Publications by authors named "Bruce W Eckloff"

54 Publications

RNA-Seq Reveals Differences in Expressed Tumor Mutation Burden in Colorectal and Endometrial Cancers with and without Defective DNA-Mismatch Repair.

J Mol Diagn 2021 05 4;23(5):555-564. Epub 2021 Feb 4.

Division of Laboratory Genetics and Experimental Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota. Electronic address:

Tumor mutation burden (TMB) is an emerging biomarker of immunotherapy response. RNA sequencing in FFPE tissue samples was used for determining TMB in microsatellite-stable (MSS) and microsatellite instability-high (MSI-H) tumors in patients with colorectal or endometrial cancer. Tissue from tumors and paired normal tissue from 46 MSI-H and 12 MSS cases were included. Of the MSI-H tumors, 29 had defective DNA mismatch-repair mutations, and 17 had MLH1 promoter hypermethylation. TMB was measured using the expressed somatic nucleotide variants (eTMB). A method of accurate measurement of eTMB was developed that removes FFPE-derived artifacts by leveraging mutation signatures. There was a significant difference in the median eTMB values observed between MSI-H and MSS cases: 27.3 versus 6.7 mutations/megabase (mut/Mb) (P = 3.5 × 10). Among tumors with defective DNA-mismatch repair, those with mismatch-repair mutations had a significantly higher median eTMB than those with hypermethylation: 28.1 versus 17.5 mut/Mb (P = 0.037). Multivariate analysis showed that MSI status, tumor type (endometrial or colorectal), and age were significantly associated with eTMB. Additionally, using whole-exome sequencing in a subset of these patients, it was determined that DNA TMB correlated well with eTMB (Spearman correlation coefficient, 0.83). These results demonstrate that RNA sequencing can be used for measuring eTMB in FFPE tumor specimens.
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http://dx.doi.org/10.1016/j.jmoldx.2021.01.008DOI Listing
May 2021

Next-Generation Sequencing of CYP2C19 in Stent Thrombosis: Implications for Clopidogrel Pharmacogenomics.

Cardiovasc Drugs Ther 2021 06;35(3):549-559

Department of Cardiovascular Medicine, Mayo Clinic, 200 First St SW, Rochester, MN, 55905, USA.

Purpose: Describe CYP2C19 sequencing results in the largest series of clopidogrel-treated cases with stent thrombosis (ST), the closest clinical phenotype to clopidogrel resistance. Evaluate the impact of CYP2C19 genetic variation detected by next-generation sequencing (NGS) with comprehensive annotation and functional studies.

Methods: Seventy ST cases on clopidogrel identified from the PLATO trial (n = 58) and Mayo Clinic biorepository (n = 12) were matched 1:1 with controls for age, race, sex, diabetes mellitus, presentation, and stent type. NGS was performed to cover the entire CYP2C19 gene. Assessment of exonic variants involved measuring in vitro protein expression levels. Intronic variants were evaluated for potential splicing motif variations.

Results: Poor metabolizers (n = 4) and rare CYP2C19*8, CYP2C19*15, and CYP2C19*11 alleles were identified only in ST cases. CYP2C19*17 heterozygote carriers were observed more frequently in cases (n = 29) than controls (n = 18). Functional studies of CYP2C19 exonic variants (n = 11) revealed 3 cases and only 1 control carrying a deleterious variant as determined by in vitro protein expression studies. Greater intronic variation unique to ST cases (n = 169) compared with controls (n = 84) was observed with predictions revealing 13 allele candidates that may lead to a potential disruption of splicing and a loss-of-function effect of CYP2C19 in ST cases.

Conclusion: NGS detected CYP2C19 poor metabolizers and paradoxically greater number of so-called rapid metabolizers in ST cases. Rare deleterious exonic variation occurs in 4%, and potentially disruptive intronic alleles occur in 16% of ST cases. Additional studies are required to evaluate the role of these variants in platelet aggregation and clopidogrel metabolism.
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http://dx.doi.org/10.1007/s10557-020-06988-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7779664PMC
June 2021

Recurrent mutations in ALK-negative anaplastic large cell lymphoma.

Blood 2019 06 17;133(26):2776-2789. Epub 2019 May 17.

Division of Hematology, Mayo Clinic, Rochester, MN.

Anaplastic large cell lymphomas (ALCLs) represent a relatively common group of T-cell non-Hodgkin lymphomas (T-NHLs) that are unified by similar pathologic features but demonstrate marked genetic heterogeneity. ALCLs are broadly classified as being anaplastic lymphoma kinase (ALK) or ALK, based on the presence or absence of rearrangements. Exome sequencing of 62 T-NHLs identified a previously unreported recurrent mutation in the musculin gene, , exclusively in ALK ALCLs. Additional sequencing for a total of 238 T-NHLs confirmed the specificity of for ALK ALCL and further demonstrated that 14 of 15 mutated cases (93%) had coexisting rearrangements. Musculin is a basic helix-loop-helix (bHLH) transcription factor that heterodimerizes with other bHLH proteins to regulate lymphocyte development. The E116K mutation localized to the DNA binding domain of musculin and permitted formation of musculin-bHLH heterodimers but prevented their binding to authentic target sequence. Functional analysis showed MSC acted in a dominant-negative fashion, reversing wild-type musculin-induced repression of and cell cycle inhibition. Chromatin immunoprecipitation-sequencing and transcriptome analysis identified the cell cycle regulatory gene as a direct transcriptional target of musculin. MSC reversed E2F2-induced cell cycle arrest and promoted expression of the CD30-IRF4-MYC axis, whereas its expression was reciprocally induced by binding of IRF4 to the promoter. Finally, ALCL cells expressing were preferentially targeted by the BET inhibitor JQ1. These findings identify a novel recurrent mutation as a key driver of the CD30-IRF4-MYC axis and cell cycle progression in a unique subset of ALCLs.
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http://dx.doi.org/10.1182/blood.2019000626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6598380PMC
June 2019

NUP155 insufficiency recalibrates a pluripotent transcriptome with network remodeling of a cardiogenic signaling module.

BMC Syst Biol 2018 05 30;12(1):62. Epub 2018 May 30.

Genetics and Genomics Group, Sanford Research, 2301 E. 60th Street N, Sioux Falls, SD, 57104, USA.

