MAB21L2, c.151 C>G, p.(Arg51Gly); the mutation was present in all five family members with the disease and appeared de novo in the first affected generation of the three-generational pedigree. MAB21L2 encodes a protein similar to C. elegans mab-21 cell fate-determining factor; the molecular function of MAB21L2 is largely unknown. To further evaluate the role of MAB21L2, zebrafish mutants carrying a p.(Gln48Serfs*5) frameshift truncation (Q48Sfs*5mab21l2) and a p.(Arg51_Phe52del) in-frame deletion (R51_F52delmab21l2) were developed with TALEN technology. Homozygous zebrafish embryos from both lines developed variable lens and coloboma phenotypes: Q48Sfs*5mab21l2 embryos demonstrated severe lens and retinal defects with complete lethality while R51_F52delmab21l2 mutants displayed a milder lens phenotype and severe coloboma with a small number of fish surviving to adulthood. Protein studies showed decreased stability for the human p.(Arg51Gly) and zebrafish p.(Arg51_Phe52del) mutant proteins and predicted a complete loss-of-function for the zebrafish p.(Gln48Serfs*5) frameshift truncation. Additionally, in contrast to wild-type human MAB21L2 transcript, mutant p.(Arg51Gly) mRNA failed to efficiently rescue the ocular phenotype when injected into Q48Sfs*5mab21l2 embryos, suggesting this allele is functionally deficient. Histology, immunohistochemistry, and in situ hybridization experiments identified retinal invagination defects, an increase in cell death, abnormal proliferation patterns, and altered expression of several ocular markers in the mab21l2 mutants. These findings support the identification of MAB21L2 as a novel factor involved in human coloboma and highlight the power of genome editing manipulation in model organisms for analysis of the effects of whole exome variation in humans.Ocular coloboma results from abnormal embryonic development and is often associated with additional ocular and systemic features. Coloboma is a highly heterogeneous disorder with many cases remaining unexplained. Whole exome sequencing from two cousins affected with dominant coloboma with microcornea, cataracts, and skeletal dysplasia identified a novel heterozygous allele in  MAB21L2. We used genome editing to disrupt the mab21l2 gene in zebrafish, which led to similar eye conditions in affected fish, providing additional evidence for the role of this gene in eye development. The function of MAB21L2 is largely unknown; further study of the pathways affected by MAB21L2 deficiency and study of the zebrafish generated by this project will provide more information about the role of this gene in eye development.Coloboma is a hole or gap in one or more of the structures of the eye. Coloboma occurs when the eye is not formed properly during prenatal development. It is often associated with additional eye abnormalities and can result in significant loss of vision. Identification of the genetic causes of coloboma provides more information about how the eye develops. We used whole exome sequencing in an affected family to identify mutations in a new gene associated with dominant coloboma in humans,  Coloboma is a congenital segmental ocular defect which can affect one or more structures of the eye; typical coloboma results in an inferior deficiency of iris, chorioretinal, and/or optic nerve tissue –3. OculaSOX2, OTX2, PAX2, PAX6, CHD7 and SALL2; secreted signaling factor-encoding gene SHH; members of the transforming growth factor-beta (TGF-beta) superfamily GDF6 and GDF3; members of the retinoic acid synthesis pathway STRA6 and ALDH1A3; as well as membrane porphyrin transporter ABCB6, and a member of the HIPPO growth control pathway, YAP1 [A number of genes have been associated with coloboma including transcriptional regulators ay, YAP1 . Due to ay, YAP1 –21. Sincay, YAP1 ,22. Receay, YAP1 .MAB21L2 in human coloboma and further investigate its role in vertebrate ocular development by generation and analysis of zebrafish mab21l2 mutants.In this manuscript we present evidence for the role of The proband (Patient 1) was diagnosed with bilateral microcornea, iris and chorioretinal coloboma, corectopia, nystagmus and cataract ; the catMAB21L2 missense mutation c.151 C>G, p.(Arg51Gly), was present in all five affected individuals  and absent in the unaffected parents (a low ‘G’ peak at position c.151 was seen in the mother’s sample (I-2) in addition to the wild-type ‘C’ on sequencing of two independent PCR reactions, suggesting that she is likely to have low-level mosaicism for this mutation)  and having the highest Genomic Evolutionary Rate Profiling (GERP++) score, as well as enrichment of both human MAB21L2 and mouse Mab21l2 transcripts in ocular structures based in BioGPS (http://biogps.org). The mutation was predicted to be damaging by SIFT, Polyphen2, Mutation Taster and MutationAssessor, and demonstrated high conservation scores for the affected nucleotide with a GERP++ score of 6.16 and a PhyloP score of 2.94  [Independent whole exome sequencing of DNA samples from the proband’s two affected nephews  . The MABnserved) ).MAB21L2 gene, located at 4q31.3, encodes a 359-a.a. protein similar to C. elegans mab-21 cell fate-determining factor [C. elegans mab-21  project of the UK10K Consortium study [MAB21L2 mutations to human coloboma phenotypes (<2%) and suggest no role for MAB21L2 mutations in the other ocular disorders that were examined in this study.To further explore the role of um study ,31. We iMAB21L2/mab21l2 in ocular development, we performed careful evaluation of mab21l2 expression in zebrafish using in situ hybridization. At 18-hpf, expression was seen in the presumptive eye field and midbrain , lens, spinal cord, midbrain and pharyngeal arch region ; c.150_156delCCGTTTC, p.(Arg51Serfs*4); c.151delC, p.; c.151dupC, p.(Arg51Profs*14); and c.155delT, p.(Phe52Serfs*5) frameshift mutations  and an in-frame deletion of two amino acids including the arginine at position 51, c.151_156delCGTTTC, p.(Arg51_Phe52del) (To determine the effect of he52del) , S1 Fig.mab21l2 mutants and heterozygous fish with the c.151_156delCGTTTC, p.(Arg51_Phe52del) in-frame deletion were bred to obtain homozygous embryos; the resulting embryos were examined to determine the associated phenotypes  and the c.151_156delCGTTTC, p.(Arg51_Phe52del) in-frame deletion (mab21l2R51_F52del) alleles  frameshift mutation  and cornea, discontinuous RPE , as well as generally normal eye size and lens appearance  and mab21l2R51_F52del  120-hpf homozygous fish: the cartilage elements of the pharyngeal arches, particularly the ceratohyal cartilage of the hyoid arch , were malformed and displaced  mab21l2R51_F52del homozygous larvae developed into adults. Gross morphological examination of adult fish revealed a severe ocular phenotype with anophthalmia of one eye and microphthalmia of the contralateral eye  identified an excessive number of TUNEL-positive cells in 24–72-hpf homozygous ild-type ; S3 Fig.ild-type ; at 48–7the lens ; at 48–7the lens , S3 Fig.mab21l2Q48Sfs*5 and mab21l2R51_F52del mutant embryos; the monoclonal antibody ZL-1 was used to examine zebrafish fiber cell differentiation and the sections were counterstained with wheat germ agglutinin (WGA) and/or DAPI and to visualize cell membranes and nuclei, correspondingly  was performed to compare proliferation patterns in 24–72-hpf wild-type and both ondingly .. Strongondingly  as well ondingly . In mab2y stages  with a dy stages ; no proly stages  in theset 24-hpf ; disordet 24-hpf  with patt 24-hpf ; in cont embryos . ZL-1 po embryos .; this s mutants ..mab21l2Q48Sfs*5 frameshift mutants   in the developing 48-hpf lens seemed to be somewhat downregulated in both mutants with mutant lenses also appearing smaller than wild-type lenses   and beco(24-hpf) , S5 Fig.t 24-hpf  but an at 24-hpf , S5 Fig.t 72-hpf , S5 Fig.MAB21L2 was also found to be endogenously expressed in these cells (MAB21L2 transcript encoding for wild-type and p.(Arg51Gly) mutant proteins detected by RT-PCR did not show a significant difference (To examine the effect of the p.(Arg51Gly) mutation on the MAB21L2 protein, we tested the expression, localization and protein stability of the wild-type and mutant proteins via transfections of the corresponding FLAG-tagged constructs into human lens epithelial B3 (HLE-B3) cells ; MAB21L2se cells .. Mab21lse cells . Westernse cells ; the mutse cells . Since tfference , S1 Fig.fference . Proteinfference . CalculaFunctional analysis of the p.(Gln48*) (to represent the zebrafish p.(Gln48Serfs*5) mutant) and p.(Arg51_Phe52del) proteins was also undertaken in HLE-B3 cells. The p.(Gln48*) mutant was found to be highly unstable as no detectable level of this protein (estimated to have a molecular weight of ~5.5 kDa) was identified via Western blot analysis . The p.; cellulamab21l2Q48Sfs*5 crosses with either wild type MAB21L2 mRNA or mRNA encoding for the p.(Arg51Gly) mutant; a small number of embryos were left uninjected as a control. The embryos were carefully examined for ocular anomalies at 72-hpf, divided into affected and normal groups, and then genotyped. In total, out of 104 surviving embryos injected with wild type MAB21L2, 68% (21/31) of homozygous fish developed normal eyes and lenses while out of 108 surviving embryos injected with the mutant mRNA encoding for p.(Arg51Gly), only 14% (3/22) of homozygous fish developed normal eyes and lenses; in the control group, none of the 9 uninjected homozygous embryos (out of 36 surviving) had normal eyes and lenses. The difference in the proportion of normal phenotypes seen in the homozygous mab21l2Q48Sfs*5 fish injected with wild-type mRNA versus embryos injected with either mutant mRNA or left uninjected was statistically significant (p<.0005), while the difference between the mutant RNA- injected and the uninjected embryos was not statistically significant (p>.20) by chi-squared analysis  mutant supports the pathogenic role of this MAB21L2 allele in human disease.To further test the pathogenicity of the human p.