Redefining precision radiotherapy through liquid biopsy.

Precision radiotherapy refers to the ability to deliver radiation doses with sub-millimetre accuracy. It does not however consider individual variation in tumour or normal tissue response, failing to maximise tumour control and minimise toxicity. Combining precise delivery with personalised dosing, through analysis of cell-free DNA, would redefine precision in radiotherapy.

Spatial transcriptomics identifies spatially dysregulated expression of GRM3 and USP47 in amyotrophic lateral sclerosis.

AIMS: The mechanisms underlying the selective degeneration of motor neurones in amyotrophic lateral sclerosis (ALS) are poorly understood. The aim of this study was to implement spatially resolved RNA sequencing in human post mortem cortical tissue from an ALS patient harbouring the C9orf72 hexanucleotide repeat expansion to identify dysregulated transcripts that may account for differential vulnerabilities of distinct (i) cell types and (ii) brain regions in the pathogenesis of ALS. METHODS: Using spatial transcriptomics (ST) we analysed the transcriptome of post mortem brain tissue, with spatial resolution down to 100 µm. Validation of these findings was then performed using BaseScope, an adapted, in situ hybridization technique with single-transcript single-cell-resolution, providing extensive regional and cell-type specific confirmation of these dysregulated transcripts. The validation cohort was then extended to include multiple post mortem brain regions and spinal cord tissue from an extended cohort of C9orf72, sporadic ALS (sALS) and SOD1 ALS cases. RESULTS: We identified sixteen dysregulated transcripts of proteins that have roles within six disease-related pathways. Furthermore, these complementary molecular pathology techniques converged to identify two spatially dysregulated transcripts, GRM3 and USP47, that are commonly dysregulated across sALS, SOD1 and C9orf72 cases alike. CONCLUSIONS: This study presents the first description of ST in human post mortem cortical tissue from an ALS patient harbouring the C9orf72 hexanucleotide repeat expansion. These data taken together highlight the importance of preserving spatial resolution, facilitating the identification of genes whose dysregulation may in part underlie regional susceptibilities to ALS, crucially highlighting potential therapeutic and diagnostic targets.

Transgenic rescue of defective Cd36 ameliorates insulin resistance in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) display several features of the human insulin-resistance syndromes. Cd36 deficiency is genetically linked to insulin resistance in SHR. We show that transgenic expression of Cd36 in SHR ameliorates insulin resistance and lowers serum fatty acids. Our results provide direct evidence that Cd36 deficiency can promote defective insulin action and disordered fatty-acid metabolism in spontaneous hypertension.

Quantitative trait loci for cellular defects in glucose and fatty acid metabolism in hypertensive rats.

Coronary heart disease, hypertension, non-insulin-dependent diabetes and obesity are major causes of ill health in industrial societies. Disturbances of carbohydrate and lipid metabolism are a common feature of these disorders. The bases for these disturbances and their roles in disease pathogenesis are poorly understood. The spontaneously hypertensive rat (SHR), a widely used animal model of essential hypertension, has a global defect in insulin action on glucose metabolism and shows reduced catecholamine action on lipolysis in fat cells. In our study we used cellular defects in carbohydrate and lipid metabolism to dissect the genetics of defective insulin and catecholamine action in the SHR strain. In a genome screen for loci linked to insulin and catecholamine action, we identified two quantitative trait loci (QTLs) for defective insulin action, on chromosome 4 and 12. We found that the major (and perhaps only) genetic determinant of defective control of lipolysis in SHR maps to the same region of chromosome 4. These linkage results were ascertained in at least two independent crosses. As the SHR strain manifests many of the defining features of human metabolic Syndrome X, in which hypertension associates with insulin resistance, dyslipidaemia and abdominal obesity, the identification of genes for defective insulin and catecholamine action in SHR may facilitate gene identification in this syndrome and in related human conditions, such as type-2 diabetes and familial combined hyperlipidaemia.

Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats.

