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1.  Small cell carcinoma of the ovary, hypercalcemic type, displays frequent inactivating germline and somatic mutations in SMARCA4 
Nature genetics  2014;46(5):427-429.
Small cell carcinoma of the ovary of hypercalcemic type (SCCOHT) is an extremely rare, aggressive cancer affecting children and young women. We identified germline and somatic inactivating mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 69% (9/13) of SCCOHT cases in addition to SMARCA4 protein loss in 82% (14/17) of SCCOHT tumors but in only 0.4% (2/485) of other primary ovarian tumors. These data implicate SMARCA4 in SCCOHT oncogenesis.
doi:10.1038/ng.2928
PMCID: PMC4332808  PMID: 24658001
2.  Characterization of X Chromosome Inactivation Using Integrated Analysis of Whole-Exome and mRNA Sequencing 
PLoS ONE  2014;9(12):e113036.
In females, X chromosome inactivation (XCI) is an epigenetic, gene dosage compensatory mechanism by inactivation of one copy of X in cells. Random XCI of one of the parental chromosomes results in an approximately equal proportion of cells expressing alleles from either the maternally or paternally inherited active X, and is defined by the XCI ratio. Skewed XCI ratio is suggestive of non-random inactivation, which can play an important role in X-linked genetic conditions. Current methods rely on indirect, semi-quantitative DNA methylation-based assay to estimate XCI ratio. Here we report a direct approach to estimate XCI ratio by integrated, family-trio based whole-exome and mRNA sequencing using phase-by-transmission of alleles coupled with allele-specific expression analysis. We applied this method to in silico data and to a clinical patient with mild cognitive impairment but no clear diagnosis or understanding molecular mechanism underlying the phenotype. Simulation showed that phased and unphased heterozygous allele expression can be used to estimate XCI ratio. Segregation analysis of the patient's exome uncovered a de novo, interstitial, 1.7 Mb deletion on Xp22.31 that originated on the paternally inherited X and previously been associated with heterogeneous, neurological phenotype. Phased, allelic expression data suggested an 83∶20 moderately skewed XCI that favored the expression of the maternally inherited, cytogenetically normal X and suggested that the deleterious affect of the de novo event on the paternal copy may be offset by skewed XCI that favors expression of the wild-type X. This study shows the utility of integrated sequencing approach in XCI ratio estimation.
doi:10.1371/journal.pone.0113036
PMCID: PMC4264736  PMID: 25503791
3.  Open-access synthetic spike-in mRNA-seq data for cancer gene fusions 
BMC Genomics  2014;15(1):824.
Background
Oncogenic fusion genes underlie the mechanism of several common cancers. Next-generation sequencing based RNA-seq analyses have revealed an increasing number of recurrent fusions in a variety of cancers. However, absence of a publicly available gene-fusion focused RNA-seq data impedes comparative assessment and collaborative development of novel gene fusions detection algorithms. We have generated nine synthetic poly-adenylated RNA transcripts that correspond to previously reported oncogenic gene fusions. These synthetic RNAs were spiked at known molarity over a wide range into total RNA prior to construction of next-generation sequencing mRNA libraries to generate RNA-seq data.
Results
Leveraging a priori knowledge about replicates and molarity of each synthetic fusion transcript, we demonstrate utility of this dataset to compare multiple gene fusion algorithms’ detection ability. In general, more fusions are detected at higher molarity, indicating that our constructs performed as expected. However, systematic detection differences are observed based on molarity or algorithm-specific characteristics. Fusion-sequence specific detection differences indicate that for applications where specific sequences are being investigated, additional constructs may be added to provide quantitative data that is specific for the sequence of interest.
Conclusions
To our knowledge, this is the first publicly available synthetic RNA-seq data that specifically leverages known cancer gene-fusions. The proposed method of designing multiple gene-fusion constructs over a wide range of molarity allows granular performance analyses of multiple fusion-detection algorithms. The community can leverage and augment this publicly available data to further collaborative development of analytical tools and performance assessment frameworks for gene fusions from next-generation sequencing data.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-824) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-824
PMCID: PMC4190330  PMID: 25266161
RNA-seq; Gene fusions; Cancer genomics
4.  High Density SNP Screen in A Large Multiplex Neural Tube Defect Family Refines Linkage to Loci at 7p21-Pter And 2q33.1-35 
BACKGROUND
Neural tube defects (NTDs) are considered complex with both genetic and environmental factors implicated. To date, no major causative genes have been identified in humans despite several investigations. The first genomewide screen in NTDs (Rampersaud et al. 2005) demonstrated evidence of linkage to chromosomes 7 and 10. This screen included forty-four multiplex families and consisted of 402 microsatellite markers spaced approximately 10 cM apart. Further investigation of the genomic screen data identified a single large multiplex family, pedigree 8776, as primarily driving the linkage results on chromosome 7.
