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Noise-induced hearing loss leads to anatomic and physiologic changes in primary auditory cortex (A1) and the adjacent dorsal rostral belt (RB). Since acetylcholine is known to modulate plasticity in other cortical areas, changes in A1 and RB following noise damage may be due to changes in cholinergic receptor expression. We used [ 3 H]scopolamine and [ 18 F]flubatine binding to measure muscarinic acetylcholine receptor (mAChR) and nicotinic acetylcholine receptor (nAChR) expression, respectively, in guinea pig A1 and RB 3 weeks following unilateral, left ear noise exposure, and a temporary threshold shift in hearing. [ 3 H]Scopolamine binding decreased in right A1 and RB (contralateral to noise) compared to sham controls across all cortical layers. [ 18 F]Flubatine binding showed a nonsignificant upward trend in right A1 following noise but only significantly increased in right RB and 2 layers of left RB (ipsilateral to noise). This selective response may ultimately influence cortical plasticity and function. The mechanism(s) by which cholinergic receptors are altered following noise exposure remain unknown. However, these data demonstrate noise exposure may differentially influence mAChRs that typically populate interneurons in A1 and RB more than nAChRs that are traditionally located on thalamocortical projections and provide motivation for cholinergic imaging in clinical patient populations of temporary or permanent hearing loss.

Recombinase polymerase amplification (RPA) is an isothermal amplification assay that has been ubiquitously utilized in the detection of infectious agents. Like any nucleic acid amplification technology, primer-template complementarity is critical to RPA reaction success. Mismatches arising in the primer-template complex are known to impact reaction kinetics, invalidate downstream analysis, such as nucleic acid quantification, and result in false negatives if used in a diagnostic capacity. Although the impact of specific primer-template mismatches has been well characterized for techniques such as PCR, characterization remains limited for RPA. Through our study, we systematically characterize the impact of mismatches on the RPA reaction, when located in the 3'-anchor region of the primer-template complex. Our investigation identified that the nucleotides involved, as well as position of each mismatch, influence the size of the impact, with terminal cytosine-thymine and guanine-adenine mismatches being the most detrimental. The presence of some mismatch combinations, such as a penultimate cytosine-cytosine and a terminal cytosine-adenine mismatch pairing, led to complete RPA reaction inhibition. Through the successful characterization of 315 mismatch combinations, researchers can optimize their RPA assay accordingly and seek to implement RPA technology for rapid, in-field genotyping., (Copyright © 2022 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Actinobacillus pleuropneumoniae (APP) is the causative agent of porcine pleuropneumonia, resulting in high economic impact worldwide. There are currently 19 known serovars of APP, with different ones being predominant in specific geographic regions. Outbreaks of pleuropneumonia, characterized by sudden respiratory difficulties and high mortality, can occur when infected pigs are brought into naïve herds, or by those carrying different serovars. Good biosecurity measures include regular diagnostic testing for surveillance purposes. Current gold standard diagnostic techniques lack sensitivity (bacterial culture), require expensive thermocycling machinery (PCR) and are time consuming (culture and PCR). Here we describe the development of an isothermal point-of-care diagnostic test - utilizing recombinase polymerase amplification (RPA) for the detection of APP, targeting the species-specific apxIVA gene. Our APP-RPA diagnostic test achieved a sensitivity of 10 copies/μL using a strain of APP serovar 8, which is the most prevalent serovar in the UK. Additionally, our APP-RPA assay achieved a clinical sensitivity and specificity of 84.3 and 100%, respectively, across 61 extracted clinical samples obtained from farms located in England and Portugal. Using a small subset ( n = 14) of the lung tissue samples, we achieved a clinical sensitivity and specificity of 76.9 and 100%, respectively) using lung imprints made on FTA cards tested directly in the APP-RPA reaction. Our results demonstrate that our APP-RPA assay enables a suitable rapid and sensitive screening tool for this important veterinary pathogen., Competing Interests: TN, PM, EV, and PP are employed by Ceva Animal Health Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Stringer, Li, Bossé, Forrest, Hernandez-Garcia, Tucker, Nunes, Costa, Mortensen, Velazquez, Penny, Rodriguez-Manzano, Georgiou and Langford.)

Background: Soil-transmitted helminths (STHs) are parasitic nematodes that inhabit the human intestine. They affect more than 1.5 billion people worldwide, causing physical and cognitive impairment in children. The global strategy to control STH infection includes periodic mass drug administration (MDA) based on the results of diagnostic testing among populations at risk, but the current microscopy method for detecting infection has diminished sensitivity as the intensity of infection decreases. Thus, improved diagnostic tools are needed to support decision-making for STH control programs., Methodology: We developed a nucleic acid amplification test based on recombinase polymerase amplification (RPA) technology to detect STH in stool. We designed primers and probes for each of the four STH species, optimized the assay, and then verified its performance using clinical stool samples., Principal Findings: Each RPA assay was as sensitive as a real-time polymerase chain reaction (PCR) assay in detecting copies of cloned target DNA sequences. The RPA assay amplified the target in DNA extracted from human stool samples that were positive for STH based on the Kato-Katz method, with no cross-reactivity of the non-target genomic DNA. When tested with clinical stool samples from patients with infections of light, moderate, and heavy intensity, the RPA assays demonstrated performance comparable to that of real-time PCR, with better results than Kato-Katz. This new rapid, sensitive and field-deployable method for detecting STH infections can help STH control programs achieve their goals., Conclusions: Semi-quantitation of target by RPA assay is possible and is comparable to real-time PCR. With proper instrumentation, RPA assays can provide robust, semi-quantification of STH DNA targets as an alternative field-deployable indicator to counts of helminth eggs for assessing infection intensity., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: MSF and OWS were former employees of TwistDx. Other authors have declared that no competing interests exist.

