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1.  Photoreceptor precursors derived from three-dimensional embryonic stem cell cultures integrate and mature within adult degenerate retina 
Nature biotechnology  2013;31(8):10.1038/nbt.2643.
Irreversible blindness caused by loss of photoreceptors may be amenable to cell therapy. We previously demonstrated retinal repair1 and restoration of vision through transplantation of photoreceptor precursors obtained from post-natal retinas into visually impaired adult mice2,3. Considerable progress has been made in differentiating embryonic stem cells (ESCs) in vitro toward photoreceptor lineages4-6. However, the capability of ESC-derived photoreceptors to integrate after transplantation has not been demonstrated unequivocally. Here, to isolate photoreceptor precursors fit for transplantation, we adapted a recently reported three-dimensional (3D) differentiation protocol that generates neuroretina from mouse ESCs6. We show that Rhop.GFP-selected rod precursors derived by this protocol integrate within degenerate retinae of adult mice and mature into outer segment–bearing photoreceptors. Notably, ESC-derived precursors at a developmental stage similar to postnatal days 4-8 integrate more efficiently than cells at other stages. This study shows conclusively that ESCs can provide a source of photoreceptors for retinal cell transplantation.
doi:10.1038/nbt.2643
PMCID: PMC3826328  PMID: 23873086
2.  Transcriptional Instability during Evolving Sepsis May Limit Biomarker Based Risk Stratification 
PLoS ONE  2013;8(3):e60501.
Background
Sepsis causes extensive morbidity and mortality in children worldwide. Prompt recognition and timely treatment of sepsis is critical in reducing morbidity and mortality. Genomic approaches are used to discover novel pathways, therapeutic targets and biomarkers. These may facilitate diagnosis and risk stratification to tailor treatment strategies.
Objective
To investigate the temporal gene expression during the evolution of sepsis induced multi-organ failure in response to a single organism, Neisseria meningitidis, in previously healthy children.
Method
RNA was extracted from serial blood samples (6 time points over 48 hours from presentation) from five critically ill children with meningococcal sepsis. Extracted RNA was hybridized to Affymetrix arrays. The RNA underwent strict quality control and standardized quantitation. Gene expression results were analyzed using GeneSpring software and Ingenuity Pathway Analysis.
Result
A marked variability in differential gene expression was observed between time points and between patients revealing dynamic expression changes during the evolution of sepsis. While there was evidence of time-dependent changes in expected gene networks including those involving immune responses and inflammatory pathways, temporal variation was also evident in specific “biomarkers” that have been proposed for diagnostic and risk stratification functions. The extent and nature of this variability was not readily explained by clinical phenotype.
Conclusion
This is the first study of its kind detailing extensive expression changes in children during the evolution of sepsis. This highlights a limitation of static or single time point biomarker estimation. Serial estimations or more comprehensive network approaches may be required to optimize risk stratification in complex, time-critical conditions such as evolving sepsis.
doi:10.1371/journal.pone.0060501
PMCID: PMC3609793  PMID: 23544148
3.  Characterisation and Validation of Insertions and Deletions in 173 Patient Exomes 
PLoS ONE  2012;7(12):e51292.
Recent advances in genomics technologies have spurred unprecedented efforts in genome and exome re-sequencing aiming to unravel the genetic component of rare and complex disorders. While in rare disorders this allowed the identification of novel causal genes, the missing heritability paradox in complex diseases remains so far elusive. Despite rapid advances of next-generation sequencing, both the technology and the analysis of the data it produces are in its infancy. At present there is abundant knowledge pertaining to the role of rare single nucleotide variants (SNVs) in rare disorders and of common SNVs in common disorders. Although the 1,000 genome project has clearly highlighted the prevalence of rare variants and more complex variants (e.g. insertions, deletions), their role in disease is as yet far from elucidated.
We set out to analyse the properties of sequence variants identified in a comprehensive collection of exome re-sequencing studies performed on samples from patients affected by a broad range of complex and rare diseases (N = 173). Given the known potential for Loss of Function (LoF) variants to be false positive, we performed an extensive validation of the common, rare and private LoF variants identified, which indicated that most of the private and rare variants identified were indeed true, while common novel variants had a significantly higher false positive rate. Our results indicated a strong enrichment of very low-frequency insertion/deletion variants, so far under-investigated, which might be difficult to capture with low coverage and imputation approaches and for which most of study designs would be under-powered. These insertions and deletions might play a significant role in disease genetics, contributing specifically to the underlining rare and private variation predicted to be discovered through next generation sequencing.
doi:10.1371/journal.pone.0051292
PMCID: PMC3522676  PMID: 23251486
4.  Epilepsy, Ataxia, Sensorineural Deafness, Tubulopathy, and KCNJ10 Mutations 
The New England Journal of Medicine  2009;360(19):1960-1970.
