and P. T. Magee, J. Bacteriol. 170:895-899, 1988; B. Slutsky, M. Staebell, J. Anderson, L. Risen, M. Pfaller, and D. R. Soll, J. Bacteriol. 169:189-197, 1987). We have isolated WO-1 mutants that show a marked deficiency at being able to switch from the white form to the opaque form under conditions normally favorable for this transition. Pulsed-field electrophoresis demonstrated that one of the initial two spontaneous nonswitching mutants lacked the smallest chromosome that is normally present in WO-1. The availability of a WO-1 derivative whose only functional ADE2 gene is located on this small chromosome made possible, through the induction of chromosome nondisjunction, the isolation of numerous new mutants missing this chromosome as well as mutants containing two copies of the chromosome. Mutants missing the smallest chromosome showed a greatly diminished ability to produce opaque sectors and to produce germ tubes in the presence of human serum. Mutants containing two copies of the small chromosome showed an increased ability to produce germ tubes. These results indicate that this small chromosome carries one or more genes involved in both the white-opaque switch and the yeast-hyphal switch.
MUC-2, the first described intestinal mucin gene, has become important as a prototype for secreted mucins in several organ systems. However, little is known about its protein backbone structure and hence its role in diseases such as colon cancer, ulcerative colitis, and cystic fibrosis, which are known to have mucin abnormalities. Studies in this manuscript show that MUC-2 contains two distinct regions with a high degree of internal homology, but the two regions bear no significant homology to each other. Region 1 consists mostly of 48-bp repeats which are interrupted in places by 21-24-bp segments. Several of these interrupting sequences show similarity to each other, creating larger composite repeat units. Region 1 has no length polymorphisms. Region 2 is composed of 69-bp tandem repeats arranged in an uninterrupted array of up to 115 individual units. Southern analysis of genomic DNA samples using TaqI and HinfI reveals both length and sequence polymorphisms which occur within region 2. The sequence polymorphisms have different ethnic distributions, while the length polymorphisms are due to variable numbers of tandem repeats.
The SIR1 gene product of Saccharomyces cerevisiae is one of several proteins involved in repressing transcription of the silent mating-type genes. Strains with mutations in the genes coding for these proteins are defective in mating due to derepression of the silent loci. We have found that overexpression of the SIR1 gene suppresses the mating defects of several of these mutants, including nat1 and ard1 mutants (the products of these two genes are responsible for N-terminal acetylation of a subset of yeast proteins), certain sir3 mutants, and a histone H4 mutant. The SIR1 gene has been sequenced and found to contain an open reading frame coding for a 678-amino-acid protein.