(Arg51Gly) allele, we injected embryos generated by heterozygous analysis . The effMAB21L2 wild type mRNA or mRNA encoding for the p.(Arg51Gly) mutant were injected (at the same concentrations as in rescue experiments) into wild-type embryos and observed for phenotypes up to 120-hpf. Among the 39 surviving embryos that were injected with wild-type MAB21L2, no embryos displayed an eye phenotype . Similarly, among the 61 surviving embryos that were injected with the mutant mRNA encoding for the p.(Arg51Gly) protein, no embryos showed an eye phenotype . Thus, these experiments identified no dominant-negative effect for the p.(Arg51Gly) mutant allele in zebrafish at concentrations that are sufficient for phenotypic rescue of the mab21l2-deficient phenotype.To investigate the possibility of dominant-negative effects for the mutant allele, MAB21L2/mab21l2 in vertebrate ocular development through demonstration of abnormal phenotypes associated with mutations in this gene in humans and zebrafish. Human MAB21L2, located at 4q31.3, is a member of the MAB21L gene family that consists of three members, MAB21L1-3. MAB21L proteins are related to the mab-21  nuclear factor [C. elegans [mab-21 and sma members (SMAD family) in C. elegans indicated that mab-21 is positioned downstream of sma and is negatively regulated by dbl-1, a homolog of human BMP5, thus placing mab-21 in the TGF-β signaling cascade [BMP4, BMP7, GDF3, and GDF6, lead to an overlapping spectrum of phenotypes in humans [C. elegans mutants [Mab21l2 expression was found to be upregulated in embryonic mouse lenses heterozygous for a Pax6 loss-of-function allele [In this manuscript we present evidence for the conserved function of r factor  associat elegans . The mol cascade ,36. The n humans , 37–39.  mutants  and Mab2n allele  suggestiMab21l2 ocular expression was reported in the dorsal optic vesicle and head surface ectoderm in E8.5–9.5 embryos and the neural retina, optic nerve and RPE at E12 [Mab21l2 knockout mice demonstrated a rudimentary retina and aphakia due to improper invagination of the optic vesicle as well as ventral body wall defects, improper formation of the heart and liver, and embryonic lethality [Mab21l2/mab21l2  [Mab21l2/mab21l2 transcripts in the developing lens [mab21l2 resulted in microphthalmia and incomplete retinal development including discontinuous inner and outer plexiform layers [mab21l2 in both tissues. While the phenotypes of mab21l2 morphants and mutants show overlap, some ocular features, including severe lens degeneration, coloboma, and early retinal invagination defects, appear to be more pronounced or observed primarily in genetic mutants; this may be due to incomplete disruption of mab21l2 function via morpholino, the transient nature of morpholino-induced effects, and/or genetic background differences.In mouse, E at E12 ,42; homoethality ,42. Prevmab21l1) ,43–45 whing lens ,46,47, wmab21l2 mutants generated in this study demonstrated variable retinal invagination defects. In zebrafish, retinal invagination, which occurs upon contact of the optic vesicle (formed as an extension of the forebrain neuroepithelium) with the surface ectoderm, is completed with formation of the cup-shaped optic vesicle by ~22-hpf. In mab21l2Q48Sfs*5 embryos, a shallow misshapen optic cup, especially at its ventral part, was detected in 24-hpf embryos; the lens mass  was also visibly smaller than normal at this stage. In the mab21l2R51_F52del in-frame deletion mutant, a more constricted optic cup with some ventral and dorsal retinal folding was observed, while the developing lens appeared to have normal size and shape in most embryos. The observed defects in early optic cup formation indicate a possible conserved role for mab21l2 in this process in vertebrates.Both of the mab21l2Q48Sfs*5 or mab21l2R51_F52del homozygous mutant embryos identified multiple deviations from normal patterns. The most remarkable finding was a significant increase in TUNEL staining in the developing retina and lens of mab21l2Q48Sfs*5 mutants at early embryonic stages. An increase in TUNEL staining was also observed at later stages of development and was, to a lesser degree, present in the mab21l2R51_F52del embryos carrying homozygous in-frame deletion alleles. Both increased cell death (through apoptosis and/or necroptosis) and defects in cellular proliferation have been linked to optic fissure closure defects in other genetic models of coloboma [mab21l2 deficient phenotypes. Additionally, the expression of several ocular markers including pax6b, pax2.1, foxe3 and ZL-1 was altered in mab21l2 mutants. These observed differences seem more likely to be caused by the severe disorganization of mutant ocular tissues rather than direct regulation of expression of those markers by mab21l2.Studies of proliferation and cell death in zebrafish wild-type and coloboma ; since tcoloboma , furthermab21l2Q48Sfs*5 frameshift truncation and mab21l2R51_F52del in-frame deletion mutations suggest different molecular mechanisms. The mab21l2R51_F52del in-frame deletion demonstrated milder features in comparison to the mab21l2Q48Sfs*5 mutant, such as less affected lens development, less pronounced cell death and incomplete embryonic lethality. These observations support the possibility that this mutation results in a hypomorphic allele retaining some of its normal function, particularly in relation to lens development. The severe coloboma observed in mab21l2R51_F52del embryos may point to the particular importance of domains located in the N-terminal region of mab21l2 for its proper functioning in the developing retina. Since the zebrafish p.(Arg51_Phe52del) in-frame deletion and the human p.(Arg51Gly) missense alleles affect homologous regions of the MAB21L2/mab21l2 proteins, the mechanism(s) of these mutations may be similar to each other. Understanding of these mechanisms requires additional studies into the domain structure and function of MAB21L2/mab21l2 protein.The distinct phenotypes associated with the MAB21L2 mRNA, mutant mRNA encoding for the p.(Arg51Gly) protein failed to efficiently rescue the ocular phenotype of homozygous mab21l2Q48Sfs*5 embryos, suggesting functional deficiency for the identified human mutation. In addition, overexpression of mutant MAB21L2 mRNA in zebrafish embryos resulted in no obvious ocular phenotype, implying absence of a dominant-negative effect. However, it is possible that the full impact of the p.(Arg51Gly) mutation was not discernable due to insufficient dosage, its acting later in development, or other factors; this needs to be further investigated by additional approaches, including development of zebrafish mutants carrying this specific allele.Analysis of wild-type and mutant proteins in human lens epithelial cells identified a range of protein stability defects among mutant forms with the human p.(Arg51Gly) mutant stability mildly reduced, the zebrafish p.(Arg51_Phe52del) mutant stability highly affected, and the p.(Gln48*) truncation protein (similar to the p.(Gln48Serfs*5) mutant) being entirely unstable. This analysis suggested that the zebrafish p.(Gln48Serfs*5) mutation results in a complete loss-of-function allele, since the similar mutant protein p.(Gln48*) was completely unstable and any protein produced would be missing the entire mab-21 domain. These experiments also indicated the likely importance of arginine 51 and the immediately adjacent amino acids for normal conformation and stability of the MAB21L2/mab21l2 protein because disruptions of this region resulted in decreased stability for both the human p.(Arg51Gly) and the zebrafish p.(Arg51_Phe52del) proteins. Further studies demonstrated that, in contrast to wild-type human MAB21L2 mutations suggests its involvement in skeletal development. Additional phenotypes such as shortened body/curved tail, craniofacial malformations and lethality were present in the zebrafish mab21l2 mutants while heart, liver, embryonic lethality and other anomalies were previously reported in mouse Mab21l2 mutants [MAB21L2-associated phenotypes are likely to be identified and may present with broad interfamilial variability.No abnormal phenotype was seen in heterozygous zebrafish for either the frameshift truncation or in-frame deletion mutations, while a dominant phenotype was observed in human patients with the p.(Arg51Gly) alteration. This may be explained by variability in gene dosage requirements between species  or a dif mutants ,42. Cons mutants , additioMAB21L2 were described by Rainger et al [MAB21L2 mutation affecting the C-terminal region of the protein was identified. Functional assays of the mutant alleles using an inducible HEK293 cell system expressing wild-type or mutant MAB21L2 as GFP fusion proteins suggested a possible gain-of-function effect for the dominant mutations based on observed increased protein stability; an increase in phospho-ERK1 was detected with induction of either wild-type or p.(Arg51His) mutant MAB21L2 protein [mab21l2 mutants, uncovered an early onset optic cup apoptosis phenotype in affected embryos, and demonstrated a decrease in protein stability, as well as functional deficiency and no dominant-negative effect for the p.