Detection of animal species in meat product is crucial to prevent adulterated and unnecessary contamination during processing. Gold standard is the real-time PCR assays, which can be conducted at highly equipped laboratories. Toward the development of point-of-need test, two rapid molecular assays based on recombinase polymerase amplification (RPA) for the detection of pork and horse DNA were established. Target genes are the porcine mitochondrial ND2 and equine ATP 6-8 genes. The pork and horse_RPA assays detected 16 and one DNA molecules/µl in eleven to six minutes, respectively. The myoglobin in the meat did not influence the assays performances, while the presence of high background-DNA induced a one log decrease in the sensitivity. Both assays are highly specific and identify down to 0.1% of their target DNA in meat mixtures. Both RPA assays could be used on-site as a rapid and mobile detection system to determine contamination of meat products., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jonas Kissenkötter, Susanne Böhlken-Fascher, Claus-Peter CzernyandAhmed Abd El Waheddeclare no conflict ofinterest.Matthew S. Forrest and Olaf Piepenburg were employees at TwistDx Ltd. RPA technology is subject to background IP protection., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objectives: Rapid and accurate sexually transmitted infection diagnosis can reduce onward transmission and improve treatment efficacy. We evaluated the accuracy of a 15-minute run-time recombinase polymerase amplification-based prototype point-of-care test (TwistDx) for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG)., Methods: Prospective, multicentre study of symptomatic and asymptomatic patients attending three English sexual health clinics. Research samples provided were additional self-collected vulvovaginal swab (SCVS) (female participants) and first-catch urine (FCU) aliquot (female and male participants). Samples were processed blind to the comparator (routine clinic CT/NG nucleic acid amplification test (NAAT)) results. Discrepancies were resolved using Cepheid CT/NG GeneXpert., Results: Both recombinase polymerase amplification and routine clinic NAAT results were available for 392 male and 395 female participants. CT positivity was 8.9% (35/392) (male FCU), 7.3% (29/395) (female FCU) and 7.1% (28/395) (SCVS). Corresponding NG positivity was 3.1% (12/392), 0.8% (3/395) and 0.8% (3/395). Specificity and positive predictive values were 100% for all sample types and both organisms, except male CT FCU (99.7% specificity (95% confidence interval (CI) 98.4-100.0; 356/357), 97.1% positive predictive value (95% CI 84.7-99.9; 33/34)). For CT, sensitivity was ≥94.3% for FCU and SCVS. CT sensitivity for female FCU was higher (100%; 95% CI, 88.1-100; 29/29) than for SCVS (96.4%; 95% CI, 81.7-99.9; 27/28). NG sensitivity and negative predictive values were 100% in FCU (male and female)., Conclusions: This prototype test has excellent performance characteristics, comparable to currently used NAATs, and fulfils several World Health Organization ASSURED criteria. Its rapidity without loss of performance suggests that once further developed and commercialized, this test could positively affect clinical practice and public health., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Summary: Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification method, is enhancing our ability to detect a diverse array of pathogens, thereby assisting the diagnosis of infectious diseases and the detection of microorganisms in food and water. However, new bioinformatics tools are needed to automate and improve the design of the primers and probes sets to be used in RPA, particularly to account for the high genetic diversity of circulating pathogens and cross detection of genetically similar organisms. PrimedRPA is a python-based package that automates the creation and filtering of RPA primers and probe sets. It aligns several sequences to identify conserved targets, and filters regions that cross react with possible background organisms., Availability and Implementation: PrimedRPA was implemented in Python 3 and supported on Linux and MacOS and is freely available from http://pathogenseq.lshtm.ac.uk/PrimedRPA.html., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2018. Published by Oxford University Press.)

Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technology that provides rapid and robust infectious disease pathogen detection, ideal for point-of-care (POC) diagnostics in disease-prevalent low-resource countries. We have developed and evaluated three duplex RPA assays incorporating competitive internal controls for the detection of leading bacterial meningitis pathogens. Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae singleplex RPA assays were initially developed and evaluated, demonstrating 100% specificity with limits of detection of 4.1, 8.5 and 3.9 genome copies per reaction, respectively. Each assay was further developed into internally controlled duplex RPA assays via the incorporation of internal amplification control templates. Clinical performance of each internally controlled duplex RPA assay was evaluated by testing 64 archived PCR-positive clinical samples. Compared to real-time PCR, all duplex RPA assays demonstrated 100% diagnostic specificity, with diagnostic sensitivities of 100%, 86.3% and 100% for the S. pneumoniae, N. meningitidis and H. influenzae assays, respectively. This study details the first report of internally controlled duplex RPA assays for the detection of bacterial meningitis pathogens: S. pneumoniae, N. meningitidis and H. influenzae. We have successfully demonstrated the clinical diagnostic utility of each duplex RPA assay, introducing effective diagnostic technology for POC bacterial meningitis identification in disease-prevalent developing countries., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Streptococcus pneumoniae is an important cause of microbial disease in humans. The introduction of multivalent vaccines has coincided with a dramatic decrease in the number of pneumococcal-related deaths. In spite of this, at a global level, pneumococcal infection remains an important cause of death among children under 5 years of age and in adults 65 years of age or older. In order to properly manage patients and control the spread of infection, a rapid and highly sensitive diagnostic method is needed for routine implementation, especially in resource-limited regions where pneumococcal disease is most prevalent., Methods: Using the gene encoding leader peptidase A as a molecular diagnostics target, a real-time RPA assay was designed and optimised for the detection of S. pneumoniae in whole blood. The performance of the assay was compared to real-time PCR in terms of its analytical limit of detection and specificity. The inhibitory effect of human genomic DNA on amplification was investigated. The potential clinical utility of the assay was investigated using a small number of clinical samples., Results: The RPA assay has a limit of detection equivalent to PCR (4.0 and 5.1 genome equivalents per reaction, respectively) and was capable of detecting the equivalent of <1 colony forming unit of S. pneumoniae when spiked into human whole blood. The RPA assay was 100 % inclusive (38/38 laboratory reference strains and 19/19 invasive clinical isolates) and 100 % exclusive; differentiating strains of S. pneumoniae species from other viridans group streptococci, including S. pseudopneumoniae. When applied to the analysis of a small number (n = 11) of clinical samples (blood culture positive for S. pneumoniae), the RPA assay was demonstrated to be both rapid and sensitive., Conclusions: The RPA assay developed in this work is shown to be as sensitive and as specific as PCR. In terms of reaction kinetics, the RPA assay is shown to exceed those of the PCR, with the RPA running to completion in 20 minutes and capable generating a positive signal in as little as 6 minutes. This work represents a potentially suitable assay for application in point-of-care settings.

