Mycobacterium tuberculosis population structure determines the outcome of genetics-based second-line drug resistance testing.

The global emergence of multidrug-resistant tuberculosis has highlighted the need for the development of rapid tests to identify resistance to second-line antituberculosis drugs. Resistance to fluoroquinolones and aminoglycosides develops through nonsynonymous single nucleotide polymorphisms in the gyrA and gyrB genes and the rrs gene, respectively. Using DNA sequencing as the gold standard for the detection of mutations conferring resistance, in conjunction with spoligotyping, we demonstrated heteroresistance in 25% and 16.3% of Mycobacterium tuberculosis isolates resistant to ofloxacin and amikacin, respectively. Characterization of follow-up isolates from the same patients showed that the population structure of clones may change during treatment, suggesting different phases in the emergence of resistance. The presence of underlying mutant clones was identified in isolates which failed to show a correlation between phenotypic resistance and mutation in the gyrA or rrs gene. These clones harbored previously described mutations in either the gyrA or rrs gene, suggesting that rare mutations conferring resistance to ofloxacin or amikacin may not be as important as was previously thought. We concluded that the absence of a correlation between genotypic and phenotypic resistance implies an early phase in the emergence of resistance within the patient. Thus, the diagnostic utility of genetics-based drug susceptibility tests will depend on the proportion of patients whose bacilli are in the process of acquiring resistance in the study setting. These data have implications for the interpretation of molecular and microbiological diagnostic tests for patients with drug-susceptible and drug-resistant tuberculosis who fail to respond to treatment and for those with discordant results.

The diagnostic performance of the GenoType MTBDRplus version 2 line probe assay is equivalent to that of the Xpert MTB/RIF assay.

Molecular diagnostics for Mycobacterium tuberculosis have recently been endorsed by the World Health Organization. The Xpert MTB/RIF assay was endorsed for use on patient material, regardless of smear gradation, while the GenoType MTBDRplus (version 1) has been limited for use on smear-positive patient material. In this study, we evaluated the diagnostic performance of the Xpert MTB/RIF and GenoType MTBDRplus (version 2) assays on smear-positive and smear-negative patient specimens submitted to a high-throughput diagnostic laboratory. A total of 282 consecutive specimens were subjected to the two new molecular assays, and their performance characteristics were assessed relative to the routine diagnostic standard. Both assays showed similar diagnostic performance characteristics. The sensitivities of the GenoType MTBDRplus (v2.0) and Xpert MTB/RIF assays for the detection of culture-positive M. tuberculosis were 73.1% and 71.2%, respectively, while the specificities of both assays were 100%. Both assays were able to diagnose the presence of M. tuberculosis in 57 to 58% of smear-negative cases, suggesting that the performance characteristics were dependent on bacillary load. The detection of M. tuberculosis in culture-negative specimens confirmed that molecular assays should not be used for treatment monitoring. The sensitivity and specificity for rifampin resistance detection were 100% in both assays; however, the GenoType MTBDRplus (v2.0) assay provided additional information on isoniazid susceptibility. The GenoType MTBDRplus (v2.0) assay will complement the Xpert MTB/RIF screening assay by validating rifampin susceptibility and providing information on isoniazid susceptibility. In addition, the GenoType MTBDRplus (v2.0) assay will provide pharmacogenetic information that may be critical in guiding appropriate treatment.

Dried culture spots for Xpert MTB/RIF external quality assessment: results of a phase 1 pilot study in South Africa.

Implementation of Xpert MTB/RIF requires quality assessment. A pilot program using dried culture spots (DCSs) of inactivated Mycobacterium tuberculosis is described. Of 274 DCS results received, 2.19% generated errors; the remainder yielded 100% correct Mycobacterium tuberculosis detection. The probe A cycle threshold (C(T)) variability of three DCS batches was ≤ 3.47. The study of longer-term DCS stability is ongoing.

Evidence of an X-linked or recessive genetic component to prostate cancer risk.