(Arg51Gly) mutation in zebrafish embryos.While this paper was in revision, four families with mutations in er et al . This ma protein . This di protein . In our MAB21L2 allele with the ocular phenotype in the family, the de novo appearance of the mutation in the affected progeny of unaffected parents with low-level mosaicism present in the mother, the in silico and experimental indications of the functional importance of the arginine at position 51, the development of ocular defects in mab21l2 mutant zebrafish embryos, as well as rescue of the ocular phenotype of homozygous mab21l2Q48Sfs*5 embryos with injection of wild-type but not the p.(Arg51Gly)-encoding mutant mRNA, provide strong support for the pathogenicity of the identified MAB21L2 allele and the involvement of MAB21L2 in human ocular development and disease. The developed zebrafish mab21l2 mutants will help to further explore the developmental roles and molecular function of this conserved ocular factor.Taken as a whole, the complete co-segregation of the identified http://www.UK10K.org. Access to data generated by the UK10K_Rare_Coloboma (EGA Study ID: EGAS00001000127) project of the UK10K Consortium study (http://www.UK10K.org) was obtained through a Data Access Agreement. The animal protocol was approved by the Institutional Animal Care and Use Committee at the Medical College of Wisconsin (protocol number AUA00000351_AR_5).The human study was approved by the Children’s Hospital of Wisconsin Institutional Review Board (protocol number CHW 03/56) with written informed consent obtained from each participant and/or their legal representative, as appropriate. This study also utilized data generated by the UK10K Consortium, derived from samples from UK10K_Rare_Coloboma (EGA Study ID: EGAS00001000127); a full list of the investigators who contributed to the generation of the data is available from http://www.ncbi.nlm.nih.gov/snp), NHLBI Exome Sequencing Project Exome Variant Server , and 1000 Genomes (http://www.1000genomes.org/data), as well as our own data) and possible effect on protein function; for missense variants, functional profiling was performed using SIFT, Polyphen2, Mutation Taster, MutationAssessor, and FATHMM as well as nucleotide conservation scores GERP++ and PhyloP as accessed through dbNSFP [Whole exome sequencing of DNA samples of the proband’s two affected nephews was undertaken. Library capture was completed using the Agilent Sure Select v4 capture kit  and 100 base pair paired end sequencing was performed using the Illumina HiSeq 2000 at Axeq Technologies . The obtained data were aligned using the Burrows-Wheeler Aligner (BWA) and variants were called using the SAMTOOLS analysis pipeline available through Axeq. Exome data were analyzed using the SNP & Variation Suite  to identify variants that are shared between the affected individuals and then prioritized based on their absence/rarity in the general population  were analyzed using the SNP & Variation Suite  with filtering for the MAB21L2 gene only. DNA samples from this cohort were not available for independent confirmation of the identified variants.Variant Call Format (VCF) files from the UK10K_Rare_Coloboma (EGA Study ID: EGAS00001000127) project of the UK10K Consortium study . The accession numbers for the MAB21L sequences used in the protein alignment are as follows: human MAB21L2 (AF155219.1), murine Mab21l2 (AF223425.1), zebrafish mab21l2 (AY038031.1), human MAB21L1 (NM_005584), murine Mab21l1 (AF228913.1), zebrafish mab21l1 (NM_152974.2), human MAB21L3 (NM_152367.2), murine Mab21l3 (NM_172295.4), zebrafish mab21l3 (NM_001110025.1), Drosophila melanogaster mab-21 (NP_651971.2), Caenorhabditis elegans mab-21 (NP_497940.2).Protein sequences were aligned using the Kalign multiple sequence alignment tool  was purchased from GeneCopoiea . The mutations were introduced using the Agilent QuikChange Lightning (Cat. #210519) site directed mutagenesis kit . Mutagenesis primers were designed using the online QuikChange Primer Design tool. HPLC purified mutagenesis primers were ordered from Thermo Fisher Scientific  and sequences were as follows: MAB21L2-p.R51G-Sense—5’-gaggtgcaggagcctggcttcatcagct-3’, MAB21L2-p.R51G-Antisense—5’-agctgatgaagccaggctcctgcacctc-3’, MAB21L2-p.51_52del-Sense—5’-gctcaaggagctgataggctcctgcacctc-3’, MAB21L2-p.51_52del-Antisense—5’- gaggtgcaggagcctatcagctccttgagc-3’, MAB21L2- Q48X-Sense—5’- gaagcgaggctcctacacctccacttcct-3’, MAB21L2- Q48X-Antisense—5’- aggaagtggaggtgtaggagcctcgcttc-3’.Human lens epithelial B3 (HLE-B3) cells  were maintained in MEM (Life Technologies) supplemented with 20% FBS (Life Technologies), 2 mM glutamine (Life Technologies), and 1 mM sodium pyruvate (Life Technologies). All transfections were performed by transfecting HLE-B3 cells with 15 μg of DNA and 30 μl of Lipofectamine 2000 (Life Technologies) in Opti-MEM in 100 mm petri dishes.http://bioinformatics.org/sms/prot_mw.html). The BenchMark Pre-stained Protein Ladder (Cat. #10748-010) (Life Technologies) was utilized as the protein molecular weight standard.For Western blot analysis, nuclear and cytoplasmic fractions were generated using the CelLytic NuCLEAR Extraction kit . 48 hours after transfection, cells were washed with PBS, collected, and resuspended in 0.5 ml of hypotonic lysis buffer and incubated on ice for 15 minutes, 30 μl of 10% IGEPAL was added to the cell suspension. Cells were then vortexed and spun at 10,000 xg for 30 seconds at 4°C. The supernatant (cytoplasmic fraction) was decanted and saved. The nuclear fraction was generated by adding 70 μl of nuclear extraction buffer to the pellet, incubating on ice for 25 minutes, spun at 20,000 xg for 5 minutes at 4°C and the supernatant was saved (nuclear fraction). Molecular weights for wild-type and mutant proteins were estimated using Protein Molecular Weight calculator  glycerol, 2.1% SDS, and 0.01% bromophenol blue) (Cat. #161-0737)  followed by boiling for 4 minutes at 95°C and run on a 10% Criterion Tris-HCl Gel (Cat. #345-1018) (Bio-Rad). Gels were immunobloted with anti-FLAG (Cat. #F1804) (Sigma-Aldrich) antibody at a 1:1000 dilution or anti-GAPDH (Cat#ab8245)  antibody at 1:5000 dilution. The secondary antibody was goat anti-mouse IgG HRP conjugate (Cat. #12-349)  at a 1:2500 dilution. The blots were developed using the Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific).For cellular immunofluorescence experiments cells were split 24 hours after transfection and seeded onto coverslips. After 24 hours the cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, washed twice with PBS, permeabilized with 0.25% Triton X-100 in PBS, washed twice with PBS, blocked with 10% normal donkey serum in PBS for 30 minutes at 37°C, incubated with 1:1000 anti-FLAG antibody in 10% normal donkey serum in PBS for 2 hours at 37°C, washed three times with PBS, incubated in 1:1000 Alexa Flour 568 donkey anti-mouse IgG (Cat. #A10037) (Life Technologies), washed twice with PBS, incubated for 5 minutes at room temp with 1:1000 DAPI in PBS, washed twice with PBS and mounted on glass slides.Danio rerio) maintenance and developmental staging were performed as previously described [mab21l2, pax6b, pax2 and foxe3 was studied in 18–72-hpf zebrafish embryos using transcript-specific antisense riboprobes and previously described protocols [mab21l2 probe, a 698-bp fragment covering the sequence between nucleotides 788 and 1485  and pax2.1 (AL906738) were purchased from Open Biosystems  and linearized with EcoRv and BamHI, respectively; for the foxe3 probe, a full length 1369-bp fragment covering the sequence between nucleotides 16 and 1384 (GenBank #BC163348.1) was utilized.Zebrafish . Interruption of the mab21l2 target sequence was predicted to affect the BanII restriction site (GRGCYC) located between the left and right TALENs; thus BanII digestion of the PCR-amplified genomic fragment involving this region was utilized to determine TALEN cutting efficiencies as well as to identify mutant embryos/adult fish and was followed by DNA sequencing for confirmation. Zebrafish embryos at the 1–4 cell stage were injected with TALEN RNA; initial analysis of injected larvae confirmed genome editing events and ~200 injected embryos were raised to adulthood to generate mosaic founders; these fish were then bred to produce embryos carrying germline mab21l2 mutations that were raised to adulthood, genotyped using the primers provided in TALENs were constructed following the Sanjana et al (2012) protocol . BrieflyMAB21L2 using the following primers: MAB21L2-FLAG-F 5’- CAAAGACGATGACGACAAGG-3’ and MAB21L2-rt-R 5’- GGTAGAGCACCACCTCAAATTC-3’ . The following GAPDH primers were used as a loading control: GAPDH-F 5’- CCAAGGTCATCCATGACAACT-3’ and GAPDH-R 5’- GAGGCAGGGATGATGTTCTG-3’ . Endogeneous expression of MAB21L2 transcript in the human lens epithelial cell line was detected with the following primers MAB21L2-endogenous-F, 5'-CCAGGTGGAAAACGAGAGTG-3' and MAB21L2-rt-R, 5’-GGTAGAGCACCACCTCAAATTC-3’ .To detect transcript levels in HLE-B3 cells, RNA from cells was isolated following transfection with expression plasmids containing sequences encoding for either wild type, or p.(Arg51Gly) or p.