BACKGROUND
Five children from two consanguineous families presented with epilepsy beginning in infancy and severe ataxia, moderate sensorineural deafness, and a renal salt-losing tubulopathy with normotensive hypokalemic metabolic alkalosis. We investigated the genetic basis of this autosomal recessive disease, which we call the EAST syndrome (the presence of epilepsy, ataxia, sensorineural deafness, and tubulopathy).
METHODS
Whole-genome linkage analysis was performed in the four affected children in one of the families. Newly identified mutations in a potassium-channel gene were evaluated with the use of a heterologous expression system. Protein expression and function were further investigated in genetically modified mice.
RESULTS
Linkage analysis identified a single significant locus on chromosome 1q23.2 with a lod score of 4.98. This region contained the KCNJ10 gene, which encodes a potassium channel expressed in the brain, inner ear, and kidney. Sequencing of this candidate gene revealed homozygous missense mutations in affected persons in both families. These mutations, when expressed heterologously in xenopus oocytes, caused significant and specific decreases in potassium currents. Mice with Kcnj10 deletions became dehydrated, with definitive evidence of renal salt wasting.
CONCLUSIONS
Mutations in KCNJ10 cause a specific disorder, consisting of epilepsy, ataxia, sensorineural deafness, and tubulopathy. Our findings indicate that KCNJ10 plays a major role in renal salt handling and, hence, possibly also in blood-pressure maintenance and its regulation.
doi:10.1056/NEJMoa0810276
PMCID: PMC3398803  PMID: 19420365
5.  Global analysis of gene expression in NGF-deprived sympathetic neurons identifies molecular pathways associated with cell death 
BMC Genomics  2011;12:551.
Background
Developing sympathetic neurons depend on nerve growth factor (NGF) for survival and die by apoptosis after NGF withdrawal. This process requires de novo gene expression but only a small number of genes induced by NGF deprivation have been identified so far, either by a candidate gene approach or in mRNA differential display experiments. This is partly because it is difficult to obtain large numbers of sympathetic neurons for in vitro studies. Here, we describe for the first time, how advances in gene microarray technology have allowed us to investigate the expression of all known genes in sympathetic neurons cultured in the presence and absence of NGF.
Results
We have used Affymetrix Exon arrays to study the pattern of expression of all known genes in NGF-deprived sympathetic neurons. We identified 415 up- and 813 down-regulated genes, including most of the genes previously known to be regulated in this system. NGF withdrawal activates the mixed lineage kinase (MLK)-c-Jun N-terminal kinase (JNK)-c-Jun pathway which is required for NGF deprivation-induced death. By including a mixed lineage kinase (MLK) inhibitor, CEP-11004, in our experimental design we identified which of the genes induced after NGF withdrawal are potential targets of the MLK-JNK-c-Jun pathway. A detailed Gene Ontology and functional enrichment analysis also identified genetic pathways that are highly enriched and overrepresented amongst the genes expressed after NGF withdrawal. Five genes not previously studied in sympathetic neurons - trib3, ddit3, txnip, ndrg1 and mxi1 - were validated by real time-PCR. The proteins encoded by these genes also increased in level after NGF withdrawal and this increase was prevented by CEP-11004, suggesting that these genes are potential targets of the MLK-JNK-c-Jun pathway.
Conclusions
The sympathetic neuron model is one of the best studied models of neuronal apoptosis. Overall, our microarray data gives a comprehensive overview of, and provides new information about, signalling pathways and transcription factors that are regulated by NGF withdrawal.
doi:10.1186/1471-2164-12-551
PMCID: PMC3256215  PMID: 22067274
6.  Generation of novel pharmacogenomic candidates in the response to methotrexate in juvenile idiopathic arthritis: correlation between gene expression and genotype 
Pharmacogenetics and genomics  2010;20(11):665-676.