Objective: Research investigating the association between continuity of care (CoC) and patient outcomes in mental health care is limited. A previous review (1970-2002) concluded that evidence for an association between CoC and outcomes was inconsistent and limited. This systematic review, conducted a decade later, provides an update., Methods: Searches (1950-2014) were conducted on MEDLINE and PsycINFO. Included studies used a clearly identified measure of CoC and examined its relation to an outcome among adults (ages 18-65). Only English-language publications were included., Results: A total of 984 studies were identified that measured CoC. Eighteen met inclusion criteria, and 13 found an association between CoC and an outcome. As found in the previous review, studies reported conflicting results for the most frequently examined outcomes (hospitalization, symptom severity, social functioning, and service satisfaction). Little consistency was found between studies in choice of CoC measures and outcomes. Studies varied markedly in quality. Two of the three studies rated as good quality reported significant associations between CoC and social functioning. Compared with older studies, studies published since the previous systematic review (2002-2014) found a larger proportion of significant associations., Conclusions: Little consistency was found in the way CoC was measured, which made it difficult to compare studies. Therefore, clear evidence about the association between CoC and outcomes remains limited. Results in regard to social functioning are encouraging. However, in order for conclusions to be made, researchers need to be more consistent with the measures they choose to allow comparison of studies.

Improved access to effective tests for diagnosing tuberculosis (TB) has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA) is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC) DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110) and 20 fg (IS1081)were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9) and 86.1% (95%CI: 78.1, 94.1) respectively (n = 71). Specificities were 100% and 88.6% (95% CI: 80.8, 96.1) respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2) and 70.8% (95%CI: 62.9, 78.7) were obtained (n = 90). Specificities were 95.4 (95% CI: 92.3,98.1) and 88% (95% CI: 83.6, 92.4) respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB assays could be of use for integration into a point-of-care test for use in resource constrained settings.

Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (∼4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates.

Background: The germline BRCA2 mutation is associated with increased prostate cancer (PrCa) risk. We have assessed survival in young PrCa cases with a germline mutation in BRCA2 and investigated loss of heterozygosity at BRCA2 in their tumours., Methods: Two cohorts were compared: one was a group with young-onset PrCa, tested for germline BRCA2 mutations (6 of 263 cases had a germline BRAC2 mutation), and the second was a validation set consisting of a clinical set from Manchester of known BRCA2 mutuation carriers (15 cases) with PrCa. Survival data were compared with a control series of patients in a single clinic as determined by Kaplan-Meier estimates. Loss of heterozygosity was tested for in the DNA of tumour tissue of the young-onset group by typing four microsatellite markers that flanked the BRCA2 gene, followed by sequencing., Results: Median survival of all PrCa cases with a germline BRCA2 mutation was shorter at 4.8 years than was survival in controls at 8.5 years (P=0.002). Loss of heterozygosity was found in the majority of tumours of BRCA2 mutation carriers. Multivariate analysis confirmed that the poorer survival of PrCa in BRCA2 mutation carriers is associated with the germline BRCA2 mutation per se., Conclusion: BRCA2 germline mutation is an independent prognostic factor for survival in PrCa. Such patients should not be managed with active surveillance as they have more aggressive disease.

Background: High-throughput measurement of allele-specific expression (ASE) is a relatively new and exciting application area for array-based technologies. In this paper, we explore several data sets which make use of Illumina's GoldenGate BeadArray technology to measure ASE. This platform exploits coding SNPs to obtain relative expression measurements for alleles at approximately 1500 positions in the genome., Results: We analyze data from a mixture experiment where genomic DNA samples from pairs of individuals of known genotypes are pooled to create allelic imbalances at varying levels for the majority of SNPs on the array. We observe that GoldenGate has less sensitivity at detecting subtle allelic imbalances (around 1.3 fold) compared to extreme imbalances, and note the benefit of applying local background correction to the data. Analysis of data from a dye-swap control experiment allowed us to quantify dye-bias, which can be reduced considerably by careful normalization. The need to filter the data before carrying out further downstream analysis to remove non-responding probes, which show either weak, or non-specific signal for each allele, was also demonstrated. Throughout this paper, we find that a linear model analysis of the data from each SNP is a flexible modelling strategy that allows for testing of allelic imbalances in each sample when replicate hybridizations are available., Conclusions: Our analysis shows that local background correction carried out by Illumina's software, together with quantile normalization of the red and green channels within each array, provides optimal performance in terms of false positive rates. In addition, we strongly encourage intensity-based filtering to remove SNPs which only measure non-specific signal. We anticipate that a similar analysis strategy will prove useful when quantifying ASE on Illumina's higher density Infinium BeadChips.