Background: Atrial fibrillation is a cardiac disease driven by numerous idiopathic etiologies. NUP155 is a nuclear pore complex protein that has been identified as a clinical driver of atrial fibrillation, yet the precise mechanism is unknown. The present study employs a systems biology algorithm to identify effects of NUP155 disruption on cardiogenicity in a model of stem cell-derived differentiation.

Methods: Embryonic stem (ES) cell lines (n = 5) with truncated NUP155 were cultured in parallel with wild type (WT) ES cells (n = 5), and then harvested for RNAseq. Samples were run on an Illumina HiSeq 2000. Reads were analyzed using Strand NGS, Cytoscape, DAVID and Ingenuity Pathways Analysis to deconvolute the NUP155-disrupted transcriptome. Network topological analysis identified key features that controlled framework architecture and functional enrichment.

Results: In NUP155 truncated ES cells, significant expression changes were detected in 326 genes compared to WT. These genes segregated into clusters that enriched for specific gene ontologies. Deconvolution of the collective framework into discrete sub-networks identified a module with the highest score that enriched for Cardiovascular System Development, and revealed NTRK1/TRKA and SRSF2/SC35 as critical hubs within this cardiogenic module.

Conclusions: The strategy of pluripotent transcriptome deconvolution used in the current study identified a novel association of NUP155 with potential drivers of arrhythmogenic AF. Here, NUP155 regulates cardioplasticity of a sub-network embedded within a larger framework of genome integrity, and exemplifies how transcriptome cardiogenicity in an embryonic stem cell genome is recalibrated by nucleoporin dysfunction.
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http://dx.doi.org/10.1186/s12918-018-0590-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5977756PMC
May 2018

Targeted sequencing of 36 known or putative colorectal cancer susceptibility genes.

Mol Genet Genomic Med 2017 Sep 23;5(5):553-569. Epub 2017 Jul 23.

Department of Laboratory Medicine and PathologyMayo ClinicRochesterMinnesota.

Background: Mutations in several genes predispose to colorectal cancer. Genetic testing for hereditary colorectal cancer syndromes was previously limited to single gene tests; thus, only a very limited number of genes were tested, and rarely those infrequently mutated in colorectal cancer. Next-generation sequencing technologies have made it possible to sequencing panels of genes known and suspected to influence colorectal cancer susceptibility.

Methods: Targeted sequencing of 36 known or putative CRC susceptibility genes was conducted for 1231 CRC cases from five subsets: (1) Familial Colorectal Cancer Type X ( = 153); (2) CRC unselected by tumor immunohistochemical or microsatellite stability testing ( = 548); (3) young onset (age <50 years) ( = 333); (4) proficient mismatch repair (MMR) in cases diagnosed at ≥50 years ( = 68); and (5) deficient MMR CRCs with no germline mutations in MLH1, MSH2, MSH6, or PMS2 ( = 129). Ninety-three unaffected controls were also sequenced.

Results: Overall, 29 nonsense, 43 frame-shift, 13 splice site, six initiator codon variants, one stop codon, 12 exonic deletions, 658 missense, and 17 indels were identified. Missense variants were reviewed by genetic counselors to determine pathogenicity; 13 were pathogenic, 61 were not pathogenic, and 584 were variants of uncertain significance. Overall, we identified 92 cases with pathogenic mutations in ,,,, or multiple pathogenic mutations (7.5%). Four cases with intact MMR protein expression by immunohistochemistry carried pathogenic MMR mutations.

Conclusions: Results across case subsets may help prioritize genes for inclusion in clinical gene panel tests and underscore the issue of variants of uncertain significance both in well-characterized genes and those for which limited experience has accumulated.
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http://dx.doi.org/10.1002/mgg3.317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5606870PMC
September 2017

Androgen Receptor Variant AR-V9 Is Coexpressed with AR-V7 in Prostate Cancer Metastases and Predicts Abiraterone Resistance.

Clin Cancer Res 2017 Aug 4;23(16):4704-4715. Epub 2017 May 4.

Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota.

Androgen receptor (AR) variant AR-V7 is a ligand-independent transcription factor that promotes prostate cancer resistance to AR-targeted therapies. Accordingly, efforts are under way to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The purpose of this study was to understand whether other AR variants may be coexpressed with AR-V7 and promote resistance to AR-targeted therapies. We utilized complementary short- and long-read sequencing of intact AR mRNA isoforms to characterize AR expression in CRPC models. Coexpression of AR-V7 and AR-V9 mRNA in CRPC metastases and circulating tumor cells was assessed by RNA-seq and RT-PCR, respectively. Expression of AR-V9 protein in CRPC models was evaluated with polyclonal antisera. Multivariate analysis was performed to test whether AR variant mRNA expression in metastatic tissues was associated with a 12-week progression-free survival endpoint in a prospective clinical trial of 78 CRPC-stage patients initiating therapy with the androgen synthesis inhibitor, abiraterone acetate. AR-V9 was frequently coexpressed with AR-V7. Both AR variant species were found to share a common 3' terminal cryptic exon, which rendered AR-V9 susceptible to experimental manipulations that were previously thought to target AR-V7 uniquely. AR-V9 promoted ligand-independent growth of prostate cancer cells. High AR-V9 mRNA expression in CRPC metastases was predictive of primary resistance to abiraterone acetate (HR = 4.0; 95% confidence interval, 1.31-12.2; = 0.02). AR-V9 may be an important component of therapeutic resistance in CRPC. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-0017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5644285PMC
August 2017

Integrated mate-pair and RNA sequencing identifies novel, targetable gene fusions in peripheral T-cell lymphoma.

Blood 2016 09 13;128(9):1234-45. Epub 2016 Jun 13.

Department of Laboratory Medicine and Pathology.