METHODS
To investigate this family more thoroughly, a high-density single nucleotide polymorphism (SNP) screen was performed. Two-point and multipoint linkage analyses were performed using both parametric and nonparametric methods.
RESULTS
For both the microsatellite and SNP markers, linkage analysis suggested the involvement of a locus or loci proximal to the telomeric regions of chromosomes 2q and 7p, with both regions having nonparametric lod* scores of ~3.0, yielding very similar evidence in favor of linkage.
CONCLUSIONS
The regions of strongest evidence for linkage map proximal to the telomeres on these two chromosomes. In addition to mutations and/or variants in a major gene, these loci may harbor a microdeletion and/or translocation; potentially, polygenic factors may also be involved. This single family may be promising for narrowing the search for NTD susceptibility genes.
doi:10.1002/bdra.20272
PMCID: PMC4169147  PMID: 16933213
Neural tube defects (NTDs); spina bifida; genetic mapping; linkage; genome screen
5.  In vitro-differentiated neural cell cultures progress towards donor-identical brain tissue 
Human Molecular Genetics  2013;22(17):3534-3546.
Multiple research groups have observed neuropathological phenotypes and molecular symptoms in vitro using induced pluripotent stem cell (iPSC)-derived neural cell cultures (i.e. patient-specific neurons and glia). However, the global differences/similarities that may exist between in vitro neural cells and their tissue-derived counterparts remain largely unknown. In this study, we compared temporal series of iPSC-derived in vitro neural cell cultures to endogenous brain tissue from the same autopsy donor. Specifically, we utilized RNA sequencing (RNA-Seq) to evaluate the transcriptional progression of in vitro-differentiated neural cells (over a timecourse of 0, 35, 70, 105 and 140 days), and compared this with donor-identical temporal lobe tissue. We observed in vitro progression towards the reference brain tissue, and the following three results support this conclusion: (i) there was a significant increasing monotonic correlation between the days of our timecourse and the number of actively transcribed protein-coding genes and long intergenic non-coding RNAs (lincRNAs) (P < 0.05), consistent with the transcriptional complexity of the brain; (ii) there was an increase in CpG methylation after neural differentiation that resembled the epigenomic signature of the endogenous tissue; and (iii) there was a significant decreasing monotonic correlation between the days of our timecourse and the percent of in vitro to brain-tissue differences (P < 0.05) for tissue-specific protein-coding genes and all putative lincRNAs. Taken together, these results are consistent with in vitro neural development and physiological progression occurring predominantly by transcriptional activation of downregulated genes rather than deactivation of upregulated genes.
doi:10.1093/hmg/ddt208
PMCID: PMC3736871  PMID: 23666530
6.  Whole genome sequencing reveals potential targets for therapy in patients with refractory KRAS mutated metastatic colorectal cancer 
BMC Medical Genomics  2014;7:36.
Background
The outcome of patients with metastatic colorectal carcinoma (mCRC) following first line therapy is poor, with median survival of less than one year. The purpose of this study was to identify candidate therapeutically targetable somatic events in mCRC patient samples by whole genome sequencing (WGS), so as to obtain targeted treatment strategies for individual patients.
Methods
Four patients were recruited, all of whom had received > 2 prior therapy regimens. Percutaneous needle biopsies of metastases were performed with whole blood collection for the extraction of constitutional DNA. One tumor was not included in this study as the quality of tumor tissue was not sufficient for further analysis. WGS was performed using Illumina paired end chemistry on HiSeq2000 sequencing systems, which yielded coverage of greater than 30X for all samples. NGS data were processed and analyzed to detect somatic genomic alterations including point mutations, indels, copy number alterations, translocations and rearrangements.
Results
All 3 tumor samples had KRAS mutations, while 2 tumors contained mutations in the APC gene and the PIK3CA gene. Although we did not identify a TCF7L2-VTI1A translocation, we did detect a TCF7L2 mutation in one tumor. Among the other interesting mutated genes was INPPL1, an important gene involved in PI3 kinase signaling. Functional studies demonstrated that inhibition of INPPL1 reduced growth of CRC cells, suggesting that INPPL1 may promote growth in CRC.
