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Chronic hepatitis C virus ( HCV) infection is characterized by high interindividual variability in response to pegylated interferon and ribavirin. A genetic polymorphism on chromosome 19 (rs12979860) upstream of interferon-λ3 ( IFNλ3) is associated with a twofold change in sustained virologic response rate after 48 weeks of treatment with pegylated interferon/ribavirin in HCV genotype 1 ( GT1) treatment-naïve patients. We conducted epigenetic analysis on the IFNλ3 promoter to investigate whether DNA methylation is associated with response to HCV therapy. DNA samples from HCV GT1-infected subjects receiving an interferon-free paritaprevir-containing combination regimen (N=540) and from HCV-uninfected, healthy controls (N=124) were analysed for IFNλ3 methylation levels. Methylation was strongly associated with rs12979860 allele status whether adjusting for HCV status (r=65.0%, 95% CI: [60.2%, 69.5%]), or not (r=64.4%), both with P<2.2×10−16. In HCV GT1-infected subjects, C/C genotypes had significantly lower methylation levels relative to C/T or T/T genotypes ( P<1×10−14), with each T allele resulting in a nine-unit increase in mean methylation level. Methylation levels did not correlate with response in subjects treated for 12 or 24 weeks. However, non-C/C subjects with higher methylation levels were more likely to relapse when treatment duration was 8 weeks. This analysis suggests that methylation status of the IFNλ3 promoter region may be a useful parameter that identifies patients more likely to relapse following HCV therapy; however, continuing therapy for a sufficient duration can overcome this difference. These findings may provide mechanistic insight into the role of IFNλ3 genetic variants in HCV treatment response. [ABSTRACT FROM AUTHOR]

The complete nucleotide sequence of the transferred region (T-DNA) of an octopine tumor inducing (Ti) plasmid from Agrobacterium tumefaciens (pTi15955) has been determined. A total of 24 595 nucleotides extending approximately 900 bases to either side of the outermost, T-DNA boundaries was sequenced. Computer analysis of the sequenced portion of the Ti plasmid revealed that recognition sites for 72 restriction endonucleases are present in the DNA sequence at least once; no site for EcoK exists in this DNA sequence. Two imperfect 24 base repeats border the T-DNA sequence; the left starts at position 909 and the right ends at position 23 782, giving the T-DNA region a total length, of 22 874 nucleotides. Another two similar 24 base repeats lie within T-DNA and divide it, into three distinct domains: T-left (TL-DNA) 13 175 bp of apparently eukaryotic origin; T-center (TC-DNA) 1816 bp of prokaryotic origin; and T-right (TR-DNA) 7 883 bp of eukaryotic origin. The T-DNA contains nine reported transcripts, however, 26 open reading frames longer than 300 bases that start with an ATG initiation codon were found. Fourteen open reading frames are bounded by putative eukaryotic promoters, ribosome binding sites, and poly(A) addition sites and occur only in TL-and TR-DNAs. No open reading frames showing eukaryotic promoter sequences are located within the TC-DNA. [ABSTRACT FROM AUTHOR]

Background: Polymorphisms within cytochrome P450 2D6 (CYP2D6) result in different metabolizer phenotypes, but some discordance suggests the possibility of additional unknown alleles or factors contributing to metabolism. A recent study showed a high frequency frameshift-causing deletion, CYP2D7 138delT, which converted the CYP2D7 pseudogene to a functional gene within the brain.1 The high frequency of the deletion and the resulting CYP2D7 expression could have important implications for brain-specific metabolism of psychoactive substances. Our goal was to determine the frequency of this deletion in a larger ethnically diverse population.Methods: The CYP2D7 138delT genotypes for 163 Caucasians, 95 East Asians, 50 South Asians, 68 Hispanic Latinos, and 68 African Americans were determined by Pyrosequencing.Results: The 138delT allele was observed at a frequency of 1.0% in East Asians and 0.74% in Hispanic Latinos. The deletion was not observed in the other ethnic populations.Conclusions: The very low frequency of the CYP2D7 138delT polymorphism in our panel is in contrast to the high frequency (50%) reported in the Indian population.1 Our results suggest that CYP2D7 138delT is unlikely to be highly relevant for population variation of pharmacokinetics or drug response.Clinical Pharmacology & Therapeutics (2005) 79, P42–P42; doi: 10.1016/j.clpt.2005.12.149 [ABSTRACT FROM AUTHOR]

