Publications by authors named "John P Carulli"

24 Publications

  • Page 1 of 1

Discovery and Characterization of SY-1365, a Selective, Covalent Inhibitor of CDK7.

Cancer Res 2019 07 7;79(13):3479-3491. Epub 2019 May 7.

Syros Pharmaceuticals, Inc., Cambridge, Massachusetts.

Recent studies suggest that targeting transcriptional machinery can lead to potent and selective anticancer effects in cancers dependent on high and constant expression of certain transcription factors for growth and survival. Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the CDK-activating kinase complex. Its function is required for both cell-cycle regulation and transcriptional control of gene expression. CDK7 has recently emerged as an attractive cancer target because its inhibition leads to decreased transcript levels of oncogenic transcription factors, especially those associated with super-enhancers. Here, we describe a selective CDK7 inhibitor SY-1365, which is currently in clinical trials in populations of patients with ovarian and breast cancer (NCT03134638). , SY-1365 inhibited cell growth of many different cancer types at nanomolar concentrations. SY-1365 treatment decreased MCL1 protein levels, and cancer cells with low BCL2L1 (BCL-XL) expression were found to be more sensitive to SY-1365. Transcriptional changes in acute myeloid leukemia (AML) cell lines were distinct from those following treatment with other transcriptional inhibitors. SY-1365 demonstrated substantial antitumor effects in multiple AML xenograft models as a single agent; SY-1365-induced growth inhibition was enhanced in combination with the BCL2 inhibitor venetoclax. Antitumor activity was also observed in xenograft models of ovarian cancer, suggesting the potential for exploring SY-1365 in the clinic in both hematologic and solid tumors. Our findings support targeting CDK7 as a new approach for treating transcriptionally addicted cancers. SIGNIFICANCE: These findings demonstrate the molecular mechanism of action and potent antitumor activity of SY-1365, the first selective CDK7 inhibitor to enter clinical investigation.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-0119DOI Listing
July 2019

A whole-genome sequence study identifies genetic risk factors for neuromyelitis optica.

Nat Commun 2018 05 16;9(1):1929. Epub 2018 May 16.

Program in Medical and Population Genetics, Broad Institute of Harvard and MIT, Cambridge, MA, 02142, USA.

Neuromyelitis optica (NMO) is a rare autoimmune disease that affects the optic nerve and spinal cord. Most NMO patients ( > 70%) are seropositive for circulating autoantibodies against aquaporin 4 (NMO-IgG+). Here, we meta-analyze whole-genome sequences from 86 NMO cases and 460 controls with genome-wide SNP array from 129 NMO cases and 784 controls to test for association with SNPs and copy number variation (total N = 215 NMO cases, 1244 controls). We identify two independent signals in the major histocompatibility complex (MHC) region associated with NMO-IgG+, one of which may be explained by structural variation in the complement component 4 genes. Mendelian Randomization analysis reveals a significant causal effect of known systemic lupus erythematosus (SLE), but not multiple sclerosis (MS), risk variants in NMO-IgG+. Our results suggest that genetic variants in the MHC region contribute to the etiology of NMO-IgG+ and that NMO-IgG+ is genetically more similar to SLE than MS.
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http://dx.doi.org/10.1038/s41467-018-04332-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955905PMC
May 2018

SMN deficiency in severe models of spinal muscular atrophy causes widespread intron retention and DNA damage.

Proc Natl Acad Sci U S A 2017 03 7;114(12):E2347-E2356. Epub 2017 Mar 7.

Rare Disease, Biogen, Cambridge, MA 02142

Spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease, is the leading monogenic cause of infant mortality. Homozygous loss of the gene survival of motor neuron 1 () causes the selective degeneration of lower motor neurons and subsequent atrophy of proximal skeletal muscles. The protein product, survival of motor neuron (SMN), is ubiquitously expressed and is a key factor in the assembly of the core splicing machinery. The molecular mechanisms by which disruption of the broad functions of SMN leads to neurodegeneration remain unclear. We used an antisense oligonucleotide (ASO)-based inducible mouse model of SMA to investigate the SMN-specific transcriptome changes associated with neurodegeneration. We found evidence of widespread intron retention, particularly of minor U12 introns, in the spinal cord of mice 30 d after SMA induction, which was then rescued by a therapeutic ASO. Intron retention was concomitant with a strong induction of the p53 pathway and DNA damage response, manifesting as γ-H2A.X positivity in neurons of the spinal cord and brain. Widespread intron retention and markers of the DNA damage response were also observed with SMN depletion in human SH-SY5Y neuroblastoma cells and human induced pluripotent stem cell-derived motor neurons. We also found that retained introns, high in GC content, served as substrates for the formation of transcriptional R-loops. We propose that defects in intron removal in SMA promote DNA damage in part through the formation of RNA:DNA hybrid structures, leading to motor neuron death.
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http://dx.doi.org/10.1073/pnas.1613181114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373344PMC
March 2017

JC Polyomavirus Abundance and Distribution in Progressive Multifocal Leukoencephalopathy (PML) Brain Tissue Implicates Myelin Sheath in Intracerebral Dissemination of Infection.