The human insulin-resistance syndromes, type 2 diabetes, obesity, combined hyperlipidaemia and essential hypertension, are complex disorders whose genetic basis is unknown. The spontaneously hypertensive rat (SHR) is insulin resistant and a model of these human syndromes. Quantitative trait loci (QTLs) for SHR defects in glucose and fatty acid metabolism, hypertriglyceridaemia and hypertension map to a single locus on rat chromosome 4. Here we combine use of cDNA microarrays, congenic mapping and radiation hybrid (RH) mapping to identify a defective SHR gene, Cd36 (also known as Fat, as it encodes fatty acid translocase), at the peak of linkage to these QTLs. SHR Cd36 cDNA contains multiple sequence variants, caused by unequal genomic recombination of a duplicated ancestral gene. The encoded protein product is undetectable in SHR adipocyte plasma membrane. Transgenic mice overexpressing Cd36 have reduced blood lipids. We conclude that Cd36 deficiency underlies insulin resistance, defective fatty acid metabolism and hypertriglyceridaemia in SHR and may be important in the pathogenesis of human insulin-resistance syndromes.

Genetic regulation of catecholamine synthesis, storage and secretion in the spontaneously hypertensive rat.

Understanding catecholamine metabolism is crucial for elucidating the pathogenesis of hereditary hypertension. Here we integrated transcriptional and biochemical profiling with physiologic quantitative trait locus (eQTL and pQTL) mapping in adrenal glands of the HXB/BXH recombinant inbred (RI) strains, derived from the spontaneously hypertensive rat (SHR) and normotensive Brown Norway (BN.Lx). We found simultaneous down-regulation of five heritable transcripts in the catecholaminergic pathway in young (6 weeks) SHRs. We identified cis-acting eQTLs for Dbh, Pnmt (catecholamine biosynthesis) and Vamp1 (catecholamine secretion); enzymatic activities of Dbh and Pnmt paralleled transcripts, with pQTLs for activities mirroring eQTLs. We also detected trans-regulated expression of Vmat1 and Chga (both involved in catecholamine storage), with co-localization of these trans-eQTLs to the Pnmt locus. Pnmt re-sequencing revealed promoter polymorphisms that result in decreased response of the transfected SHR promoter to glucocorticoid, compared with BN.Lx. Of physiological pertinence, Dbh activity negatively correlated with systolic blood pressure in RI strains, whereas Pnmt activity was negatively correlated with heart rate. The finding of such cis- and trans-QTLs at an age before the onset of frank hypertension suggests that these heritable changes in biosynthetic enzyme expression represent primary genetic mechanisms for regulation of catecholamine action and blood pressure control in this widely studied model of hypertension.

Differential coexpression analysis of obesity-associated networks in human subcutaneous adipose tissue.

OBJECTIVE: To use a unique obesity-discordant sib-pair study design to combine differential expression analysis, expression quantitative trait loci (eQTLs) mapping and a coexpression regulatory network approach in subcutaneous human adipose tissue to identify genes relevant to the obese state. STUDY DESIGN: Genome-wide transcript expression in subcutaneous human adipose tissue was measured using Affymetrix U133 Plus 2.0 microarrays (Affymetrix, Santa Clara, CA, USA), and genome-wide genotyping data was obtained using an Applied Biosystems (Applied Biosystems; Life Technologies, Carlsbad, CA, USA) SNPlex linkage panel. SUBJECTS: A total of 154 Swedish families ascertained through an obese proband (body mass index (BMI) >30 kg m(-2)) with a discordant sibling (BMI>10 kg m(-2) less than proband). RESULTS: Approximately one-third of the transcripts were differentially expressed between lean and obese siblings. The cellular adhesion molecules (CAMs) KEGG grouping contained the largest number of differentially expressed genes under cis-acting genetic control. By using a novel approach to contrast CAMs coexpression networks between lean and obese siblings, a subset of differentially regulated genes was identified, with the previously GWAS obesity-associated neuronal growth regulator 1 (NEGR1) as a central hub. Independent analysis using mouse data demonstrated that this finding of NEGR1 is conserved across species. CONCLUSION: Our data suggest that in addition to its reported role in the brain, NEGR1 is also expressed in subcutaneous adipose tissue and acts as a central 'hub' in an obesity-related transcript network.