Objectives
Little is known about mechanisms of efficacy of methotrexate (MTX) in childhood arthritis, or genetic influences upon response to MTX. The aims of this study were to use gene expression profiling to identify novel pathways/genes altered by MTX and then investigate these genes for genotype associations with response to MTX treatment.
Methods
Gene expression profiling before and after MTX treatment was performed on 11 children with juvenile idiopathic arthritis (JIA) treated with MTX, in whom response at 6 months of treatment was defined. Genes showing the most differential gene expression after treatment were selected for SNP genotyping. Genotype frequencies were compared between non-responders and responders (ACR-Ped70). An independent cohort was available for validation.
Results
Gene expression profiling before and after MTX treatment revealed 1222 differentially expressed probes sets (fold change >1.7, p< 0.05) and 1065 when restricted to full responder cases only. Six highly differentially expressed genes were analysed for genetic association to response to MTX. Three SNPs in the SLC16A7 gene showed significant association with MTX response. One SNP showed validated association in an independent cohort.
Conclusions
This study is the first, to our knowledge, to evaluate gene expression profiles in children with JIA before and after MTX, and to analyse genetic variation in differentially expressed genes. We have identified a gene which may contribute to genetic variability in MTX response in JIA, and established as proof of principle that genes which are differentially expressed at mRNA level after drug administration may also be good candidates for genetic analysis.
doi:10.1097/FPC.0b013e32833f2cd0
PMCID: PMC2963015  PMID: 20827233
Methotrexate (MTX); pharmacogenomics; treatment; juvenile idiopathic arthritis (JIA); autoimmunity; transcriptional profiling; microarray
7.  Genome-wide analysis of alternative splicing in medulloblastoma identifies splicing patterns characteristic of normal cerebellar development 
Cancer research  2011;71(6):2045-2055.
Alternative splicing is an important mechanism for the generation of protein diversity at a post-transcriptional level. Modifications in the splicing patterns of several genes have been shown to contribute to the malignant transformation of different tissue types. In this study, we used the Affymetrix Exon arrays to investigate patterns of differential splicing between paediatric medulloblastomas and normal cerebellum on a genome-wide scale. Of the 1262 genes identified as potentially generating tumour-associated splice forms, we selected 14 examples of differential splicing of known cassette exons and successfully validated 11 of them by RT-PCR. The pattern of differential splicing of three validated events was characteristic for the molecular subset of Sonic Hedgehog (Shh)-driven medulloblastomas, suggesting that their unique gene signature includes the expression of distinctive transcript variants. Generally, we observed that tumour and normal fetal cerebellar samples shared significantly lower exon inclusion rates compared to normal adult cerebellum. We investigated whether tumour-associated splice forms were expressed in primary cultures of Shh-dependent mouse cerebellar granule cell precursors (GCPs) and found that Shh caused a decrease in the cassette exon inclusion rate of five out of the seven tested genes. Furthermore, we observed a significant increase in exon inclusion between post-natal days 7 and 14 of mouse cerebellar development, at the time when GCPs mature into post-mitotic neurons. We conclude that inappropriate splicing frequently occurs in human medulloblastomas and may be linked to the activation of developmental signalling pathways and a failure of cerebellar precursor cells to differentiate.
doi:10.1158/0008-5472.CAN-10-2519
PMCID: PMC3060131  PMID: 21248070
Medulloblastoma; alternative splicing; exon arrays; Sonic Hedgehog; Granule Cell Precursor
8.  ChIP-on-chip analysis reveals angiopoietin 2 (Ang2, ANGPT2) as a novel target of steroidogenic factor-1 (SF-1, NR5A1) in the human adrenal gland 
The FASEB Journal  2011;25(4):1166-1175.