PLoS One 2016 18;11(5):e0155897. Epub 2016 May 18.

Neurology, Biogen Inc, Cambridge, MA, United States of America.

Over half of adults are seropositive for JC polyomavirus (JCV), but rare individuals develop progressive multifocal leukoencephalopathy (PML), a demyelinating JCV infection of the central nervous system. Previously, PML was primarily seen in immunosuppressed patients with AIDS or certain cancers, but it has recently emerged as a drug safety issue through its association with diverse immunomodulatory therapies. To better understand the relationship between the JCV life cycle and PML pathology, we studied autopsy brain tissue from a 70-year-old psoriasis patient on the integrin alpha-L inhibitor efalizumab following a ~2 month clinical course of PML. Sequence analysis of lesional brain tissue identified PML-associated viral mutations in regulatory (non-coding control region) DNA, capsid protein VP1, and the regulatory agnoprotein, as well as 9 novel mutations in capsid protein VP2, indicating rampant viral evolution. Nine samples, including three gross PML lesions and normal-appearing adjacent tissues, were characterized by histopathology and subject to quantitative genomic, proteomic, and molecular localization analyses. We observed a striking correlation between the spatial extent of demyelination, axonal destruction, and dispersion of JCV along white matter myelin sheath. Our observations in this case, as well as in a case of PML-like disease in an immunocompromised rhesus macaque, suggest that long-range spread of polyomavirus and axonal destruction in PML might involve extracellular association between virus and the white matter myelin sheath.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0155897PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4871437PMC
July 2017

Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways.

Science 2015 Mar 19;347(6229):1436-41. Epub 2015 Feb 19.

Department of Basic and Clinical Neuroscience, King's College London, Institute of Psychiatry, Psychology and Neuroscience, London SE5 8AF, UK.

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment. We report the results of a moderate-scale sequencing study aimed at increasing the number of genes known to contribute to predisposition for ALS. We performed whole-exome sequencing of 2869 ALS patients and 6405 controls. Several known ALS genes were found to be associated, and TBK1 (the gene encoding TANK-binding kinase 1) was identified as an ALS gene. TBK1 is known to bind to and phosphorylate a number of proteins involved in innate immunity and autophagy, including optineurin (OPTN) and p62 (SQSTM1/sequestosome), both of which have also been implicated in ALS. These observations reveal a key role of the autophagic pathway in ALS and suggest specific targets for therapeutic intervention.
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http://dx.doi.org/10.1126/science.aaa3650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4437632PMC
March 2015

Rescue of gene-expression changes in an induced mouse model of spinal muscular atrophy by an antisense oligonucleotide that promotes inclusion of SMN2 exon 7.

Genomics 2015 Apr 31;105(4):220-8. Epub 2015 Jan 31.

Division of Genetics and Genomics, Biogen Idec, 12 Cambridge Center, Cambridge, MA 02142, USA. Electronic address:

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by disruption of the survival motor neuron 1 (SMN1) gene, partly compensated for by the paralogous gene SMN2. Exon 7 inclusion is critical for full-length SMN protein production and occurs at a much lower frequency for SMN2 than for SMN1. Antisense oligonucleotide (ASO)-mediated blockade of an intron 7 splicing silencer was previously shown to promote inclusion of SMN2 exon 7 in SMA mouse models and mediate phenotypic rescue. However, downstream molecular consequences of this ASO therapy have not been defined. Here we characterize the gene-expression changes that occur in an induced model of SMA and show substantial rescue of those changes in central nervous system tissue upon intracerebroventricular administration of an ASO that promotes inclusion of exon 7, with earlier administration promoting greater rescue. This study offers a robust reference set of preclinical pharmacodynamic gene expression effects for comparison of other investigational therapies for SMA.
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http://dx.doi.org/10.1016/j.ygeno.2015.01.007DOI Listing
April 2015

Newborn blood spot screening test using multiplexed real-time PCR to simultaneously screen for spinal muscular atrophy and severe combined immunodeficiency.

Clin Chem 2015 Feb 11;61(2):412-9. Epub 2014 Dec 11.

Newborn Screening and Molecular Biology Branch, Division of Laboratory Sciences, Centers for Disease Control and Prevention, Atlanta, GA;

Background: Spinal muscular atrophy (SMA) is a motor neuron disorder caused by the absence of a functional survival of motor neuron 1, telomeric (SMN1) gene. Type I SMA, a lethal disease of infancy, accounts for the majority of cases. Newborn blood spot screening (NBS) to detect severe combined immunodeficiency (SCID) has been implemented in public health laboratories in the last 5 years. SCID detection is based on real-time PCR assays to measure T-cell receptor excision circles (TREC), a byproduct of T-cell development. We modified a multiplexed real-time PCR TREC assay to simultaneously determine the presence or absence of the SMN1 gene from a dried blood spot (DBS) punch in a single reaction well.

