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Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq.
Treutlein B, Brownfield DG, Wu AR, Neff NF, Mantalas GL, Espinoza FH, Desai TJ, Krasnow MA, Quake SR
Abstract -

The mammalian lung is a highly branched network in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that mediate gas exchange. Although this transformation has been studied by marker expression analysis and fate-mapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types are still incompletely known, in part because of the limited number of lineage markers and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations. Here we show that single-cell transcriptome analysis circumvents these problems and enables direct measurement of the various cell types and hierarchies in the developing lung. We used microfluidic single-cell RNA sequencing (RNA-seq) on 198 individual cells at four different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups by using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or the previous purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell-type diversity in the distal lung and led to the discovery of many previously unknown cell-type markers, including transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full life cycle of the alveolar type?2 cell lineage. This single-cell genomics approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.

Illumina Summary -

The mammalian lung cell progenitor cells develop along distinct lineages. To study the development of progenitor cells into mature alveolar cell types, this study set out to study the gene expression from single cells in development. Using microfluidic single-cell transcriptome analysis by Illumina sequencing on 198 individual cells at four different stages of differentiation, the authors classified cells into distinct groups based on expression and elucidated the full life cycle of the alveolar type 2 cell lineage.

Nature 509 371-5 2014
Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling.
Tome JM, Ozer A, Pagano JM, Gheba D, Schroth GP, Lis JT
Abstract -

RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.

Illumina Summary -

The interactions of RNA and protein are critical to gene expression and regulation. This paper presents a high-throughput sequencing RNA affinity profiling assay (HiTS-RAP) with a protocol that does not rely on antibodies. The authors demonstrated their method by measuring the affinity of specific aptamers to their respective targets using Illumina Genome Analyzer for sequencing. HiTS-RAP will facilitate genome-wide, direct, quantitative measurement of RNA affinity for regulatory proteins.

Nat Methods 2014
Towards error-free profiling of immune repertoires.
Shugay M, Britanova OV, Merzlyak EM, Turchaninova MA, Mamedov IZ, Tuganbaev TR, Bolotin DA, Staroverov DB, Putintseva EV, Plevova K, Linnemann C, Shagin D, Pospisilova S, Lukyanov S, Schumacher TN, Chudakov DM
Abstract -

Deep profiling of antibody and T cell-receptor repertoires by means of high-throughput sequencing has become an attractive approach for adaptive immunity studies, but its power is substantially compromised by the accumulation of PCR and sequencing errors. Here we report MIGEC (molecular identifier groups-based error correction), a strategy for high-throughput sequencing data analysis. MIGEC allows for nearly absolute error correction while fully preserving the natural diversity of complex immune repertoires.

Illumina Summary -

Next-generation sequencing has opened up new possibilities for analyzing genomic diversity in studies of the adaptive immune system. This paper presents MIGEC an improved analysis method for determining T cell-receptor repertoires by Illumina sequencing. Using uniqe barcode labelling combined with two-stage PCR amplification MIGEC allows for nearly absolute error correction while fully preserving the natural diversity of complex immune repertoires.

Nat Methods 2014
Germline and somatic SMARCA4 mutations characterize small cell carcinoma of the ovary, hypercalcemic type.
Witkowski L, Carrot-Zhang J, Albrecht S, Fahiminiya S, Hamel N, Tomiak E, Grynspan D, Saloustros E, Nadaf J, Rivera B, Gilpin C, Castellsagué E, Silva-Smith R, Plourde F, Wu M, Saskin A, Arseneault M, Karabakhtsian RG, Reilly EA, Ueland FR, Margiolaki A, Pavlakis K, Castellino SM, Lamovec J, Mackay HJ, Roth LM, Ulbright TM, Bender TA, Georgoulias V, Longy M, Berchuck A, Tischkowitz M, Nagel I, Siebert R, Stewart CJ, Arseneau J, McCluggage WG, Clarke BA, Riazalhosseini Y, Hasselblatt M, Majewski J, Foulkes WD
Abstract -

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is the most common undifferentiated ovarian malignancy in women under 40 years of age. We sequenced the exomes of six individuals from three families with SCCOHT. After discovering segregating deleterious germline mutations in SMARCA4 in all three families, we tested DNA from a fourth affected family, which also carried a segregating SMARCA4 germline mutation. All the familial tumors sequenced harbored either a somatic mutation or loss of the wild-type allele. Immunohistochemical analysis of these cases and additional familial and non-familial cases showed loss of SMARCA4 (BRG1) protein in 38 of 40 tumors overall. Sequencing of cases with available DNA identified at least one germline or somatic deleterious SMARCA4 mutation in 30 of 32 cases. Additionally, the SCCOHT cell line BIN-67 had biallelic deleterious mutations in SMARCA4. Our findings identify alterations in SMARCA4 as the major cause of SCCOHT, which could lead to improvements in genetic counseling and new treatment approaches.