We used data from a population-based cohort study of blacks, Hispanics, Japanese and whites to examine the frequency of prevalent prostate and breast cancer by family history status of first-degree relatives (parents and siblings). Independent of race, the age-adjusted relative risk for prevalent prostate cancer in subjects with affected brothers was approximately two times that in subjects with affected fathers (P < 0.00005). No such excess risk for breast cancer was observed among subjects with affected sisters compared to those with affected mothers (age- and race-adjusted relative risk = 1.10, P = 0.34). The magnitude of the relative risk for prostate cancer in sibling- versus parent-affected groups was significantly different from that of the comparable relative risk for breast cancer (P < 0.00005). An excess risk of prostate cancer in men with affected brothers compared to those with affected fathers is consistent with the hypothesis of an X-linked, or recessive, model of inheritance.

5-Methylcytosine as an endogenous mutagen in the human LDL receptor and p53 genes.

Direct genomic sequencing revealed that cytosine residues known to have undergone a germ-line mutation in the low density lipoprotein receptor gene or somatic mutations in the p53 tumor suppressor gene were methylated in all normal human tissues analyzed. Thus, these mutations should be scored as transitions from 5-methylcytosine to thymine rather than from cytosine to thymine. Methylated cytosines occur exclusively at CpG dinucleotides, which, although markedly underrepresented in human DNA, are sites for more than 30 percent of all known disease-related point mutations. Thus, 5-methylcytosine functions as an endogenous mutagen and carcinogen in humans, in that methylation seems to increase the potential for mutation at cytosine residues at least by a factor of 10.

Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris.

OBJECTIVE: Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. RESULTS: The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489-2780 mg L-1), and were secreted at moderate to high percentages of the total extracellular protein (51-94%). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5-6-fold, reaching enzyme activity levels (1020-202 U L-1) that could allow their direct application.

Two common low density lipoprotein receptor gene mutations cause familial hypercholesterolemia in Afrikaners.

Familial hypercholesterolemia (FH), an autosomal dominant disease caused by mutations in the LDL receptor gene, is five times more frequent in the Afrikaner population of South Africa than it is in the population of the United States and Europe. It has been proposed that the high frequency is due to a founder effect. In this paper, we characterized 24 mutant LDL receptor alleles from 12 Afrikaner individuals homozygous for FH. We identified two mutations that together makeup greater than 95% of the mutant LDL receptor genes represented in our sample. Both mutations were basepair substitutions that result in single-amino acid changes. Each mutation can be detected readily with the polymerase chain reaction and restriction analysis. The finding of two common LDL receptor mutations in the Afrikaner FH homozygotes predicts that these mutations will predominate in the Afrikaner population and that the high frequency of FH is due to a founder effect. The increased incidence of ischemic heart disease in the Afrikaner population may in part be due to the high frequency of these two mutations in the LDL receptor gene.

Chylomicron remnant clearance from the plasma is normal in familial hypercholesterolemic homozygotes with defined receptor defects.

The retinyl palmitate fat tolerance test was used to measure chylomicron remnant clearance in 10 normal subjects (apolipoprotein E [apo E] isotypes 3 or 4 only), 6 normolipidemic apo E2/2 homozygotes and 5 familial hypercholesterolemic homozygotes. Skin fibroblasts with fully upregulated LDL receptors from the latter subjects degraded rabbit 125I-beta VLDL in vitro at rates ranging from less than 10-48% of normal. Experiments in vivo revealed no significant differences between the normal and homozygous familial hypercholesterolemic (FHH) subjects in chylomicron remnant clearance assessed on the basis of "areas under the curves" for retinyl palmitate levels present in post-prandial serum, chylomicron remnants (Sf. less than 1,000), or chylomicrons (Sf. greater than 1,000). Remnant clearance was greatly decreased at all times in the apo E2/2 homozygotes, indicative of an important degree of flux control exerted by a receptor-mediated step involving apo E as ligand. The absence of any excess remnant accumulation in FHH subjects with varying "impairment" of LDL receptor-mediated degradation of apo E-containing lipoproteins, permits the conclusion that chylomicron remnants are initially cleared from the plasma by apo E-recognizing receptors which are genetically distinct from LDL receptors.