Peripheral T-cell lymphomas (PTCLs) represent a heterogeneous group of T-cell malignancies that generally demonstrate aggressive clinical behavior, often are refractory to standard therapy, and remain significantly understudied. The most common World Health Organization subtype is PTCL, not otherwise specified (NOS), essentially a "wastebasket" category because of inadequate understanding to assign cases to a more specific diagnostic entity. Identification of novel fusion genes has contributed significantly to improving the classification, biologic understanding, and therapeutic targeting of PTCLs. Here, we integrated mate-pair DNA and RNA next-generation sequencing to identify chromosomal rearrangements encoding expressed fusion transcripts in PTCL, NOS. Two of 11 cases had novel fusions involving VAV1, encoding a truncated form of the VAV1 guanine nucleotide exchange factor important in T-cell receptor signaling. Fluorescence in situ hybridization studies identified VAV1 rearrangements in 10 of 148 PTCLs (7%). These were observed exclusively in PTCL, NOS (11%) and anaplastic large cell lymphoma (11%). In vitro, ectopic expression of a VAV1 fusion promoted cell growth and migration in a RAC1-dependent manner. This growth was inhibited by azathioprine, a clinically available RAC1 inhibitor. We also identified novel kinase gene fusions, ITK-FER and IKZF2-ERBB4, as candidate therapeutic targets that show similarities to known recurrent oncogenic ITK-SYK fusions and ERBB4 transcript variants in PTCLs, respectively. Additional novel and potentially clinically relevant fusions also were discovered. Together, these findings identify VAV1 fusions as recurrent and targetable events in PTCLs and highlight the potential for clinical sequencing to guide individualized therapy approaches for this group of aggressive malignancies.
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http://dx.doi.org/10.1182/blood-2016-03-707141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009513PMC
September 2016

Target-enrichment sequencing and copy number evaluation in inherited polyneuropathy.

Neurology 2016 05 13;86(19):1762-71. Epub 2016 Apr 13.

From the Departments of Neurology, Peripheral Nerve Division (W.W., P.J.D., C.J.K.), Department of Health Science Research (C.W., S.B., J.M.E.), Laboratory Medicine and Pathology (D.B.D., E.C.T., P.A.L., Y.W., C.J.K.), Medical Genome Facility (B.W.E., Y.W.), and Medical Genetics (C.J.K., D.B.D.), Mayo Clinic, Rochester, MN; Department of Neurology (W.W.), China-Japan Friendship Hospital, Beijing, China; and Department of Neurology (S.S.S.), Perelman School of Medicine, University of Pennsylvania, Philadelphia.

Objective: To assess the efficiency of target-enrichment next-generation sequencing (NGS) with copy number assessment in inherited neuropathy diagnosis.

Methods: A 197 polyneuropathy gene panel was designed to assess for mutations in 93 patients with inherited or idiopathic neuropathy without known genetic cause. We applied our novel copy number variation algorithm on NGS data, and validated the identified copy number mutations using CytoScan (Affymetrix). Cost and efficacy of this targeted NGS approach was compared to earlier evaluations.

Results: Average coverage depth was ∼760× (median = 600, 99.4% > 100×). Among 93 patients, 18 mutations were identified in 17 cases (18%), including 3 copy number mutations: 2 PMP22 duplications and 1 MPZ duplication. The 2 patients with PMP22 duplication presented with bulbar and respiratory involvement and had absent extremity nerve conductions, leading to axonal diagnosis. Average onset age of these 17 patients was 25 years (2-61 years), vs 45 years for those without genetic discovery. Among those with onset age less than 40 years, the diagnostic yield of targeted NGS approach is high (27%) and cost savings is significant (∼20%). However, the cost savings for patients with late onset age and without family history is not demonstrated.

Conclusions: Incorporating copy number analysis in target-enrichment NGS approach improved the efficiency of mutation discovery for chronic, inherited, progressive length-dependent polyneuropathy diagnosis. The new technology is facilitating a simplified genetic diagnostic algorithm utilizing targeted NGS, clinical phenotypes, age at onset, and family history to improve diagnosis efficiency. Our findings prompt a need for updating the current practice parameters and payer guidelines.
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http://dx.doi.org/10.1212/WNL.0000000000002659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862246PMC
May 2016

Prevalence of Pathogenic Mutations in Cancer Predisposition Genes among Pancreatic Cancer Patients.

Cancer Epidemiol Biomarkers Prev 2016 Jan 19;25(1):207-11. Epub 2015 Oct 19.

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota. Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota.

The prevalence of germline pathogenic mutations in a comprehensive panel of cancer predisposition genes is not well-defined for patients with pancreatic ductal adenocarcinoma (PDAC). To estimate the frequency of mutations in a panel of 22 cancer predisposition genes, 96 patients unselected for a family history of cancer who were recruited to the Mayo Clinic Pancreatic Cancer patient registry over a 12-month period were screened by next-generation sequencing. Fourteen pathogenic mutations in 13 patients (13.5%) were identified in eight genes: four in ATM, two in BRCA2, CHEK2, and MSH6, and one in BARD1, BRCA1, FANCM, and NBN. These included nine mutations (9.4%) in established pancreatic cancer genes. Three mutations were found in patients with a first-degree relative with PDAC, and 10 mutations were found in patients with first- or second-degree relatives with breast, pancreas, colorectal, ovarian, or endometrial cancers. These results suggest that a substantial proportion of patients with PDAC carry germline mutations in predisposition genes associated with other cancers and that a better understanding of pancreatic cancer risk will depend on evaluation of families with broad constellations of tumors. These findings highlight the need for recommendations governing germline gene-panel testing of patients with pancreatic cancer.
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http://dx.doi.org/10.1158/1055-9965.EPI-15-0455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4754121PMC
January 2016

Draft Genome Sequences of Nine Pseudomonas aeruginosa Strains, Including Eight Clinical Isolates.

Genome Announc 2015 Oct 8;3(5). Epub 2015 Oct 8.

Department of Surgery, Mayo Clinic, Rochester, Minnesota, USA Center for Individualized Medicine, Mayo Clinic, Rochester, Minnesota, USA Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota, USA

We report on nine draft genomes of Pseudomonas aeruginosa isolates, assembled using a hybrid paired-end and Nextera mate-pair library approach. Eight are of clinical origin, and one is the ATCC 27853 strain. We also report their multilocus sequence types.
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http://dx.doi.org/10.1128/genomeA.01154-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4599088PMC
October 2015

Recessive MYH6 Mutations in Hypoplastic Left Heart With Reduced Ejection Fraction.

Circ Cardiovasc Genet 2015 Aug 17;8(4):564-71. Epub 2015 Jun 17.

From the Cardiovascular Genetics Research Laboratory (J.L.T., T.M.O.), Division of Pediatric Cardiology, Department of Pediatric and Adolescent Medicine (M.Y.Q., P.W.O'L., T.M.O.), Division of Cardiovascular Diseases, Department of Internal Medicine (T.M.O.), Departments of Health Sciences Research and Biomedical Statistics and Informatics (M.T.Z., J.M.E.), Medical Genome Facility (B.W.E., E.D.W.), and Department of Biochemistry and Molecular Biology (E.D.W.), Mayo Clinic, Rochester, MN.