Illumina Summary -

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is the most common undifferentiated ovarian malignancy in women under 40 years of age. This study used Illumina HiSeq for whole-exome sequencing of DNA from FFPE samples of well-characterized SCCOHT samples to determine common genetic variants. The authors discovered a loss of SMARCA4 (BRG1) protein in 38 of 40 tumors. Sequencing of cases with available DNA identified at least one germline or somatic deleterious SMARCA4 mutation in 30 of 32 cases. The authors hypothesise the alterations of the chromatin-remodeling gene SMARCA4 are a major driver of SCCOHT.

Nat Genet 46 438-43 2014
Identification of genomic alterations in oesophageal squamous cell cancer.
Song Y, Li L, Ou Y, Gao Z, Li E, Li X, Zhang W, Wang J, Xu L, Zhou Y, Ma X, Liu L, Zhao Z, Huang X, Fan J, Dong L, Chen G, Ma L, Yang J, Chen L, He M, Li M, Zhuang X, Huang K, Qiu K, Yin G, Guo G, Feng Q, Chen P, Wu Z, Wu J, Ma L, Zhao J, Luo L, Fu M, Xu B, Chen B, Li Y, Tong T, Wang M, Liu Z, Lin D, Zhang X, Yang H, Wang J, Zhan Q
Abstract -

Oesophageal cancer is one of the most aggressive cancers and is the sixth leading cause of cancer death worldwide. Approximately 70% of global oesophageal cancer cases occur in China, with oesophageal squamous cell carcinoma (ESCC) being the histopathological form in the vast majority of cases (>90%). Currently, there are limited clinical approaches for the early diagnosis and treatment of ESCC, resulting in a 10% five-year survival rate for patients. However, the full repertoire of genomic events leading to the pathogenesis of ESCC remains unclear. Here we describe a comprehensive genomic analysis of 158 ESCC cases, as part of the International Cancer Genome Consortium research project. We conducted whole-genome sequencing in 17 ESCC cases and whole-exome sequencing in 71 cases, of which 53 cases, plus an additional 70 ESCC cases not used in the whole-genome and whole-exome sequencing, were subjected to array comparative genomic hybridization analysis. We identified eight significantly mutated genes, of which six are well known tumour-associated genes (TP53, RB1, CDKN2A, PIK3CA, NOTCH1, NFE2L2), and two have not previously been described in ESCC (ADAM29 and FAM135B). Notably, FAM135B is identified as a novel cancer-implicated gene as assayed for its ability to promote malignancy of ESCC cells. Additionally, MIR548K, a microRNA encoded in the amplified 11q13.3-13.4 region, is characterized as a novel oncogene, and functional assays demonstrate that MIR548K enhances malignant phenotypes of ESCC cells. Moreover, we have found that several important histone regulator genes (MLL2 (also called KMT2D), ASH1L, MLL3 (KMT2C), SETD1B, CREBBP and EP300) are frequently altered in ESCC. Pathway assessment reveals that somatic aberrations are mainly involved in the Wnt, cell cycle and Notch pathways. Genomic analyses suggest that ESCC and head and neck squamous cell carcinoma share some common pathogenic mechanisms, and ESCC development is associated with alcohol drinking. This study has explored novel biological markers and tumorigenic pathways that would greatly improve therapeutic strategies for ESCC.

Illumina Summary -

Approximately 70% of global oesophageal cancer cases occur in China, with oesophageal squamous cell carcinoma (ESCC) being the histopathological form in the vast majority of cases (>90%). In this study, the authors performed a comprehensive genomic analysis of 158 ESCC cases as part of the International Cancer Genome Consortium research project. Using whole-genome sequencing for 17 cases and whole-exome sequencing for 71 cases and combining with array comparative genomic hybridization (arrayCGH) eight significantly mutated genes were reported of which two novel genes for ESCC: ADAM29 and FAM135B.