Progressive increases in the methylation status and heterochromatinization of the myoD CpG island during oncogenic transformation.

Alterations in DNA methylation patterns are one of the earliest and most common events in tumorigenesis. Overall levels of genomic methylation often decrease during transformation, but localized regions of increased methylation have been observed in the same tumors. We have examined changes in the methylation status of the muscle determination gene myoD, which contains a CpG island, as a function of oncogenic transformation. This CpG island underwent de novo methylation during immortalization of 10T1/2 cells, and progressively more sites became methylated during the subsequent transformation of the cells to oncogenicity. The greatest increase in methylation occurred in the middle of the CpG island in exon 1 during transformation. Interestingly, no methylation was apparent in the putative promoter of myoD in either the 10T1/2 cell line or its transformed derivative. The large number of sites in the CpG island that became methylated during transformation was correlated with heterochromatinization of myoD as evidenced by a decreased sensitivity to cleavage of DNA in nuclei by MspI. A site in the putative promoter also became insensitive to MspI digestion in nuclei, suggesting that the chromatin structural changes extended beyond the areas of de novo methylation. Unlike Lyonized genes on the inactive X chromosome, whose timing of replication is shifted to late S phase, myoD replicated early in S phase in the transformed cell line. Methylation analysis of myoD in DNAs from several human tumors, which presumably do not express the gene, showed that hypermethylation also frequently occurs during carcinogenesis in vivo. Thus, the progressive increase in methylation of myoD during immortalization and transformation coinciding with a change in chromatin structure, as illustrated by the in vitro tumorigenic model, may represent a common mechanism in carcinogenesis for permanently silencing the expression of genes which can influence cell growth and differentiation.

Multiple signal input and output domains of the 160-kilodalton nuclear receptor coactivator proteins.

Members of the 160-kDa nuclear receptor coactivator family (p160 coactivators) bind to the conserved AF-2 activation function found in the hormone binding domains of nuclear receptors (NR) and are potent transcriptional coactivators for NRs. Here we report that the C-terminal region of p160 coactivators glucocorticoid receptor interacting protein 1 (GRIP1), steroid receptor coactivator 1 (SRC-1a), and SRC-1e binds the N-terminal AF-1 activation function of the androgen receptor (AR), and p160 coactivators can thereby enhance transcriptional activation by AR. While they all interact efficiently with AR AF-1, these same coactivators have vastly different binding strengths with and coactivator effects on AR AF-2. p160 activation domain AD1, which binds secondary coactivators CREB binding protein (CBP) and p300, was previously implicated as the principal domain for transmitting the activating signal to the transcription machinery. We identified a new highly conserved motif in the AD1 region which is important for CBP/p300 binding. Deletion of AD1 only partially reduced p160 coactivator function, due to signaling through AD2, another activation domain located at the C-terminal end of p160 coactivators. C-terminal coactivator fragments lacking AD1 but containing AD2 and the AR AF-1 binding site served as efficient coactivators for full-length AR and AR AF-1. The two signal input domains (one that binds NR AF-2 domains and one that binds AF-1 domains of some but not all NRs) and the two signal output domains (AD1 and AD2) of p160 coactivators played different relative roles for two different NRs: AR and thyroid hormone receptor.

Association of prostate cancer risk with genetic polymorphisms in vitamin D receptor and androgen receptor.