The nuclear receptor steroidogenic factor-1 (SF-1, NR5A1) is a key regulator of adrenal and gonadal biology. Disruption of SF-1 can lead to disorders of adrenal development, while increased SF-1 dosage has been associated with adrenocortical tumorigenesis. We aimed to identify a novel subset of SF-1 target genes in the adrenal by using chromatin immunoprecipitation (ChIP) microarrays (ChIP-on-chip) combined with systems analysis. SF-1 ChIP-on-chip was performed in NCI-H295R human adrenocortical cells using promoter tiling arrays, leading to the identification of 445 gene loci where SF-1-binding regions were located from 10 kb upstream to 3 kb downstream of a transcriptional start. Network analysis of genes identified as putative SF-1 targets revealed enrichment for angiogenic process networks. A 1.1-kb SF-1-binding region was identified in the angiopoietin 2 (Ang2, ANGPT2) promoter in a highly repetitive region, and SF-1-dependent activation was confirmed in luciferase assays. Angiogenesis is paramount in adrenal development and tumorigenesis, but until now a direct link between SF-1 and vascular remodeling has not been established. We have identified Ang2 as a potentially important novel target of SF-1 in the adrenal gland, indicating that regulation of angiogenesis might be an important additional mechanism by which SF-1 exerts its actions in the adrenal gland.—Ferraz-de-Souza, B., Lin, L., Shah, S., Jina, N., Hubank, M., Dattani, M. T., Achermann, J. C. ChIP-on-chip analysis reveals angiopoietin 2 (Ang2, ANGPT2) as a novel target of steroidogenic factor-1 (SF-1, NR5A1) in the human adrenal gland.
doi:10.1096/fj.10-170522
PMCID: PMC3058709  PMID: 21163858
adrenal development; adrenal tumorigenesis; transcriptional regulation; angiogenesis; NCI-H295R adrenocortical cells
9.  Sterol O-Acyltransferase 1 (SOAT1, ACAT) Is a Novel Target of Steroidogenic Factor-1 (SF-1, NR5A1, Ad4BP) in the Human Adrenal 
We used up- and down-regulation of the nuclear receptor steroidogenic factor-1 (SF-1, NR5A1, Ad4BP) to identify new components of adrenal function and steroidogenesis.
Context:
Steroidogenic factor-1 (SF-1, NR5A1, Ad4BP) is a master regulator of adrenal development and steroidogenesis. Defects in several known targets of SF-1 can cause adrenal disorders in humans.
Objective:
We aimed to identify novel targets of SF-1 in the human adrenal. These factors could be important regulators of adrenal development and steroidogenesis and potential candidates for adrenal dysfunction.
Design:
A gene discovery strategy was developed based on bidirectional manipulation of SF-1. Overexpression or knockdown of SF-1 in NCI-H295R human adrenocortical cells was used to identify a subset of positively-regulated SF-1 targets.
Results:
This approach identified well-established SF-1 target genes (STAR, CYP11A) and several novel genes (VSNL1, ZIM2, PEG3, SOAT1, and MTSS1). Given its role in cholesterol metabolism, sterol O-acyltransferase 1 (SOAT1, previously referred to as acyl-Coenzyme A:cholesterol acyltransferase 1, ACAT) was studied further and found to be expressed in the developing human fetal adrenal cortex. We hypothesized that impaired SOAT1 activity could result in adrenal insufficiency through reduced cholesteryl ester reserves or through toxic destruction of the adrenal cells during development. Therefore, mutational analysis of SOAT1 in a cohort of 43 patients with unexplained adrenal insufficiency was performed but failed to reveal significant coding sequence changes.
Conclusions:
Our reverse discovery approach led to the identification of novel SF-1 targets and defined SOAT1 as an important factor in human adrenal steroidogenesis. SF-1–dependent up-regulation of SOAT1 may be important for maintaining readily-releasable cholesterol reserves needed for active steroidogenesis and during episodes of recurrent stress.
doi:10.1210/jc.2010-2021
PMCID: PMC3124353  PMID: 21239516
10.  New targets of urocortin-mediated cardioprotection 
The urocortin (UCN) hormones UCN1 and UCN2 have been shown previously to confer significant protection against myocardial ischaemia/reperfusion (I/R) injury; however, the molecular mechanisms underlying their action are poorly understood. To further define the transcriptional effect of UCNs that underpins their cardioprotective activity, a microarray analysis was carried out using an in vivo rat coronary occlusion model of I/R injury. Infusion of UCN1 or UCN2 before the onset of reperfusion resulted in the differential regulation of 66 and 141 genes respectively, the majority of which have not been described previously. Functional analysis demonstrated that UCN-regulated genes are involved in a wide range of biological responses, including cell death (e.g. X-linked inhibitor of apoptosis protein), oxidative stress (e.g. nuclear factor erythroid derived 2-related factor 1/nuclear factor erythroid derived 2-like 1) and metabolism (e.g. Prkaa2/AMPK). In addition, both UCN1 and UCN2 were found to modulate the expression of a host of genes involved in G-protein-coupled receptor (GPCR) signalling including Rac2, Gnb1, Dab2ip (AIP1), Ralgds, Rnd3, Rap1a and PKA, thereby revealing previously unrecognised signalling intermediates downstream of CRH receptors. Moreover, several of these GPCR-related genes have been shown previously to be involved in mitogen-activated protein kinase (MAPK) activation, suggesting a link between CRH receptors and induction of MAPKs. In addition, we have shown that both UCN1 and UCN2 significantly reduce free radical damage following myocardial infarction, and comparison of the UCN gene signatures with that of the anti-oxidant tempol revealed a significant overlap. These data uncover novel gene expression changes induced by UCNs, which will serve as a platform to further understand their mechanism of action in normal physiology and cardioprotection.