Nature 509 91-5 2014
Integrated exome and transcriptome sequencing reveals ZAK isoform usage in gastric cancer.
Liu J, McCleland M, Stawiski EW, Gnad F, Mayba O, Haverty PM, Durinck S, Chen YJ, Klijn C, Jhunjhunwala S, Lawrence M, Liu H, Wan Y, Chopra V, Yaylaoglu MB, Yuan W, Ha C, Gilbert HN, Reeder J, Pau G, Stinson J, Stern HM, Manning G, Wu TD, Neve RM, de Sauvage FJ, Modrusan Z, Seshagiri S, Firestein R, Zhang Z
Abstract -

Gastric cancer is the second leading cause of worldwide cancer mortality, yet the underlying genomic alterations remain poorly understood. Here we perform exome and transcriptome sequencing and SNP array assays to characterize 51 primary gastric tumours and 32 cell lines. Meta-analysis of exome data and previously published data sets reveals 24 significantly mutated genes in microsatellite stable (MSS) tumours and 16 in microsatellite instable (MSI) tumours. Over half the patients in our collection could potentially benefit from targeted therapies. We identify 55 splice site mutations accompanied by aberrant splicing products, in addition to mutation-independent differential isoform usage in tumours. ZAK kinase isoform TV1 is preferentially upregulated in gastric tumours and cell lines relative to normal samples. This pattern is also observed in colorectal, bladder and breast cancers. Overexpression of this particular isoform activates multiple cancer-related transcription factor reporters, while depletion of ZAK in gastric cell lines inhibits proliferation. These results reveal the spectrum of genomic and transcriptomic alterations in gastric cancer, and identify isoform-specific oncogenic properties of ZAK.

Illumina Summary -

Gastric cancer is the fourth most common cancer and the second most common cause of cancer-related death worldwide. This study examined the underlying genomic alterations associated with the development of the disease using Whole exome data sequencing on Illumina HiSeq2000. The authors subclassified the 51 cancer samples as microsatellite stable (MSS) and microsatellite instable (MSI) and seeked out significantly mutated genes in each class. They found 24 significantly mutated genes in MSS and 16 in MSI tumours and found that over half of the patients could potentially benefit from targeted therapies.

Nat Commun 5 3830 2014
An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage.
Newman AM, Bratman SV, To J, Wynne JF, Eclov NC, Modlin LA, Liu CL, Neal JW, Wakelee HA, Merritt RE, Shrager JB, Loo BW, Alizadeh AA, Diehn M
Abstract -

Circulating tumor DNA (ctDNA) is a promising biomarker for noninvasive assessment of cancer burden, but existing ctDNA detection methods have insufficient sensitivity or patient coverage for broad clinical applicability. Here we introduce cancer personalized profiling by deep sequencing (CAPP-Seq), an economical and ultrasensitive method for quantifying ctDNA. We implemented CAPP-Seq for non-small-cell lung cancer (NSCLC) with a design covering multiple classes of somatic alterations that identified mutations in >95% of tumors. We detected ctDNA in 100% of patients with stage II-IV NSCLC and in 50% of patients with stage I, with 96% specificity for mutant allele fractions down to ~0.02%. Levels of ctDNA were highly correlated with tumor volume and distinguished between residual disease and treatment-related imaging changes, and measurement of ctDNA levels allowed for earlier response assessment than radiographic approaches. Finally, we evaluated biopsy-free tumor screening and genotyping with CAPP-Seq. We envision that CAPP-Seq could be routinely applied clinically to detect and monitor diverse malignancies, thus facilitating personalized cancer therapy.

Illumina Summary -

Circulating tumor DNA (ctDNA) is a promising biomarker for noninvasive assessment of cancer burden. This paper presents a method for ctDNA profiling by deep sequencing: CAPP-Seq. The method was implemented for non-small-cell lung cancer (NSCLC) with Illumina HiSeq sequencing technology by selectively capturing and sequencing regions of recurrent cancer mutations. The authors detected ctDNA in 100% of patients with stage II-IV NSCLC and in 50% of patients with stage I, with 96% specificity for mutant allele fractions down to ~0.02%.