Method: An SMN1 assay using a locked nucleic acid probe was initially developed with cell culture and umbilical cord blood (UCB) DNA extracts, and then integrated into the TREC assay. DBS punches were placed in 96-well arrays, washed, and amplified directly using reagents specific for TREC, a reference gene [ribonuclease P/MRP 30kDa subunit (RPP30)], and the SMN1 gene. The assay was tested on DBS made from UCB units and from peripheral blood samples of SMA-affected individuals and their family members.

Results: DBS made from SMA-affected individuals showed no SMN1-specific amplification, whereas DBS made from all unaffected carriers and UCB showed SMN1 amplification above a well-defined threshold. TREC and RPP30 content in all DBS were within the age-adjusted expected range.

Conclusions: SMA caused by the absence of SMN1 can be detected from the same DBS punch used to screen newborns for SCID.
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http://dx.doi.org/10.1373/clinchem.2014.231019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906865PMC
February 2015

Whole Genome Sequencing Reveals a Chromosome 9p Deletion Causing DOCK8 Deficiency in an Adult Diagnosed with Hyper IgE Syndrome Who Developed Progressive Multifocal Leukoencephalopathy.

J Clin Immunol 2015 Jan 12;35(1):92-6. Epub 2014 Nov 12.

Translational Sciences, Biogen Idec, Cambridge, MA, USA.

Purpose: A 30 year-old man with a history of recurrent skin infections as well as elevated serum IgE and eosinophils developed neurological symptoms and had T2-hyperintense lesions observed in cerebral MRI. The immune symptoms were attributed to Hyper IgE syndrome (HIES) and the neurological symptoms with presence of JC virus in cerebrospinal fluid were diagnosed as Progressive Multifocal Leukoencephalopathy (PML). The patient was negative for STAT3 mutations. To determine if other mutations explain HIES and/or PML in this subject, his DNA was analyzed by whole genome sequencing.

Methods: Whole genome sequencing was completed to 30X coverage, and whole genome SNP typing was used to complement these data. The methods revealed single nucleotide variants, structural variants, and copy number variants across the genome. Genome-wide data were analyzed for homozygous or compound heterozygous null mutations for all protein coding genes. Mutations were confirmed by PCR and/or Sanger sequencing.

Results: Whole genome analysis revealed deletions near the telomere of both copies of chromosome 9p. Several genes, including DOCK8, were impacted by the deletions but it was unclear whether each chromosome had identical or distinct deletions. PCR across the impacted region combined with Sanger sequencing of selected fragments confirmed a homozygous deletion from position 10,211 to 586,751.

Conclusion: While several genes are impacted by the deletion, DOCK8 deficiency is the most probable cause of HIES in this patient. DOCK8 deficiency may have also predisposed the patient to develop PML.
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http://dx.doi.org/10.1007/s10875-014-0114-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4306731PMC
January 2015

Development of a multi-biomarker disease activity test for rheumatoid arthritis.

PLoS One 2013 9;8(4):e60635. Epub 2013 Apr 9.

Arthritis and Immunology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States of America.

Background: Disease activity measurement is a key component of rheumatoid arthritis (RA) management. Biomarkers that capture the complex and heterogeneous biology of RA have the potential to complement clinical disease activity assessment.

Objectives: To develop a multi-biomarker disease activity (MBDA) test for rheumatoid arthritis.

Methods: Candidate serum protein biomarkers were selected from extensive literature screens, bioinformatics databases, mRNA expression and protein microarray data. Quantitative assays were identified and optimized for measuring candidate biomarkers in RA patient sera. Biomarkers with qualifying assays were prioritized in a series of studies based on their correlations to RA clinical disease activity (e.g. the Disease Activity Score 28-C-Reactive Protein [DAS28-CRP], a validated metric commonly used in clinical trials) and their contributions to multivariate models. Prioritized biomarkers were used to train an algorithm to measure disease activity, assessed by correlation to DAS and area under the receiver operating characteristic curve for classification of low vs. moderate/high disease activity. The effect of comorbidities on the MBDA score was evaluated using linear models with adjustment for multiple hypothesis testing.

Results: 130 candidate biomarkers were tested in feasibility studies and 25 were selected for algorithm training. Multi-biomarker statistical models outperformed individual biomarkers at estimating disease activity. Biomarker-based scores were significantly correlated with DAS28-CRP and could discriminate patients with low vs. moderate/high clinical disease activity. Such scores were also able to track changes in DAS28-CRP and were significantly associated with both joint inflammation measured by ultrasound and damage progression measured by radiography. The final MBDA algorithm uses 12 biomarkers to generate an MBDA score between 1 and 100. No significant effects on the MBDA score were found for common comorbidities.

Conclusion: We followed a stepwise approach to develop a quantitative serum-based measure of RA disease activity, based on 12-biomarkers, which was consistently associated with clinical disease activity levels.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060635PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3621826PMC
October 2013

Sequencing and analysis of JC virus DNA from natalizumab-treated PML patients.

J Infect Dis 2011 Jul;204(2):237-44

Biogen Idec Inc., Cambridge, MA 02142, USA.