(Arg51_Phe52del) MAB21L2 alleles using TRIzol Reagent (Cat. #15596-026) (Life Technologies) 48-hours after transfection. Each sample was treated with DNase I (Cat. #18068) and reverse transcribed in triplicate using the SuperScript III Reverse Transcriptase system (Cat. #18080) (Life Technologies). Following reverse transcription the samples were treated with RNase H (Cat. #18021). Samples were amplified for mab21l2 transcript levels in zebrafish RNA, three independent samples, each containing two embryos of either homozygous mab21l2Q48Sfs*5 and mab21l2R51_F52del mutants or wild type, were isolated using the RNAqueous-Micro Total RNA Isolation Kit (Cat. #AM1931) (Life Technologies). Each sample was reverse transcribed as described previously. Samples were amplified for mab21l2 using the following primers: mab21l2-zf-T-F 5’-TCTTTTCCTGGGAGTTGTGC-3’ and mab21l2-zf-T-R 5’- CCCCATCTGGTTCAGGTAAA-3’ . Because MAB21L2/mab21l2 represents a single exon gene, a minus-reverse transcriptase control was included in each RT-PCR experiment to assure absence of contaminating DNA in each RNA sample. Amplification of the zebrafish gene for translation elongation factor 1 alpha (loading control) was performed with the following primers: ef1ax1-2F 5’- TCTCTCAATCTTGAAACTTATCAATCA-3’ and ef1ax3R 5’- AACACCCAGGCGTACTTGAA-3’ . The 1 Kb Plus DNA Ladder was utilized as nucleic acid standard (Cat. #10787-018) (Life Technologies). The PCR products were analyzed by electrophoresis and bands were quantitated using Image J software.To detect For rescue and overexpression experiments, mRNA was generated using the T7 mMESSAGE mMACHINE Transcription kit (Cat. #AM1344) (Life Technologies). 300 pg of mRNA was injected into 1–4 cell stage embryos produced by wild-type or c.141_153delCCAAGAGCCCCGT heterozygous mutant crosses. The injected embryos were observed for up to 5 days of fertilization, separated into groups based on observed presence/absence of ocular phenotype and then genotyped.S1 Table(DOC)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FigC) Agarose gels representing RT-PCR expression analysis of recombinant FLAG-MAB21L2 wild-type and mutant transcripts (left) as well as endogenous MAB21L2 transcript in human lens epithelial cells (HLE-B3). RT minus (RT-) control refers to a mock reverse transcription containing all the RT-PCR reagents, except the reverse transcriptase, to demonstrate the absence of contaminating DNA in the corresponding RNA samples; ef1a and GAPDH amplification was performed for loading control in zebrafish and human samples correspondingly; the reactions were run on the same gel but irrelevant lanes were removed when needed; M- molecular marker, black arrow indicates mab21l2-, FLAG-MAB21L2 or MAB21L2-specific bands of 350, 305 and 384 bp, correspondingly; black arrowhead points to ef1a- and GAPDH- specific products of 205 and 148 bp, respectively.(B) Agarose gel analysis of zebrafish mab21l2 transcript- specific amplification from 48-hpf homozygous zebrafish wild-type and mutant embryos; ((TIF)Click here for additional data file.S2 FigA wild-type fish from the same progeny is shown at the top and followed by 7 compound heterozygous embryos. Please note small eye (black arrowheads) in all mutant embryos and shortened tail (black arrows) in 4 out of 7 fish.(TIF)Click here for additional data file.S3 Figmab21l2 mutants with remarkably high levels in the mab21l2Q48Sfs*5 embryos (E-H) and moderately increased levels in the mab21l2R51_F52del embryos (I-L); arrowheads indicate sites of increased TUNEL staining in the eye and brain; le, lens; r, retina; m, midbrain.An increase in TUNEL staining was observed in both (TIF)Click here for additional data file.S4 FigOverlay images of the PCNA and DAPI immunostaining are shown in (TIF)Click here for additional data file.S5 Figpax2.1 pattern is abnormal in 72-hpf mutant embryos , arrowhead in D1 shows abnormal areas of pax2.1-positive cells in the central retina and arrowheads in E1 show broad and intense expression in the region of optic fissure; foxe3 expression is decreased in 48-hpf mutants ; le, lens; of, optic fissure.The ZL-1 staining is absent in 24-hpf (A1) but present in 48–72-hpf mutant embryos , (TIF)Click here for additional data file.


Previous observations indicate that complete regression of metastatic melanoma can be mediated by CD8 lymphocytes that predominantly recognize unique tumor specific mutations predicted in-silico to bind in context of HLA-A*02:01 [Tumor specific mutations were initially identified by conducting whole exome sequencing and RNA sequencing on tumors and matched normal tissue. Putative minimal epitopes for each of the patients' HLA alleles were generated using in-silico MHC-peptide binding prediction algorithms (IEDB: cut off percentile rank < 2 and ann IC50 < 300nm). Candidate peptide epitopes were then pulsed onto autologous CD14+ monocytes and cultured with tumor infiltrating lymphocytes (TIL); mutation specific reactivity was detected using IFN-γ ELISA or by analysis of 4-1BB up-regulation using FACS.Three unique neoantigens were identified as the targets of each of the 2 melanoma patients' T cell populations that were screened using this approach. Between 1 and 6 unique neoantigens, totaling 17, were identified as targets of TIL derived from 7 of the 11 patients with common epithelial cancers who were evaluated. These included 3 colorectal cancer, 2 lung cancer, 1 ovarian cancer, and 1 cholangiocarcinoma patients. No mutated target antigens were identified from three patients with colon cancer and one patient with ovarian cancer using this approach. Recognized epitopes were predicted to be presented in the context of the following class I alleles: A*01:01, A*02:01, A*03:01, A*11:01 A*30:02, B*07:02, B*40:01, B*08:01, B*57:01, C*03:03, C*06:02, C*07:01, C*07:02, C*14:02, C*16:01. Five out of the 23 mutated minimal epitopes were predicted to be the top binding candidates for the corresponding alleles, and 20 out of the 24 peptides were among the top ten of predicted binders.These observations indicate that this approach represents an efficient method for identifying cancer neoantigens restricted by multiple HLA class I alleles in patients with a variety of metastatic malignancies that include melanoma and common epithelial cancers such as lung, gastrointestinal and ovarian cancers. These studies should allow the development of novel cancer therapies based upon the adoptive transfer of enriched populations of TIL or autologous T cell receptor transduced T cells that recognize patient-specific mutated antigens.


In January 2015, British Columbia, Canada, reported avian influenza A(H7N9) virus infection in 2 travelers returning from China who sought outpatient care for typical influenza-like illness. There was no further spread, but serosurvey findings showed broad population susceptibility to H7N9 virus. Travel history and timely notification are critical to emerging pathogen detection and response. Since February 2013, >600 human cases of avian influenza A(H7N9) infection have been reported from eastern China, where the virus is considered enzootic in poultry , Canada, traveled in Hong Kong ; Taipei, Taiwan (January 3–6); and Fujian Province, China (January 7–11), returning home on January 12. They recalled seeing live poultry and copious droppings while visiting Fujian on January 8 but recollected no other poultry contact.Around January 3–7, the previously healthy woman experienced mild cough, sore throat, and hoarseness. She recovered, but influenza-like illness (ILI), including fever, cough, myalgia, and fatigue, developed on January 14. On January 15, she sought outpatient care. A healthcare worker (HCW) collected a nasal swab specimen, which was sent to the BC Public Health Microbiology and Reference Laboratory (BC-PHMRL), where most influenza testing is centralized in BC. On January 16, the sample tested positive for influenza A virus by reverse transcription PCR (RT-PCR), and the HCW was informed; the next business day (January 19), the patient was prescribed oseltamivir.t] 23.43), the specimen could not be subtyped for human influenza H1 or H3 virus by matrix gene–based RT-PCR. Further subtyping using RT-PCR–based targets for the hemagglutinin gene indicated an H7 virus. Sequence analysis of a matrix gene fragment showed 99% identity with H7N9 and H9N2 viruses, the latter of which is known to have donated internal genes to H7N9 virus . On January 29, NML confirmed H7N9 virus infection.On January 13, a day before the woman became ill, her husband, who had a history of asthma, had onset of ILI symptoms  and visited the same HCW. The HCW prescribed doxycycline but did not collect a specimen. On January 19, after his wife received a diagnosis of influenza, the man was prescribed oseltamivir. A throat swab specimen collected from him on January 23 was RT-PCR–positive for influenza A  and on January 28 (day 15 after ILI onset) for the man (Ct 30.21).Neither patient experienced conjunctivitis, which has been reported with H7N7 and H7N3 infections , including 1 who received oseltamivir prophylaxis. All contacts remained asymptomatic. Passengers on the flight taken by the patients while they were asymptomatic were not included in active surveillance because >10 days had elapsed . Phylogenetic analysis of the hemagglutinin and neuraminidase genes showed that the strain clustered with 2014 and 2015 H7N9 human isolates from Jiangsu, Zhejiang, and Fujian Provinces, China, and 2014 chicken isolates from Jiangxi, China, belonging to clade W2-C (>4-fold antibody rise) to H7N9 virus; the HCW had no detectable H7N9 antibody (Antibody titers to H7N9 and recent human influenza A(H3N2) and A(H1N1)pdm09 strains in paired serum samples from the case-patients and HCW were measured by hemagglutination inhibition assay at NML  (6). At antibody .,>60 years of age in 2013 had low-level titers (>10 but <40), but no other samples had detectable H7N9 virus antibody  (antibody . Three son assay .–The onset of ILI in 2 BC patients 5–6 days after observing poultry in China is consistent with the median 5-day incubation period reported elsewhere for H7N9 virus (,,,,Reported human cases of H7N9 infection have mostly been in older men, and two thirds have been categorized as severe (,,Despite broad susceptibility and instances of household or familial transmission, H7N9 virus has not demonstrated easy person-to-person spread. Poultry exposure remains the major risk factor for human H7N9 infection (