Nat Med 2014
XBP1 promotes triple-negative breast cancer by controlling the HIF1a pathway.
Chen X, Iliopoulos D, Zhang Q, Tang Q, Greenblatt MB, Hatziapostolou M, Lim E, Tam WL, Ni M, Chen Y, Mai J, Shen H, Hu DZ, Adoro S, Hu B, Song M, Tan C, Landis MD, Ferrari M, Shin SJ, Brown M, Chang JC, Liu XS, Glimcher LH
Abstract -

Cancer cells induce a set of adaptive response pathways to survive in the face of stressors due to inadequate vascularization. One such adaptive pathway is the unfolded protein (UPR) or endoplasmic reticulum (ER) stress response mediated in part by the ER-localized transmembrane sensor IRE1 (ref. 2) and its substrate XBP1 (ref. 3). Previous studies report UPR activation in various human tumours, but the role of XBP1 in cancer progression in mammary epithelial cells is largely unknown. Triple-negative breast cancer (TNBC)--a form of breast cancer in which tumour cells do not express the genes for oestrogen receptor, progesterone receptor and HER2 (also called ERBB2 or NEU)--is a highly aggressive malignancy with limited treatment options. Here we report that XBP1 is activated in TNBC and has a pivotal role in the tumorigenicity and progression of this human breast cancer subtype. In breast cancer cell line models, depletion of XBP1 inhibited tumour growth and tumour relapse and reduced the CD44(high)CD24(low) population. Hypoxia-inducing factor 1a (HIF1a) is known to be hyperactivated in TNBCs. Genome-wide mapping of the XBP1 transcriptional regulatory network revealed that XBP1 drives TNBC tumorigenicity by assembling a transcriptional complex with HIF1a that regulates the expression of HIF1a targets via the recruitment of RNA polymerase II. Analysis of independent cohorts of patients with TNBC revealed a specific XBP1 gene expression signature that was highly correlated with HIF1a and hypoxia-driven signatures and that strongly associated with poor prognosis. Our findings reveal a key function for the XBP1 branch of the UPR in TNBC and indicate that targeting this pathway may offer alternative treatment strategies for this aggressive subtype of breast cancer.

Illumina Summary -

Cancer cells induce a set of adaptive response pathways to survive in the face of stressors due to inadequate vascularization. In this study, one such mechanism, the unfolded protein or endoplasmatic reticulum stress response, mediated in part by IRE1 and XBP1 was examined in triple-negative breast cancer. Using Illumina HiSeq the authors performed chromatin immuno-precipitation sequencing (ChIP-seq) to determine the genome-wide regulatory network of XBP1. The XBP1 pathway activation correlates with poor patient survival indicating that targeting this pathway may improve the effectiveness of tumor therapies.

Nature 508 103-7 2014
Massively parallel functional annotation of 3' untranslated regions.
Zhao W, Pollack JL, Blagev DP, Zaitlen N, McManus MT, Erle DJ
Abstract -

Functional characterization of noncoding sequences is crucial for understanding the human genome and learning how genetic variation contributes to disease. 3' untranslated regions (UTRs) are an important class of noncoding sequences, but their functions remain largely uncharacterized. We developed a method for massively parallel functional annotation of sequences from 3' UTRs (fast-UTR) and used this approach to measure the effects of a total of >450 kilobases of 3' UTR sequences from >2,000 human genes on steady-state mRNA abundance, mRNA stability and protein production. We found widespread regulatory effects on mRNA that were coupled to effects on mRNA stability and protein production. Furthermore, we discovered 87 novel cis-regulatory elements and measured the effects of genetic variation within known and novel 3' UTR motifs. This work shows how massively parallel approaches can improve the functional annotation of noncoding sequences, advance our understanding of cis-regulatory mechanisms and quantify the effects of human genetic variation.

Illumina Summary -

3' untranslated regions (UTRs) are an important class of noncoding sequences, but their functions remain largely uncharacterized. This paper introduces a new method, fast-UTR, for massively parallel functional annotation of 3'UTR sequences. The method uses a BTV reporter plasmid system with an enhanced GFP reporter transgene and a multiple cloning site (MCS). Pools of 200-mers w 3'UTR segments were synthesized and inserted into BTV at the MCS. The authors transfected BEAS-2B immortalized human bronchial epithelial cells and used DNA and RNA sequencing by Illumina HiSeq to characterize the 3'UTR sequences and their effect on transcription. This work shows how massively parallel approaches can quantify the effect of human genetic variation in noncoding sequences.