Background: Imprinted genes show expression from one parental allele only and are important for development and behaviour. This extreme mode of allelic imbalance has been described for approximately 56 human genes. Imprinting status is often disrupted in cancer and dysmorphic syndromes. More subtle variation of gene expression, that is not parent-of-origin specific, termed 'allele-specific gene expression' (ASE) is more common and may give rise to milder phenotypic differences. Using two allele-specific high-throughput technologies alongside bioinformatics predictions, normal term human placenta was screened to find new imprinted genes and to ascertain the extent of ASE in this tissue., Results: Twenty-three family trios of placental cDNA, placental genomic DNA (gDNA) and gDNA from both parents were tested for 130 candidate genes with the Sequenom MassArray system. Six genes were found differentially expressed but none imprinted. The Illumina ASE BeadArray platform was then used to test 1536 SNPs in 932 genes. The array was enriched for the human orthologues of 124 mouse candidate genes from bioinformatics predictions and 10 human candidate imprinted genes from EST database mining. After quality control pruning, a total of 261 informative SNPs (214 genes) remained for analysis. Imprinting with maternal expression was demonstrated for the lymphocyte imprinted gene ZNF331 in human placenta. Two potential differentially methylated regions (DMRs) were found in the vicinity of ZNF331. None of the bioinformatically predicted candidates tested showed imprinting except for a skewed allelic expression in a parent-specific manner observed for PHACTR2, a neighbour of the imprinted PLAGL1 gene. ASE was detected for two or more individuals in 39 candidate genes (18%)., Conclusions: Both Sequenom and Illumina assays were sensitive enough to study imprinting and strong allelic bias. Previous bioinformatics approaches were not predictive of new imprinted genes in the human term placenta. ZNF331 is imprinted in human term placenta and might be a new ubiquitously imprinted gene, part of a primate-specific locus. Demonstration of partial imprinting of PHACTR2 calls for re-evaluation of the allelic pattern of expression for the PHACTR2-PLAGL1 locus. ASE was common in human term placenta.

Benzene is an established cause of leukemia, and possibly lymphoma, in humans, but the underlying molecular pathways remain largely undetermined. We used two microarray platforms to identify global gene expression changes associated with well-characterized occupational benzene exposure in the peripheral blood mononuclear cells (PBMC) of a population of shoe-factory workers. Differential expression of 2692 genes (Affymetrix) and 1828 genes (Illumina) was found and the concordance was 50% (based on an average fold-change > or =1.3 from the two platforms), with similar expression ratios among the concordant genes. Four genes (CXCL16, ZNF331, JUN and PF4), which we previously identified by microarray and confirmed by real-time PCR, were among the top 100 genes identified by both platforms in the current study. Gene ontology analysis showed overrepresentation of genes involved in apoptosis among the concordant genes while pathway analysis identified pathways related to lipid metabolism. The two-platform approach allows for robust changes in the PBMC transcriptome of benzene-exposed individuals to be identified.

Genome-wide associations have shown a lot of promise in dissecting the genetics of complex traits in humans with single variants, yet a large fraction of the genetic effects is still unaccounted for. Analyzing genetic interactions between variants (epistasis) is one of the potential ways forward. We investigated the abundance and functional impact of a specific type of epistasis, namely the interaction between regulatory and protein-coding variants. Using genotype and gene expression data from the 210 unrelated individuals of the original four HapMap populations, we have explored the combined effects of regulatory and protein-coding single nucleotide polymorphisms (SNPs). We predict that about 18% (1,502 out of 8,233 nsSNPs) of protein-coding variants are differentially expressed among individuals and demonstrate that regulatory variants can modify the functional effect of a coding variant in cis. Furthermore, we show that such interactions in cis can affect the expression of downstream targets of the gene containing the protein-coding SNP. In this way, a cis interaction between regulatory and protein-coding variants has a trans impact on gene expression. Given the abundance of both types of variants in human populations, we propose that joint consideration of regulatory and protein-coding variants may reveal additional genetic effects underlying complex traits and disease and may shed light on causes of differential penetrance of known disease variants., Competing Interests: The authors have declared that no competing interests exist.