Ultra-small superparamagnetic iron oxide nanoparticles (USPIO) are promising contrast agents for magnetic resonance imaging (MRI). USPIO mediated proton relaxation rate enhancement is strongly dependent on compartmentalization of the agent and can vary depending on their intracellular or extracellular location in the tumor microenvironment. We compared the T1- and T2-enhancement pattern of intracellular and extracellular USPIO in mouse models of cancer and pilot data from patients. A better understanding of these MR signal effects will enable non-invasive characterizations of the composition of the tumor microenvironment.Six 4T1 and six MMTV-PyMT mammary tumors were grown in mice and imaged with ferumoxytol-enhanced MRI. R1 relaxation rates were calculated for different tumor types and different tumor areas and compared with histology. The transendothelial leakage rate of ferumoxytol was obtained by our measured relaxivity of ferumoxytol and compared between different tumor types, using a t-test. Additionally, 3 patients with malignant sarcomas were imaged with ferumoxytol-enhanced MRI. T1- and T2-enhancement patterns were compared with histopathology in a descriptive manner as a proof of concept for clinical translation of our observations.-3 mL/min/100cm3) compared to MMTV-PyMT tumors (1.0±0.9x10-3 mL/min/100 cm3). Likewise, ferumoxytol imaging in patients showed similar findings with high T1 signal in areas of tumor necrosis and low signal in areas of intracellularly compartmentalized iron.4T1 tumors showed central areas of high signal on T1 and low signal on T2 weighted MR images, which corresponded to extracellular nanoparticles in a necrotic core on histopathology. MMTV-PyMT tumors showed little change on T1 but decreased signal on T2 weighted images, which correlated to compartmentalized nanoparticles in tumor associated macrophages. Only 4T1 tumors demonstrated significantly increased R1 relaxation rates of the tumor core compared to the tumor periphery (p<0.001). Transendothelial USPIO leakage was significantly higher for 4T1 tumors (3.4±0.9x10Differential T1- and T2-enhancement patterns of USPIO in tumors enable conclusions about their intracellular and extracellular location. This information can be used to characterize the composition of the tumor microenvironment. Ultra small superparamagnetic iron oxide nanoparticles (USPIO) have been developed as contrast agents for magnetic resonance (MR) imaging –5. USPIOHigher relaxivity (magnetic moment) leading to higher sensitivity .Specific tumor delivery via the enhanced permeability and retention (EPR) effect .Possibility to chemically link diagnostic pharmacophores for targeted delivery .Possibility to add other contrastophores for multi-modality imaging , 10.Possibility to link therapeutic pharmacophores for simultaneous imaging and therapy –13.Several USPIO compounds are currently translated to clinical applications –18. It iin vivo may enable discrimination of different histopathological compartments within tumor tissue. While the T1- and T2-enhancement pattern of intracellular and extracellular USPIO has been studied extensively in vitro [in vivo. Thus, the goal of our study was to compare the T1- and T2-enhancement pattern of intracellular and extracellular USPIO in mouse models of cancer and pilot data from patients. A better understanding of MR signal effects of intracellular and extracellular USPIO in tissues will facilitate non-invasive USPIO localization in vivo, enable conclusions about the composition of the tumor microenvironment and form the basis for related quantitative analyses.Therefore, we hypothesized that regional differences in T1- and T2-relaxivities of USPIO in vitro , 25, to -1s-1, an R2 relaxivity of 83 mM-1s-1 (at 0.47 T and 39°C) and a plasma half-life of 14.5 hours in humans [Ferumoxytol  is a colloidal solution of USPIO nanoparticles, which are composed of an iron oxide core and a semisynthetic carbohydrate coating of polyglucose sorbitol carboxymethyl ether with a hydrodynamic diameter of 30 nm . Ferumoxn humans .-1mM-1 and R2 relaxivity of 40 s-1mM-1 measured at 1.5 T and 40°C [GEH121333 is a new core/shell USPIO agent with an iron oxide core and a hydroxyphosphonate-PEG shell. GEH121333 nanoparticles have an average hydrodynamic diameter of 22 nm, as measured by differential Light Scattering (DLS) with monodispersity, as measured by Field Flow Fractionation (FFF). GEH121333 nanoparticles have an R1 relaxivity of 17 sand 40°C .-1s-1, an R2 relaxivity of 4.1 mM-1s-1 (at 0.47 T and 37°C) and a plasma half-life of 1.6 hours [Gd-DTPA  is a standard, low molecular weight, gadolinium chelate with an R1 relaxivity of 3.8 mM.6 hours . A dose Animal experiments were approved by Institutional Animal Care and Use Committee (IACUC) at the University of California San Francisco and Administrative Panel on Laboratory Animal Care (APLAC) at Stanford University and all animals were kept and treated in accordance to NIH and institutional guidelines. Two tumor models were used: To mimic a slow-growing malignancy, a transgenic MMTV-PymT model was used , 33. MMTSix animals with 4T1 tumors and six animals with MMTV PyMT tumors underwent MR imaging under isoflurane anesthesia, using a 2 T Omega CSI-II animal scanner  and a custom birdcage radiofrequency coil with a circulating deuterium oxide warming pad. MR images were obtained before, immediately after, 1 hour after and 24 hours post injection (p.i.) of ferumoxytol using following sequences: (i) T1 weighted spin echo (SE) 500/12 ms (TR/TE) sequences, (ii) T2* weighted gradient echo (GE) 240/10 ms (TR/TE) flip angle 30 degrees sequences and (iii) inversion recovery (IR) 3000/1500 ms (TR/TE) with increasing inversion times (70–3370 ms in increments of 300 ms). (i) and (ii) were acquired with a slice thickness of 2 mm, a field of view of 3x3 cm, and a matrix of 128x128, (iii) had same slice thickness and field of view but a matrix of 64x64. Ferumoxytol was injected at a dose of 0.5 mmol Fe/kg body weight (27.92 mg Fe/kg) in 100 μl normal saline via an indwelling tail vein catheter placed before animal imaging.Inversion recovery fast GE images were used to calculate maps of relaxation rate for T1, R1 (1/T1), for each slice covering the tumor by fitting the image pixel values to the following equation: SI(TI) = 1–2 exp (-TI ˣ R1), where TI is inversion time. Regions of interest were drawn on the R1 maps to measure the entire tumor in all slices and the resultant data were averaged by weighting the individual slice values by the number of tumor pixels in that slice.The trans-endothelial permeability of ferumoxytol across tumor micro vessels was calculated from 4T1 and MMTV-PymT tumors. We assume that the change in MR signal enhancement in tumor is a combination of signal change from contrast in the blood as well as in the interstitial space and that the blood concentration of ferumoxytol is roughly constant during the first hour after intravenous administration . DividinTo evaluate, whether the variable size of ferumoxytol nanoparticles was the reason for the observed differences in tumor enhancement, we performed an additional experiment in six mice MMTV-PyMT tumors. The tumors were allowed to grow up to a size of 1.5 cm, which lead to formation of a central necrosis as confirmed by fluid-equivalent central hyperintense tumor areas on T2-weighted MR images. MR images were obtained on a 7T animal MR scanner before iron oxide injection and at 24 and 48 h post injection  using the following pulse sequences: T1-weighted 3-dimensional (3D) fast spoiled gradient recalled acquisition (FSPGR) 6.6ms/1.4ms/2-30° (repetition time/echo time/flip angle) and T2*-weighted 3D FGRE 70ms/1.6–20.6ms/20°. All MR acquisitions were performed with a field of view of 4.5x4.5 cm and a slice thickness of 0.6 mm. MR data were analyzed by one independent investigator at Stanford using custom research software tool . T1 and T2* relaxation times of tumors were calculated based on multi-flip-angle FSPGR and multi-echo FGRE images, converted to relaxation rates . Statistical comparisons of ΔR1 relaxation rates of 4T1 and MMTV-PymT tumors was performed using a t-test with a p < 0.05 considered to be significant.After the last imaging sequence, animals were sacrificed and tumors were explanted and immersed in formalin. Sections were dehydrated and embedded in paraffin for cutting. Routine H&E staining was performed on sections to detect necrosis versus viable tumor tissue. Necrotic areas were identified by microscopic appearance of coagulative necrosis with sheets of homogenous clusters and dying and dead cells whereas viable tumor tissue was identified by microscopic appearance of intact cells. Histologic distribution of iron on Prussian blue stain was used to differentiate between compartmentalized and free iron. Blue staining in a compartmentalized fashion indicated intracellular iron whereas diffuse blue staining not in a cellular distribution indicated extracellular iron.Formalin-fixed paraffin embedded (FFPE) whole tumor tissue sections of GEH121333 injected animals were deparaffinized with Histochoice clearing agent , rehydrated by a series of alcohol washes, and processed for iron stains and antigen retrieval. Immunohistochemistry (IHC) detection of tumor associated macrophages (TAM) and GEH121333 in tumor tissue was performed by an antigen retrieval method developed specifically for FFPE tissues. In brief, slides were incubated in citrate buffer for 20 minutes followed by 0.3% Triton X-100/EDTA for 20 minutes