Nat Biotechnol 32 387-91 2014
Fibroblast growth factor receptor 2 tyrosine kinase fusions define a unique molecular subtype of cholangiocarcinoma.
Arai Y, Totoki Y, Hosoda F, Shirota T, Hama N, Nakamura H, Ojima H, Furuta K, Shimada K, Okusaka T, Kosuge T, Shibata T
Abstract -

Cholangiocarcinoma is an intractable cancer, with limited therapeutic options, in which the molecular mechanisms underlying tumor development remain poorly understood. Identification of a novel driver oncogene and applying it to targeted therapies for molecularly defined cancers might lead to improvements in the outcome of patients. We performed massively parallel whole transcriptome sequencing in eight specimens from cholangiocarcinoma patients without KRAS/BRAF/ROS1 alterations and identified two fusion kinase genes, FGFR2-AHCYL1 and FGFR2-BICC1. In reverse-transcriptase polymerase chain reaction (RT-PCR) screening, the FGFR2 fusion was detected in nine patients with cholangiocarcinoma (9/102), exclusively in the intrahepatic subtype (9/66, 13.6%), rarely in colorectal (1/149) and hepatocellular carcinoma (1/96), and none in gastric cancer (0/212). The rearrangements were mutually exclusive with KRAS/BRAF mutations. Expression of the fusion kinases in NIH3T3 cells activated MAPK and conferred anchorage-independent growth and in vivo tumorigenesis of subcutaneous transplanted cells in immune-compromised mice. This transforming ability was attributable to its kinase activity. Treatment with the fibroblast growth factor receptor (FGFR) kinase inhibitors BGJ398 and PD173074 effectively suppressed transformation. Conclusion: FGFR2 fusions occur in 13.6% of intrahepatic cholangiocarcinoma. The expression pattern of these fusions in association with sensitivity to FGFR inhibitors warrant a new molecular classification of cholangiocarcinoma and suggest a new therapeutic approach to the disease. (Hepatology 2014;59:1427-1434).

Illumina Summary -

Cholangiocarcinoma (CC) is an intractable cancer, with limited therapeutic options. CC is the second most common primary hepatic malignancy after hepatocellular carcinoma, and is associated with hepatitis virus infection. In this study CC was examined by massively parallel whole transcriptome sequencing in eight specimens from cholangiocarcinoma patients without KRAS/BRAF/ROS1 alterations. The authors identified two fusion kinase genes, FGFR2-AHCYL1 and FGFR2-BICC1 and showed by expressing the fusion kinases in NIH3T3 cells that Treatment with the fibroblast growth factor receptor (FGFR) kinase inhibitors BGJ398 and PD173074 effectively suppressed transformation.

Hepatology 59 1427-34 2014
Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability.
Tatton-Brown K, Seal S, Ruark E, Harmer J, Ramsay E, Del Vecchio Duarte S, Zachariou A, Hanks S, O'Brien E, Aksglaede L, Baralle D, Dabir T, Gener B, Goudie D, Homfray T, Kumar A, Pilz DT, Selicorni A, Temple IK, Van Maldergem L, Yachelevich N, van Montfort R, Rahman N
Abstract -

Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations by sequencing DNMT3A in a further 142 individuals with overgrowth. The mutations alter residues in functional DNMT3A domains, and protein modeling suggests that they interfere with domain-domain interactions and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing methylation during embryogenesis and is commonly somatically mutated in acute myeloid leukemia. Thus, DNMT3A joins an emerging group of epigenetic DNA- and histone-modifying genes associated with both developmental growth disorders and hematological malignancies.

Illumina Summary -

The genetic control of growth during human development is highly complex and is influenced by genetic variation. In this study the authors investigated the exomes of ten proband-parent trios to identify new genetic causes of human overgrowth. Using Illumina HiSeq for exome sequencing and validation of individual sites by Sanger sequencing, the authors identified two de novo mutations in DNMT3A. A follow-up study of DNMT3A in 142 individuals identified a further 11 de novo mutations in DNMT3A. Interestingly, DNMT3A joins an emerging family of genes with dual roles in the pathogenesis of syndromic overgrowth and myeloid neoplasms. The authors will follow-up with a longitudinal study of cancer incidence in individuals with DNMT3A mutations.