Background: Non-Hodgkin lymphoma (NHL) is the fifth most common cancer in the U.S. and few causes have been identified. Genetic association studies may help identify environmental risk factors and enhance our understanding of disease mechanisms., Methodology/principal Findings: 768 coding and haplotype tagging SNPs in 146 genes were examined using Illumina GoldenGate technology in a large population-based case-control study of NHL in the San Francisco Bay Area (1,292 cases 1,375 controls are included here). Statistical analyses were restricted to HIV- participants of white non-Hispanic origin. Genes involved in steroidogenesis, immune function, cell signaling, sunlight exposure, xenobiotic metabolism/oxidative stress, energy balance, and uptake and metabolism of cholesterol, folate and vitamin C were investigated. Sixteen SNPs in eight pathways and nine haplotypes were associated with NHL after correction for multiple testing at the adjusted q<0.10 level. Eight SNPs were tested in an independent case-control study of lymphoma in Germany (494 NHL cases and 494 matched controls). Novel associations with common variants in estrogen receptor 1 (ESR1) and in the vitamin C receptor and matrix metalloproteinase gene families were observed. Four ESR1 SNPs were associated with follicular lymphoma (FL) in the U.S. study, with rs3020314 remaining associated with reduced risk of FL after multiple testing adjustments [odds ratio (OR) = 0.42, 95% confidence interval (CI) = 0.23-0.77) and replication in the German study (OR = 0.24, 95% CI = 0.06-0.94). Several SNPs and haplotypes in the matrix metalloproteinase-3 (MMP3) and MMP9 genes and in the vitamin C receptor genes, solute carrier family 23 member 1 (SLC23A1) and SLC23A2, showed associations with NHL risk., Conclusions/significance: Our findings suggest a role for estrogen, vitamin C and matrix metalloproteinases in the pathogenesis of NHL that will require further validation.

Genetic variation influences gene expression, and this variation in gene expression can be efficiently mapped to specific genomic regions and variants. Here we have used gene expression profiling of Epstein-Barr virus-transformed lymphoblastoid cell lines of all 270 individuals genotyped in the HapMap Consortium to elucidate the detailed features of genetic variation underlying gene expression variation. We find that gene expression is heritable and that differentiation between populations is in agreement with earlier small-scale studies. A detailed association analysis of over 2.2 million common SNPs per population (5% frequency in HapMap) with gene expression identified at least 1,348 genes with association signals in cis and at least 180 in trans. Replication in at least one independent population was achieved for 37% of cis signals and 15% of trans signals, respectively. Our results strongly support an abundance of cis-regulatory variation in the human genome. Detection of trans effects is limited but suggests that regulatory variation may be the key primary effect contributing to phenotypic variation in humans. We also explore several methodologies that improve the current state of analysis of gene expression variation.

Extensive studies are currently being performed to associate disease susceptibility with one form of genetic variation, namely, single-nucleotide polymorphisms (SNPs). In recent years, another type of common genetic variation has been characterized, namely, structural variation, including copy number variants (CNVs). To determine the overall contribution of CNVs to complex phenotypes, we have performed association analyses of expression levels of 14,925 transcripts with SNPs and CNVs in individuals who are part of the International HapMap project. SNPs and CNVs captured 83.6% and 17.7% of the total detected genetic variation in gene expression, respectively, but the signals from the two types of variation had little overlap. Interrogation of the genome for both types of variants may be an effective way to elucidate the causes of complex phenotypes and disease in humans.

Benzene is a recognized hematotoxicant and carcinogen that produces genotoxic damage. DNA double-strand breaks (DSB) are one of the most severe DNA lesions caused directly and indirectly by benzene metabolites. DSB may lead to chromosome aberrations, apoptosis and hematopoietic progenitor cell suppression. We hypothesized that genetic polymorphisms in genes involved in DNA DSB repair may modify benzene-induced hematotoxicity. We analyzed one or more single nucleotide polymorphisms (SNPs) in each of seven candidate genes (WRN, TP53, NBS1, BRCA1, BRCA2, XRCC3 and XRCC4) in a study of 250 workers exposed to benzene and 140 controls in China. Four SNPs in WRN (Ex4 -16 G > A, Ex6 +9 C > T, Ex20 -88 G > T and Ex26 -12 T > G), one SNP in TP53 (Ex4 +119 C > G) and one SNP in BRCA2 (Ex11 +1487 A > G) were associated with a statistically significant decrease in total white blood cell (WBC) counts among exposed workers. The SNPs in WRN and TP53 remained significant after accounting for multiple comparisons. One or more SNPs in WRN had broad effects on WBC subtypes, with significantly decreased granulocyte, total lymphocyte, CD4(+)-T cell, CD8(+)-T cell and monocyte counts. Haplotypes of WRN were associated with decreased WBC counts among benzene-exposed subjects. Likewise, subjects with TP53 Ex4 +119 C > G variant had reduced granulocyte, CD4(+)-T cell and B cell counts. The effect of BRCA2 Ex11 +1487 A > G polymorphism was limited to granulocytes. These results suggest that genetic polymorphisms in WRN, TP53 and BRCA2 that maintain genomic stability impact benzene-induced hematotoxicity.

Background and Objectives: The risk of non-Hodgkin's lymphoma (NHL) has been associated with inflammation. One possible mechanism may involve oxidative stress as reactive oxygen species (ROS) can generate pro-inflammatory signals. Anti-oxidant enzymes including superoxide dismutase, glutathione peroxidase and catalase protect against the harmful effects of ROS. Genetic variation in the genes coding for these enzymes (SOD2, GPX1, and CAT, respectively) alters ROS production and therefore may provide a mechanism for the relationship between inflammation and NHL., Design and Methods: Data from two population-based, case-control studies of lymphoma in the UK (700 cases and 915 controls) and USA (1593 cases and 2517 controls) were pooled to analyze polymorphisms in genes involved in the oxidative stress response (SOD2 Val16Ala, CAT C-262T and GPX1 Pro197Leu)., Results: No associations were observed between SOD2 Val16Ala and CAT C-262T and total NHL, diffuse large-B cell lymphoma or follicular lymphoma. However, when we looked at marginal zone lymphoma, a specific subtype of lymphoma characterised by inflammation, we found that homozygosity for the SOD2 16Ala allele was associated with a decreased risk among UK study participants. The GPX1 197Leu allele was weakly associated with NHL and follicular lymphoma., Interpretation and Conclusion: Analysis of genetic variation in oxidative stress genes in two lymphoma case-control studies suggests a possible role for oxidative stress in the risk of NHL. The risk modification is seen predominantly for marginal zone lymphomas which frequently arise in the context of chronic inflammation. However, in order to clarify the role of oxidative stress in the etiology of NHL analyses of additional polymorphisms and haplotypes in these and other genes involved in the oxidative stress response are needed.