BACKGROUND: Prostate cancer is an increasingly common disease for which there are few well-established risk factors. Family history data suggest a genetic component; however, the majority of prostate cancer cases cannot be explained by a single-gene model. Prostate cell division is influenced by two steroid hormones, testosterone and vitamin D, the action of each being mediated by its respective receptor. The genes for the two receptors are candidates in a multigenic model for prostate cancer susceptibility. PURPOSE: We examined genetic polymorphisms in two steroid receptors, the androgen receptor (AR) and the vitamin D receptor (VDR), in a case-control pilot study of prostate cancer. METHODS: Fifty-seven non-Hispanic white case patients with prostate cancer and 169 non-Hispanic white control subjects were genotyped for a previously described microsatellite (CAG repeats) in the AR gene and for a newly discovered poly-A microsatellite in the 3'-untranslated region (3'UTR) of the VDR gene. To compare genotypes with respect to prostate cancer risk, we estimated odds ratios (ORs) by using logistic regression. ORs were also estimated separately for advanced and localized cases of disease. All P values resulted from two-sided tests. RESULTS: Both the AR and the VDR polymorphisms were associated, individually and after mutual adjustment, with prostate cancer. Adjusted ORs (95% confidence intervals [CIs]) for prostate cancer were 2.10 (95% CI = 1.11-3.99) for individuals carrying an AR CAG allele with fewer than 20 repeats versus an allele with 20 or more repeats and 4.61 (95% CI = 1.34-15.82) for individuals carrying at least one long (A18 to A22) VDR poly-A allele versus two short (A14 to A17) poly-A alleles. For both the AR and VDR genes, the at-risk genotypes were more strongly associated with advanced disease than with localized disease. CONCLUSIONS: In this pilot study, genetic variation in both the VDR and the AR genes was associated with prostate cancer, and both genes appear to preferentially confer risk for advanced disease. These two genetic risk factors, if confirmed, are among the strongest risk factors yet identified for prostate cancer. IMPLICATIONS: These results are consistent with a multigenic model of prostate cancer susceptibility. On the basis of the joint effect of several genetic loci, one might ultimately be able to construct a risk profile to predict advanced disease, so that men whose disease is unlikely to progress to an advanced stage can possibly be spared aggressive treatment.

The CAG and GGC microsatellites of the androgen receptor gene are in linkage disequilibrium in men with prostate cancer.

The androgen receptor genotype was determined in the white blood cell DNA of 45 African-American, 39 non-Hispanic white, and 39 Asian (Chinese, Japanese) normal subjects and 68 patients with prostate cancer (57 whites), all of whom were residents of Los Angeles County. For each subject, we measured the number of repeats in the polymorphic CAG and GGC microsatellites of exon 1 of the androgen receptor gene. In normal subjects, the distributions of CAG and GGC microsatellites differed significantly among the races (two-sided P = 0.046 and < 0.0005, respectively). The prevalence of short CAG alleles (< 22 repeats) was highest (75%) in African-American males with the highest risk for prostate cancer, intermediate (62%) in intermediate-risk non-Hispanic whites, and lowest (49%) in Asians at very low risk for prostate cancer. High-risk African-Americans also had the lowest frequency (20%) of the GGC allele with 16 repeats; the comparable values for intermediate-risk whites and low-risk Asians were 57% and 70%, respectively. Consistent with the interracial variation in CAG and GGC distributions, there was an excess of white patients with < 22 CAG and not-16 GGC repeats relative to white controls (relative risk, 2.1; one-sided P = 0.08). We observed no association (linkage) between the two microsatellites among normal subjects. On the other hand, there was a statistically significant negative association between the numbers of CAG and GGC repeats among the prostate cancer patients studied (two-sided P = 0.008). Among the 47 subjects with short CAG alleles (< 22 repeats), 43% had long GGC alleles (> 16 repeats) whereas only 15% of the 20 subjects with long CAG alleles (> or = 22 repeats) had long GGC alleles. Overall, our data suggest a possible association between CAG and GGC microsatellites of the androgen receptor gene and prostate cancer development.

A polymorphism in the CYP17 gene increases the risk of breast cancer.

We conducted a case-control study to determine whether a polymorphism in the CYP17 gene was associated with risk of breast cancer. We found an increased risk of advanced breast cancer in women carrying an A2 allele. The odds ratio was 2.5 [95% confidence interval (CI), 1.07-5.94] for regional or metastatic disease. Among controls, the A1/A1 genotype was associated with a later age at menarche. The reduced risk of breast cancer associated with a later age of menarche was largely limited to A1/A1 women: odds ratio, 0.47 (CI, 0.22-0.98) for breast cancer and later age at menarche among A1 homozygotes compared with 0.80 (CI, 0.51-1.27) for A1/A2 and A2/A2 genotypes. These findings suggest that the CYP17 genotype may be a biomarker for the onset of ovulation and advanced breast cancer risk.