doi:10.1677/JME-09-0148
PMCID: PMC3069736  PMID: 20501665
11.  Prox1 maintains muscle structure and growth in the developing heart 
Development (Cambridge, England)  2008;136(3):495-505.
Impaired cardiac muscle growth and aberrant myocyte arrangement underlie congenital heart disease and cardiomyopathy. We show that cardiac-specific inactivation of the homeobox transcription factor Prox1 results in disruption of the expression and localisation of sarcomeric proteins, gross myofibril disarray and growth retarded hearts. Furthermore, we demonstrate that Prox1 is required for direct transcriptional regulation of structural proteins α-actinin, N-RAP and Zyxin which collectively function to maintain an actin-α-actinin interaction as the fundamental association of the sarcomere. Aspects of abnormal heart development and manifestation of a subset of muscular-based disease have previously been attributed to mutations in key structural proteins. Our study demonstrates an essential requirement for direct transcriptional regulation of sarcomere integrity, in the context of enabling fetal cardiomyocyte hypertrophy, maintenance of contractile function and progression towards inherited or acquired myopathic disease.
doi:10.1242/dev.030007
PMCID: PMC2655234  PMID: 19091769
Prox1; heart development; myocardium; sarcomere; hypertrophy; myopathy
12.  Coordinated oncogenic transformation and inhibition of host immune responses by the PAX3-FKHR fusion oncoprotein 
The Journal of Experimental Medicine  2005;202(10):1399-1410.
Tumors have evolved elaborate mechanisms for evading immune detection, such as production of immunoinhibitory cytokines and down-regulation of major histocompatibility complex (MHC) expression. We have studied PAX3-FKHR as an example of an oncogenic fusion protein associated with an aggressive metastatic cancer. We show that PAX3-FKHR alters expression of genes that are normally regulated by Janus kinase/signal transducer and activator of transcription (STAT) signaling pathways. This occurs as a result of a specific interaction between PAX3-FKHR and the STAT3 transcription factor, which results in a dramatic reduction in tumor MHC expression, and an alteration in local cytokine concentrations to inhibit surrounding inflammatory cells and immune detection. Collectively, these data show that an oncogenic transcription factor can promote tumor growth and tissue invasion while inhibiting local inflammatory and immune responses. This is the first time that an immunomodulatory role has been described for an oncogenic fusion protein.
doi:10.1084/jem.20050730
PMCID: PMC2212991  PMID: 16287709
13.  Microarray analysis of pediatric ependymoma identifies a cluster of 112 candidate genes including four transcripts at 22q12.1-q13.31, 2 
Neuro-Oncology  2005;7(1):20-31.
Ependymomas are glial cell–derived tumors characterized by varying degrees of chromosomal abnormalities and variability in clinical behavior. Cytogenetic analysis of pediatric ependymoma has failed to identify consistent patterns of abnormalities, with the exception of monosomy of 22 or structural abnormalities of 22q. In this study, a total of 19 pediatric ependymoma samples were used in a series of expression profiling, quantitative real-time PCR (Q-PCR), and loss of heterozygosity experiments to identify candidate genes involved in the development of this type of pediatric malignancy. Of the 12,627 genes analyzed, a subset of 112 genes emerged as being abnormally expressed when compared to three normal brain controls. Genes with increased expression included the oncogene WNT5A; the p53 homologue p63 and several cell cycle, cell adhesion, and proliferation genes. Underexpressed genes comprised the NF2 interacting gene SCHIP-1 and the adenomatous polyposis coli (APC)-associated gene EB1 among others. We validated the abnormal expression of six of these genes by Q-PCR. The subset of differentially expressed genes also included four underexpressed transcripts mapping to 22q12.3-13.3. By Q-PCR we show that one of these genes, CBX7 (22q13.1), was deleted in 55% of cases. Other genes mapping to cytogenetic hot spots included two overexpressed and three underexpressed genes mapping to 1q31-41 and 6q21-q24.3, respectively. These genes represent candidate genes involved in ependymoma tumorigenesis. To the authors’ knowledge, this is the first time microarray analysis and Q-PCR have been linked to identify heterozygous/homozygous deletions.