Genetic variation in innate immunity may alter host-pathogen defence mechanisms and promote aberrant immune responses and non-Hodgkin lymphoma (NHL). To test this hypothesis, we investigated polymorphisms in innate immune genes in a pooled analysis of two population-based case-control studies of NHL from the San Francisco Bay Area (308 cases, 684 controls) and UK (596 cases, 758 controls). The caspase recruitment domain-containing protein 1007fs homozygote variant was positively associated with NHL risk (odds ratios (OR) = 3.1, 95% confidence intervals (CI) 1.1-8.8), whereas the toll-like receptor 4 1063A>G variant allele was inversely associated with diffuse large cell lymphoma (OR = 0.67, 95% CI 0.45-0.99). These results suggest that variation in innate immune genes may alter NHL susceptibility.

The exploration of quantitative variation in human populations has become one of the major priorities for medical genetics. The successful identification of variants that contribute to complex traits is highly dependent on reliable assays and genetic maps. We have performed a genome-wide quantitative trait analysis of 630 genes in 60 unrelated Utah residents with ancestry from Northern and Western Europe using the publicly available phase I data of the International HapMap project. The genes are located in regions of the human genome with elevated functional annotation and disease interest including the ENCODE regions spanning 1% of the genome, Chromosome 21 and Chromosome 20q12-13.2. We apply three different methods of multiple test correction, including Bonferroni, false discovery rate, and permutations. For the 374 expressed genes, we find many regions with statistically significant association of single nucleotide polymorphisms (SNPs) with expression variation in lymphoblastoid cell lines after correcting for multiple tests. Based on our analyses, the signal proximal (cis-) to the genes of interest is more abundant and more stable than distal and trans across statistical methodologies. Our results suggest that regulatory polymorphism is widespread in the human genome and show that the 5-kb (phase I) HapMap has sufficient density to enable linkage disequilibrium mapping in humans. Such studies will significantly enhance our ability to annotate the non-coding part of the genome and interpret functional variation. In addition, we demonstrate that the HapMap cell lines themselves may serve as a useful resource for quantitative measurements at the cellular level., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Benzene is a recognized hematotoxin and leukemogen but its mechanism of action and the role of genetic susceptibility are still unclear. Cytokines, chemokines, and cellular adhesion molecules are soluble proteins that play an important regulatory role in hematopoiesis. We therefore hypothesized that variation in these genes could influence benzene-induced hematotoxicity. We analyzed common, well-studied single-nucleotide polymorphisms (SNPs) in 20 candidate genes drawn from these pathways in a study of 250 workers exposed to benzene and 140 unexposed controls in China. After accounting for multiple comparisons, SNPs in five genes were associated with a statistically significant decrease in total WBC counts among exposed workers [IL-1A (-889C>T), IL-4 (-1098T>G), IL-10 (-819T>C), IL-12A (8685G>A), and VCAM1 (-1591T>C)], and one SNP [CSF3 (Ex4-165C>T)] was associated with an increase in WBC counts. The adhesion molecule VCAM1 variant was particularly noteworthy as it was associated with a decrease in B cells, natural killer cells, CD4+ T cells, and monocytes. Further, VCAM1 (-1591T>C) and CSF3 (Ex4-165C>T) were associated, respectively, with decreased (P = 0.041) and increased (P = 0.076) CFU-GEMM progenitor cell colony formation in 29 benzene-exposed workers. This is the first report to provide evidence that SNPs in genes that regulate hematopoiesis influence benzene-induced hematotoxicity.

Expression of prolactin and of prolactin and estrogen receptors in lymphocytes, bone marrow, and lymphoma cell lines suggests that hormonal modulation may influence lymphoma risk. Prolactin and estrogen promote the proliferation and survival of B cells, factors that may increase non-Hodgkin lymphoma risk, and effects of estrogen may be modified by catechol-O-methyltransferase (COMT), an enzyme that alters estrogenic activity. Cytochrome P450 17A1 (CYP17A1), a key enzyme in estrogen biosynthesis, has been associated with increased cancer risk and may affect lymphoma susceptibility. We studied the polymorphisms prolactin (PRL) -1149G>T, CYP17A1 -34T>C, and COMT 108/158Val>Met, and predicted haplotypes among a subset of participants (n = 308 cases, n = 684 controls) in a San Francisco Bay Area population-based non-Hodgkin lymphoma study (n = 1,593 cases, n = 2,515 controls) conducted from 1988 to 1995. Oral contraceptive and other hormone use also was analyzed. Odds ratios (OR) for non-Hodgkin lymphoma and follicular lymphoma were reduced for carriers of the PRL -1149TT genotype [OR, 0.64; 95% confidence interval (95% CI), 0.41-1.0; OR, 0.53; 95% CI, 0.26-1.0, respectively]. Diffuse large-cell lymphoma risk was increased for those with CYP17A1 polymorphisms including CYP17A1 -34CC (OR, 2.0; 95% CI, 1.1-3.5). ORs for all non-Hodgkin lymphoma and follicular lymphoma among women were decreased for COMT IVS1 701A>G [rs737865; variant allele: OR, 0.53; 95% CI, 0.34-0.82; OR, 0.42; 95% CI, 0.23-0.78, respectively]. Compared with never users of oral contraceptives, a 35% reduced risk was observed among oral contraceptive users in the total population. Reduced ORs for all non-Hodgkin lymphoma were observed with use of exogenous estrogens among genotyped women although 95% CIs included unity. These results suggest that PRL, CYP17A1, and COMT may be relevant genetic loci for non-Hodgkin lymphoma and indicate a possible role for prolactin and estrogen in lymphoma pathogenesis.