Background: Progressive multifocal leukoencephalopathy (PML) in natalizumab-treated MS patients is linked to JC virus (JCV) infection. JCV sequence variation and rearrangements influence viral pathogenicity and tropism. To better understand PML development, we analyzed viral DNA sequences in blood, CSF and/or urine of natalizumab-treated PML patients.

Methods: Using biofluid samples from 17 natalizumab-treated PML patients, we sequenced multiple isolates of the JCV noncoding control region (NCCR), VP1 capsid coding region, and the entire 5 kb viral genome.

Results: Analysis of JCV from multiple biofluids revealed that individuals were infected with a single genotype. Across our patient cohort, multiple PML-associated NCCR rearrangements and VP1 mutations were present in CSF and blood, but absent from urine-derived virus. NCCR rearrangements occurred in CSF of 100% of our cohort. VP1 mutations were observed in blood or CSF in 81% of patients. Sequencing of complete JCV genomes demonstrated that NCCR rearrangements could occur without VP1 mutations, but VP1 mutations were not observed without NCCR rearrangement.

Conclusions: These data confirm that JCV in natalizumab-PML patients is similar to that observed in other PML patient groups, multiple genotypes are associated with PML, individual patients appear to be infected with a single genotype, and PML-associated mutations arise in patients during PML development.
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http://dx.doi.org/10.1093/infdis/jir256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3114470PMC
July 2011

Progressive multifocal leukoencephalopathy (PML) development is associated with mutations in JC virus capsid protein VP1 that change its receptor specificity.

J Infect Dis 2011 Jul;204(1):103-14

Biogen IDEC Inc., Cambridge, MA, USA.

Progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease caused by JC virus (JCV) infection of oligodendrocytes, may develop in patients with immune disorders following reactivation of chronic benign infection. Mutations of JCV capsid viral protein 1 (VP1), the capsid protein involved in binding to sialic acid cell receptors, might favor PML onset. Cerebrospinal fluid sequences from 37/40 PML patients contained one of several JCV VP1 amino acid mutations, which were also present in paired plasma but not urine sequences despite the same viral genetic background. VP1-derived virus-like particles (VLPs) carrying these mutations lost hemagglutination ability, showed different ganglioside specificity, and abolished binding to different peripheral cell types compared with wild-type VLPs. However, mutants still bound brain-derived cells, and binding was not affected by sialic acid removal by neuraminidase. JCV VP1 substitutions are acquired intrapatient and might favor JCV brain invasion through abrogation of sialic acid binding with peripheral cells, while maintaining sialic acid-independent binding with brain cells.
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http://dx.doi.org/10.1093/infdis/jir198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3307153PMC
July 2011

A genome-wide screen of gene-gene interactions for rheumatoid arthritis susceptibility.

Hum Genet 2011 May 6;129(5):473-85. Epub 2011 Jan 6.

Center for Population Studies and the Framingham Heart Study, National Heart, Lung, and Blood Institute/NIH, 73 Mt. Wayte Avenue, Framingham, MA 01702, USA.

The objective of the study was to identify interacting genes contributing to rheumatoid arthritis (RA) susceptibility and identify SNPs that discriminate between RA patients who were anti-cyclic citrullinated protein positive and healthy controls. We analyzed two independent cohorts from the North American Rheumatoid Arthritis Consortium. A cohort of 908 RA cases and 1,260 controls was used to discover pairwise interactions among SNPs and to identify a set of single nucleotide polymorphisms (SNPs) that predict RA status, and a second cohort of 952 cases and 1,760 controls was used to validate the findings. After adjusting for HLA-shared epitope alleles, we identified and replicated seven SNP pairs within the HLA class II locus with significant interaction effects. We failed to replicate significant pairwise interactions among non-HLA SNPs. The machine learning approach "random forest" applied to a set of SNPs selected from single-SNP and pairwise interaction tests identified 93 SNPs that distinguish RA cases from controls with 70% accuracy. HLA SNPs provide the most classification information, and inclusion of non-HLA SNPs improved classification. While specific gene-gene interactions are difficult to validate using genome-wide SNP data, a stepwise approach combining association and classification methods identifies candidate interacting SNPs that distinguish RA cases from healthy controls.
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http://dx.doi.org/10.1007/s00439-010-0943-zDOI Listing
May 2011

Convergent Random Forest predictor: methodology for predicting drug response from genome-scale data applied to anti-TNF response.

Genomics 2009 Dec 20;94(6):423-32. Epub 2009 Aug 20.

Biogen IDEC 14 Cambridge Ctr, Cambridge, MA 02142, USA.