doi:10.1215/S1152851704000596
PMCID: PMC1871622  PMID: 15701279
14.  Effects of ADMA upon Gene Expression: An Insight into the Pathophysiological Significance of Raised Plasma ADMA 
PLoS Medicine  2005;2(10):e264.
Background
Asymmetric dimethylarginine (ADMA) is a naturally occurring inhibitor of nitric oxide synthesis that accumulates in a wide range of diseases associated with endothelial dysfunction and enhanced atherosclerosis. Clinical studies implicate plasma ADMA as a major novel cardiovascular risk factor, but the mechanisms by which low concentrations of ADMA produce adverse effects on the cardiovascular system are unclear.
Methods and Findings
We treated human coronary artery endothelial cells with pathophysiological concentrations of ADMA and assessed the effects on gene expression using U133A GeneChips (Affymetrix). Changes in several genes, including bone morphogenetic protein 2 inducible kinase (BMP2K), SMA-related protein 5 (Smad5), bone morphogenetic protein receptor 1A, and protein arginine methyltransferase 3 (PRMT3; also known as HRMT1L3), were confirmed by Northern blotting, quantitative PCR, and in some instances Western blotting analysis to detect changes in protein expression. To determine whether these changes also occurred in vivo, tissue from gene deletion mice with raised ADMA levels was examined. More than 50 genes were significantly altered in endothelial cells after treatment with pathophysiological concentrations of ADMA (2 μM). We detected specific patterns of changes that identify pathways involved in processes relevant to cardiovascular risk and pulmonary hypertension. Changes in BMP2K and PRMT3 were confirmed at mRNA and protein levels, in vitro and in vivo.
Conclusion
Pathophysiological concentrations of ADMA are sufficient to elicit significant changes in coronary artery endothelial cell gene expression. Changes in bone morphogenetic protein signalling, and in enzymes involved in arginine methylation, may be particularly relevant to understanding the pathophysiological significance of raised ADMA levels. This study identifies the mechanisms by which increased ADMA may contribute to common cardiovascular diseases and thereby indicates possible targets for therapies.
Pathophysiological concentrations of asymmetric dimethylarginine elicit significant changes in coronary artery endothelial cell gene expression and highlight specific molecular pathways for further investigation.
doi:10.1371/journal.pmed.0020264
PMCID: PMC1240048  PMID: 16190779
15.  Biologic predictors of extension of oligoarticular juvenile idiopathic arthritis as determined from synovial fluid cellular composition and gene expression 
Arthritis and Rheumatism  2010;62(3):896-907.
Objective
To identify biomarkers in the first synovial fluid (SF) aspirate obtained from children with oligoarticular juvenile idiopathic arthritis (JIA), which could be used to identify children whose disease is likely to extend to a more severe phenotype.
Methods
Patients with recent-onset oligoarticular JIA were identified and grouped according to those whose mild disease persisted (persistent disease) or those whose disease would extend from a mild to more severe phenotype (extended-to-be disease) at 1 year after diagnosis. Flow cytometry was used to delineate differences in the mononuclear cell populations between the first blood sample and first SF aspirate from the same patient and between outcome (persistent versus extended-to-be) groups. Proportions of lymphocytes in the joint were modeled on chemotaxis of lymphocytes to CCL5, using Transwell migration assays. Levels of CCL5 in the SF were quantified by enzyme-linked immunosorbent assay. RNA profiles of SF mononuclear cells were compared between groups using the Affymetrix GeneChip hybridization protocol and hierarchical clustering analyses.