Introduction: The acceleration of biological aging is a risk factor for Alzheimer's disease (AD). Here, we performed weighted gene co-expression network analysis (WGCNA) to identify modules and dysregulated genesinvolved in biological aging in AD., Methods: We performed WGCNA to identify modules associated with biological clocks and hub genes of the module with the highest module significance. In addition, we performed differential expression analysis and association analysis with AD biomarkers., Results: WGCNA identified five modules associated with biological clocks, with the module designated as "purple" showing the strongest association. Functional enrichment analysis revealed that the purple module was related to cell migration and death. Ten genes were identified as hub genes in purple modules, of which CX3CR1 was downregulated in AD and low levels of CX3CR1 expression were associated with AD biomarkers., Conclusion: Network analysis identified genes associated with biological clocks, which suggests the genetic architecture underlying biological aging in AD., Highlights: Examine links between Alzheimer's disease (AD) peripheral transcriptome and biological aging changes.Weighted gene co-expression network analysis (WGCNA) found five modules related to biological aging.Among the hub genes of the module, CX3CR1 was downregulated in AD.The CX3CR1 expression level was associated with cognitive performance and brain atrophy., Competing Interests: Qingqin S. Li is an employee of Janssen Research & Development, LLC, an operating company of Johnson & Johnson, and holds company equity. The authors declare that they have no non‐financial competing interests. Author disclosures are available in the supporting information., (© 2022 The Authors. Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring published by Wiley Periodicals, LLC on behalf of Alzheimer's Association.)

With the rapid advancement of sequencing technologies, next generation sequencing (NGS) analysis has been widely applied in cancer genomics research. More recently, NGS has been adopted in clinical oncology to advance personalized medicine. Clinical applications of precision oncology require accurate tests that can distinguish tumor-specific mutations from artifacts introduced during NGS processes or data analysis. Therefore, there is an urgent need to develop best practices in cancer mutation detection using NGS and the need for standard reference data sets for systematically measuring accuracy and reproducibility across platforms and methods. Within the SEQC2 consortium context, we established paired tumor-normal reference samples and generated whole-genome (WGS) and whole-exome sequencing (WES) data using sixteen library protocols, seven sequencing platforms at six different centers. We systematically interrogated somatic mutations in the reference samples to identify factors affecting detection reproducibility and accuracy in cancer genomes. These large cross-platform/site WGS and WES datasets using well-characterized reference samples will represent a powerful resource for benchmarking NGS technologies, bioinformatics pipelines, and for the cancer genomics studies., (© 2021. The Author(s).)

The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Background: Alzheimer's disease (AD) is a chronic progressive neurodegenerative disease impacting an estimated 44 million adults worldwide. The causal pathology of AD (accumulation of amyloid-beta and tau), precedes hallmark symptoms of dementia by more than a decade, necessitating development of early diagnostic markers of disease onset, particularly for new drugs that aim to modify disease processes. To evaluate differentially methylated positions (DMPs) as novel blood-based biomarkers of AD, we used a subset of 653 individuals with peripheral blood (PB) samples in the Alzheimer's disease Neuroimaging Initiative (ADNI) consortium. The selected cohort of AD, mild cognitive impairment (MCI), and age-matched healthy controls (CN) all had imaging, genetics, transcriptomics, cerebrospinal protein markers, and comprehensive clinical records, providing a rich resource of concurrent multi-omics and phenotypic information on a well-phenotyped subset of ADNI participants., Results: In this manuscript, we report cross-diagnosis differential peripheral DNA methylation in a cohort of AD, MCI, and age-matched CN individuals with longitudinal DNA methylation measurements. Epigenome-wide association studies (EWAS) were performed using a mixed model with repeated measures over time with a P value cutoff of 1 × 10 -5 to test contrasts of pairwise differential peripheral methylation in AD vs CN, AD vs MCI, and MCI vs CN. The most highly significant differentially methylated loci also tracked with Mini Mental State Examination (MMSE) scores. Differentially methylated loci were enriched near brain and neurodegeneration-related genes (e.g., BDNF, BIN1, APOC1) validated using the genotype tissue expression project portal (GTex)., Conclusions: Our work shows that peripheral differential methylation between age-matched subjects with AD relative to healthy controls will provide opportunities to further investigate and validate differential methylation as a surrogate of disease. Given the inaccessibility of brain tissue, the PB-associated methylation marks may help identify the stage of disease and progression phenotype, information that would be central to bringing forward successful drugs for AD.