Conclusions
Our study further supports potential molecularly defined therapeutic contexts that might provide insights into treatment strategies for refractory mCRC. New insights into the role of INPPL1 in colon tumor cell growth have also been identified. Continued development of appropriate targeted agents towards specific events may be warranted to help improve outcomes in CRC.
doi:10.1186/1755-8794-7-36
PMCID: PMC4074842  PMID: 24943349
Metastatic colorectal cancer; Whole genome sequencing; KRAS mutations
8.  Integrated Genomic Characterization Reveals Novel, Therapeutically Relevant Drug Targets in FGFR and EGFR Pathways in Sporadic Intrahepatic Cholangiocarcinoma 
PLoS Genetics  2014;10(2):e1004135.
Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.
Author Summary
Cholangiocarcinoma is a cancer that affects the bile ducts. Unfortunately, many patients diagnosed with cholangiocarcinoma have disease that cannot be treated with surgery or has spread to other parts of the body, thus severely limiting treatment options. New advances in drug treatment have enabled treatment of these cancers with “targeted therapy” that exploits an error in the normal functioning of a tumor cell, compared to other cells in the body, thus allowing only tumor cells to be killed by the drug. We sought to identify changes in the genetic material of cholangiocarcinoma patient tumors in order to identify potential errors in cellular functioning by utilizing cutting edge genetic sequencing technology. We identified three patient tumors possessing an FGFR2 gene that was aberrantly fused to another gene. Two of these patients were able to receive targeted therapy for FGFR2 with resulting tumor shrinkage. A fourth tumor contained an error in a gene that controls a very important cellular mechanism in cancer, termed epidermal growth factor pathway (EGFR). This patient received therapy targeting this mechanism and also demonstrated response to treatment. Thus, we have been able to utilize cutting edge technology with targeted drug treatment to personalize medical treatment for cancer in cholangiocarcinoma patients.
doi:10.1371/journal.pgen.1004135
PMCID: PMC3923676  PMID: 24550739
9.  Whole Genome Analyses of a Well-Differentiated Liposarcoma Reveals Novel SYT1 and DDR2 Rearrangements 
PLoS ONE  2014;9(2):e87113.
Liposarcoma is the most common soft tissue sarcoma, but little is known about the genomic basis of this disease. Given the low cell content of this tumor type, we utilized flow cytometry to isolate the diploid normal and aneuploid tumor populations from a well-differentiated liposarcoma prior to array comparative genomic hybridization and whole genome sequencing. This work revealed massive highly focal amplifications throughout the aneuploid tumor genome including MDM2, a gene that has previously been found to be amplified in well-differentiated liposarcoma. Structural analysis revealed massive rearrangement of chromosome 12 and 11 gene fusions, some of which may be part of double minute chromosomes commonly present in well-differentiated liposarcoma. We identified a hotspot of genomic instability localized to a region of chromosome 12 that includes a highly conserved, putative L1 retrotransposon element, LOC100507498 which resides within a gene cluster (NAV3, SYT1, PAWR) where 6 of the 11 fusion events occurred. Interestingly, a potential gene fusion was also identified in amplified DDR2, which is a potential therapeutic target of kinase inhibitors such as dastinib, that are not routinely used in the treatment of patients with liposarcoma. Furthermore, 7 somatic, damaging single nucleotide variants have also been identified, including D125N in the PTPRQ protein. In conclusion, this work is the first to report the entire genome of a well-differentiated liposarcoma with novel chromosomal rearrangements associated with amplification of therapeutically targetable genes such as MDM2 and DDR2.
doi:10.1371/journal.pone.0087113
PMCID: PMC3914808  PMID: 24505276
10.  Induction of Pluripotent Stem Cells from Autopsy Donor-Derived Somatic Cells 
Neuroscience letters  2011;502(3):219-224.
Human induced pluripotent stem cells (iPSCs) have become an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors' complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative diseases are commonly misdiagnosed in live human subjects. Postmortem histopathological examination of a donor's brain, combined with premortem clinical criteria, is often the most robust approach to correctly classify an individual as a disease-specific case or unaffected control. In this study, we describe iPSCs generated from a skin biopsy collected postmortem during the rapid autopsy of a 75-year-old male, whole body donor, defined as an unaffected neurological control by both clinical and histopathological criteria. These iPSCs were established in a feeder-free system by lentiviral transduction of the Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. Selected iPSC clones expressed both nuclear and surface antigens recognized as pluripotency markers of human embryonic stem cells (hESCs) and were able to differentiate in vitro into neurons and glia. Statistical analysis also demonstrated that fibroblast proliferation was significantly affected by biopsy site, but not donor age (within an elderly cohort). These results provide evidence that autopsy donor-derived fibroblasts can be successfully reprogrammed into iPSCs, and may provide an advantageous approach for generating iPSC-based neurological disease models.
doi:10.1016/j.neulet.2011.07.048
PMCID: PMC3195418  PMID: 21839145
induced pluripotent stem cells; genetic disease models; diagnostics; neurodegenerative diseases; postmortem; autopsy; neural differentiation
11.  A Pilot Study Using Next-Generation Sequencing in Advanced Cancers: Feasibility and Challenges 
PLoS ONE  2013;8(10):e76438.
