Genetic structure of a linear population of Beta vulgaris ssp maritima (sea beet) revealed by isozyme and RFLP analysis

Knowledge of the genetic structure of plant populations is necessary for the understanding of the dynamics of major ecological processes. It also has applications in conservation biology and risk assessment for genetically modified crops. This paper reports the genetic structure of a linear population of sea beet, Beta vulgaris ssp. maritima (the wild relative of sugar beet), on Furzey Island, Poole Harbour. The relative spatial positions of the plants were accurately mapped and the plants were scored for variation at isozyme and RFLP loci. Structure was analysed by repeated subdivision of the population to find the average size of a randomly mating group. Estimates of F-ST between randomly mating units were then made, and gave patterns consistent with the structure of the population being determined largely by founder effects. The implications of these results for the monitoring of transgene spread in wild sea beet populations are discussed.

Tissue biomarker development in a multicentre trial context: a feasibility study on the PETACC3 stage II and III colon cancer adjuvant treatment trial.

PURPOSE: We evaluated the feasibility of biomarker development in the context of multicenter clinical trials. EXPERIMENTAL DESIGN: Formalin-fixed, paraffin-embedded (FFPE) tissue samples were collected from a prospective adjuvant colon cancer trial (PETACC3). DNA was isolated from tumor as well as normal tissue and used for analysis of microsatellite instability, KRAS and BRAF genotyping, UGT1A1 genotyping, and loss of heterozygosity of 18 q loci. Immunohistochemistry was used to test expression of TERT, SMAD4, p53, and TYMS. Messenger RNA was retrieved and tested for use in expression profiling experiments. RESULTS: Of the 3,278 patients entered in the study, FFPE blocks were obtained from 1,564 patients coming from 368 different centers in 31 countries. In over 95% of the samples, genomic DNA tests yielded a reliable result. Of the immmunohistochemical tests, p53 and SMAD4 staining did best with reliable results in over 85% of the cases. TERT was the most problematic test with 46% of failures, mostly due to insufficient tissue processing quality. Good quality mRNA was obtained, usable in expression profiling experiments. CONCLUSIONS: Prospective clinical trials can be used as framework for biomarker development using routinely processed FFPE tissues. Our results support the notion that as a rule, translational studies based on FFPE should be included in prospective clinical trials.

ZNRD1 (zinc ribbon domain-containing 1) is a host cellular factor that influences HIV-1 replication and disease progression.

BACKGROUND: Human immunodeficiency virus (HIV) takes advantage of multiple host proteins to support its own replication. The gene ZNRD1 (zinc ribbon domain-containing 1) has been identified as encoding a potential host factor that influenced disease progression in HIV-positive individuals in a genomewide association study and also significantly affected HIV replication in a large-scale in vitro short interfering RNA (siRNA) screen. Genes and polymorphisms identified by large-scale analysis need to be followed up by means of functional assays and resequencing efforts to more precisely map causal genes. METHODS: Genotyping and ZNRD1 gene resequencing for 208 HIV-positive subjects (119 who experienced long-term nonprogression [LTNP] and 89 who experienced normal disease progression) was done by either TaqMan genotyping assays or direct sequencing. Genetic association analysis was performed with the SNPassoc package and Haploview software. siRNA and short hairpin RNA (shRNA) specifically targeting ZNRD1 were used to transiently or stably down-regulate ZNRD1 expression in both lymphoid and nonlymphoid cells. Cells were infected with X4 and R5 HIV strains, and efficiency of infection was assessed by reporter gene assay or p24 assay. RESULTS: Genetic association analysis found a strong statistically significant correlation with the LTNP phenotype (single-nucleotide polymorphism rs1048412; [Formula: see text]), independently of HLA-A10 influence. siRNA-based functional analysis showed that ZNRD1 down-regulation by siRNA or shRNA impaired HIV-1 replication at the transcription level in both lymphoid and nonlymphoid cells. CONCLUSION: Genetic association analysis unequivocally identified ZNRD1 as an independent marker of LTNP to AIDS. Moreover, in vitro experiments pointed to viral transcription as the inhibited step. Thus, our data strongly suggest that ZNRD1 is a host cellular factor that influences HIV-1 replication and disease progression in HIV-positive individuals.