and blocked against non-specific binding by incubating with blocking solution (10% goat serum/PBS) for 1–2 hours at room temperature. A mixture of two primary antibodies containing (1) anti-PEG, rabbit-derived  and (2) rat anti-mouse F4/80  was applied on the sections at a dilution of 1:100 and left overnight at 4°C in PBS/10% goat serum. For indirect detection of bound primary antibodies, species-specific Cy3 or Cy5-conjugated secondary antibodies  were used at a dilution of 1:200. The sections were further stained with 4',6-diamidino-2-phenylindole (DAPI) to identify cell nuclei.As a proof-of-concept for clinical translation of the observed findings, we investigated four patients with bone tumors with ferumoxytol-enhanced MRI as a part of a larger clinical trial aimed at improving tumor staging with iron oxide based contrast agents . We seleBoth MMTV-PymT and 4T1 tumors appeared homogenous and featureless on pre contrast T1 and T2* weighted MR images Figs .Ferumoxytol injection caused significant long-lasting signal enhancement of iliac vessels in both MMTV-PymT and 4T1 animals on T1-weighted MR scans at 15 min and 24 hours p.i. . Both MM-1s-1. Plasma ΔR1 was 125.8±10.1 s-1 5 minutes p.i., 114.3±12.4 s-1 60 minutes p.i. and 3.89±3.7 s-1 24 hours p.i. Corresponding iron concentrations in plasma were 10.6±0.9 mM Fe at 5 minutes, 9.65±1.0 mM Fe at 60 minutes and 0.33±0.31 mM Fe at 24 hours. Assuming a mono-exponential loss of Fe in blood, we obtained a blood clearance rate of 0.15 h-1. As we could not faithfully measure blood in tumor secondary to the high ferumoxytol concentration in the blood, we used blood concentration curves to determine tumor leakage rates. This yielded a trans-endothelial leakage of 3.4±0.9x10-3 mL/min/100 cm3 for 4T1 tumors and 1.0±0.9x10-3 mL/min/100 cm3 for MMTV-PyMT tumors.The R1 relaxivity of ferumoxytol in 0.9% normal saline at 2 T and room temperature was 11.85 mM (Fe)The average ΔR1 and ΔR2 in tumor and necrosis after 1 hour, 24 hour and 48 hour post injection are calculated and shown in Histological correlations showed a markedly heterogeneity in 4T1 tumor with a core comprised of cellular debris and leukocytes consistent with necrosis and a periphery comprised of tumor cells . In the In accordance with results described above, intravenous injection of the monodisperse iron oxide nanoparticle compound GEH121333 demonstrated significant positive (bright) T1-enhancement and significant negative (dark) T2-signal enhancement of central tumor areas , while pHistological correlations showed an abundance of free (extracellular) iron oxide nanoparticles in central necrosis. The necrotic areas were outlined by a rim of macrophages, which contained iron oxides. More peripheral highly cellular tumor areas demonstrated iron oxide nanoparticles in macrophages and few interstitial iron oxides.As a proof-of-concept for clinical translation of our observations, we investigated MR signal effects of ferumoxytol nanoparticles in four patients with malignant bone sarcomas. We used the iliac vessels as an internal standard for a proton-rich environment with extracellular USPIO and we used hematopoietic bone marrow as an internal standard for a highly cellular tissue with abundant macrophages and intracellular USPIO compartmentalization. The iliac vessels demonstrated significant T1-enhancement, while normal bone marrow demonstrated lack of T1-enhancement, consistent with our hypothesis that extracellular iron oxides in proton rich environment exhibit strong T1-signal effects while intracellularly compartmentalized iron oxides show diminished T1-signal effects. No difference between these differently compartmentalized iron oxides could be noted on T2-weighted MR scans.Accordingly, necrotic or cystic tumor areas with histopathological proven micro vessel supply, such as early tumor necrosis and telangiectatic tumor areas, demonstrated significant T1-enhancement. Solid tumor tissue demonstrated minimal or no T1-enhancement Figs  and 7. Oin vivo findings are in accordance with previous in vitro studies on MR signal effects of intracellular and extracellular iron oxides [Data showed that T1 weighted MR images provide important information about intracellular and extracellular compartmentalization of iron oxide nanoparticles in tumors. Ferumoxytol nanoparticles in tumor necrosis demonstrated marked T1 and T2-enhancement at 24 after injection, while intracellularly compartmentalized ferumoxytol nanoparticles demonstrated predominant T2, with little or no T1 enhancement. These n oxides .Our data indicate that ferumoxytol-enhanced MRI may be more sensitive for the detection of an early necrosis compared to T2 weighted or Gd-DTPA enhanced T1 weighted MRI scans. The typical lack of contrast enhancement of tumor necrosis with Gd-DTPA is based on lack of tumor perfusion and the typical hyper-intensity on T2 weighted images is based on increased proton content, both of which may be only noted in advanced, liquefied necrosis . Ferumoxet al. and Moore et al. postulated that USPIO-induced T1 and T2-enhancement in solid tumors on relatively early postcontrast scans is largely determined by the tumor’s blood volume, which is in the order of 5% for most cancers [Our data also showed in accordance with others that ferumoxytol causes little or no T1-enhancement in solid tumors. Turetschek  cancers , 24, 26. cancers .We acknowledge limitations of our study. In our animal model, we utilized a dose of ferumoxytol, which was fivefold higher than the FDA approved dose of iron oxide nanoparticles for patients (0.5 versus 0.1 mmol Fe/kg). Given the faster bio distribution and higher uptake efficiency for USPIO in rodent spleen and liver, these higher doses are recommended for rodent studies in order to provide similar contrast agent kinetics compared to humans , 27. OurIn summary, we have shown that extracellular iron oxides in early tumor necrosis lead to strong T1 and T2-enhancement while compartmentalized intracellular iron oxides in macrophages are characterized by predominant T2-enhancement with little or no T1-enhancement. This information is important for the interpretation of iron oxide enhanced MRI scans of malignant tumors, especially if conclusions are drawn between iron oxide MR enhancement and presence or quantities of macrophages in target tissues.


Kip1 expression and cleaved caspase-3 are increased, while cyclin E expression is decreased, in S100A4-deficient pancreatic tumors in vivo. S100A4-deficient tumors have lower expression of vascular endothelial growth factor, suggesting reduced angiogenesis. Biochemical assays revealed that S100A4 activates Src and focal adhesion kinase (FAK) signaling events, and inhibition of both kinases is required to maximally block the tumorigenic potential of pancreatic cancer cells. These findings support that S100A4 plays an important role in pancreatic cancer progression in vivo and S100A4 promotes tumorigenic phenotypes of pancreatic cancer cells through the Src-FAK mediated dual signaling pathway.S100A4 expression is associated with poor clinical outcomes of patients with pancreatic cancer. The effects of loss or gain of S100A4 were examined in pancreatic cancer cell lines. S100A4 downregulation remarkably reduces cell migration and invasion, inhibits proliferation, and induces apoptosis in pancreatic tumor cells. S100A4 downregulation results in significant cell growth inhibition and apoptosis in response to TGF-β1, supporting a non-canonical role of S100A4 in pancreatic cancer. The role of S100A4 in tumor progression was studied by using an orthotopic human pancreatic cancer xenograft mouse model. Tumor mass is remarkably decreased in animals injected with S100A4-deficient pancreatic tumor cells. P27 Whether S100A4 directly contributes to pancreatic cancer progression in vivo or is just a secondary effect of other changes during pancreatic cancer progression remains to be answered.S100A4, also known as metastasin (Mts1)1343463811713Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase, that is upregulated in many types of cancers, including pancreatic cancers192120222425262722in vitro by modulating S100A4 expression levels (loss and gain approaches), and pancreatic cancer growth in vivo by using an orthotopic human pancreatic cancer xenograft mouse model. The findings provide evidence that S100A4 facilitates pancreatic cancer progression through promoting cell migration and invasion, anchorage-independent growth, angiogenesis, and tumor survival. S100A4 also plays an important role in protecting pancreatic cancer cells against transforming growth factor beta (TGF-β)-induced growth inhibition and apoptosis. S100A4 enables intracellular FAK and Src signaling events that operate as a dual signaling pathway and underlie the tumorigenic potential of pancreatic carcinoma cells. Together, these results indicate that S100A4 would be an attractive therapeutic target in pancreatic cancer.In this study, we examined the effects of S100A4 on the aggressive characteristics of pancreatic cancer cells S100A4 protein expression in human pancreatic tumor samples is more than that in non-tumor control samples as reported by others7The silencing of S100A4 resulted in decreased cell invasion  , TGF-β1 remarkably inhibited cell growth , supportKip inhibits cyclin-mediated cell progression. p27Kip has been shown to regulate breast cancer cell functions3536Kip1 expression  in these S100A4-deficient S2VP10 cells (Cyclins (such as cyclin E) regulate cell cycle transition from G1 to S phase, and cyclin-dependent kinase inhibitor p27pression , and indpression . TGF-β1 10 cells , support10 cells , suggestin vivo requirements for