Benzene is an industrial chemical and component of gasoline that is an established cause of leukemia. To better understand the risk benzene poses, we examined the effect of benzene exposure on peripheral blood mononuclear cell (PBMC) gene expression in a population of shoe-factory workers with well-characterized occupational exposures using microarrays and real-time polymerase chain reaction (PCR). PBMC RNA was stabilized in the field and analyzed using a comprehensive human array, the U133A/B Affymetrix GeneChip set. A matched analysis of six exposed-control pairs was performed. A combination of robust multiarray analysis and ordering of genes using paired t-statistics, along with bootstrapping to control for a 5% familywise error rate, was used to identify differentially expressed genes in a global analysis. This resulted in a set of 29 known genes being identified that were highly likely to be differentially expressed. We also repeated these analyses on a smaller subset of 508 cytokine probe sets and found that the expression of 19 known cytokine genes was significantly different between the exposed and the control subjects. Six genes were selected for confirmation by real-time PCR, and of these, CXCL16, ZNF331, JUN, and PF4 were the most significantly affected by benzene exposure, a finding that was confirmed in a larger data set from 28 subjects. The altered expression was not caused by changes in the makeup of the PBMC fraction. Thus, microarray analysis along with real-time PCR confirmation reveals that altered expressions of CXCL16, ZNF331, JUN, and PF4 are potential biomarkers of benzene exposure.

'Omic' technologies include genomics, transcriptomics (gene expression profiling), proteomics and metabolomics. We are utilizing these new technologies in an effort to develop novel biomarkers of exposure, susceptibility and response to benzene. Advances in genomics allow one to study hundreds to thousands of single nucleotide polymorphisms simultaneously on small quantities of DNA using array-based technologies. We are currently utilizing these technologies to examine genetic variation in pathways relating to biotransformation, DNA repair, folate metabolism and immune response with the goal of finding biomarkers of susceptibility to benzene hematotoxicity. Transcriptomics is used to measure the full complement of activated genes, mRNAs or transcripts in a particular tissue at a particular time typically using microarray technology. We have applied microarrays to the study of global gene expression in the peripheral blood cells of benzene-exposed workers. More than 100 genes were identified as being potentially differentially expressed, with genes related to apoptosis and immune function being the most significantly affected. Initial studies employing proteomics have also shown that several proteins are altered in the serum of exposed compared to control subjects and these proteins are potential biomarkers of benzene exposure. Omic technologies therefore have significant potential in generating novel biomarkers of exposure, susceptibility and response to benzene.

We investigated the association between seven polymorphisms in four candidate genes involved in vitamin D and androgen metabolism with early-onset prostate cancer (CaP) risk. The polymorphisms were genotyped in 288 UK males who were diagnosed with CaP at the age of 55 y or younger and up to 700 population-based controls. An additional 50 cases (not selected for age) and 76 controls were also genotyped. Short (< or =22 repeats) AR (CAG)(n) repeats were associated with a significantly reduced risk of early onset CaP (OR 0.68, 95% CI 0.50-0.91) compared with men with long (> 22) repeats. Men homozygous for the leucine variant of SRD5A2 p.89V > L were also found to be at a significantly increased risk of CaP compared with men who were homozygous for the valine allele (OR 1.84, 95% CI 1.15-2.98). No associations were found with the AR (GGC)(n), CYP17 Msp A1 I, VDR Taq I, SRD5A2 (TA)(n) and p.49A >T polymorphisms and CaP risk. These findings suggest that common polymorphisms in the AR and SRD5A2 genes may be associated with early-onset CaP in British men.

Folate metabolism plays an essential role in DNA synthesis and methylation processes. Deviations in the flux of folate due to genetic variation could result in selective growth and genomic instability and affect susceptibility to various cancers including lymphoma. To test this hypothesis, genetic polymorphisms in the folate metabolic pathway were investigated using DNA from a population-based case-control study of non-Hodgkin lymphoma (NHL) conducted in the San Francisco Bay Area between 1988 and 1995. The polymorphisms examined and haplotypes generated included thymidylate synthase (TYMS 28-bp triple repeat [3R]-->double repeat [2R], 1494del6, IVS6 -68C>T, 1122A>G, and 1053C>T); 5,10-methylenetetrahydrofolate reductase (MTHFR 677C>T and 1298A>C); serine hydroxymethyltransferase (SHMT1 C1420T); reduced folate carrier (RFC G80A); and methionine synthase (MTR A2756G), making the present study the largest and most comprehensive to date to evaluate associations between genetic polymorphisms in folatemetabolizing genes and NHL risk. The TYMS 6 base pair (bp)(-)6bp- (homozygous for 6bp deletion), IVS6 -68C>T, and 1053C>T genotypes (all in complete linkage disequilibrium) were all inversely associated with NHL (TYMS; odds ratio [OR] = 0.57; 0.34-0.94), particularly with diffuse large cell lymphoma (DLCL; OR = 0.29; 0.10-0.82). Further, the MTR 2756AG/GG and the MTHFR 677TT genotypes were associated with increased risk for NHL (OR = 1.3; 0.99-1.7) and follicular lymphoma (FL; OR = 1.8; 0.98-3.1), respectively. We did not observe any significant differences in genotype frequencies of the SHMT1 and RFC polymorphisms between the cases and controls. The associations of DLCL and FL with TYMS 1494del6 and MTHFR 677TT genotypes, respectively, suggest that folate metabolism may play an important role in the pathogenesis of specific subtypes of NHL.