Nat Genet 46 385-8 2014
2'-OMe-phosphorodithioate-modified siRNAs show increased loading into the RISC complex and enhanced anti-tumour activity.
Wu SY, Yang X, Gharpure KM, Hatakeyama H, Egli M, McGuire MH, Nagaraja AS, Miyake TM, Rupaimoole R, Pecot CV, Taylor M, Pradeep S, Sierant M, Rodriguez-Aguayo C, Choi HJ, Previs RA, Armaiz-Pena GN, Huang L, Martinez C, Hassell T, Ivan C, Sehgal V, Singhania R, Han HD, Su C, Kim JH, Dalton HJ, Kovvali C, Keyomarsi K, McMillan NA, Overwijk WW, Liu J, Lee JS, Baggerly KA, Lopez-Berestein G, Ram PT, Nawrot B, Sood AK
Abstract -

Improving small interfering RNA (siRNA) efficacy in target cell populations remains a challenge to its clinical implementation. Here, we report a chemical modification, consisting of phosphorodithioate (PS2) and 2'-O-Methyl (2'-OMe) MePS2 on one nucleotide that significantly enhances potency and resistance to degradation for various siRNAs. We find enhanced potency stems from an unforeseen increase in siRNA loading to the RNA-induced silencing complex, likely due to the unique interaction mediated by 2'-OMe and PS2. We demonstrate the therapeutic utility of MePS2 siRNAs in chemoresistant ovarian cancer mouse models via targeting GRAM domain containing 1B (GRAMD1B), a protein involved in chemoresistance. GRAMD1B silencing is achieved in tumours following MePS2-modified siRNA treatment, leading to a synergistic anti-tumour effect in combination with paclitaxel. Given the previously limited success in enhancing siRNA potency with chemically modified siRNAs, our findings represent an important advance in siRNA design with the potential for application in numerous cancer types.

Illumina Summary -

Small interfering RNAs (siRNAs) are a promising therapeutic mechanism for modulation of gene expression. This study presents how a chemical modification of phosphorodithioate (PS2) and 2'-O-Methyl (2'-OMe) MePS2 on one nucleotide significantly enhances potency and resistance to degradation. The authors used the Illumina human HT-12 v4 BeadChip array for analysis of gene expression and pathway enrichment analysis. The authors demonstrated the therapeutic utility of MePS2 siRNAs in chemoresistant ovarian cancer mouse models via targeting GRAM domain containing 1B (GRAMD1B), a protein involved in chemoresistance.

Nat Commun 5 3459 2014
Recurrent PTPRB and PLCG1 mutations in angiosarcoma.
Behjati S, Tarpey PS, Sheldon H, Martincorena I, Van Loo P, Gundem G, Wedge DC, Ramakrishna M, Cooke SL, Pillay N, Vollan HK, Papaemmanuil E, Koss H, Bunney TD, Hardy C, Joseph OR, Martin S, Mudie L, Butler A, Teague JW, Patil M, Steers G, Cao Y, Gumbs C, Ingram D, Lazar AJ, Little L, Mahadeshwar H, Protopopov A, Al Sannaa GA, Seth S, Song X, Tang J, Zhang J, Ravi V, Torres KE, Khatri B, Halai D, Roxanis I, Baumhoer D, Tirabosco R, Amary MF, Boshoff C, McDermott U, Katan M, Stratton MR, Futreal PA, Flanagan AM, Harris A, Campbell PJ
Abstract -

Angiosarcoma is an aggressive malignancy that arises spontaneously or secondarily to ionizing radiation or chronic lymphoedema. Previous work has identified aberrant angiogenesis, including occasional somatic mutations in angiogenesis signaling genes, as a key driver of angiosarcoma. Here we employed whole-genome, whole-exome and targeted sequencing to study the somatic changes underpinning primary and secondary angiosarcoma. We identified recurrent mutations in two genes, PTPRB and PLCG1, which are intimately linked to angiogenesis. The endothelial phosphatase PTPRB, a negative regulator of vascular growth factor tyrosine kinases, harbored predominantly truncating mutations in 10 of 39 tumors (26%). PLCG1, a signal transducer of tyrosine kinases, encoded a recurrent, likely activating p.Arg707Gln missense variant in 3 of 34 cases (9%). Overall, 15 of 39 tumors (38%) harbored at least one driver mutation in angiogenesis signaling genes. Our findings inform and reinforce current therapeutic efforts to target angiogenesis signaling in angiosarcoma.