Susceptibility to prostate cancer: interaction between genotypes at the androgen receptor and prostate-specific antigen loci.

The androgen receptor (AR) regulates gene transcription by binding to androgen response elements in target gene promoters. The prostate-specific antigen (PSA) gene has a polymorphic androgen response element sequence with two alleles, A and G. We hypothesize that allelic differences in AR-driven PSA expression may influence prostate cancer risk. To test this hypothesis, we assayed PSA genotype for 57 prostate cancer cases and 156 controls from our previous pilot study in which prostate cancer risk was associated with the AR "CAG-short" genotype. Odds ratios (ORs) were estimated relating prostate cancer risk to AR and PSA genotypes, singly and in combination. Subjects with the PSA GG genotype were at significantly increased risk for advanced, but not for localized, prostate cancer (OR, 2.90; 95% confidence interval, 1.24-6.78). When cross-classifying subjects by AR and PSA genotypes, subjects with either a CAG-short allele (and not PSA GG) or with the PSA GG genotype (and not CAG-short) had a modest, statistically insignificant increase in prostate cancer risk overall. However, subjects with both a short CAG allele and PSA genotype GG had a more than 5-fold increase in prostate cancer risk (OR, 5.08; 95% confidence interval, 1.59-16.25). All of the ORs were substantially greater for advanced prostate cancer. Studies with larger numbers of advanced cases will be needed to confirm these results. These results indicate that polymorphism in the PSA gene promoter influences prostate cancer risk, and that the allelic variation in promoter activity may be androgen-dependent. Furthermore, these results support a multigenic etiology for prostate cancer.

Building a multigenic model of breast cancer susceptibility: CYP17 and HSD17B1 are two important candidates.

We conducted a nested case-control study to evaluate whether polymorphisms in two genes involved in estrogen metabolism, CYP17 and HSD17B1, were useful in developing a breast cancer risk model that could help discriminate women who are at higher risk of breast cancer. If polymorphisms in these genes affect the level of circulating estrogens, they may directly influence breast cancer risk. The base population for this study is a multiethnic cohort study that includes African-American, Non-Latina White, Japanese, Latina, and Native Hawaiian women. For this analysis, 1508 randomly selected controls and 850 incident breast cancer cases of the first four ethnic groups who agreed to provide a blood specimen were included (76 and 80% response rates, respectively). The CYP17 A2 allele and the HSD17B1 A allele were considered "high-risk" alleles. Subjects were then classified according to number of high-risk alleles. After adjusting for age, weight, and ethnicity, we found that carrying one or more high-risk alleles increases the risk of advanced breast cancer in a dose-response fashion. The risk among women carrying four high-risk alleles was 2.21 [95% confidence interval (CI), 0.98-5.00; P for trend = 0.03] compared with those who carried none. This risk was largely limited to women who were not taking hormone replacement therapy (relative risk, 2.60; 95% CI, 0.95-7.14) and was most pronounced among those weighing 170 pounds or less (RR, 3.05; 95% CI, 1.29-7.25). These findings suggest that breast cancer risk has a strong genetic component and supports the theory that the underlying mechanism of "complex traits" can be understood using a multigenic model of candidate genes.

Risk of endometrial cancer and estrogen replacement therapy history by CYP17 genotype.

Common variants among genes coding for enzymes in sex steroid biosynthetic pathways may influence the risk of endometrial cancer. We examined the association between endometrial cancer risk and estrogen replacement therapy (ERT) by CYP17 genotype using 51 incident cases and 391 randomly selected controls from a multiethnic cohort in Hawaii and Los Angeles, California. The relative risk of endometrial cancer was calculated for ever use versus never use of ERT by CYP17 genotype (TT, TC, and CC). We found that women who reported ever taking ERT were more than twice as likely to develop endometrial cancer as women who never took ERT [odds ratio (OR), 2.24; 95% confidence interval (CI), 1.19-4.23]. Among these women, the risk of endometrial cancer was higher for women homozygous for the CYP17 T allele (OR, 4.10; 95% CI, 1.64-10.3), but not for women with the C allele (OR, 1.31; 95% CI, 0.53-3.21). These preliminary findings suggest that CYP17 or other variants in estrogen biosynthesis or metabolism pathways may be potential markers of endometrial cancer susceptibility due to ERT.