Purpose
New anticancer agents that target a single cell surface receptor, up-regulated or amplified gene product, or mutated gene, have met with some success in treating advanced cancers. However, patients' tumors still eventually progress on these therapies. If it were possible to identify a larger number of targetable vulnerabilities in an individual's tumor, multiple targets could be exploited with the use of specific therapeutic agents, thus possibly giving the patient viable therapeutic alternatives.
Experimental Design
In this exploratory study, we used next-generation sequencing technologies (NGS) including whole genome sequencing (WGS), and where feasible, whole transcriptome sequencing (WTS) to identify genomic events and associated expression changes in advanced cancer patients.
Results
WGS on paired tumor and normal samples from nine advanced cancer patients and WTS on six of these patients' tumors was completed. One patient's treatment was based on targets and pathways identified by NGS and the patient had a short-lived PET/CT response with a significant reduction in his tumor-related pain. To design treatment plans based on information garnered from NGS, several challenges were encountered: NGS reporting delays, communication of results to out-of-state participants and their treating oncologists, and chain of custody handling for fresh biopsy samples for Clinical Laboratory Improvement Amendments (CLIA) target validation.
Conclusion
While the initial effort was a slower process than anticipated due to a variety of issues, we demonstrate the feasibility of using NGS in advanced cancer patients so that treatments for patients with progressing tumors may be improved.
doi:10.1371/journal.pone.0076438
PMCID: PMC3813699  PMID: 24204627
12.  Long insert whole genome sequencing for copy number variant and translocation detection 
Nucleic Acids Research  2013;42(2):e8.
As next-generation sequencing continues to have an expanding presence in the clinic, the identification of the most cost-effective and robust strategy for identifying copy number changes and translocations in tumor genomes is needed. We hypothesized that performing shallow whole genome sequencing (WGS) of 900–1000-bp inserts (long insert WGS, LI-WGS) improves our ability to detect these events, compared with shallow WGS of 300–400-bp inserts. A priori analyses show that LI-WGS requires less sequencing compared with short insert WGS to achieve a target physical coverage, and that LI-WGS requires less sequence coverage to detect a heterozygous event with a power of 0.99. We thus developed an LI-WGS library preparation protocol based off of Illumina’s WGS library preparation protocol and illustrate the feasibility of performing LI-WGS. We additionally applied LI-WGS to three separate tumor/normal DNA pairs collected from patients diagnosed with different cancers to demonstrate our application of LI-WGS on actual patient samples for identification of somatic copy number alterations and translocations. With the evolution of sequencing technologies and bioinformatics analyses, we show that modifications to current approaches may improve our ability to interrogate cancer genomes.
doi:10.1093/nar/gkt865
PMCID: PMC3902897  PMID: 24071583
13.  Germline Mutations in HOXB13 and Prostate-Cancer Risk 
The New England journal of medicine  2012;366(2):141-149.
BACKGROUND
Family history is a significant risk factor for prostate cancer, although the molecular basis for this association is poorly understood. Linkage studies have implicated chromosome 17q21-22 as a possible location of a prostate-cancer susceptibility gene.
METHODS
We screened more than 200 genes in the 17q21-22 region by sequencing germline DNA from 94 unrelated patients with prostate cancer from families selected for linkage to the candidate region. We tested family members, additional case subjects, and control subjects to characterize the frequency of the identified mutations.
RESULTS
Probands from four families were discovered to have a rare but recurrent mutation (G84E) in HOXB13 (rs138213197), a homeobox transcription factor gene that is important in prostate development. All 18 men with prostate cancer and available DNA in these four families carried the mutation. The carrier rate of the G84E mutation was increased by a factor of approximately 20 in 5083 unrelated subjects of European descent who had prostate cancer, with the mutation found in 72 subjects (1.4%), as compared with 1 in 1401 control subjects (0.1%) (P = 8.5×10−7). The mutation was significantly more common in men with early-onset, familial prostate cancer (3.1%) than in those with late-onset, nonfamilial prostate cancer (0.6%) (P = 2.0×10−6).