Background: The molecular underpinnings of hypoplastic left heart are poorly understood. Staged surgical palliation has dramatically improved survival, yet eventual failure of the systemic right ventricle necessitates cardiac transplantation in a subset of patients. We sought to identify genetic determinants of hypoplastic left heart with latent right ventricular dysfunction in individuals with a Fontan circulation.

Methods And Results: Evaluation of cardiac structure and function by echocardiography in patients with hypoplastic left heart and their first-degree relatives identified 5 individuals with right ventricular ejection fraction ≤40% after Fontan operation. Whole genome sequencing was performed on DNA from 21 family members, filtering for genetic variants with allele frequency <1% predicted to alter protein structure or expression. Secondary family-based filtering for de novo and recessive variants revealed rare inherited missense mutations on both paternal and maternal alleles of MYH6, encoding myosin heavy chain 6, in 2 patients who developed right ventricular dysfunction 3 to 11 years postoperatively. Parents and siblings who were heterozygous carriers had normal echocardiograms. Protein modeling of the 4 highly conserved amino acid substitutions, residing in both head and tail domains, predicted perturbation of protein structure and function.

Conclusions: In contrast to dominant MYH6 mutations with variable penetrance identified in other congenital heart defects and dilated cardiomyopathy, this study reveals compound heterozygosity for recessive MYH6 mutations in patients with hypoplastic left heart and reduced systemic right ventricular ejection fraction. These findings implicate a shared molecular basis for the developmental arrest and latent myopathy of left and right ventricles, respectively.
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http://dx.doi.org/10.1161/CIRCGENETICS.115.001070DOI Listing
August 2015

Homozygous/Compound Heterozygous Triadin Mutations Associated With Autosomal-Recessive Long-QT Syndrome and Pediatric Sudden Cardiac Arrest: Elucidation of the Triadin Knockout Syndrome.

Circulation 2015 Jun 28;131(23):2051-60. Epub 2015 Apr 28.

From Department of Molecular Pharmacology & Experimental Therapeutics, Windland Smith Rice Sudden Death Genomics Laboratory (H.M.A., D.J.T., M.L.W., M.J.A.), Department of Medicine/Division of Cardiovascular Diseases (D.J.T., M.L.W., M.J.A.), Medical Genome Facility (B.W.E.), and Department of Pediatrics/Division of Pediatric Cardiology (M.J.A.), Mayo Clinic, Rochester, MN; and Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo College of Medicine, Rochester, MN (S.M., J.M.E.).

Background: Long-QT syndrome (LQTS) may result in syncope, seizures, or sudden cardiac arrest. Although 16 LQTS-susceptibility genes have been discovered, 20% to 25% of LQTS remains genetically elusive.

Methods And Results: We performed whole-exome sequencing child-parent trio analysis followed by recessive and sporadic inheritance modeling and disease-network candidate analysis gene ranking to identify a novel underlying genetic mechanism for LQTS. Subsequent mutational analysis of the candidate gene was performed with polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing on a cohort of 33 additional unrelated patients with genetically elusive LQTS. After whole-exome sequencing and variant filtration, a homozygous p.D18fs*13 TRDN-encoded triadin frameshift mutation was discovered in a 10-year-old female patient with LQTS with a QTc of 500 milliseconds who experienced recurrent exertion-induced syncope/cardiac arrest beginning at 1 year of age. Subsequent mutational analysis of TRDN revealed either homozygous or compound heterozygous frameshift mutations in 4 of 33 unrelated cases of LQTS (12%). All 5 TRDN-null patients displayed extensive T-wave inversions in precordial leads V1 through V4, with either persistent or transient QT prolongation and severe disease expression of exercise-induced cardiac arrest in early childhood (≤3 years of age) and required aggressive therapy. The overall yield of TRDN mutations was significantly greater in patients ≤10 years of age (5 of 10, 50%) compared with older patients (0 of 24, 0%; P=0.0009).

Conclusions: We identified TRDN as a novel underlying genetic basis for recessively inherited LQTS. All TRDN-null patients had strikingly similar phenotypes. Given the recurrent nature of potential lethal arrhythmias, patients fitting this phenotypic profile should undergo cardiac TRDN genetic testing.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.115.015397DOI Listing
June 2015

Comprehensive assessment of genetic variants within TCF4 in Fuchs' endothelial corneal dystrophy.

Invest Ophthalmol Vis Sci 2014 Aug 28;55(9):6101-7. Epub 2014 Aug 28.

Department of Ophthalmology, Mayo Clinic, Rochester, Minnesota, United States.

Purpose: The single nucleotide variant (SNV), rs613872, in the transcription factor 4 (TCF4) gene was previously found to be strongly associated (P = 6 × 10(-26)) with Fuchs' endothelial corneal dystrophy (FECD). Subsequently, an intronic expansion of the repeating trinucleotides, TGC, was found to be even more predictive of disease. We performed comprehensive sequencing of the TCF4 gene region in order to identify the best marker for FECD within TCF4 and to identify other novel variants that may be associated with FECD.

Methods: Leukocyte DNA was isolated from 68 subjects with FECD and 16 unaffected individuals. A custom capture panel was used to isolate the region surrounding the two previously validated markers of FECD. Sequencing of the TCF4 coding region, introns and flanking sequence, spanning 465 kb was performed at >1000× average coverage using the Illumina HiSequation 2000.

Results: TGC expansion (>50 repeats) was present in 46 (68%) FECD-affected subjects and one (6%) normal subject. A total of 1866 variants, including 1540 SNVs, were identified. Only two previously reported SNVs resided in the TCF4 coding region, neither of which segregated with disease. No variant, including TGC expansion, correlated perfectly with disease status. Trinucleotide repeat expansion was a better predictor of disease than any other variant.

Conclusions: Complete sequencing of the TCF4 genomic region revealed no single causative variant for FECD. The intronic trinucleotide repeat expansion within TCF4 continues to be more strongly associated with FECD than any other genetic variant.
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http://dx.doi.org/10.1167/iovs.14-14958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4179444PMC
August 2014

Clinical biomarkers of pulmonary carcinoid tumors in never smokers via profiling miRNA and target mRNA.