Aims: The selective endothelin (ET) A receptor antagonist atrasentan has been shown to lower albuminuria in North American and Asian patients with type 2 diabetes and nephropathy. As drug responses to many drugs may differ between North American and Asian populations, we assessed the influence of geographical region on the albuminuria and fluid retention response to atrasentan., Materials and Methods: Two 12-week double-blind randomised controlled trials were performed with atrasentan 0.75 or 1.25 mg/d vs placebo in patients with type 2 diabetes and nephropathy. The efficacy endpoint was the percentage change in albuminuria. Bodyweight change, a proxy of fluid retention, was used as a safety endpoint. Pharmacodynamics were determined in Asians (N = 77) and North Americans (N = 134). Atrasentan plasma concentration was measured in 161 atrasentan-treated patients., Results: Mean albuminuria reduction in Asian, compared to North American, patients was, respectively, -34.4% vs -26.3% for 0.75 mg/d ( P  = .44) and -48.0% vs -28.9% for 1.25 mg/d ( P  = .035). Bodyweight gain did not differ between North American and Asian populations. Atrasentan plasma concentrations were higher in Asians compared to North Americans and correlated with albuminuria response (7.2% albuminuria reduction per doubling atrasentan concentration; P  = .024). Body surface area (β = -1.09 per m 2 ; P  < .001) and bilirubin, as a marker of hepatic organic anion transporter activity, (β = 0.69 per mg/dL increment; P  = .010) were independent determinants of atrasentan plasma concentration; correction by body surface area and bilirubin left no significant difference in plasma concentration between Asian and North American populations., Conclusion: The higher exposure and albuminuria reduction of atrasentan in Asian patients is not associated with more fluid retention, suggesting that Asian patients are less sensitive to atrasentan-induced sodium retention., (© 2016 John Wiley & Sons Ltd.)

Cytochrome P450 2D7 (CYP2D7) has long been considered a pseudogene. A recent report described an indel polymorphism (CYP2D7 138delT) that causes a frameshift generating an open reading frame and functional protein. This polymorphism was observed in 6 of 12 samples from an Indian population. Individuals with the 138delT polymorphism expressed CYP2D7 protein from a brain-specific, alternatively spliced transcript (J Biol Chem 279:27383-27389, 2004). The unexpectedly high frequency of the variant allele and resulting CYP2D7 expression could have important implications for brain-specific metabolism of CYP2D substrates including many psychoactive drugs. However, the 138delT variant has not been detected in other studies (Pharmacogenetics 11:45-55, 2001; Biochem Biophys Res Commun 336:1241-1250, 2005). Our goal was to determine the frequency of this variant in a larger, ethnically diverse population. CYP2D7 138delT genotypes for 163 Caucasians, 95 East Asians, 50 Indians, 68 Hispanic Latinos, and 68 African Americans were determined by Pyrosequencing. The 138delT allele was observed at a frequency of 1.0% in East Asians and 0.74% in Hispanic Latinos. The deletion was not observed in Indians or the other ethnic populations. In addition, in each of the three samples with 138delT, the putative brain-specific transcript contains a premature stop codon that would preclude protein expression. The low frequency of the CYP2D7 138delT polymorphism in our ethnically diverse sample, and particularly the absence from 50 Indian samples, is in contrast to the high frequency previously reported. Our results suggest that CYP2D7 138delT is unlikely to be highly relevant for population variation of pharmacokinetics or drug response.

The nucleotide sequence of 42 775 bp of the vir-region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955 is reported here. Although the nucleotide sequences of several parts of this region from this or closely related plasmids have been published previously, the present work establishes for the first time the complete arrangement of all the essential virulence genes and their intergenic regions of an octopine Ti plasmid. The disruption of some of the intergenic areas by insertion (IS) elements is typical for the octopine Ti plasmids. Several new ORFs were identified, including ORFs immediately downstream of virD4 and virE2, which probably represent new genes involved in virulence.

Adenosine kinase is ubiquitous in eukaryotes and is a key enzyme in the regulation of the intracellular levels of adenosine, an important physiological effector of many cells and tissues. In this paper we report the cloning of cDNAs encoding adenosine kinase from both rat and human tissues. Two distinct forms of adenosine kinase mRNA were identified in human tissues. Sequence variation between the two forms is restricted to the extreme 5'-end of the adenosine kinase mRNA, including a portion of the coding region, and is consistent with differential splicing of a single transcriptional product. We have expressed both forms in E. coli and produced soluble active enzyme which catalyzes the phosphorylation of adenosine with high specific activity in vitro and is susceptible to known adenosine kinase inhibitors.