CONCLUSIONS
The novel HOXB13 G84E variant is associated with a significantly increased risk of hereditary prostate cancer. Although the variant accounts for a small fraction of all prostate cancers, this finding has implications for prostate-cancer risk assessment and may provide new mechanistic insights into this common cancer. (Funded by the National Institutes of Health and others.)
doi:10.1056/NEJMoa1110000
PMCID: PMC3779870  PMID: 22236224
14.  IDENTIFICATION OF GENETIC VARIANTS USING BARCODED MULTIPLEXED SEQUENCING 
Nature methods  2008;5(10):887-893.
We developed a generalized framework for multiplexed resequencing of targeted regions of the human genome on the Illumina Genome Analyzer using degenerate indexed DNA sequence barcodes ligated to fragmented DNA prior to sequencing. Using this method, the DNA of multiple HapMap individuals was simultaneously sequenced at several ENCODE (ENCyclopedia of DNA Elements) regions. We then evaluated the use of Bayes factors for discovering and genotyping polymorphisms from aligned sequenced reads. If we required that predicted polymorphisms be either previously identified by dbSNP or be visually evident upon reinspection of archived ENCODE traces, we observed a false-positive rate of 11.3% using strict thresholds (Ks>1,000) for predicting variants and 69.6% for lax thresholds (Ks>10). Conversely, false-negative rates ranged from 10.8% to 90.8%, with those at stricter cut-offs occurring at lower coverage (< 10 aligned reads). These results suggest that >90% of genetic variants are discoverable using multiplexed sequencing provided sufficient coverage at the polymorphic base.
doi:10.1038/nmeth.1251
PMCID: PMC3171277  PMID: 18794863
15.  Cerebellar Telomere Length and Psychiatric Disorders 
Behavior genetics  2010;40(2):250-254.
We tested whether telomere length is altered in the brains of patients diagnosed with major depression (MD), bipolar disorder (BD) and schizophrenia (SZ) by measuring mean telomere length (mTL) with real-time PCR. The samples are cerebellar gray matter from 46 SZ, 46 BP, and 15 MD patients, and 48 healthy controls. We found no difference in mTL between SZ and controls, BD and controls, MD and controls, or all cases and controls; no correlation between mTL and age was observed, either. This suggests that brain gray matter is unlikely to be related to the telomere length shortening reported in blood of psychiatric patients. White matter deserves further investigation as it has been reported to have a different mTL dynamic from gray matter. Since mTL has been reported to be a heritable quantitative trait, we also carried out genome-wide mapping of genetic factors for mTL, treating mTL as a quantitative trait. No association survived correction of multiple testing for the number of SNPs studied. The previously reported rs2630578 (BICD1) association was not replicated. This suggests that telomere length of cerebellar gray matter is determined by multiple loci with “weak effects.”
doi:10.1007/s10519-010-9338-0
PMCID: PMC3053383  PMID: 20127402
Mean telomere length; Bipolar disorder; Major depression; Schizophrenia; Mapping; Quantitative trait
16.  Identification of somatic mutations in cancer through Bayesian-based analysis of sequenced genome pairs 
BMC Genomics  2013;14:302.
Background
The field of cancer genomics has rapidly adopted next-generation sequencing (NGS) in order to study and characterize malignant tumors with unprecedented resolution. In particular for cancer, one is often trying to identify somatic mutations – changes specific to a tumor and not within an individual’s germline. However, false positive and false negative detections often result from lack of sufficient variant evidence, contamination of the biopsy by stromal tissue, sequencing errors, and the erroneous classification of germline variation as tumor-specific.
Results
We have developed a generalized Bayesian analysis framework for matched tumor/normal samples with the purpose of identifying tumor-specific alterations such as single nucleotide mutations, small insertions/deletions, and structural variation. We describe our methodology, and discuss its application to other types of paired-tissue analysis such as the detection of loss of heterozygosity as well as allelic imbalance. We also demonstrate the high level of sensitivity and specificity in discovering simulated somatic mutations, for various combinations of a) genomic coverage and b) emulated heterogeneity.
Conclusion
We present a Java-based implementation of our methods named Seurat, which is made available for free academic use. We have demonstrated and reported on the discovery of different types of somatic change by applying Seurat to an experimentally-derived cancer dataset using our methods; and have discussed considerations and practices regarding the accurate detection of somatic events in cancer genomes. Seurat is available at https://sites.google.com/site/seuratsomatic.
doi:10.1186/1471-2164-14-302
PMCID: PMC3751438  PMID: 23642077
Cancer genomics; Next generation sequencing; Somatic mutation detection
17.  Plasma cytokine profiling in sibling pairs discordant for autism spectrum disorder 
Objective
Converging lines of evidence point to the existence of immune dysfunction in autism spectrum disorder (ASD), which could directly affect several key neurodevelopmental processes. Previous studies have shown higher cytokine levels in patients with autism compared with matched controls or subjects with other developmental disorders. In the current study, we used plasma-cytokine profiling for 25 discordant sibling pairs to evaluate whether these alterations occur within families with ASD.