Characterization of the macrophage transcriptome in glomerulonephritis-susceptible and -resistant rat strains.

Crescentic glomerulonephritis (CRGN) is a major cause of rapidly progressive renal failure for which the underlying genetic basis is unknown. Wistar-Kyoto (WKY) rats show marked susceptibility to CRGN, whereas Lewis rats are resistant. Glomerular injury and crescent formation are macrophage dependent and mainly explained by seven quantitative trait loci (Crgn1-7). Here, we used microarray analysis in basal and lipopolysaccharide (LPS)-stimulated macrophages to identify genes that reside on pathways predisposing WKY rats to CRGN. We detected 97 novel positional candidates for the uncharacterized Crgn3-7. We identified 10 additional secondary effector genes with profound differences in expression between the two strains (>5-fold change, <1% false discovery rate) for basal and LPS-stimulated macrophages. Moreover, we identified eight genes with differentially expressed alternatively spliced isoforms, by using an in-depth analysis at the probe level that allowed us to discard false positives owing to polymorphisms between the two rat strains. Pathway analysis identified several common linked pathways, enriched for differentially expressed genes, which affect macrophage activation. In summary, our results identify distinct macrophage transcriptome profiles between two rat strains that differ in susceptibility to glomerulonephritis, provide novel positional candidates for Crgn3-7 and define groups of genes that play a significant role in differential regulation of macrophage activity.

Gene copy number variation and common human disease.

Variation in gene copy number is increasingly recognized as a common, heritable source of inter-individual differences in genomic sequence. The role of copy number variation is well established in the pathogenesis of rare genomic disorders. More recently, germline and somatic copy number variation have been shown to be important pathogenic factors in a range of common diseases, including infectious, autoimmune and neuropsychiatric diseases and cancer. In this review, we describe the range of methods available for measuring copy number variants (CNVs) in individuals and populations, including the limitations of presently available assays, and highlight some key examples of common diseases in which CNVs have been shown clearly to have a pathogenic role. Although there has been major progress in this field in the last 5 years, understanding the full contribution of CNVs to the genetic basis of common diseases will require further studies, with more accurate CNV assays and larger cohorts than have presently been completed.

Copy number variation in the human genome and its implication in autoimmunity.

The causes of autoimmune disease remain poorly defined. However, it is known that genetic factors contribute to disease susceptibility. Hitherto, studies have focused upon single nucleotide polymorphisms as both tools for mapping and as probable causal variants. Recent studies, using genome-wide analytical techniques, have revealed that, in the genome, segments of DNA ranging in size from kilobases to megabases can vary in copy number. These changes of DNA copy number represent an important element of genomic polymorphism in humans and in other species and may therefore make a substantial contribution to phenotypic variation and population differentiation. Furthermore, copy number variation (CNV) in genomic regions harbouring dosage-sensitive genes may cause or predispose to a variety of human genetic diseases. Several recent studies have reported an association between CNV and autoimmunity in humans such as systemic lupus, psoriasis, Crohn's disease, rheumatoid arthritis and type 1 diabetes. The use of novel analytical techniques facilitates the study of complex human genomic structures such as CNV, and allows new susceptibility loci for autoimmunity to be found that are not readily mappable by single nucleotide polymorphism-based association analyses alone.

EMAAS: an extensible grid-based rich internet application for microarray data analysis and management.