Background: Transition mutations (A-G) at residue 2143, cognate to position 2058 in the Escherichia coli 23S rRNA gene, have been shown to confer resistance to macrolides in Helicobacter pylori. This study reports the finding that transversion mutations (A-C) can occur at 2143 as well., Materials and Methods: Three clarithromycin-resistant H. pylori isolated from three different patients after treatment with clarithromycin were analyzed for point mutations by cycle sequencing of a 163-bp amplified region surrounding residue 2143 within the conserved loop of the 23S rRNA gene., Results: Nucleotide sequence comparisons of a 163-bp amplified product revealed that A-C transversion mutations occurred at position 2143. H. pylori isolated from the patients prior to treatment were susceptible to clarithromycin and displayed no polymorphism at 2143., Conclusion: This is the first report to show that A-C transversion mutations at position 2143 can confer resistance to clarithromycin in H. pylori and further support the role that mutations at position 2143 play in conferring macrolide resistance in H. pylori.

Partial cDNA clones generated by RT-PCR were used as probes to clone the cDNAs encoding the human alpha 4 and beta 2 neuronal nicotinic acetylcholine receptor (nAChR) subunits. The 2.1-kb alpha 4 cDNA shows 84 and 76% identity to the rat and chicken cDNA sequences, respectively. The deduced amino-acid sequence shares 89 and 84% similarity, respectively, with the corresponding rat and chicken proteins, with most of the divergence occurring in the cytoplasmic domain. The 1721-nucleotide beta 2 sequence was identical to the human beta 2 sequence previously reported. Transfection of the alpha 4 and beta 2 clones into HEK293 cells resulted in the formation of binding sites that display high affinity towards [3H] cytisine, a characteristic of the alpha 4 beta 2 subtype produced in vivo.

The 11.3 kb plasmid pSE101 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site and into the chromosome of Streptomyces lividans at many sites. Multisite integration in S. lividans was also observed when a 1.9 kb segment of pSE101 containing attP and adjacent plasmid sequence was used to transform a pSE101- S. lividans host. Nucleotide sequencing of this segment revealed the presence of a complete open reading frame (ORF) designated int, encoding a putative polypeptide of 448 amino acids that shows similarities to site-specific recombinases of the integrase family. Sequencing of the 1.3 kb segment upstream of int revealed the presence of three additional ORFs: the one most distal to int encodes a putative 76 amino acid basic polypeptide analogous to the Xis proteins of a number of bacteriophages. Nucleotide sequencing of attP, and the attB, attL and attR sites from Sac. erythraea revealed a 46 bp sequence common to all sites with no duplications of chromosomal sequences in the integrated state. A putative structural gene for a tRNA(Thr) was found to overlap the 46 bp common sequence at attB. Sequencing of four pSE101 integration sites (attB') and corresponding attL' and attR' sites in S. lividans showed that the 46 bp sequence was present at each attR' site, whereas only the first three bases, CTT, were retained at each attL' and attB' site. A feature common to the four attB' sites and to attB is a highly conserved 21 bp segment with inverted repeats flanking the CTT sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

The entire nucleotide sequence of human T-cell lymphotropic virus type II (HTLV-II) from a previously described isolate of patient NRA (HTLV-IINRA) was determined. Clones encoding the 5' LTR and gag, pol, env and tax/rex open reading frames were subcloned and sequenced on both strands. The provirus consisted of 8957 nucleotides and showed 95.2% homology with the HTLV-IIMo prototype at the nucleotide level. Less than 5% amino acid variation between HTLV-IINRA and HTLV-IIMo was observed for coding regions. Although isolate HTLV-IINRA had an additional 25 amino acids at the 3' end of tax/rex, this region was 96% homologous with the 5' end of HTLV-IIMo 3' LTR. To further investigate HTLV-II variability, a portion of the env gp46 gene derived from 9 HTLV-II infected persons was amplified by polymerase chain reaction and sequenced. Sequence was obtained for 320 nucleotides corresponding to HTLV-IIMo positions 5291 to 5610. Isolates similar to the HTLV-IIMo and HTLV-IINRA prototypes were identified, and sequences were highly conserved.

Cells of the mononuclear phagocyte system are the predominant cell producing HIV-1 in most tissues including the central nervous system (CNS), spinal cord, lung and skin; infection is associated with dementia, neuropathy, pneumonitis, and dermatitis respectively. Different HIV-1 isolates vary markedly in their ability to infect mononuclear phagocytes productively. Here we describe molecular clones of a CNS-derived isolate, HIV-1(JR-FL), which can replicate efficiently in mononuclear phagocytes. Analysis by polymerase chain reaction of early events after infection indicates that the early phase of viral replication before reverse transcription determines tropism. Genetic mapping of the macrophage-tropic phenotype by construction of recombinant viruses indicates that mononuclear phagocyte infectivity can be determined by a 157-amino-acid region of the gp 120 glycoprotein of HIV-1(JR-FL). Significantly, this region is upstream from the previously defined CD4-binding domain. We propose that at least one determinant for mononuclear phagocyte tropism involves target cell interactions with regions of gp120 distinct from the CD4-binding domain.