Methods
Plasma-cytokine profiling was conducted using an array-based multiplex sandwich ELISA for simultaneous quantitative measurement of 40 unique targets. We also analyzed the correlations between cytokine levels and clinically relevant quantitative traits (Vineland Adaptive Behavior Scale in Autism (VABS) composite score, Social Responsiveness Scale (SRS) total T score, head circumference, and full intelligence quotient (IQ)). In addition, because of the high phenotypic heterogeneity of ASD, we defined four subgroups of subjects (those who were non-verbal, those with gastrointestinal issues, those with regressive autism, and those with a history of allergies), which encompass common and/or recurrent endophenotypes in ASD, and tested the cytokine levels in each group.
Results
None of the measured parameters showed significant differences between children with ASD and their related typically developing siblings. However, specific target levels did correlate with quantitative clinical traits, and these were significantly different when the ASD subgroups were analyzed. It is notable that these differences seem to be attributable to a predisposing immunogenetic background, as no other significant differences were noticed between discordant sibling pairs. Interleukin-1β appears to be the cytokine most involved in quantitative traits and clinical subgroups of ASD.
Conclusions
In the present study, we found a lack of significant differences in plasma-cytokine levels between children with ASD and in their related non-autistic siblings. Thus, our results support the evidence that the immune profiles of children with autism do not differ from their typically developing siblings. However, the significant association of cytokine levels with the quantitative traits and the clinical subgroups analyzed suggests that altered immune responses may affect core feature of ASD.
doi:10.1186/1742-2094-10-38
PMCID: PMC3616926  PMID: 23497090
18.  Genome-Wide Characterization of Pancreatic Adenocarcinoma Patients Using Next Generation Sequencing 
PLoS ONE  2012;7(10):e43192.
Pancreatic adenocarcinoma (PAC) is among the most lethal malignancies. While research has implicated multiple genes in disease pathogenesis, identification of therapeutic leads has been difficult and the majority of currently available therapies provide only marginal benefit. To address this issue, our goal was to genomically characterize individual PAC patients to understand the range of aberrations that are occurring in each tumor. Because our understanding of PAC tumorigenesis is limited, evaluation of separate cases may reveal aberrations, that are less common but may provide relevant information on the disease, or that may represent viable therapeutic targets for the patient. We used next generation sequencing to assess global somatic events across 3 PAC patients to characterize each patient and to identify potential targets. This study is the first to report whole genome sequencing (WGS) findings in paired tumor/normal samples collected from 3 separate PAC patients. We generated on average 132 billion mappable bases across all patients using WGS, and identified 142 somatic coding events including point mutations, insertion/deletions, and chromosomal copy number variants. We did not identify any significant somatic translocation events. We also performed RNA sequencing on 2 of these patients' tumors for which tumor RNA was available to evaluate expression changes that may be associated with somatic events, and generated over 100 million mapped reads for each patient. We further performed pathway analysis of all sequencing data to identify processes that may be the most heavily impacted from somatic and expression alterations. As expected, the KRAS signaling pathway was the most heavily impacted pathway (P<0.05), along with tumor-stroma interactions and tumor suppressive pathways. While sequencing of more patients is needed, the high resolution genomic and transcriptomic information we have acquired here provides valuable information on the molecular composition of PAC and helps to establish a foundation for improved therapeutic selection.
doi:10.1371/journal.pone.0043192
PMCID: PMC3468610  PMID: 23071490
19.  Cancer of the ampulla of Vater: analysis of the whole genome sequence exposes a potential therapeutic vulnerability 
Genome Medicine  2012;4(7):56.
Background
Recent advances in the treatment of cancer have focused on targeting genomic aberrations with selective therapeutic agents. In rare tumors, where large-scale clinical trials are daunting, this targeted genomic approach offers a new perspective and hope for improved treatments. Cancers of the ampulla of Vater are rare tumors that comprise only about 0.2% of gastrointestinal cancers. Consequently, they are often treated as either distal common bile duct or pancreatic cancers.