BACKGROUND: Microarray experimentation requires the application of complex analysis methods as well as the use of non-trivial computer technologies to manage the resultant large data sets. This, together with the proliferation of tools and techniques for microarray data analysis, makes it very challenging for a laboratory scientist to keep up-to-date with the latest developments in this field. Our aim was to develop a distributed e-support system for microarray data analysis and management. RESULTS: EMAAS (Extensible MicroArray Analysis System) is a multi-user rich internet application (RIA) providing simple, robust access to up-to-date resources for microarray data storage and analysis, combined with integrated tools to optimise real time user support and training. The system leverages the power of distributed computing to perform microarray analyses, and provides seamless access to resources located at various remote facilities. The EMAAS framework allows users to import microarray data from several sources to an underlying database, to pre-process, quality assess and analyse the data, to perform functional analyses, and to track data analysis steps, all through a single easy to use web portal. This interface offers distance support to users both in the form of video tutorials and via live screen feeds using the web conferencing tool EVO. A number of analysis packages, including R-Bioconductor and Affymetrix Power Tools have been integrated on the server side and are available programmatically through the Postgres-PLR library or on grid compute clusters. Integrated distributed resources include the functional annotation tool DAVID, GeneCards and the microarray data repositories GEO, CELSIUS and MiMiR. EMAAS currently supports analysis of Affymetrix 3' and Exon expression arrays, and the system is extensible to cater for other microarray and transcriptomic platforms. CONCLUSION: EMAAS enables users to track and perform microarray data management and analysis tasks through a single easy-to-use web application. The system architecture is flexible and scalable to allow new array types, analysis algorithms and tools to be added with relative ease and to cope with large increases in data volume.

Copy number variation of Fc gamma receptor genes and disease predisposition.

Naturally occurring variation in gene copy number is increasingly recognized as a major source of inter-individual differences in phenotype and is an important susceptibility factor for genetically complex diseases. Several studies provide evidence of copy number variation at genes involved in inflammation and immunity highlighting their possible contribution to the inter-individual variation observed in immune responses. This review will explore copy number variation at the Fc gamma receptor cluster and its relevance in the pathogenesis of common human diseases.

Genetics of Cd36 and the clustering of multiple cardiovascular risk factors in spontaneous hypertension.

Disorders of carbohydrate and lipid metabolism have been reported to cluster in patients with essential hypertension and in spontaneously hypertensive rats (SHRs). A deletion in the Cd36 gene on chromosome 4 has recently been implicated in defective carbohydrate and lipid metabolism in isolated adipocytes from SHRs. However, the role of Cd36 and chromosome 4 in the control of blood pressure and systemic cardiovascular risk factors in SHRs is unknown. In the SHR. BN-Il6/Npy congenic strain, we have found that transfer of a segment of chromosome 4 (including Cd36) from the Brown Norway (BN) rat onto the SHR background induces reductions in blood pressure and ameliorates dietary-induced glucose intolerance, hyperinsulinemia, and hypertriglyceridemia. These results demonstrate that a single chromosome region can influence a broad spectrum of cardiovascular risk factors involved in the hypertension metabolic syndrome. However, analysis of Cd36 genotypes in the SHR and stroke-prone SHR strains indicates that the deletion variant of Cd36 was not critical to the initial selection for hypertension in the SHR model. Thus, the ability of chromosome 4 to influence multiple cardiovascular risk factors, including hypertension, may depend on linkage of Cd36 to other genes trapped within the differential segment of the SHR. BN-Il6/Npy strain.

Cd36 and molecular mechanisms of insulin resistance in the stroke-prone spontaneously hypertensive rat.