Biomarker development for prediction of patient response to therapy is one of the goals of molecular profiling of human tissues. Due to the large number of transcripts, relatively limited number of samples, and high variability of data, identification of predictive biomarkers is a challenge for data analysis. Furthermore, many genes may be responsible for drug response differences, but often only a few are sufficient for accurate prediction. Here we present an analysis approach, the Convergent Random Forest (CRF) method, for the identification of highly predictive biomarkers. The aim is to select from genome-wide expression data a small number of non-redundant biomarkers that could be developed into a simple and robust diagnostic tool. Our method combines the Random Forest classifier and gene expression clustering to rank and select a small number of predictive genes. We evaluated the CRF approach by analyzing four different data sets. The first set contains transcript profiles of whole blood from rheumatoid arthritis patients, collected before anti-TNF treatment, and their subsequent response to the therapy. In this set, CRF identified 8 transcripts predicting response to therapy with 89% accuracy. We also applied the CRF to the analysis of three previously published expression data sets. For all sets, we have compared the CRF and recursive support vector machines (RSVM) approaches to feature selection and classification. In all cases the CRF selects much smaller number of features, five to eight genes, while achieving similar or better performance on both training and independent testing sets of data. For both methods performance estimates using cross-validation is similar to performance on independent samples. The method has been implemented in R and is available from the authors upon request: [email protected]
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http://dx.doi.org/10.1016/j.ygeno.2009.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476397PMC
December 2009

CRIPTO3, a presumed pseudogene, is expressed in cancer.

Biochem Biophys Res Commun 2008 Dec 1;377(1):215-20. Epub 2008 Oct 1.

Department of Drug Discovery, Biogen Idec Inc, 14 Cambridge Center, Cambridge, MA 02142, USA.

Cripto is a cell surface protein highly expressed in certain solid tumors, and overexpression of Cripto protein is oncogenic. Cripto-1 protein is encoded by CRIPTO1 gene. CRIPTO3, a presumed pseudogene, has an open reading frame with six amino acid differences from Cripto-1. We show that CRIPTO3 mRNA is the CRIPTO message expressed in many cancer samples. A CRIPTO3 SAGE tag was found in several cancer SAGE libraries, while the CRIPTO1 tag was found in ES cell libraries. In vitro experiments indicate both Cripto-1 and Cripto-3 proteins are functional in the Nodal-dependent signal pathway. Our data indicate that CRIPTO3 is an expressed gene, particularly in certain cancers, and suggest a potentially novel mechanism of oncogenesis through activation of a retrogene.
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http://dx.doi.org/10.1016/j.bbrc.2008.09.113DOI Listing
December 2008

Experimental comparison and cross-validation of Affymetrix HT plate and cartridge array gene expression platforms.

Genomics 2008 Nov 17;92(5):359-65. Epub 2008 Sep 17.

Biogen Idec, Inc., 14 Cambridge Center, Cambridge, MA 02142, USA.

The successful use of gene expression microarrays in basic research studies has spawned interest in the use of this technology for clinical trial and population-based studies, but cost, complexity of sample processing and tracking, and limitations of sample throughput have restricted their use for these very large-scale investigations. The Affymetrix GeneChip Plate Array System addresses these concerns and could facilitate larger studies if the data prove to be comparable to industry-standard cartridge arrays. Here we present a comparative evaluation of performance between Affymetrix GeneChip Human 133A cartridge and plate arrays with an emphasis on the assessment of systematic variation and its impact on log ratio data. This study utilized two standardized control RNAs on four independent lots of plate and cartridge arrays. We found that HT plate arrays showed improved specificity and were more reproducible over a wide intensity range, but cartridge arrays exhibit better sensitivity. Not surprisingly, artifactual changes due to positional effects were detectable on plate arrays, but were generally small in number and magnitude and in practice may be removed using standard fold-change and p-value thresholds. Overall, log ratio data between cartridges and plate arrays were remarkably concordant. We conclude that HT arrays offer significant improvements over cartridge arrays for large-scale studies.
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http://dx.doi.org/10.1016/j.ygeno.2008.06.010DOI Listing
November 2008

Genome-wide association scan identifies candidate polymorphisms associated with differential response to anti-TNF treatment in rheumatoid arthritis.

Mol Med 2008 Sep-Oct;14(9-10):575-81

Biogen Idec Inc., Cambridge, Massachusetts, USA.

The prediction of response (or non-response) to anti-TNF treatment for rheumatoid arthritis (RA) is a pressing clinical problem. We conducted a genome-wide association study using the Illumina HapMap300 SNP chip on 89 RA patients prospectively followed after beginning anti-TNF therapy as part of Autoimmune Biomarkers Collaborative Network (ABCoN [Autoimmune Bio-markers Collaborative Network]) patient cohort. Response to therapy was determined by the change in Disease Activity Score (DAS28) observed after 14 wks. We used a two-part analysis that treated the change in DAS28 as a continuous trait and then incorporated it into a dichotomous trait of "good responder" and "nonresponder" by European League Against Rheumatism (EULAR) criteria. We corrected for multiple tests by permutation, and adjusted for potential population stratification using EIGENSTRAT. Multiple single nucleotide polymorphism (SNP) markers showed significant associations near or within loci including: the v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) gene on chromosome 20; the type I interferon gene IFNk on chromosome 9; and in a locus on chromosome 7 that includes the paraoxonase I (PON1) gene. An SNP in the IL10 promoter (rs1800896) that was previously reported as associated with anti-TNF response was weakly associated with response in this cohort. Replications of these results in independent and larger data sets clearly are required. We provide a reference list of candidate SNPs (P < 0.01) that can be investigated in future pharmacogenomic studies.
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http://dx.doi.org/10.2119/2008-00056.LiuDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2276142PMC
November 2008

Several regions in the major histocompatibility complex confer risk for anti-CCP-antibody positive rheumatoid arthritis, independent of the DRB1 locus.