Genomewide association study of atazanavir pharmacokinetics and hyperbilirubinemia in AIDS Clinical Trials Group protocol A5202.

BACKGROUND: Atazanavir-associated hyperbilirubinemia can cause premature discontinuation of atazanavir and avoidance of its initial prescription. We used genomewide genotyping and clinical data to characterize determinants of atazanavir pharmacokinetics and hyperbilirubinemia in AIDS Clinical Trials Group protocol A5202. METHODS: Plasma atazanavir pharmacokinetics and indirect bilirubin concentrations were characterized in HIV-1-infected patients randomized to atazanavir/ritonavir-containing regimens. A subset had genomewide genotype data available. RESULTS: Genomewide assay data were available from 542 participants, of whom 475 also had data on estimated atazanavir clearance and relevant covariates available. Peak bilirubin concentration and relevant covariates were available for 443 participants. By multivariate analysis, higher peak on-treatment bilirubin levels were found to be associated with the UGT1A1 rs887829 T allele (P=6.4×10), higher baseline hemoglobin levels (P=4.9×10), higher baseline bilirubin levels (P=6.7×10), and slower plasma atazanavir clearance (P=8.6×10). For peak bilirubin levels greater than 3.0 mg/dl, the positive predictive value of a baseline bilirubin level of 0.5 mg/dl or higher with hemoglobin concentrations of 14 g/dl or higher was 0.51, which increased to 0.85 with rs887829 TT homozygosity. For peak bilirubin levels of 3.0 mg/dl or lower, the positive predictive value of a baseline bilirubin level less than 0.5 mg/dl with a hemoglobin concentration less than 14 g/dl was 0.91, which increased to 0.96 with rs887829 CC homozygosity. No polymorphism predicted atazanavir pharmacokinetics at genomewide significance. CONCLUSION: Atazanavir-associated hyperbilirubinemia is best predicted by considering UGT1A1 genotype, baseline bilirubin level, and baseline hemoglobin level in combination. Use of ritonavir as a pharmacokinetic enhancer may have abrogated genetic associations with atazanavir pharmacokinetics.

Polymorphisms in toll-like receptor 4 and toll-like receptor 9 influence viral load in a seroincident cohort of HIV-1-infected individuals.

OBJECTIVES: Toll-like receptors (TLRs) are innate immune sensors that are integral to resisting chronic and opportunistic infections. Mounting evidence implicates TLR polymorphisms in susceptibilities to various infectious diseases, including HIV-1. We investigated the impact of TLR single nucleotide polymorphisms (SNPs) on clinical outcome in a seroincident cohort of HIV-1-infected volunteers. DESIGN: We analyzed TLR SNPs in 201 antiretroviral treatment-naive HIV-1-infected volunteers from a longitudinal seroincident cohort with regular follow-up intervals (median follow-up 4.2 years, interquartile range 4.4). Participants were stratified into two groups according to either disease progression, defined as peripheral blood CD4(+) T-cell decline over time, or peak and setpoint viral load. METHODS: Haplotype tagging SNPs from TLR2, TLR3, TLR4, and TLR9 were detected by mass array genotyping, and CD4(+) T-cell counts and viral load measurements were determined prior to antiretroviral therapy initiation. The association of TLR haplotypes with viral load and rapid progression was assessed by multivariate regression models using age and sex as covariates. RESULTS: Two TLR4 SNPs in strong linkage disequilibrium [1063 A/G (D299G) and 1363 C/T (T399I)] were more frequent among individuals with high peak viral load compared with low/moderate peak viral load (odds ratio 6.65, 95% confidence interval 2.19-20.46, P < 0.001; adjusted P = 0.002 for 1063 A/G). In addition, a TLR9 SNP previously associated with slow progression was found less frequently among individuals with high viral setpoint compared with low/moderate setpoint (odds ratio 0.29, 95% confidence interval 0.13-0.65, P = 0.003, adjusted P = 0.04). CONCLUSION: This study suggests a potentially new role for TLR4 polymorphisms in HIV-1 peak viral load and confirms a role for TLR9 polymorphisms in disease progression.