Insulin resistance is of pathogenic importance in several common human disorders including type 2 diabetes, hypertension, obesity and hyperlipidemia, but the underlying mechanisms are unknown. The spontaneously hypertensive rat (SHR) is a model of these human insulin resistance syndromes. Quantitative trait loci (QTLs) for SHR defects in glucose and fatty acid metabolism, hypertriglyceridemia, and hypertension map to a single region on rat chromosome 4. Genetic analysis of an SHR derived from a National Institutes of Health colony led to the identification of a causative mutation in the SHR Cd36. We have investigated glucose and fatty acid metabolism in the stroke-prone SHR (SHRSP). We demonstrate defects in insulin action on 2-deoxy-D-glucose transport (SHRSP 3.3 +/- 1.5 vs. 21.0 +/- 7.4 pmol x min(-1) x [20 microl packed cells](-1), SHRSP vs. WKY, respectively, P = 0.01) and inhibition of catecholamine-stimulated lipolysis (P < 0.05 at all concentrations of insulin) in adipocytes isolated from SHRSP. In contrast, basal levels of catecholamine-stimulated nonesterified free fatty acid (NEFA) release and plasma levels of NEFA are similar in SHRSP and WKY. These results are in agreement with the data on the SHR.4 congenic strain, which suggested that the QTL containing Cd36 mutations accounted for the entire defect in basal catecholamine action but only for approximately 40% of the SHR defect in insulin action. In the SHR, both abnormalities appear consequent of defective Cd36 expression. Because Cd36 sequence and expression are apparently normal in SHRSP, it is likely that the molecular mechanism for defective insulin action in this strain is caused by a gene(s) different than Cd36.

Insulin-like growth factor I gene expression in the rat ovary is confined to the granulosa cells of developing follicles.

The in vivo intraovarian synthesis of insulin-like growth factor-I (IGF-I) has been studied in the rat by Northern blot, dot blot hybridization, and in situ hybridization histochemistry. Ovarian IGF-I mRNA transcript sizes (7.0 kilobases (kb), 1.6 kb, and a group from 0.4-0.9 kb) were similar to those in liver and other tissues. The proportion of ovarian IGF-I to tubulin messenger RNA (mRNA) was increased to 176% of control values by treatment with diethylstilbestrol, while the ratio in liver was decreased to 64.4%. In situ hybridization identifies the major in vivo site of IGF-I synthesis in the ovary as the granulosa cells of developing follicles. IGF-I mRNA was present in the granulosa cells of developing preantral and antral follicles, but was not seen in atretic follicles or corpora lutea. In preovulatory follicles high levels of IGF-I mRNA were confined to the antral cell layers and to the cells of the cumulus oophorus. High levels of tubulin gene expression within follicles were seen in a similar distribution to that for IGF-I but 80-90% of corpora lutea also strongly expressed the tubulin gene. Interstitial cells, including thecal cells, express the tubulin gene at low levels but do not express the IGF-I gene. The distribution of IGF-I mRNA is the same as that previously observed for mitotically active granulosa cells, and therefore offers strong support for the view that IGF-I in the ovary acts by an autocrine-paracrine mechanism to promote granulosa cell replication.

Genes encoding atrial and brain natriuretic peptides as candidates for sensitivity to brain ischemia in stroke-prone hypertensive rats.

-Previous studies suggested that atrial natriuretic peptide gene (Anp) and brain natriuretic peptide gene (Bnp) are plausible candidate genes for susceptibility to stroke and for sensitivity to brain ischemia in the stroke-prone spontaneously hypertensive rat (SHRSP). We performed structural and functional analyses of these 2 genes in SHRSP from Glasgow colonies (SHRSPGla) and Wistar-Kyoto rats from Glasgow colonies (WKYGla) and developed a radiation hybrid map of the relevant region of rat chromosome 5. Sequencing of the coding regions of the Anp and Bnp genes revealed no difference between the 2 strains. Expression studies in brain tissue showed no differences at baseline and at 24 hours after middle cerebral artery occlusion. Plasma concentrations of atrial natriuretic peptide (ANP) did not differ between the SHRSPGla and WKYGla, whereas concentrations of brain natriuretic peptide were significantly higher in the SHRSPGla as compared with the WKYGla (n=11 to 14; 163+/-21 pg/mL and 78+/-14 pg/mL; 95% confidence interval 31 to 138, P=0.003). We did not detect any attenuation of endothelium-dependent relaxations to bradykinin or ANP in middle cerebral arteries from the SHRSPGla; indeed the sensitivity to ANP was significantly increased in arteries harvested from this strain (WKYGla: n=8; pD2=7. 3+/-0.2 and SHRSPGla: n=8; pD2=8.2+/-0.15; P<0.01). Moreover, radiation hybrid mapping and fluorescence in situ hybridization allowed us to map the Anf marker in the telomeric position of rat chromosome 5 in close proximity to D5Rat48, D5Rat47, D5Mgh15, and D5Mgh16. These results exclude Anp and Bnp as candidate genes for the sensitivity to brain ischemia and pave the way to further congenic and physical mapping strategies.