Breast cancer susceptibility gene 1 (BRCAI) is a coactivator of the androgen receptor.

In the present study, the role of BRCA1 in ligand-dependent androgen receptor (AR) signaling was assessed. In transfected prostate and breast cancer cell lines, BRCA1 enhanced AR-dependent transactivation of a probasin-derived reporter gene. The effects of BRCA1 were mediated through the NH2-terminal activation function (AF-1) of the receptor. Cotransfection of p160 coactivators markedly potentiated BRCA1-mediated enhancement of AR signaling. In addition, BRCA1 was shown to interact physically with both the AR and the p160 coactivator, glucocorticoid receptor interacting protein 1. These findings suggest that BRCA1 may directly modulate AR signaling and, therefore, may have implications regarding the proliferation of normal and malignant androgen-regulated tissues.

Cytochrome P450c17alpha gene (CYP17) polymorphism predicts use of hormone replacement therapy.

We investigated whether a polymorphism in the cytochrome P450c17alpha gene (CYP17), which is associated with higher endogenous hormone levels, influences the use of hormone replacement therapy (HRT). The study included 749 postmenopausal women ages 44-75 years at baseline randomly selected from a larger multiethnic cohort. African-American, Japanese, Latina, and white women were included in the study. Women who carry the CYP17 A2/A2 genotype were about half as likely as women with the A1/A1 genotype to be current HRT users (odds ratio = 0.52; 95% confidence interval, 0.31-0.86). This association was present in all four racial/ethnic groups and for women above and below the median weight of 150 pounds. These findings suggest that the actual risk of breast cancer associated with HRT use may be higher than previously reported.

Association of prostate cancer with vitamin D receptor haplotypes in African-Americans.

In previous studies, allelic variation in the 3' end of the vitamin D receptor gene was associated with increased risk of prostate cancer in white men. Several polymorphisms, including a BsmI restriction site and a poly(A) microsatellite, can be used interchangeably to mark the unidentified locus in whites. In African-Americans, however, these markers are not interchangeable, due to weaker linkage disequilibrium in this genomic region in this population. Here, we genotyped both the BsmI and poly(A) markers for 151 African-American prostate cancer cases (102 localized and 49 advanced) and 174 African-American male controls from a large epidemiological cohort. A direct haplotyping procedure was devised to determine BsmI/poly(A) haplotypes for double heterozygotes so that haplotypes could be used as allelic markers in standard logistic regression analyses. Using BsmI alone, b alleles were associated with a 2-fold decrease in risk of advanced prostate cancer. The association was, however, confined to haplotypes carrying a long (L) allele of the poly(A) microsatellite. BL and bL haplotypes were associated with increased and decreased risk, respectively, whereas neither BS nor bS haplotypes were associated with prostate cancer risk. An allelic variant that confers increased risk of advanced prostate cancer appears to be associated with the BsmI/poly(A) BL haplotype in African-Americans.

Cytochrome P450c17alpha gene (CYP17) polymorphism is associated with serum estrogen and progesterone concentrations.

An increased level of serum estrogen is one marker of breast cancer risk. We have recently reported that increased risk of advanced breast cancer is associated with a common allele of the cytochrome P450c17alpha gene (CYP17), designated A2. We now show that CYP17 genotype is associated with serum hormone levels among 83 young, nulliparous women. Serum estradiol (E2) levels measured around day 11 of the menstrual cycle were 11 and 57% higher (P = 0.04), respectively, among women hetero- and homozygous for the CYP17 A2 allele compared to A1/A1 women. Similarly, around cycle day 22, E2 levels were 7 and 28% higher (P = 0.06), and progesterone levels were 24 and 30% higher (P = 0.04), respectively. These data provide direct evidence of genetic control of serum hormone levels.