Methods
We analyzed DNA from a resected cancer of the ampulla of Vater and whole blood DNA from a 63 year-old man who underwent a pancreaticoduodenectomy by whole genome sequencing, achieving 37× and 40× coverage, respectively. We determined somatic mutations and structural alterations.
Results
We identified relevant aberrations, including deleterious mutations of KRAS and SMAD4 as well as a homozygous focal deletion of the PTEN tumor suppressor gene. These findings suggest that these tumors have a distinct oncogenesis from either common bile duct cancer or pancreatic cancer. Furthermore, this combination of genomic aberrations suggests a therapeutic context for dual mTOR/PI3K inhibition.
Conclusions
Whole genome sequencing can elucidate an oncogenic context and expose potential therapeutic vulnerabilities in rare cancers.
doi:10.1186/gm357
PMCID: PMC3580412  PMID: 22762308
20.  Paired Tumor and Normal Whole Genome Sequencing of Metastatic Olfactory Neuroblastoma 
PLoS ONE  2012;7(5):e37029.
Background
Olfactory neuroblastoma (ONB) is a rare cancer of the sinonasal tract with little molecular characterization. We performed whole genome sequencing (WGS) on paired normal and tumor DNA from a patient with metastatic-ONB to identify the somatic alterations that might be drivers of tumorigenesis and/or metastatic progression.
Methodology/Principal Findings
Genomic DNA was isolated from fresh frozen tissue from a metastatic lesion and whole blood, followed by WGS at >30X depth, alignment and mapping, and mutation analyses. Sanger sequencing was used to confirm selected mutations. Sixty-two somatic short nucleotide variants (SNVs) and five deletions were identified inside coding regions, each causing a non-synonymous DNA sequence change. We selected seven SNVs and validated them by Sanger sequencing. In the metastatic ONB samples collected several months prior to WGS, all seven mutations were present. However, in the original surgical resection specimen (prior to evidence of metastatic disease), mutations in KDR, MYC, SIN3B, and NLRC4 genes were not present, suggesting that these were acquired with disease progression and/or as a result of post-treatment effects.
Conclusions/Significance
This work provides insight into the evolution of ONB cancer cells and provides a window into the more complex factors, including tumor clonality and multiple driver mutations.
doi:10.1371/journal.pone.0037029
PMCID: PMC3359355  PMID: 22649506
21.  Assessing and Managing Risk when Sharing Aggregate Genetic Variant Data 
Nature Reviews. Genetics  2011;12(10):730-736.
Preface
Access to genetic data across studies is an important aspect of identifying new genetic associations through genome-wide association studies (GWAS). Meta-analysis across multiple GWAS with combined cohort sizes of tens of thousands of individuals often uncovers many more genome-wide associated loci than the original individual studies, which emphasizes the importance of tools and mechanisms for data sharing. However, even sharing summary-level data, such as allele frequencies, inherently carries some degree of privacy risk to study participants. Here we discuss mechanisms and resources for sharing data from GWAS, particularly focusing on approaches for assessing and quantifying privacy risks to participants from sharing of summary-level data.
doi:10.1038/nrg3067
PMCID: PMC3349221  PMID: 21921928
22.  Amyloid pathway-based candidate gene analysis of [11C]PiB-PET in the Alzheimer’s Disease Neuroimaging Initiative (ADNI) cohort 
Brain Imaging and Behavior  2012;6(1):1-15.
Amyloid imaging with [11C]Pittsburgh Compound-B (PiB) provides in vivo data on plaque deposition in those with, or at risk for, Alzheimer’s disease (AD). We performed a gene-based association analysis of 15 quality-controlled amyloid-pathway associated candidate genes in 103 Alzheimer’s Disease Neuroimaging Initiative participants. The mean normalized PiB uptake value across four brain regions known to have amyloid deposition in AD was used as a quantitative phenotype. The minor allele of an intronic SNP within DHCR24 was identified and associated with a lower average PiB uptake. Further investigation at whole-brain voxel-wise level indicated that non-carriers of the minor allele had higher PiB uptake in frontal regions compared to carriers. DHCR24 has been previously shown to confer resistance against beta-amyloid and oxidative stress-induced apoptosis, thus our findings support a neuroprotective role. Pathway-based genetic analysis of targeted molecular imaging phenotypes appears promising to help elucidate disease pathophysiology and identify potential therapeutic targets.
doi:10.1007/s11682-011-9136-1
PMCID: PMC3256261  PMID: 21901424
Alzheimer’s disease; ADNI; Pathway-based gene analysis; PiB-PET; Endophenotype; Voxel-based analysis
23.  Amyloid pathway-based candidate gene analysis of [11C]PiB-PET in the Alzheimer’s Disease Neuroimaging Initiative (ADNI) cohort 
Brain Imaging and Behavior  2011;6(1):1-15.