Cell Biosci 2014 9;4:35. Epub 2014 Jul 9.

Department of Health Sciences Research, Mayo Clinic College of Medicine, Rochester, Minnesota, USA.

Background: miRNAs play key regulatory roles in cellular pathological processes. We aimed to identify clinically meaningful biomarkers in pulmonary carcinoid tumors (PCTs), a member of neuroendocrine neoplasms, via profiling miRNAs and mRNAs.

Results: From the total of 1145 miRNAs, we obtained 16 and 17 miRNAs that showed positive and negative fold changes (FCs, tumors vs. normal tissues) in the top 1% differentially expressed miRNAs, respectively. We uncovered the target genes that were predicted by at least two prediction tools and overlapped by at least one-half of the top miRNAs, which yielded 44 genes (FC<-2) and 56 genes (FC>2), respectively. Higher expressions of CREB5, PTPRB and COL4A3 predicted favorable disease free survival (Hazard ratio: 0.03, 0.19 and 0.36; P value: 0.03, 0.03 and 0.08). Additionally, 79 mutated genes have been found in nine PCTs where TP53 was the only repeated mutation.

Conclusion: We identified that the expressions of three genes have clinical implications in PCTs. The biological functions of these biomarkers warrant further studies.
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http://dx.doi.org/10.1186/2045-3701-4-35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4124500PMC
August 2014

Conserved recurrent gene mutations correlate with pathway deregulation and clinical outcomes of lung adenocarcinoma in never-smokers.

BMC Med Genomics 2014 Jun 4;7:32. Epub 2014 Jun 4.

Department of Health Sciences Research, Mayo Clinic, 200 First St SW, Rochester, MN 55905, USA.

Background: Novel and targetable mutations are needed for improved understanding and treatment of lung cancer in never-smokers.

Methods: Twenty-seven lung adenocarcinomas from never-smokers were sequenced by both exome and mRNA-seq with respective normal tissues. Somatic mutations were detected and compared with pathway deregulation, tumor phenotypes and clinical outcomes.

Results: Although somatic mutations in DNA or mRNA ranged from hundreds to thousands in each tumor, the overlap mutations between the two were only a few to a couple of hundreds. The number of somatic mutations from either DNA or mRNA was not significantly associated with clinical variables; however, the number of overlap mutations was associated with cancer subtype. These overlap mutants were preferentially expressed in mRNA with consistently higher allele frequency in mRNA than in DNA. Ten genes (EGFR, TP53, KRAS, RPS6KB2, ATXN2, DHX9, PTPN13, SP1, SPTAN1 and MYOF) had recurrent mutations and these mutations were highly correlated with pathway deregulation and patient survival.

Conclusions: The recurrent mutations present in both DNA and RNA are likely the driver for tumor biology, pathway deregulation and clinical outcomes. The information may be used for patient stratification and therapeutic target development.
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http://dx.doi.org/10.1186/1755-8794-7-32DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060138PMC
June 2014

Mate pair sequencing of oropharyngeal squamous cell carcinomas reveals that HPV integration occurs much less frequently than in cervical cancer.

J Clin Virol 2014 Mar 28;59(3):195-200. Epub 2013 Dec 28.

Division of Experimental Pathology, Mayo Clinic, Rochester, MN, United States. Electronic address:

Background: Human papillomavirus (HPV) is now recognized to be very important in the pathogenesis of oropharyngeal squamous cell carcinoma (OPSCC). It is not clear yet whether the physical status of HPV in OPSCC is similar to what is found in cervical cancer.

Study Design: We performed genome-wide mate pair next generation sequencing from 20 OPSCCs patients, thirteen of which were positive for HPV16 to determine the HPV physical status and its relationship to HPV oncogene E6 and E7 expression.

Results: This high throughput approach detected HPV integration events and also determined the bridged HPV coverage in each sample. Two of the HPV16-positive OPSCCs had HPV integration and one of the HPV16-negative OPSCCs had an HPV26 integration. We mapped the site of integration in the HPV genome in all integration events and the integrations were located at E1, E5, E6 and L2 region respectively. One HPV positive OPSCC had two integration events but also had a very high bridged HPV coverage, while the other two just had HPV integrated into the human genome.

Conclusion: Our results are thus different from what is routinely observed in cervical cancer where HPV is almost always integrated into the human genome with loss of episomal HPV sequences. Thus more investigation should be carried out to study how episomal HPV alone can contribute to the development of most OPSCCs.
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http://dx.doi.org/10.1016/j.jcv.2013.12.006DOI Listing
March 2014

Gene expression, single nucleotide variant and fusion transcript discovery in archival material from breast tumors.

PLoS One 2013 22;8(11):e81925. Epub 2013 Nov 22.

Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida, United States of America.

Advantages of RNA-Seq over array based platforms are quantitative gene expression and discovery of expressed single nucleotide variants (eSNVs) and fusion transcripts from a single platform, but the sensitivity for each of these characteristics is unknown. We measured gene expression in a set of manually degraded RNAs, nine pairs of matched fresh-frozen, and FFPE RNA isolated from breast tumor with the hybridization based, NanoString nCounter (226 gene panel) and with whole transcriptome RNA-Seq using RiboZeroGold ScriptSeq V2 library preparation kits. We performed correlation analyses of gene expression between samples and across platforms. We then specifically assessed whole transcriptome expression of lincRNA and discovery of eSNVs and fusion transcripts in the FFPE RNA-Seq data. For gene expression in the manually degraded samples, we observed Pearson correlations of >0.94 and >0.80 with NanoString and ScriptSeq protocols, respectively. Gene expression data for matched fresh-frozen and FFPE samples yielded mean Pearson correlations of 0.874 and 0.783 for NanoString (226 genes) and ScriptSeq whole transcriptome protocols respectively, p<2x10(-16). Specifically for lincRNAs, we observed superb Pearson correlation (0.988) between matched fresh-frozen and FFPE pairs. FFPE samples across NanoString and RNA-Seq platforms gave a mean Pearson correlation of 0.838. In FFPE libraries, we detected 53.4% of high confidence SNVs and 24% of high confidence fusion transcripts. Sensitivity of fusion transcript detection was not overcome by an increase in depth of sequencing up to 3-fold (increase from ~56 to ~159 million reads). Both NanoString and ScriptSeq RNA-Seq technologies yield reliable gene expression data for degraded and FFPE material. The high degree of correlation between NanoString and RNA-Seq platforms suggests discovery based whole transcriptome studies from FFPE material will produce reliable expression data. The RiboZeroGold ScriptSeq protocol performed particularly well for lincRNA expression from FFPE libraries, but detection of eSNV and fusion transcripts was less sensitive.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081925PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3838386PMC
August 2014

Novel TRAF1-ALK fusion identified by deep RNA sequencing of anaplastic large cell lymphoma.