Results
Compared with peripheral blood mononuclear cells, SF mononuclear cells displayed an expansion of CD8+ T cells, reduced proportion of B cells, and expansion of CD16− natural killer cells. The lower CD4:CD8 ratio in the SF was recapitulated in vitro by the observed migration of blood T cells in response to CCL5. Synovial CCL5 levels were higher in children whose disease extended to a more severe phenotype. The CD4:CD8 ratio in the SF was significantly lower in patients with extended-to-be oligoarticular JIA (0.57 compared with 0.90 in the persistent disease group, difference 0.33, 95% confidence interval 0.04–0.62; P = 0.009). Gene expression profiling revealed that 344 genes were >1.5-fold differentially expressed between outcome groups (P < 0.05), and these included genes associated with inflammation and macrophage differentiation, which showed increased levels in patients with extended disease at 1 year, and genes associated with immune regulation, which showed increased levels in patients with persistent disease at 1 year.
Conclusion
Analyses of the proportions of synovial lymphocytes, levels of CCL5, and differential gene expression yielded potential biomarkers with which to predict the likelihood of extension of oligoarticular JIA to a more severe disease phenotype.
doi:10.1002/art.27284
PMCID: PMC2860766  PMID: 20127724
16.  Genetic complexity in hypertrophic cardiomyopathy revealed by high-throughput sequencing 
Journal of Medical Genetics  2013;50(4):228-239.
Background
Clinical interpretation of the large number of rare variants identified by high throughput sequencing (HTS) technologies is challenging. The aim of this study was to explore the clinical implications of a HTS strategy for patients with hypertrophic cardiomyopathy (HCM) using a targeted HTS methodology and workflow developed for patients with a range of inherited cardiovascular diseases. By comparing the sequencing results with published findings and with sequence data from a large-scale exome sequencing screen of UK individuals, we sought to quantify the strength of the evidence supporting causality for detected candidate variants.
Methods and results
223 unrelated patients with HCM (46±15 years at diagnosis, 74% males) were studied. In order to analyse coding, intronic and regulatory regions of 41 cardiovascular genes, we used solution-based sequence capture followed by massive parallel resequencing on Illumina GAIIx. Average read-depth in the 2.1 Mb target region was 120. Rare (frequency<0.5%) non-synonymous, loss-of-function and splice-site variants were defined as candidates. Excluding titin, we identified 152 distinct candidate variants in sarcomeric or associated genes (89 novel) in 143 patients (64%). Four sarcomeric genes (MYH7, MYBPC3, TNNI3, TNNT2) showed an excess of rare single non-synonymous single-nucleotide polymorphisms (nsSNPs) in cases compared to controls. The estimated probability that a nsSNP in these genes is pathogenic varied between 57% and near certainty depending on the location. We detected an additional 94 candidate variants (73 novel) in desmosomal, and ion-channel genes in 96 patients (43%).
Conclusions
This study provides the first large-scale quantitative analysis of the prevalence of sarcomere protein gene variants in patients with HCM using HTS technology. Inclusion of other genes implicated in inherited cardiac disease identifies a large number of non-synonymous rare variants of unknown clinical significance.
doi:10.1136/jmedgenet-2012-101270
PMCID: PMC3607113  PMID: 23396983
Hypertrophic Cardiomyopathy; Genetics; High-throughput sequencing
17.  Albuminuria is associated with too few glomeruli and too much testosterone 
Kidney International  2013;83(6):1118-1129.
Normally, the glomerular filtration barrier almost completely excludes circulating albumin from entering the urine. Genetic variation and both pre- and postnatal environmental factors may affect albuminuria in humans. Here we determine whether glomerular gene expression in mouse strains with naturally occurring variations in albuminuria would allow identification of proteins deregulated in relatively ‘leaky' glomeruli. Albuminuria increased in female B6 to male B6 to female FVB/N to male FVB/N mice, whereas the number of glomeruli/kidney was the exact opposite. Testosterone administration led to increased albuminuria in female B6 but not female FVB/N mice. A common set of 39 genes, many expressed in podocytes, were significantly differentially expressed in each of the four comparisons: male versus female B6 mice, male versus female FVB/N mice, male FVB/N versus male B6 mice, and female FVB/N versus female B6 mice. The transcripts encoded proteins involved in oxidation/reduction reactions, ion transport, and enzymes involved in detoxification. These proteins may represent novel biomarkers and even therapeutic targets for early kidney and cardiovascular disease.
doi:10.1038/ki.2013.45
PMCID: PMC3674403  PMID: 23447063
albuminuria; gender difference; glomerulus; nephron number; podocyte

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