Mol Med 2008 May-Jun;14(5-6):293-300

The Feinstein Institute for Medical Research, North Shore LIJ Health System, Manhasset, New York 11030, United States of America.

Recent evidence suggests that additional risk loci for RA are present in the major histocompatibility complex (MHC), independent of the class II HLA-DRB1 locus. We have now tested a total of 1,769 SNPs across 7.5Mb of the MHC located from 6p22.2 (26.03 Mb) to 6p21.32 (33.59 Mb) derived from the Illumina 550K Beadchip (Illumina, San Diego, CA, USA). For an initial analysis in the whole dataset (869 RA CCP + cases, 1,193 controls), the strongest association signal was observed in markers near the HLA-DRB1 locus, with additional evidence for association extending out into the Class I HLA region. To avoid confounding that may arise due to linkage disequilibrium with DRB1 alleles, we analyzed a subset of the data by matching cases and controls by DRB1 genotype (both alleles matched 1:1), yielding a set of 372 cases with 372 controls. This analysis revealed the presence of at least two regions of association with RA in the Class I region, independent of DRB1 genotype. SNP alleles found on the conserved A1-B8-DR3 (8.1) haplotype show the strongest evidence of positive association (P ~ 0.00005) clustered in the region around the HLA-C locus. In addition, we identified risk alleles that are not present on the 8.1 haplotype, with maximal association signals (P ~ 0.001-0.0027) located near the ZNF311 locus. This latter association is enriched in DRB1*0404 individuals. Finally, several additional association signals were found in the extreme centromeric portion of the MHC, in regions containing the DOB1, TAP2, DPB1, and COL11A2 genes. These data emphasize that further analysis of the MHC is likely to reveal genetic risk factors for rheumatoid arthritis that are independent of the DRB1 shared epitope alleles.
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http://dx.doi.org/10.2119/2007-00123.LeeDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2255558PMC
August 2008

STAT4 and the risk of rheumatoid arthritis and systemic lupus erythematosus.

N Engl J Med 2007 Sep;357(10):977-86

National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, MD, USA.

Background: Rheumatoid arthritis is a chronic inflammatory disease with a substantial genetic component. Susceptibility to disease has been linked with a region on chromosome 2q.

Methods: We tested single-nucleotide polymorphisms (SNPs) in and around 13 candidate genes within the previously linked chromosome 2q region for association with rheumatoid arthritis. We then performed fine mapping of the STAT1-STAT4 region in a total of 1620 case patients with established rheumatoid arthritis and 2635 controls, all from North America. Implicated SNPs were further tested in an independent case-control series of 1529 patients with early rheumatoid arthritis and 881 controls, all from Sweden, and in a total of 1039 case patients and 1248 controls from three series of patients with systemic lupus erythematosus.

Results: A SNP haplotype in the third intron of STAT4 was associated with susceptibility to both rheumatoid arthritis and systemic lupus erythematosus. The minor alleles of the haplotype-defining SNPs were present in 27% of chromosomes of patients with established rheumatoid arthritis, as compared with 22% of those of controls (for the SNP rs7574865, P=2.81x10(-7); odds ratio for having the risk allele in chromosomes of patients vs. those of controls, 1.32). The association was replicated in Swedish patients with recent-onset rheumatoid arthritis (P=0.02) and matched controls. The haplotype marked by rs7574865 was strongly associated with lupus, being present on 31% of chromosomes of case patients and 22% of those of controls (P=1.87x10(-9); odds ratio for having the risk allele in chromosomes of patients vs. those of controls, 1.55). Homozygosity of the risk allele, as compared with absence of the allele, was associated with a more than doubled risk for lupus and a 60% increased risk for rheumatoid arthritis.

Conclusions: A haplotype of STAT4 is associated with increased risk for both rheumatoid arthritis and systemic lupus erythematosus, suggesting a shared pathway for these illnesses.
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http://dx.doi.org/10.1056/NEJMoa073003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2630215PMC
September 2007

TRAF1-C5 as a risk locus for rheumatoid arthritis--a genomewide study.

N Engl J Med 2007 Sep 5;357(12):1199-209. Epub 2007 Sep 5.

Broad Institute of Harvard and the Massachusetts Institute of Technology, Cambridge, MA, USA.

Background: Rheumatoid arthritis has a complex mode of inheritance. Although HLA-DRB1 and PTPN22 are well-established susceptibility loci, other genes that confer a modest level of risk have been identified recently. We carried out a genomewide association analysis to identify additional genetic loci associated with an increased risk of rheumatoid arthritis.