Female sterility associated with increased clonal propagation suggests a unique combination of androdioecy and asexual reproduction in populations of Cardamine amara (Brassicaceae).

BACKGROUND AND AIMS: The coexistence of hermaphrodites and female-sterile individuals, or androdioecy, has been documented in only a handful of plants and animals. This study reports its existence in the plant species Cardamine amara (Brassicaceae), in which female-sterile individuals have shorter pistils than seed-producing hermaphrodites. METHODS: Morphological analysis, in situ manual pollination, microsatellite genotyping and differential gene expression analysis using Arabidopsis microarrays were used to delimit variation between female-sterile individuals and hermaphrodites. KEY RESULTS: Female sterility in C. amara appears to be caused by disrupted ovule development. It was associated with a 2.4- to 2.9-fold increase in clonal propagation. This made the pollen number of female-sterile genets more than double that of hermaphrodite genets, which fulfils a condition of co-existence predicted by simple androdioecy theories. When female-sterile individuals were observed in wild androdioecious populations, their ramet frequencies ranged from 5 to 54 %; however, their genet frequencies ranged from 11 to 29 %, which is consistent with the theoretically predicted upper limit of 50 %. CONCLUSIONS: The results suggest that a combination of sexual reproduction and increased asexual proliferation by female-sterile individuals probably explains the invasion and maintenance of female sterility in otherwise hermaphroditic populations. To our knowledge, this is the first report of the coexistence of female sterility and hermaphrodites in the Brassicaceae.

Genome-wide re-sequencing of multidrug-resistant Mycobacterium leprae Airaku-3.

Genotyping and molecular characterization of drug resistance mechanisms in Mycobacterium leprae enables disease transmission and drug resistance trends to be monitored. In the present study, we performed genome-wide analysis of Airaku-3, a multidrug-resistant strain with an unknown mechanism of resistance to rifampicin. We identified 12 unique non-synonymous single-nucleotide polymorphisms (SNPs) including two in the transporter-encoding ctpC and ctpI genes. In addition, two SNPs were found that improve the resolution of SNP-based genotyping, particularly for Venezuelan and South East Asian strains of M. leprae.

Genetic heterogeneity of the Coppock-like cataract: a mutation in CRYBB2 on chromosome 22q11.2.

PURPOSE: To identify the genetic defect for the Coppock-like cataract (CCL) affecting a Swiss family, which defect was unlinked to the chromosome 2q33-35 CCL locus. METHODS: A large family was characterized for linkage analysis by slit lamp examination or by the review of drawings made before cataract extraction. The affection status was attributed before genotyping, and the genotyping was masked to the affection status. Two-point and multipoint linkage analyses were performed using the MLINK and the LINKMAP components of the LINKAGE program package (ver. 5.1), respectively. Mutational analysis of candidate genes was performed by a combination of direct cycle sequencing and an amplification refractory mutation system assay. RESULTS: Ten individuals were affected with the CCL phenotype. The disease was autosomal dominant and appeared to be fully penetrant. A new CCL locus was identified on chromosome 22q11.2 within a 11.67-cM interval (maximum lod score [Zmax] = 4.14; theta = 0). Mutational analysis of the CRYBB2 candidate gene identified a disease-causing mutation in exon 6. This sequence change was identical with that previously described to be associated with the cerulean cataract, a clinically distinct entity. CONCLUSIONS: The CCL phenotype is genetically heterogeneous with a second gene on chromosome 22q11.2, CRYBB2. The CCL and the cerulean cataract are two distinct clinical entities associated with the same genetic defect. This work provides evidence for a modifier factor that influences cataract formation and that remains to be identified.

Estimating the net contribution of interleukin-28B variation to spontaneous hepatitis C virus clearance.