We have evaluated the use of allele-specific PCR (AS PCR) on DNA pools as a tool for screening inherited genetic variants that may be associated with risk of adult acute myeloid leukemia (AML). Two DNA pools were constructed, one of 444 AML cases, and another of 823 matched controls. The pools were validated using individual genotyping data for GSTP1 and LTalpha variants. Allele frequencies for variants in GSTP1 and LTalpha were estimated using quantitative AS PCR, and when compared to individual genotyping data, a high degree of concordance was seen. AS primer pairs were designed for nine candidate genetic variants in DNA repair and cell cycle/apoptotic regulatory genes, including Cyclin D1 [codon 870 splice site variant (A>G)]; BRCA1, P871L; ERCC2, K751Q; FAS -1377 (G>A); hMLH1 -93 (G>A) and V219I; p21, S31R; and the XRCC1 R194W and R399Q variants. For six of these assays, there was at least 95% concordance between AS PCR genotyping and an alternative approach carried out on individual samples. Furthermore, these six AS PCR assays all accurately estimated allele frequencies in the pools that had been calculated using individual genotyping data. A significant disease association was seen with AML for the -1377 variant in FAS (odds ratio 1.76, 95% confidence interval 1.26-2.44). These data suggest that quantitative AS PCR can be used as an efficient screening technique for disease associations of genetic variants in DNA pools made from case-control studies.

In a population-based case-control study, obesity was associated with elevated odds ratios (ORs) for non-Hodgkin lymphoma (NHL), and the two major subtypes, diffuse large cell (DLCL) and follicular lymphoma (FL). Those who were obese (body mass index >/= 30) were up to three times more likely to develop NHL or its major subtypes than persons with body mass index of 20 to <25. Obesity-related genetic factors including common polymorphisms in the leptin gene (LEP A19G and G-2548A) and its receptor (LEPR Q223R) were investigated in DNA available for 376 patients and 805 controls. Leptin is an adipocyte-derived hormone that regulates food intake and modulates immune and inflammatory responses through its receptor. Among those with the LEP 19G allele, an increased risk estimate was found for all NHL [OR = 1.6, confidence interval (CI) 1.1-2.3], DLCL (OR = 1.6, CI 0.86-3.0), and FL lymphoma (OR = 1.9, CI 0.98-3.6). Gene-gene interaction existed between the -G2548A and LEPR Q223R polymorphisms. Specifically, among those with LEPR 223RR, the risk estimate for NHL was increased in LEP -2548GA (OR = 1.7, CI 0.88-3.1) and LEP -2548AA (OR = 2.3,CI 1.1-4.6) relative to LEP -2548GG genotypes. These results suggest that genetic interactions between leptin and its receptor may promote immune dysfunction associated with obesity and NHL and that the emerging obesity epidemic is consistent with the increasing incidence of NHL in developed countries.

Two hundred and ninety-one infertile couples were studied over an 8-year period. Anovulation was the most common cause of infertility, being the causative factor in half of the patients. Ovulation induction with clomiphene or cyclic hormone therapy readily achieved pregnancy in the majority of these patients. The distribution of other etiologic factors was fairly uniform, but treatment was much less successful. Thorough evaluation of both partners is advised both for treatment purposes and as a more accurate guide to the prognosis for conception. Furthermore, even in the presence of oligospermia, ovulation induction may be successful in achieving a pregnancy.

We treated 21 anovulatory infertile patients with subcutaneous pulsatile gonadotropin-releasing hormone (GnRH) administered via a syringe pump. Response to treatment was assessed by urinary estrogen excretion and ultrasound measurement of follicular growth. Ten patients ovulated and 8 subsequently conceived, for a total of 10 pregnancies. Human chorionic gonadotropin (hCG) was not administered routinely, but two patients required hCG to induce follicular rupture. The majority of the patients who conceived had a body mass index (BMI) of less than 21 and a luteinizing hormone (LH)/follicle-stimulating hormone ratio of less than 1. Conversely, those patients with either elevated BMI or LH or both generally failed to respond satisfactorily to this treatment. It is suggested that pulsatile GnRH is most likely to succeed in inducing ovulation if the BMI is less than 21 and the LH is normal, but is unlikely to be successful if there is both an elevated LH and a BMI of greater than 25. Between these two extremes, the response is variable and a therapeutic trial may be appropriate.

Sixty-four patients who had been treated for anovulatory infertility and subsequently conceived were reviewed. Information was obtained on subsequent menstrual pattern, contraception used, treatment required to achieve a further pregnancy and the interval to any such pregnancy. Almost half the group had regular cycles following delivery but 2 patients had a recurrence of post oral contraceptive amenorrhoea. Most patients conceived without treatment within 2 years of the initial pregnancy but 10 again required ovulation induction. It is concluded that there is no long-term irreversible suppression of hypothalamic function in this group of patients and, once a pregnancy has been achieved, the prognosis for future fertility without therapy is good.