S100A4 were tested by inoculating S2VP10 cells (expressing either S100A4 #1 or control shRNAs) into the tail of the pancreas of female SCID mice3 versus 823 mm3, p < 0.01). The S100A4-deficient tumors showed increased p27Kip1 expression and decreased cyclin E expression compared with the control tumors (Kip1) and decreased cell growth signaling protein (cyclin E) suggest that the cellular proliferation pathways are impaired in S100A4-deficient tumors. The levels of cleaved caspase-3 and cleaved PARP were increased in S100A4-deficient tumors relative to the control tumors  also did not completely inhibit FAK activation , indicatin vivo. S100A4 expression is necessary to protect pancreatic cancer cells against TGFβ-induced growth inhibition and apoptosis. S100A4 promotes activation of Src and FAK, which form a dual signaling pathway and contribute to the tumorigenic capacity of pancreatic cancer cells.This study demonstrates the critical role of S100A4 in tumorigenic potential of pancreatic cancer cells, such as migration, invasion, growth, and survival. S100A4 downregulation in tumor cells greatly impairs their ability to develop tumors in vivo. S100A4 likely promotes pancreatic cancer progression in vivo through multiple mechanisms. In vitro S100A4 plays an important role in pancreatic cancer cell lines in cell migration and invasion, anchorage-independent growth, proliferation, and survival (Kip1 and decreased cyclin E) and increased apoptotic signaling (cleaved caspase-3 and PARP)  . Our in Interestingly, while TGF-β1 has no effect on control pancreatic tumor cells, TGF-β1 is able to induce significant growth inhibition and apoptosis in S100A4-deficent pancreatic tumor cells . The finin vivo in a pancreatic cancer mouse model  activation reduces FAK (or Src) activation . This isin vivo, and provides novel insights into the signaling mechanisms by which S100A4 promotes tumorigenic capacity of pancreatic carcinoma cells. S100A4 expression is important for pancreatic tumor cells to avoid TGF-β1-induced growth inhibition and apoptosis. S100A4 enables FAK- and Src-mediated signaling that operate as a dual signaling pathway and underlie the tumorigenic potential of pancreatic carcinoma cells. These findings suggest alternative strategies to the current therapeutic approaches targeting FAK and Src. The inhibition could be achieved by disabling the S100A4 function or by inactivating Src and FAK pathways. These approaches may be advantageous for treatment of aggressive or advanced pancreatic cancer, which are not manageable by most of the current therapies.In summary, the current work supports the role of S100A4 in pancreatic cancer progression The following antibodies were purchased: S100A4 ; phospho-FAK [pY397-FAK] ; p27Kip1, cyclin E, Src, and FAK ; phospho-SrcY418, cleaved caspase-3, cleaved Poly-(ADP-ribose) polymerase (PARP), ; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) . TGF-β1 and S100A4 were purchased from R&D Systems . Dasatinib (Src kinase inhibitor) was purchased from LC Laboratories . PF573228 (FAK inhibitor) was purchased from Calbiochem . Chemicals were purchased from Sigma-Aldrich  and Fisher Scientific . TGF-β1 and S100A4 were purchased from R&D Systems . The pan-caspase inhibitor Z-VAD-fmk was purchased from BD PharMingen . Chemicals were purchased from Sigma-Aldrich  and Fisher Scientific .Propagation and maintenance of MIA PaCa-2 and S2-VP10 cells were described previouslyThe studies and protocols have obtained approvals from local IRB. All experiments were performed in accordance with relevant guidelines and regulations. De-identified tumor tissues (n = 5), and non-tumor control tissues including matched adjacent non-tumor tissues (n = 2) and non-tumor tissue (n = 1), were obtained through UAB tissue procurement program.5 cells/30 μl which was injected into the tail of the pancreas after the following procedures. A sterile cotton tipped applicator was used to cover the injection site for 30s to prevent peritoneal leakage. The organs were returned to the abdomen and the skin and peritoneum were closed in a single layer closure with 5-0 Prolene sutures. Animals recovered on a warming blanket and received liquid acetaminophen for 24 hours with food and water ad libitum. Animals were euthanized at day 21 post xenograft procedure. Primary tumors in the pancreas were excised and the final tumor volume was calculated by using the modified ellipsoidal formula: tumor volume = 1/2 × L × W × H . The L, W, and H were determined by using external caliper. The ellipsoid volume formulas (1/2 × L × W × H) is one of the best approaches for estimating tumor mass in animal models51All animal interventions were approved by local IACUC. All experiments were performed in accordance with relevant guidelines and regulations. The model was performed as described by us previouslyThe replication incompetent lentiviral vectors expressing short hairpin RNA (shRNA) for silencing S100A4 (two shRNAs) and lentiviral vectors expressing control non-targeting shRNA were from OpenBioSystem Thermo Scientific . Pancreatic cancer cells were transfected with lentiviral vectors according to manufacturer's instructions and as described previouslyAd-Y397F is an adenoviral vector containing the hemaglutinin (HA)-tagged phospho-FAK mutant cDNA . Y397F-FAK is where tyrosine 397 of FAK is replaced by phenylalanine (Y397F is unable to be phosphorylated/activated). The construction of the adenoviral vector used similar procedures as described previouslyHuman pancreatic tumor cells or pancreatic tumor tissues were lysed in RIPA lysis buffer with 1% deoxycholate, 1.0% Triton X-100, and 0.1% SDS in 0.01 M Tris base (pH 7.4), 0.15 M NaCl, and containing the following inhibitors, 100 μM phenylmethanesulfonyl fluoride (PMSF), 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 100 μM sodium vanadate, essentially as described previouslyIn vitro invasion assay was performed with the kit with matrigel-coated inserts according to the manufacturer's instructions . 1 × 105 cells/well was added to the upper compartments of the invasion chamber. The values obtained were calculated using the number of invaded cells from three filters after 24 hours. Final results were pooled from two to three individual experiments.The wound closure monolayer/scratch motility assay and invasion assay were performed as described previouslyQuantitative RT-PCR was performed as described by us previously54The Annexin V assay with propidium iodide labeling was performed with apoptosis kit  on pancreatic tumor cells with indicated treatment as per the manufacturer's instructions and described previously by us5 cells were plated per well, treated with vehicle or TGF-β1 (4 ng/ml), and the total number of cells per well were counted after 24 hours in five replicates per condition. Experiments were repeated at least twice.Cell proliferation assays were done in 6-well plates in serum-free medium (DMEM with 1% BSA) as previously describedAnchorage-independent growth assays were performed as described previouslyAnalysis of S100A4, FAK, and Src on human pancreatic tumor tissue sections were performed as described previouslyCell cycle analysis by flow cytometry was performed as described previously by usThe VEGF ELISA was performed as per the instructions with the Quantikine Human VEGF Immunoassay ELISA Kit . Similar ELISA procedures were described by us previouslyData were analyzed using the student t-test analysis  for differences between two groups, and findings were expressed as mean + SE. Experiments were repeated three to four times. A p value < 0.05 was considered to be statistically significant.P.C., W.G. and Q.D. wrote the main manuscript text and P.C., Y.Y., J.S., A.L., M.H. and G.-Q.C. largely contributed to the animal studies. X.H., D.J.B., J.D.C., Q.T., D.C., Q.L. and Y.Y.L. largely contributed to cell signaling studies in cells and tissues. All authors contributed to the figures and supplemental figures and reviewed the manuscript.Supplementary Figure and Legend


Bacillus thuringiensis in the insect host is a powerful model to investigate the role of quorum sensing in natural conditions. It is tuned by communication systems regulators belonging to the RNPP family and directly regulated by re-internalized signaling peptides. One such RNPP regulator, NprR, acts in the presence of its cognate signaling peptide NprX as a transcription factor, regulating a set of genes involved in the survival of these bacteria in the insect cadaver. Here, we demonstrate that, in the absence of NprX and independently of its transcriptional activator function, NprR negatively controls sporulation. NprR inhibits expression of Spo0A-regulated genes by preventing the KinA-dependent phosphorylation of the phosphotransferase Spo0F, thus delaying initiation of the sporulation process. This NprR function displays striking similarities with the Rap proteins, which also belong to the RNPP family, but are devoid of DNA-binding domain and indirectly control gene expression via protein-protein interactions in Bacilli. Conservation of the Rap residues directly interacting with Spo0F further suggests a common inhibition of the sporulation phosphorelay. The crystal structure of apo NprR confirms that NprR displays a highly flexible Rap-like structure. We propose a molecular regulatory mechanism in which key residues of the bifunctional regulator NprR are directly and alternatively involved in its two functions. NprX binding switches NprR from a dimeric inhibitor of sporulation to a tetrameric transcriptional activator involved in the necrotrophic lifestyle of B. thuringiensis. NprR thus tightly coordinates sporulation and