Illumina Summary -

Angiosarcoma is an aggressive malignancy that arises spontaneously or secondarily to ionizing radiation or chronic lymphoedema. To explore the genomic drivers of angiosarcoma, this study explored 36 angiosarcomas by whole-genome, whole-exome or targeted sequencing of cancer-related genes using a combination of Illumina HiSeq2000 and HiSeq2500. The authors found that 15 of 39 tumors (38%) harbored at least one driver mutation in angiogenesis signaling genes.

Nat Genet 46 376-9 2014
Systematic evaluation of coding variation identifies a candidate causal variant in TM6SF2 influencing total cholesterol and myocardial infarction risk.
Holmen OL, Zhang H, Fan Y, Hovelson DH, Schmidt EM, Zhou W, Guo Y, Zhang J, Langhammer A, Løchen ML, Ganesh SK, Vatten L, Skorpen F, Dalen H, Zhang J, Pennathur S, Chen J, Platou C, Mathiesen EB, Wilsgaard T, Njølstad I, Boehnke M, Chen YE, Abecasis GR, Hveem K, Willer CJ
Abstract -

Blood lipid levels are heritable, treatable risk factors for cardiovascular disease. We systematically assessed genome-wide coding variation to identify new genes influencing lipid traits, fine map known lipid loci and evaluate whether low-frequency variants with large effects exist for these traits. Using an exome array, we genotyped 80,137 coding variants in 5,643 Norwegians. We followed up 18 variants in 4,666 Norwegians and identified ten loci with coding variants associated with a lipid trait (P < 5 × 10(-8)). One variant in TM6SF2 (encoding p.Glu167Lys), residing in a known genome-wide association study locus for lipid traits, influences total cholesterol levels and is associated with myocardial infarction. Transient TM6SF2 overexpression or knockdown of Tm6sf2 in mice alters serum lipid profiles, consistent with the association observed in humans, identifying TM6SF2 as a functional gene within a locus previously known as NCAN-CILP2-PBX4 or 19p13. This study demonstrates that systematic assessment of coding variation can quickly point to a candidate causal gene.

Illumina Summary -

Blood lipid levels are heritable, treatable risk factors for cardiovascular disease. In search for the missing genetic heritability for myocardial infarction (MI) this study systematically assessed genome-wide coding variation using a combination of Illumina HumanExome Beadchip and exome sequencing on Illumina HiSeq. One variant in TM6SF2 (encoding p.Glu167Lys), residing in a known genome-wide association study locus for lipid traits and known to influence total cholesterol levels was found to associate with myocardial infarction. This study demonstrates that systematic assessment of coding variation can quickly point to a candidate causal gene.

Nat Genet 46 345-51 2014
Ancestry estimation and control of population stratification for sequence-based association studies.
Wang C, Zhan X, Bragg-Gresham J, Kang HM, Stambolian D, Chew EY, Branham KE, Heckenlively J, Fulton R, Wilson RK, Mardis ER, Lin X, Swaroop A, Zöllner S, Abecasis GR
Abstract -

Estimating individual ancestry is important in genetic association studies where population structure leads to false positive signals, although assigning ancestry remains challenging with targeted sequence data. We propose a new method for the accurate estimation of individual genetic ancestry, based on direct analysis of off-target sequence reads, and implement our method in the publicly available LASER software. We validate the method using simulated and empirical data and show that the method can accurately infer worldwide continental ancestry when used with sequencing data sets with whole-genome shotgun coverage as low as 0.001×. For estimates of fine-scale ancestry within Europe, the method performs well with coverage of 0.1×. On an even finer scale, the method improves discrimination between exome-sequenced study participants originating from different provinces within Finland. Finally, we show that our method can be used to improve case-control matching in genetic association studies and to reduce the risk of spurious findings due to population structure.

Illumina Summary -

Estimating individual ancestry is important in genetic association studies, however assigning ancestry remains challenging with targeted sequencing data. This study proposes a new method for ancestry estimation based on analysing off-target sequence reads. The authors demonstrated their method using publicly available sequencing data sets and have implemented the method in the publicly available LASER software.

Nat Genet 46 409-15 2014