Genes Chromosomes Cancer 2013 Nov 2;52(11):1097-102. Epub 2013 Sep 2.

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.

Chromosomal translocations leading to expression of abnormal fusion proteins play a major role in the pathogenesis of various hematologic malignancies. The recent development of high-throughput, "deep" sequencing has allowed discovery of novel translocations leading to a rapid increase in understanding these diseases. Translocations involving the anaplastic lymphoma kinase (ALK) gene leading to ALK fusion proteins originally were discovered in anaplastic large cell lymphomas (ALCLs). Among ALCLs, NPM1-ALK fusions are most common and lead to nuclear localization of the fusion protein. Here, we present a 50-year-old male with ALCL demonstrating cytoplasmic ALK immunoreactivity only, suggesting the presence of a non-NPM1 fusion partner. We performed deep RNA sequencing of tumor tissue from this patient and identified a novel transcript fusing Exon 6 of TRAF1 to Exon 20 of ALK. The TRAF1-ALK fusion transcript was confirmed at the mRNA level by Sanger sequencing and the fusion protein was visualized by Western blot. The discovery of this TRAF1-ALK fusion expands the diversity of known ALK fusion partners and highlights the power of deep sequencing for fusion transcript discovery. © 2013 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/gcc.22104DOI Listing
November 2013

Contribution of FKBP5 genetic variation to gemcitabine treatment and survival in pancreatic adenocarcinoma.

PLoS One 2013 1;8(8):e70216. Epub 2013 Aug 1.

Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota, USA.

Purpose: FKBP51, (FKBP5), is a negative regulator of Akt. Variability in FKBP5 expression level is a major factor contributing to variation in response to chemotherapeutic agents including gemcitabine, a first line treatment for pancreatic cancer. Genetic variation in FKBP5 could influence its function and, ultimately, treatment response of pancreatic cancer.

Experimental Design: We set out to comprehensively study the role of genetic variation in FKBP5 identified by Next Generation DNA resequencing on response to gemcitabine treatment of pancreatic cancer by utilizing both tumor and germline DNA samples from 43 pancreatic cancer patients, including 19 paired normal-tumor samples. Next, genotype-phenotype association studies were performed with overall survival as well as with FKBP5 gene expression in tumor using the same samples in which resequencing had been performed, followed by functional genomics studies.

Results: In-depth resequencing identified 404 FKBP5 single nucleotide polymorphisms (SNPs) in normal and tumor DNA. SNPs with the strongest associations with survival or FKBP5 expression were subjected to functional genomic study. Electromobility shift assay showed that the rs73748206 "A(T)" SNP altered DNA-protein binding patterns, consistent with significantly increased reporter gene activity, possibly through its increased binding to Glucocorticoid Receptor (GR). The effect of rs73748206 was confirmed on the basis of its association with FKBP5 expression by affecting the binding to GR in lymphoblastoid cell lines derived from the same patients for whom DNA was used for resequencing.

Conclusion: This comprehensive FKBP5 resequencing study provides insights into the role of genetic variation in variation of gemcitabine response.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0070216PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3731355PMC
March 2014

Genetic alterations associated with progression from pancreatic intraepithelial neoplasia to invasive pancreatic tumor.

Gastroenterology 2013 Nov 2;145(5):1098-1109.e1. Epub 2013 Aug 2.

Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota.

Background & Aims: Increasing grade of pancreatic intraepithelial neoplasia (PanIN) has been associated with progression to pancreatic ductal adenocarcinoma (PDAC). However, the mechanisms that control progression from PanINs to PDAC are not well understood. We investigated the genetic alterations involved in this process.

Methods: Genomic DNA samples from laser-capture microdissected PDACs and adjacent PanIN2 and PanIN3 lesions from 10 patients with pancreatic cancer were analyzed by exome sequencing.

Results: Similar numbers of somatic mutations were identified in PanINs and tumors, but the mutational load varied greatly among cases. Ten of the 15 isolated PanINs shared more than 50% of somatic mutations with associated tumors. Mutations common to tumors and clonally related PanIN2 and PanIN3 lesions were identified as genes that could promote carcinogenesis. KRAS and TP53 frequently were altered in PanINs and tumors, but few other recurrently modified genes were detected. Mutations in DNA damage response genes were prevalent in all samples. Genes that encode proteins involved in gap junctions, the actin cytoskeleton, the mitogen-activated protein kinase signaling pathway, axon guidance, and cell-cycle regulation were among the earliest targets of mutagenesis in PanINs that progressed to PDAC.

Conclusions: Early stage PanIN2 lesions appear to contain many of the somatic gene alterations required for PDAC development.
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http://dx.doi.org/10.1053/j.gastro.2013.07.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3926442PMC
November 2013

The genomic landscape of small intestine neuroendocrine tumors.

J Clin Invest 2013 Jun 15;123(6):2502-8. Epub 2013 May 15.

Division of Medical Oncology, Mayo Clinic, Rochester, Minnesota 55905, USA.

Small intestine neuroendocrine tumors (SI-NETs) are the most common malignancy of the small bowel. Several clinical trials target PI3K/Akt/mTOR signaling; however, it is unknown whether these or other genes are genetically altered in these tumors. To address the underlying genetics, we analyzed 48 SI-NETs by massively parallel exome sequencing. We detected an average of 0.1 somatic single nucleotide variants (SNVs) per 106 nucleotides (range, 0-0.59), mostly transitions (C>T and A>G), which suggests that SI-NETs are stable cancers. 197 protein-altering somatic SNVs affected a preponderance of cancer genes, including FGFR2, MEN1, HOOK3, EZH2, MLF1, CARD11, VHL, NONO, and SMAD1. Integrative analysis of SNVs and somatic copy number variations identified recurrently altered mechanisms of carcinogenesis: chromatin remodeling, DNA damage, apoptosis, RAS signaling, and axon guidance. Candidate therapeutically relevant alterations were found in 35 patients, including SRC, SMAD family genes, AURKA, EGFR, HSP90, and PDGFR. Mutually exclusive amplification of AKT1 or AKT2 was the most common event in the 16 patients with alterations of PI3K/Akt/mTOR signaling. We conclude that sequencing-based analysis may provide provisional grouping of SI-NETs by therapeutic targets or deregulated pathways.
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http://dx.doi.org/10.1172/JCI67963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3668835PMC
June 2013

Identification of novel variants in colorectal cancer families by high-throughput exome sequencing.