Bioactivity of growth hormone releasing hormone (1-29) analogues after SC injection in man.

The bioactivity of growth hormone releasing hormone 1-29 [GHRH(1-29)NH2] has been compared with that of an agonist analogue [Ac-D-Tyr1,D-Ala2]-GHRH(1-29)NH2, in normal male volunteers. Using a submaximal dose of 3 micrograms/kg administered subcutaneously, peak growth hormone (GH) response and area under the GH curve were similar for the native and agonist analogue. In addition, no significant differences were found in peak GHRH(1-29) immunoreactivity, area under the GHRH(1-29) curves or plasma disappearance rates of the two peptides. The results suggest that, in keeping with the relative activities of other "superactive" analogues tested so far, the greatly enhanced activity of [Ac-D-Tyr1,D-Ala2]-GHRH(1-29)NH2 observed in the rat is not found in humans. It is possible that this species difference is due to differences in the interaction of GHRH peptides with the rat and the human somatotroph GHRH receptor.

Genetic study of the CD36 gene in a French diabetic population.

OBJECTIVES: CD36 is a multifunctional membrane receptor widely expressed in different tissues which binds and internalizes oxidized low-density lipoprotein. In rodents, CD36 gene variations modulate glucose homeostasis and contribute to metabolic syndrome associated with type 2 diabetes but the effects in human are unknown. METHODS: We screened the entire coding sequence of the CD36 gene in 272 individuals and we genotyped both rare and frequent variants in 454 T2D subjects and 221 controls. RESULTS: We detected five mutations, P191P and N247S were only found each in one family and did not segregate with diabetes, the three others (A/C-178 in the promoter, A/G-10 in intron 3 and (GGGTTGAGA) insertion in intron 13) being equally frequent in diabetic subjects and in controls. However, adiponectin levels, a marker for insulin sensitivity, were significantly associated with the -178 A/C promoter variant allele (p=0.003, p corrected for multiple testing=0.036), possibly reflecting association with insulin-resistance in the French population. CONCLUSION: Thus, the -178 A/C SNP promoter mutation in the CD36 gene represents a putative genetic marker for insulin-resistance in the French population, although it does not appear to contribute to the genetic risk for T2D.

Serum IGF-I levels and growth failure in juvenile chronic arthritis.

The association between growth failure and serum IGF-I levels has been assessed in 32 children with Juvenile Chronic Arthritis (JCA) aged 5-16 years. A spectrum from normal growth to severe growth failure was included in the study population. Height Standard Deviation Score (SDS) ranged from -5.79 to +1.41 (median -1.22) and Height Velocity from 0.72-8.85 cm/yr (median 3.81 cm/yr). Known risk factors for growth failure (disease activity, steroid treatment, vertebral collapse) were confirmed. Additionally, height SDS was significantly correlated with serum IGF-I levels (rs = 0.49; p = 0.008); height velocity was significantly, although less strongly correlated with IGF-I levels (rs = 0.41; p = 0.027). There was no correlation between IGF-I levels and either of two indices of nutritional status, or between IGF-I levels and current steroid dose. The correlation of serum IGF-I with parameters of growth failure may be due to either insufficient secretion of growth hormone (GH) or defective GH action. In view of the recently increased availability of GH for treatment of short stature, it is important to distinguish between these two mechanisms.