Amyloid imaging with [11 C]Pittsburgh Compound-B (PiB) provides in vivo data on plaque deposition in those with, or at risk for, Alzheimer’s disease (AD). We performed a gene-based association analysis of 15 quality-controlled amyloid-pathway associated candidate genes in 103 Alzheimer’s Disease Neuroimaging Initiative participants. The mean normalized PiB uptake value across four brain regions known to have amyloid deposition in AD was used as a quantitative phenotype. The minor allele of an intronic SNP within DHCR24 was identified and associated with a lower average PiB uptake. Further investigation at whole-brain voxel-wise level indicated that non-carriers of the minor allele had higher PiB uptake in frontal regions compared to carriers. DHCR24 has been previously shown to confer resistance against beta-amyloid and oxidative stress-induced apoptosis, thus our findings support a neuroprotective role. Pathway-based genetic analysis of targeted molecular imaging phenotypes appears promising to help elucidate disease pathophysiology and identify potential therapeutic targets.
doi:10.1007/s11682-011-9136-1
PMCID: PMC3256261  PMID: 21901424
Alzheimer’s disease; ADNI; Pathway-based gene analysis; PiB-PET; Endophenotype; Voxel-based analysis
24.  Association of CR1, CLU and PICALM with Alzheimer's disease in a cohort of clinically characterized and neuropathologically verified individuals 
Human Molecular Genetics  2010;19(16):3295-3301.
In this study, we assess 34 of the most replicated genetic associations for Alzheimer's disease (AD) using data generated on Affymetrix SNP 6.0 arrays and imputed at over 5.7 million markers from a unique cohort of over 1600 neuropathologically defined AD cases and controls (1019 cases and 591 controls). Testing the top genes from the AlzGene meta-analysis, we confirm the well-known association with APOE single nucleotide polymorphisms (SNPs), the CLU, PICALM and CR1 SNPs recently implicated in unusually large data sets, and previously implicated CST3 and ACE SNPs. In the cases of CLU, PICALM and CR1, as well as in APOE, the odds ratios we find are slightly larger than those previously reported in clinical samples, consistent with what we believe to be more accurate classification of disease in the clinically characterized and neuropathologically confirmed AD cases and controls.
doi:10.1093/hmg/ddq221
PMCID: PMC2908469  PMID: 20534741
25.  Genome-Wide Association of Bipolar Disorder Suggests an Enrichment of Replicable Associations in Regions near Genes 
PLoS Genetics  2011;7(6):e1002134.
Although a highly heritable and disabling disease, bipolar disorder's (BD) genetic variants have been challenging to identify. We present new genotype data for 1,190 cases and 401 controls and perform a genome-wide association study including additional samples for a total of 2,191 cases and 1,434 controls. We do not detect genome-wide significant associations for individual loci; however, across all SNPs, we show an association between the power to detect effects calculated from a previous genome-wide association study and evidence for replication (P = 1.5×10−7). To demonstrate that this result is not likely to be a false positive, we analyze replication rates in a large meta-analysis of height and show that, in a large enough study, associations replicate as a function of power, approaching a linear relationship. Within BD, SNPs near exons exhibit a greater probability of replication, supporting an enrichment of reproducible associations near functional regions of genes. These results indicate that there is likely common genetic variation associated with BD near exons (±10 kb) that could be identified in larger studies and, further, provide a framework for assessing the potential for replication when combining results from multiple studies.
Author Summary
Bipolar disorder (BD) is a highly heritable disease that has been difficult to characterize genetically. We have genotyped 1,190 BD cases and 401 controls to find regions of the genome associated with BD. After combining these data with previously existing genotyped samples, we did not find any genome-wide significant associations. However, when we used an additional study to prioritize loci for replication and meta-analysis purposes, we found that we were more likely to see an association in our sample with variants for which we had the highest power. We quantified this effect using logistic regression and saw a strong association between power to detect an effect based on an initial study's results and replication P-value in a second study (P = 1.5×10−7), supporting the presence of shared genetic risk factors across the studies. Moreover, this association was stronger when we restricted analysis to SNPs near coding regions, and it was further enriched when SNPs had the same direction of effect in both studies. This result supports the presence of genetic factors underlying BD near exons whose collective effect results in a detectable signal and provides a framework for assessing the potential for replication when combining results from multiple studies.
doi:10.1371/journal.pgen.1002134
PMCID: PMC3128104  PMID: 21738484

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