Cancer Epidemiol Biomarkers Prev 2013 Jul 1;22(7):1239-51. Epub 2013 May 1.

Department of Health Sciences Research, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.

Background: Colorectal cancer (CRC) in densely affected families without Lynch Syndrome may be due to mutations in undiscovered genetic loci. Familial linkage analyses have yielded disparate results; the use of exome sequencing in coding regions may identify novel segregating variants.

Methods: We completed exome sequencing on 40 affected cases from 16 multicase pedigrees to identify novel loci. Variants shared among all sequenced cases within each family were identified and filtered to exclude common variants and single-nucleotide variants (SNV) predicted to be benign.

Results: We identified 32 nonsense or splice-site SNVs, 375 missense SNVs, 1,394 synonymous or noncoding SNVs, and 50 indels in the 16 families. Of particular interest are two validated and replicated missense variants in CENPE and KIF23, which are both located within previously reported CRC linkage regions, on chromosomes 1 and 15, respectively.

Conclusions: Whole-exome sequencing identified DNA variants in multiple genes. Additional sequencing of these genes in additional samples will further elucidate the role of variants in these regions in CRC susceptibility.

Impact: Exome sequencing of familial CRC cases can identify novel rare variants that may influence disease risk.
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http://dx.doi.org/10.1158/1055-9965.EPI-12-1226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3704223PMC
July 2013

Natriuretic peptide receptor-3 gene (NPR3): nonsynonymous polymorphism results in significant reduction in protein expression because of accelerated degradation.

Circ Cardiovasc Genet 2013 Apr 14;6(2):201-10. Epub 2013 Mar 14.

Division of Cardiovascular Diseases, Department of Internal Medicine, Mayo Clinic, Rochester, MN 55905, USA.

BACKGROUND- The primary role of natriuretic peptide receptor-3 (NPR3) or NPR-C is in the clearance of natriuretic peptides that play an important role in modulating intravascular volume and vascular tone. Genetic variation in NPR3 has been associated with variation in blood pressure and obesity. Despite the importance of NPR3, sequence variation in the gene has not been addressed using DNA from different ethnic populations. We set out to identify and functionally characterize genetic variation in NPR3 in 3 ethnic groups. METHODS AND RESULTS- DNA samples from 96 European American, 96 African American, and 96 Han Chinese American healthy subjects were used to resequence NPR3 exons, splice junctions, and flanking regions. We identified 105 polymorphisms, 50 of which were novel, including 8 nonsynonymous single-nucleotide polymorphisms, 7 were novel. Expression constructs were created for the nonsynonymous single-nucleotide polymorphisms. HEK293 cells were transfected with constructs for wild type and variant allozymes; and recombinant proteins were measured by quantitative Western blot analysis. The most significant change in NPR3 protein was observed for the Arg146 variant allozyme, with 20% of wild-type protein, primarily because of autophagy-dependent degradation. NPR3 structural modeling confirmed that the Arg146 variant protein was not compatible with wild-type conformation and could result in protein misfolding or instability. CONCLUSIONS- Multiple novel NPR3 genetic polymorphisms were identified in 3 ethnic groups. The Arg146 allozyme displayed a significant decrease in protein quantity because of degradation mediated predominantly by autophagy. This genetic variation could have a significant effect on the metabolism of natriuretic peptides with potential clinical implications.
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http://dx.doi.org/10.1161/CIRCGENETICS.112.964742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3685298PMC
April 2013

Multi-platform analysis of microRNA expression measurements in RNA from fresh frozen and FFPE tissues.

PLoS One 2013 31;8(1):e52517. Epub 2013 Jan 31.

Medical Genome Facility Gene Expression Core, Mayo Clinic, Rochester, Minnesota, United States of America.

MicroRNAs play a role in regulating diverse biological processes and have considerable utility as molecular markers for diagnosis and monitoring of human disease. Several technologies are available commercially for measuring microRNA expression. However, cross-platform comparisons do not necessarily correlate well, making it difficult to determine which platform most closely represents the true microRNA expression level in a tissue. To address this issue, we have analyzed RNA derived from cell lines, as well as fresh frozen and formalin-fixed paraffin embedded tissues, using Affymetrix, Agilent, and Illumina microRNA arrays, NanoString counting, and Illumina Next Generation Sequencing. We compared the performance within- and between the different platforms, and then verified these results with those of quantitative PCR data. Our results demonstrate that the within-platform reproducibility for each method is consistently high and although the gene expression profiles from each platform show unique traits, comparison of genes that were commonly detectable showed that detection of microRNA transcripts was similar across multiple platforms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052517PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561362PMC
September 2013

FKBP5 genetic variation: association with selective serotonin reuptake inhibitor treatment outcomes in major depressive disorder.

Pharmacogenet Genomics 2013 Mar;23(3):156-66

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota 55905, USA.

Objectives: FKBP51 (51 kDa immunophilin) acts as a modulator of the glucocorticoid receptor and a negative regulator of the Akt pathway. Genetic variation in FKBP5 plays a role in antidepressant response. The aim of this study was to comprehensively assess the role of genetic variation in FKBP5, identified by both Sanger and Next Generation DNA resequencing, as well as genome-wide single nucleotide polymorphisms (SNPs) associated with FKBP5 expression in the response to the selective serotonin reuptake inhibitor (SSRI) treatment of major depressive disorder.