Methods: We genotyped 317,503 single-nucleotide polymorphisms (SNPs) in a combined case-control study of 1522 case subjects with rheumatoid arthritis and 1850 matched control subjects. The patients were seropositive for autoantibodies against cyclic citrullinated peptide (CCP). We obtained samples from two data sets, the North American Rheumatoid Arthritis Consortium (NARAC) and the Swedish Epidemiological Investigation of Rheumatoid Arthritis (EIRA). Results from NARAC and EIRA for 297,086 SNPs that passed quality-control filters were combined with the use of Cochran-Mantel-Haenszel stratified analysis. SNPs showing a significant association with disease (P<1x10(-8)) were genotyped in an independent set of case subjects with anti-CCP-positive rheumatoid arthritis (485 from NARAC and 512 from EIRA) and in control subjects (1282 from NARAC and 495 from EIRA).

Results: We observed associations between disease and variants in the major-histocompatibility-complex locus, in PTPN22, and in a SNP (rs3761847) on chromosome 9 for all samples tested, the latter with an odds ratio of 1.32 (95% confidence interval, 1.23 to 1.42; P=4x10(-14)). The SNP is in linkage disequilibrium with two genes relevant to chronic inflammation: TRAF1 (encoding tumor necrosis factor receptor-associated factor 1) and C5 (encoding complement component 5).

Conclusions: A common genetic variant at the TRAF1-C5 locus on chromosome 9 is associated with an increased risk of anti-CCP-positive rheumatoid arthritis.
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http://dx.doi.org/10.1056/NEJMoa073491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2636867PMC
September 2007

Application of functional genomic technologies in a mouse model of retinal degeneration.

Genomics 2005 Mar;85(3):309-21

Research Molecular Discovery, Biogen Idec, Inc., 14 Cambridge Center, Cambridge, MA 02142, USA.

Generation of tissue-specific, normalized and subtracted cDNA libraries has the potential to characterize the expression of rare transcriptional units not represented on Affymetrix GeneChips. Initial sequence analysis of our murine cDNA clone collections showed that as much as 86, 45, and 30% of clones are not represented on the Affymetrix Mu11k, MG-U74, and MG-430 chip sets, respectively. A detailed study that compared EST sequences of a subtracted library generated from mouse retina to those of MG-430 consensus sequences was undertaken, using UniGene build 124 as the common reference. A set of 1111 nonredundant transcript regions, not represented on the commercial array, was identified. These clusters were used as the primary filter for analyzing a data set produced by assaying samples from the Pde6b(rd1) mouse model of retinal degeneration on a 12,325-feature retinal cDNA microarray. QRT-PCR validated eight unique transcripts identified by microarray. Seven of the transcripts showed retina-specific expression. Full-length cloning strategies were applied to two of the ESTs. The genes discovered by this approach are the full-length mouse homologue of guanylate cyclase 2F (GUCY2F) and a carboxy-truncated splice variant of retinal S-antigen (SAG), known as regulators of the visual phototransduction G-protein-coupled receptor-mediated signaling pathway. These sequences have been assigned GenBank Accession Nos. and , respectively.
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http://dx.doi.org/10.1016/j.ygeno.2004.11.001DOI Listing
March 2005

Murine peptidoglycan recognition proteins PglyrpIalpha and PglyrpIbeta are encoded in the epidermal differentiation complex and are expressed in epidermal and hematopoietic tissues.

Genomics 2004 Jun;83(6):1151-63

Department of Discovery Biology, Biogen Idec, Inc., 14 Cambridge Center, Cambridge, MA 02142, USA.

Peptidoglycan recognition proteins (PGRPs or PGLYRPs) are pattern recognition molecules that are found in insects and mammals and are critical for innate immune responses. PGRPs bind peptidoglycan, a ubiquitous component of bacterial cell walls, and are involved in killing bacteria, degrading peptidoglycan, and initiating host defense reactions. Relatively little is known about the four mammalian PGRPs. In this article, we report the sequences of mouse PglyrpIalpha and PglyrpIbeta and provide details of their expression in wild-type mouse tissues. PglyrpIalpha and PglyrpIbeta are encoded within the epidermal differentiation complex on mouse chromosome 3F. Both genes are expressed in epidermal and hematopoietic tissues. PglyrpIbeta is expressed in each of 16 tissues tested, while PglyrpIalpha expression is limited to fewer tissues, including the lung and spleen as well as several tissues of the digestive system. Both proteins are expressed in epithelial cells throughout the gut, and immunohistochemical staining shows expression in salivary glands, the squamous epithelium of the stomach, and the villi of the jejunum. Immunohistochemical staining further shows expression of both PglyrpIalpha and PglyrpIbeta in macrophages in the spleen. PglyrpIalpha is not expressed in resting RAW264.7 macrophage-like cells, but is induced by stimulation with lipopolysaccharide. PglyrpIbeta is constitutively expressed in RAW264.7 cells and is unaffected by lipopolysaccharide or peptidoglycan stimulation. Computational and experimental data suggest that these proteins are secreted. This work provides a step toward understanding the roles of PglyrpIalpha and PglyrpIbeta in host defense and chronic inflammatory conditions induced by bacteria or their components.
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http://dx.doi.org/10.1016/j.ygeno.2004.01.003DOI Listing
June 2004

Kirrel2, a novel immunoglobulin superfamily gene expressed primarily in beta cells of the pancreatic islets.