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. Induction of vir gene expression by plant signal molecules is solely dependent on the virulence loci virA and virG. This study focused on the virA locus of the octopine type Ti plasmid pTi15955. The nucleic acid sequence of a 5.7-kilobase fragment encompassing virA was determined. Genetic analysis of this region revealed that virA contains one open reading frame coding for a protein of 91 639 daltons. Immunodetection with antibodies raised against a 35-kDa VirA fusion protein produced in E. coli identified by the VirA product in wild-type Agrobacterium cells. Moreover, it is shown that the VirA protein is located in the cytoplasmic membrane fraction of Agrobacterium. These data confirm the proposed regulatory function of VirA whereby VirA acts as a membrane sensor protein to identify plant signal molecules in the environment. The proposed sensory function of VirA strikingly resembles the function of the chemotaxis receptor proteins of E. coli.

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. Induction of vir gene expression by plant signal molecules is solely dependent on the virulence loci virA and virG. This study focused on the virA locus of the octopine type Ti plasmid pTi15955. The nucleic acid sequence of a 5.7-kilobase fragment encompassing virA was determined. Genetic analysis of this region revealed that virA contains one open reading frame coding for a protein of 91 639 daltons. Immunodetection with antibodies raised against a 35-kDa VirA fusion protein produced in E. coli identified the VirA product in wild-type Agrobacterium cells. Moreover, it is shown that the VirA protein is located in the cytoplasmic membrane fraction of Agrobacterium. These data confirm the proposed regulatory function of VirA whereby VirA acts as a membrane sensor protein to identify plant signal molecules in the environment. The proposed sensory function of VirA strikingly resembles the function of the chemotaxis receptor proteins of E. coli.

A recombinant plasmid (pcPvNGS-01) containing sequences related to glutamine synthetase (GS) has been identified from a cDNA library constructed from poly (A)+ RNA isolated from root nodules of Phaseolus vulgaris L. The identification of this recombinant relied on the observations that: (a) the clone hybridized strongly to purified GS mRNA; (b) in hybrid-select translation experiments, the clone selected mRNA that produced a polypeptide identical in molecular weight to purified GS subunits which was immunoprecipitated with anti-GS-antiserum; and (c) the translated nucleotide sequence of the cloned cDNA was homologous to a partial amino acid sequence of higher plant GS. The cloned cDNA hybridized to poly (A)+ RNA of different mobilities from leaves, roots, and nodules of P. vulgaris. In RNA "dot" blots washed at different stringencies, differences were observed both in the relative amounts of GS mRNA in different tissues and in the strength of their hybridization to the cDNA probe. The cloned probe hybridized to several fragments of restricted P. vulgaris DNA but not to DNA from Rhizobium phaseoli. These results suggest that GS is coded for by a small multigene family showing organ-specific expression.

We have isolated and characterized a gene encoding the phytochrome polypeptide of Avena. Based on nucleotide sequence identity with previously sequenced cDNA clones this gene is designated as type 3 (phy3). The gene is about 5.9 kb long with six exons and five introns, one each of the latter in the 5' and 3'-untranslated regions. The largest exon encodes the entire 74-kDa, chromophore-bearing, N-terminal domain of the photo-receptor postulated to be directly involved in its mechanism of action. The transcription start point, identified by mung-bean nuclease digestion, is located 24 to 35 bp downstream from a tandem TATA box. Sequence elements homologous to a number of motifs implicated as upstream regulatory elements in other genes are present in the 5'-flanking DNA of phy3. Particularly intriguing are three elements at positions -140, -470 and -650. These elements share homology with the 'GT' motif postulated to be a component of the light-regulatory element of genes encoding the small subunit of ribulose bisphosphate carboxylase.

The complete nucleotide sequence of the virB locus, from the octopine Ti plasmid of Agrobacterium tumefaciens strain 15955, has been determined. In the large virB-operon (9600 nucleotides) we have identified eleven open reading frames, designated virB1 to virB11. From DNA sequence analysis it is proposed that nearly all VirB products, i.e. VirB1 to VirB9, are secreted or membrane associated proteins. Interestingly, both a membrane protein (VirB4) and a potential cytoplasmic protein (VirB11) contain the consensus amino acid sequence of ATP-binding proteins. In view of the conjugative T-DNA transfer model, the VirB proteins are suggested to act at the bacterial surface and there play an important role in directing T-DNA transfer to plant cells.