The identification of associations between interleukin-28B (IL-28B) variants and the spontaneous clearance of hepatitis C virus (HCV) raises the issues of causality and the net contribution of host genetics to the trait. To estimate more precisely the net effect of IL-28B genetic variation on HCV clearance, we optimized genotyping and compared the host contributions in multiple- and single-source cohorts to control for viral and demographic effects. The analysis included individuals with chronic or spontaneously cleared HCV infections from a multiple-source cohort (n = 389) and a single-source cohort (n = 71). We performed detailed genotyping in the coding region of IL-28B and searched for copy number variations to identify the genetic variant or haplotype carrying the strongest association with viral clearance. This analysis was used to compare the effects of IL-28B variation in the two cohorts. Haplotypes characterized by carriage of the major alleles at IL-28B single-nucleotide polymorphisms (SNPs) were highly overrepresented in individuals with spontaneous clearance versus those with chronic HCV infections (66.1% versus 38.6%, P = 6 × 10(-9) ). The odds ratios for clearance were 2.1 [95% confidence interval (CI) = 1.6-3.0] and 3.9 (95% CI = 1.5-10.2) in the multiple- and single-source cohorts, respectively. Protective haplotypes were in perfect linkage (r(2) = 1.0) with a nonsynonymous coding variant (rs8103142). Copy number variants were not detected. We identified IL-28B haplotypes highly predictive of spontaneous HCV clearance. The high linkage disequilibrium between IL-28B SNPs indicates that association studies need to be complemented by functional experiments to identify single causal variants. The point estimate for the genetic effect was higher in the single-source cohort, which was used to effectively control for viral diversity, sex, and coinfections and, therefore, offered a precise estimate of the net host genetic contribution.

Pigs Transgenic For Human Dominant Mutations Of The GUCY2D Gene

Purpose: Studies on large animal models are an important step to test new therapeutical strategies before human application. Considering the importance of cone function for human vision and the paucity of large animal models for cone dystrophies having an enriched cone region, we propose to develop a pig model for cone degeneration. With a lentiviral-directed transgenesis, we obtained pigs transgenic for a cone-dominant mutant gene described in a human cone dystrophy.Methods: Lentiviral vectors encoding the human double mutant GUCY2DE837D/R838S cDNA under the control of a region of the pig arrestin-3 promoter (Arr3) was produced and used for lentiviral-derived transgenesis in pigs. PCR-genotyping and southern blotting determined the genotype of pigs born after injection of the vector at the zygote stage. Retina function analysis was performed by ERG and behavioral tests at 11, 24 and 54 weeks of age. OCT and histological analyses were performed to describe the retina morphology.Results: The ratio of transgenic pigs born after lentiviral-directed transgenesis was close to 50%. Transgenic pigs with 3 to 5 transgene copies per cell clearly present a reduced photopic response from 3 months of age on. Except for one pig, which has 6 integrated transgene copies, no dramatic decrease in general mobility was observed even at 6 months of age. OCT examinations reveal no major changes in the ONL structure of the 6-months old pigs. The retina morphology was well conserved in the 2 pigs sacrificed (3 and 6 months old) except a noticeable displacement of some cone nuclei in the outer segment layer.Conclusions: Lentiviral-directed transgenesis is a rapid and straightforward method to engineer transgenic pigs. Some Arr3-GUCY2DE837D/R838S pigs show signs of retinal dysfunction but further work is needed to describe the progression of the disease in this model.

The vitamin D receptor gene bAt (CCA) haplotype impairs the response to pegylated-interferon/ribavirin-based therapy in chronic hepatitis C patients.

BACKGROUND: Chronic hepatitis C infection is a major cause of end-stage liver disease. Therapy outcome is influenced by 25-OH vitamin D deficiency. To further address this observation, our study investigates the impact of the vitamin D receptor (NR1I1) haplotype and combined effects of plasma vitamin D levels in a well-described cohort of hepatitis C patients. METHODS: A total of 155 chronic hepatitis C patients were recruited from the Swiss Hepatitis C Cohort Study for NR1I1 genotyping and plasma 25-OH vitamin D level measurement. NR1I1 genotype data and combined effects of plasma 25-OH vitamin D level were analysed regarding therapy response (sustained virological response). RESULTS: A strong association was observed between therapy non-response and the NR1I1 CCA (bAt) haplotype consisting of rs1544410 (BsmI) C, rs7975232 (ApaI) C and rs731236 (TaqI) A alleles. Of the HCV patients carrying the CCA haplotype, 50.3% were non-responders (odds ratio [OR] 1.69, 95% CI 1.07, 2.67; P=0.028). A similar association was observed for the combinational CCCCAA genotype (OR 2.94, 95% CI 1.36, 6.37; P=0.007). The combinational CCCCAA genotype was confirmed as an independent risk factor for non-response in multivariate analysis (OR 2.50, 95% CI 1.07, 5.87; P=0.034). Analysing combined effects, a significant impact of low 25-OH vitamin D levels on sustained virological response were only seen in patients with the unfavourable NR1I1 CCA (bAt) haplotype (OR for non-SVR 3.55; 95% CI 1.005, 12.57; P=0.049). CONCLUSIONS: NR1I1 vitamin D receptor polymorphisms influence response to pegylated-interferon/ribavirin-based therapy in chronic hepatitis C and exert an additive genetic predisposition to previously described low 25-OH vitamin D serum levels.