Methods: We identified 657 SNPs in FKBP5 by Next Generation sequencing of 96 DNA samples from white patients, and 149 SNPs were selected for the genotyping together with 235 SNPs that were trans-associated with variation in FKBP5 expression in lymphoblastoid cells. A total of 529 DNA samples from the Mayo Clinic PGRN-SSRI Pharmacogenomic trial for which genome-wide SNPs had already been obtained were genotyped for these 384 SNPs, and associations with treatment outcomes were determined. The most significant SNPs were genotyped using 96 DNA samples from white non-Hispanic patients of the NIMH-supported Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study to attempt replication, followed by functional genomic studies.

Results: Genotype-phenotype association analysis indicated that rs352428 was associated with both 8-week treatment response in the Mayo study (odds ratio=0.49; P=0.003) and 6-week response in the STAR*D replication study (odds ratio=0.74; P=0.05). The electrophoresis mobility shift assay and the reporter gene assay confirmed the possible role of this SNP in transcription regulation.

Conclusion: This comprehensive FKBP5 sequence study provides insight into the role of common genetic polymorphisms that might influence SSRI treatment outcomes in major depressive disorder patients.
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http://dx.doi.org/10.1097/FPC.0b013e32835dc133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3784025PMC
March 2013

Mate pair sequencing of whole-genome-amplified DNA following laser capture microdissection of prostate cancer.

DNA Res 2012 Oct 18;19(5):395-406. Epub 2012 Sep 18.

Department of Molecular Medicine, Mayo Clinic, Medical Sciences Building 2, 200 First St., SW, Rochester, MN 55905, USA.

High-throughput next-generation sequencing provides a revolutionary platform to unravel the precise DNA aberrations concealed within subgroups of tumour cells. However, in many instances, the limited number of cells makes the application of this technology in tumour heterogeneity studies a challenge. In order to address these limitations, we present a novel methodology to partner laser capture microdissection (LCM) with sequencing platforms, through a whole-genome amplification (WGA) protocol performed in situ directly on LCM engrafted cells. We further adapted current Illumina mate pair (MP) sequencing protocols to the input of WGA DNA and used this technology to investigate large genomic rearrangements in adjacent Gleason Pattern 3 and 4 prostate tumours separately collected by LCM. Sequencing data predicted genome coverage and depths similar to unamplified genomic DNA, with limited repetition and bias predicted in WGA protocols. Mapping algorithms developed in our laboratory predicted high-confidence rearrangements and selected events each demonstrated the predicted fusion junctions upon validation. Rearrangements were additionally confirmed in unamplified tissue and evaluated in adjacent benign-appearing tissues. A detailed understanding of gene fusions that characterize cancer will be critical in the development of biomarkers to predict the clinical outcome. The described methodology provides a mechanism of efficiently defining these events in limited pure populations of tumour tissue, aiding in the derivation of genomic aberrations that initiate cancer and drive cancer progression.
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http://dx.doi.org/10.1093/dnares/dss021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3473372PMC
October 2012

Genome-wide analysis reveals recurrent structural abnormalities of TP63 and other p53-related genes in peripheral T-cell lymphomas.

Blood 2012 Sep 1;120(11):2280-9. Epub 2012 Aug 1.

Center for Individualized Medicine, Mayo Clinic, Rochester, MN 55905, USA.

Peripheral T-cell lymphomas (PTCLs) are aggressive malignancies of mature T lymphocytes with 5-year overall survival rates of only ∼ 35%. Improvement in outcomes has been stymied by poor understanding of the genetics and molecular pathogenesis of PTCL, with a resulting paucity of molecular targets for therapy. We developed bioinformatic tools to identify chromosomal rearrangements using genome-wide, next-generation sequencing analysis of mate-pair DNA libraries and applied these tools to 16 PTCL patient tissue samples and 6 PTCL cell lines. Thirteen recurrent abnormalities were identified, of which 5 involved p53-related genes (TP53, TP63, CDKN2A, WWOX, and ANKRD11). Among these abnormalities were novel TP63 rearrangements encoding fusion proteins homologous to ΔNp63, a dominant-negative p63 isoform that inhibits the p53 pathway. TP63 rearrangements were seen in 11 (5.8%) of 190 PTCLs and were associated with inferior overall survival; they also were detected in 2 (1.2%) of 164 diffuse large B-cell lymphomas. As TP53 mutations are rare in PTCL compared with other malignancies, our findings suggest that a constellation of alternate genetic abnormalities may contribute to disruption of p53-associated tumor suppressor function in PTCL.
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http://dx.doi.org/10.1182/blood-2012-03-419937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5070713PMC
September 2012

Human liver methionine cycle: MAT1A and GNMT gene resequencing, functional genomics, and hepatic genotype-phenotype correlation.

Drug Metab Dispos 2012 Oct 17;40(10):1984-92. Epub 2012 Jul 17.

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA.

The "methionine cycle" plays a critical role in the regulation of concentrations of (S)-adenosylmethionine (AdoMet), the major biological methyl donor. We set out to study sequence variation in genes encoding the enzyme that synthesizes AdoMet in liver, methionine adenosyltransferase 1A (MAT1A) and the major hepatic AdoMet using enzyme, glycine N-methyltransferase (GNMT), as well as functional implications of that variation. We resequenced MAT1A and GNMT using DNA from 288 subjects of three ethnicities, followed by functional genomic and genotype-phenotype correlation studies performed with 268 hepatic biopsy samples. We identified 44 and 42 polymorphisms in MAT1A and GNMT, respectively. Quantitative Western blot analyses for the human liver samples showed large individual variation in MAT1A and GNMT protein expression. Genotype-phenotype correlation identified two genotyped single-nucleotide polymorphisms (SNPs), reference SNP (rs) 9471976 (corrected p = 3.9 × 10(-10)) and rs11752813 (corrected p = 1.8 × 10(-5)), and 42 imputed SNPs surrounding GNMT that were significantly associated with hepatic GNMT protein levels (corrected p values < 0.01). Reporter gene studies showed that variant alleles for both genotyped SNPs resulted in decreased transcriptional activity. Correlation analyses among hepatic protein levels for methionine cycle enzymes showed significant correlations between GNMT and MAT1A (p = 1.5 × 10(-3)) and between GNMT and betaine homocysteine methyltransferase (p = 1.6 × 10(-7)). Our discovery of SNPs that are highly associated with hepatic GNMT protein expression as well as the "coordinate regulation" of methionine cycle enzyme protein levels provide novel insight into the regulation of this important human liver biochemical pathway.
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http://dx.doi.org/10.1124/dmd.112.046953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463826PMC
October 2012
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