Genomics 2003 Aug;82(2):130-42

Gene Discovery Department, Biogen, Inc., 12 Cambridge Center, Cambridge, MA 02142, USA.

A novel immunoglobulin superfamily (Igsf) protein gene was discovered by computational analysis of human draft genomic DNA, and multiple cDNA clones were obtained. The protein encoded by this gene contains five Ig domains, one transmembrane domain, and an intracellular domain. It has significant similarity with several known Igsf proteins, including Drosophila RST (irregular chiasm C-roughest) protein and mammalian KIRREL (kin of irregular chiasm C-roughest), NEPH1, and NPHS1 (nephrin) proteins. All these proteins have multiple Ig domains, possess properties of cell adhesion molecules, and play important roles in organ development. RT-PCR and Northern blot results indicate this gene is predominantly expressed in pancreas, and public sequence databases indicate there is also expression in the nervous system. We have named this gene Kirrel2 (kin of irregular chiasm-like 2), to reflect its similarity to irregular chiasm C-roughest and Kirrel. Four splice forms of Kirrel2 were observed, including two that we cloned from pancreas mRNA as well as two GenBank entries, one from the brain and one from a retinoblastoma cell line. A partial cDNA clone of the mouse orthologue was obtained by RT-PCR from mouse brain, and the inferred protein sequence has 85% sequence identity to the human protein. Immunohistochemical staining results indicate that the KIRREL2 protein is conserved from rodents to primates, and it is highly expressed in pancreatic islets. RT-PCR results on mouse pancreatic cell lines indicate that expression in the pancreas is restricted to beta cells. Thus, KIRREL2 protein is a beta-cell-expressed Ig domain protein and may be involved in pancreas development or beta cell function.
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http://dx.doi.org/10.1016/s0888-7543(03)00110-1DOI Listing
August 2003

A mutation in the LDL receptor-related protein 5 gene results in the autosomal dominant high-bone-mass trait.

Am J Hum Genet 2002 Jan 3;70(1):11-9. Epub 2001 Dec 3.

Department of Human Genetics, Genome Therapeutics Corporation, Waltham, MA 02453, USA.

Osteoporosis is a complex disease that affects >10 million people in the United States and results in 1.5 million fractures annually. In addition, the high prevalence of osteopenia (low bone mass) in the general population places a large number of people at risk for developing the disease. In an effort to identify genetic factors influencing bone density, we characterized a family that includes individuals who possess exceptionally dense bones but are otherwise phenotypically normal. This high-bone-mass trait (HBM) was originally localized by linkage analysis to chromosome 11q12-13. We refined the interval by extending the pedigree and genotyping additional markers. A systematic search for mutations that segregated with the HBM phenotype uncovered an amino acid change, in a predicted beta-propeller module of the low-density lipoprotein receptor-related protein 5 (LRP5), that results in the HBM phenotype. During analysis of >1,000 individuals, this mutation was observed only in affected individuals from the HBM kindred. By use of in situ hybridization to rat tibia, expression of LRP5 was detected in areas of bone involved in remodeling. Our findings suggest that the HBM mutation confers a unique osteogenic activity in bone remodeling, and this understanding may facilitate the development of novel therapies for the treatment of osteoporosis.
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http://dx.doi.org/10.1086/338450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC419982PMC
January 2002

High throughput analysis of differential gene expression.

J Cell Biochem 1998 ;72 Suppl 30-31(S30-31):286-296

Department of Human Genetics, Genome Therapeutics Corporation, Waltham, Massachusetts 02154.

Elucidation of the changes in gene expression associated with biological processes is a central problem in biology. Advances in molecular and computational biology have led to the development of powerful, high-thoughput methods for the analysis of differential gene expression. These tools have opened up new opportunities in disciplines ranging from cell and developmental biology to drug development and pharmacogenomics. In this review, the attributes of five commonly used differential gene expression methods are discussed: expressed sequence tag (EST) sequencing, cDNA microarray hybridization, subtractive cloning, differential display, and serial analysis of gene expression (SAGE). The application of EST sequencing and microarray hybridization is illustrated by the discovery of novel genes associated with osteoblast differentiation. The application of subtractive cloning is presented as a tool to identify genes regulated in vivo by the transcription factor pax-6. These and other examples illustrate the power of genomics for discovering novel genes that are important in biology and which also represent new targets for drug development. The central theme of the review is that each of the approaches to identifying differentially expressed genes is useful, and that the experimental context and subsequent evaluation of differentially expressed genes are the critical features that determine success. J. Cell. Biochem. Suppls. 30/31:286-296, 1998. © 1998 Wiley-Liss, Inc.
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http://dx.doi.org/10.1002/(SICI)1097-4644(1998)72:30/31+<286::AID-JCB35>3.0.CO;2-DDOI Listing
January 1998
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