Genome-wide association study of virologic response with efavirenz-containing or abacavir-containing regimens in AIDS clinical trials group protocols.

BACKGROUND: Efavirenz and abacavir are components of recommended first-line regimens for HIV-1 infection. We used genome-wide genotyping and clinical data to explore genetic associations with virologic failure among patients randomized to efavirenz-containing or abacavir-containing regimens in AIDS Clinical Trials Group (ACTG) protocols. PARTICIPANTS AND METHODS: Virologic response and genome-wide genotype data were available from treatment-naive patients randomized to efavirenz-containing (n=1596) or abacavir-containing (n=786) regimens in ACTG protocols 384, A5142, A5095, and A5202. RESULTS: Meta-analysis of association results across race/ethnic groups showed no genome-wide significant associations (P<5×10) with virologic response for either efavirenz or abacavir. Our sample size provided 80% power to detect a genotype relative risk of 1.8 for efavirenz and 2.4 for abacavir. Analyses focused on CYP2B genotypes that define the lowest plasma efavirenz exposure stratum did not show associations nor did analysis limited to gene sets predicted to be relevant to efavirenz and abacavir disposition. CONCLUSION: No single polymorphism is associated strongly with virologic failure with efavirenz-containing or abacavir-containing regimens. Analyses to better consider context, and that minimize confounding by nongenetic factors, may show associations not apparent here.

Restriction site-associated DNA sequencing, genotyping error estimation and de novo assembly optimization for population genetic inference.

Restriction site-associated DNA sequencing (RADseq) provides researchers with the ability to record genetic polymorphism across thousands of loci for nonmodel organisms, potentially revolutionizing the field of molecular ecology. However, as with other genotyping methods, RADseq is prone to a number of sources of error that may have consequential effects for population genetic inferences, and these have received only limited attention in terms of the estimation and reporting of genotyping error rates. Here we use individual sample replicates, under the expectation of identical genotypes, to quantify genotyping error in the absence of a reference genome. We then use sample replicates to (i) optimize de novo assembly parameters within the program Stacks, by minimizing error and maximizing the retrieval of informative loci; and (ii) quantify error rates for loci, alleles and single-nucleotide polymorphisms. As an empirical example, we use a double-digest RAD data set of a nonmodel plant species, Berberis alpina, collected from high-altitude mountains in Mexico.

Restriction site-associated DNA sequencing, genotyping error estimation and de novo assembly optimization for population genetic inference.

Restriction site-associated DNA sequencing (RADseq) provides researchers with the ability to record genetic polymorphism across thousands of loci for nonmodel organisms, potentially revolutionizing the field of molecular ecology. However, as with other genotyping methods, RADseq is prone to a number of sources of error that may have consequential effects for population genetic inferences, and these have received only limited attention in terms of the estimation and reporting of genotyping error rates. Here we use individual sample replicates, under the expectation of identical genotypes, to quantify genotyping error in the absence of a reference genome. We then use sample replicates to (i) optimize de novo assembly parameters within the program Stacks, by minimizing error and maximizing the retrieval of informative loci; and (ii) quantify error rates for loci, alleles and single-nucleotide polymorphisms. As an empirical example, we use a double-digest RAD data set of a nonmodel plant species, Berberis alpina, collected from high-altitude mountains in Mexico.