necrotrophism, ensuring survival and dissemination of the bacteria during host infection.Bacteria use quorum sensing to coordinate adaptation properties, cell fate or commitment to sporulation. The infectious cycle of  Bacillus thuringiensis is an entomopathogenic bacterium used worldwide as biopesticide. Its life cycle in insect larvae, which includes virulence, necrotrophism and sporulation, is regulated by cell-cell communication systems involving sensor proteins directly regulated by re-internalized peptide pheromones. After toxaemia caused by pore-forming Cry toxins, the PlcR sensor activates the production of virulence factors leading to insect death. B. thuringiensis then shifts to a necrotrophic lifestyle preceding sporulation. Previously, we showed that this process is regulated by the sensor NprR, which, in the presence of its cognate signaling peptide NprX, adopts a tetrameric conformation allowing its binding to specific DNA sequences and transcription of genes involved in survival of the bacteria in insect cadavers. Here, we demonstrate that, in the absence of NprX, NprR is a dimer, which negatively controls sporulation, independently of its transcription factor activity. We show that NprR prevents the phosphorylation of the phosphoprotein Spo0F and inhibits the phosphorylation cascade regulating sporulation. This demonstrates that NprX binding switches the bifunctional sensor NprR from a dimeric sporulation inhibitor to a tetrameric transcription factor. By establishing a close coordination between cell density, necrotrophism and sporulation, this communication system benefits a pathogenic bacterium feeding on death matter like B. thuringiensis. NprR is found in all strains of the B. cereus group, including B. anthracis and B. cereus involved in food poisoning. Our results may provide new insights for controlling the development and the survival of these undesirable bacteria. Sporulating bacteria have developed a number of sophisticated mechanisms devoted to the temporal and spatial coordination of cell fate and gene expression. Regulatory mechanisms, as two-component systems, contribute to maintain this tight coordination by tuning gene expression and protein activation in response to a large variety of environmental stimuli including nutrient limitation and population density . In GramBacillus subtilis  with a C-terminal 6xHis tag. The recombinant protein was purified as already described for the full-length wild-type NprR  grown at 37°C in the presence of 50 μg/ml ampicillin and 50 μg/ml kanamycin in M63 medium. At OD600nm of 1 the M63 medium was supplemented with 10 g/l of Studier amino acid and 2.5 g/l of selenomethionine. Finally, expression was induced at OD600nm of 1.5 by adding 1 mM IPTG and the culture was incubated for 4 hours at 37°C. The labelled protein was purified following the same protocol as for the unlabelled protein.Selenomethionine-labelled NprR(ΔHTH))(Y223A/F225A) was produced in B. thuringiensis was produced as an N-terminal His-tagged recombinant protein in E. coli strain NMA522 [pQE30Ωspo0F] grown at 37°C in the presence of 50 μg/ml of ampicillin. Expression was induced at OD600nm of 0.6 by adding 1 mM IPTG and the culture was incubated for 3 hours at 37°C. The protein was produced in inclusion bodies, inclusion bodies were resuspended in 25 mM Tris-HCl pH 8, 500 mM NaCl, 3 M Guanidinium Chloride and 10% Glycerol and incubated for 3 hours at 4°C. Finally Spo0F was renatured on the IMAC column following the protocol of the manufacturer. The purification was completed by a size exclusion chromatography step using a S75 16/60 column equilibrated in 20 mM Tris-HCl pH 8, 200 mM NaCl, 10% Glycerol.Spo0F from B. subtilis were produced as N-terminal His-tagged recombinant proteins in the E. coli M15 [pRep4] strain grown at 37°C in the presence of 50 μg/ml ampicillin and 50 μg/ml kanamycin. Expression was induced at OD600nm of 0.6 by adding 1 mM IPTG and the cultures were incubated overnight at 15°C. The Bs Spo0F and KinA proteins were then purified using an IMAC column following the protocol of the manufacturer (QIAGEN). The purification was completed by a size exclusion chromatography step using a S75 16/60 and a S200 16/60 column, for Spo0F and KinA respectively. The samples were stored frozen in 50 mM Tris-HCl pH 7.5, 50 mM Na2SO4, 15% Glycerol, 5 mM MgCl2 for Spo0F and KinA.Spo0F and KinA from Bt Spo0F solutions were serially diluted from about 260 μM to 8 nM in the presence of 33 nM labelled NprR and 5% glycerol in MST buffer . Data analyses were performed using the Nanotemper Analysis software.Microscale thermophoresis (MST)  was perfB. subtilis Spo0F was determined in a reaction buffer . KinA (0.1 μM) was first activated by pre-incubation (1.5 hours at 37°C) in the presence of 1 mM ATP, containing 20 μCi mmol-1 [γ-32P]-ATP. Spo0F (5.4 μM), NprR (10 μM) and NprX-8 (57 μM) were then added as indicated, and mixtures were further incubated at 37°C. Aliquots were withdrawn at different times . Samples were analyzed by 15% SDS-Tris-glycine PAGE. The gel was dried and the radioactivity detected with a PhosphorImager and the ImageQuant software (Molecular Dynamics Corp.).Phosphorylation of In order to determine the conformation of NprR carrying out the sporulation inhibitor function of the protein, we performed a large-scale screening of crystallization conditions. The truncated double mutant protein NprR(ΔHTH)(Y223A/F225A) crystallized at 18°C in 1.0 M Na citrate, 0.1 M Hepes pH 7.6. Initial needle clusters were improved using micro- and macro seeding. The crystals were flash frozen in the crystallization solution supplemented with 30% glycerol for data collection.A native data set was collected at 3.25Å resolution on beamline ID29-1 . A selenomethionine (SeMet) labelled form of the protein was used to collect anomalous diffraction data on beamline Proxima-1 . The derivative data set was collected at a wavelength of 0.97911 corresponding to the maximum of anomalous dispersion f”. Both data sets were processed with the XDS package . Sub-strWe used the Protein structure comparison service PDBeFold at European Bioinformatics Institute , to supeBacillus thuringiensis 407 used in this study is accessible in the NCBI Reference Sequence (RefSeq) Database under number NC_018877.1.The genome of S1 Fig(A) Sequence alignment of the HTH domains of NprR and PlcR. The position of the mutated MTQ motif of NprR* is underlined in blue. (B) Analysis of the PlcR/DNA interactions (PDB ID 3U3W). PlcR residues L19, T20 and Q21 from PlcR, equivalent to the MTQ motif of NprR, are highlighted in sticks and labelled.(TIF)Click here for additional data file.S2 FignprA transcription. β-Galactosidase activity of the Bt 407 wild type strain (RX PnprA’Z) and the Bt 407 ΔRX amy::nprR*-nprX mutant strain (R*X PnprA’Z) harbouring the pHT304.18-nprA’Z plasmid. (B) Effect of NprR single mutations Y223A and F225A on nprA transcription. β-Galactosidase activity of Bt 407 wild type (RX PnprA’Z) and the Bt 407 ΔRX amy::nprR[Y223A]-nprX (R[Y223A]-X PnprA’Z) and Bt 407 ΔRX amy::nprR[F225A]-nprX (R[F225A]-X PnprA’Z) mutant strains harbouring the pHT304.18-nprA’Z plasmid. (C) Effect of NprR single mutations Y165A and R343A on nprA transcription. β-Galactosidase activity of Bt 407 wild type (RX PnprA’Z) and the Bt 407 ΔRX amy::nprR[Y165A]-nprX (R[Y165A]-X PnprA’Z) and Bt 407 ΔRX amy::nprR[R343A]-nprX (R[R343A]-X PnprA’Z) mutant strains harbouring the pHT304.18-nprA’Z plasmid. The cells were grown at 37°C in HCT medium. Time zero was defined as the onset of the stationary phase.(A) NprR* lacking a functional HTH domain is unable to activate (TIF)Click here for additional data file.S3 Fig(A) Flexibility of the apo form. Apo forms of the Rap proteins. Apo-RapI (PDB ID 4I1A) and apo RapF (PDB ID 4I9E) structures. (B) Conserved dimerization mode of the Rap proteins. Closed TPR conformation of the RapF-PhrC complex (PDB ID 4I9C) and 3-helix bundle conformation of apo RapF (PDB ID 4I9E). The protein chains are shown as cartoon with cylindrical helices and coloured by spectrum from blue (Nter) to red (Cter). The conserved C-terminal type-II interface is highlighted by dots surrounding residue F360 equivalent to the NprR residue Y410. The bound peptides are displayed as black sticks.(TIF)Click here for additional data file.S4 FigThe TPR motifs are highlighted by arrows coloured by spectrum from blue to red. Conserved NprR residues D107, Y118, E188 and Y223 involved in Spo0F binding are highlighted by red stars. Residues Y165, W167, H201, H205, H206 and R343 are indicated by green triangles. Structural data from PDB files 4I9C (RapF [(TIF)Click here for additional data file.S5 Fig(A) Role of residues Y158, F159, R115 and Y117 in the dimerization interface of RapF 3-helix bundle conformation (PDB ID 4I9E). (B) Position of residues Y158 and F159 in the dimerization interface of RapF TPR conformation (PDB ID 4I9C). (C) Role of residues R115, Y117, Y153 and D297 in the TPR conformation of the RapF-PhrC complex (PDB ID 4I9C).(TIF)Click here for additional data file.S1 Table−1 and viable cells ml−1. The viable cells and heat-resistant spores were counted after 3 days in LB medium at 30°C. OD600 at t0 corresponds to the OD600 at the onset of the stationary phase. n is the number of independent sporulation efficiency measurements. Nd: Not detected. Results are given as mean ± standard error of the mean (SEM).The percentages of spores were calculated as 100 × the ratio between heat-resistant spores ml (DOCX)Click here for additional data file.S2 TableThe restriction sites are underlined. Bases in red correspond to the point mutations.(DOCX)Click here for additional data file.S3 TableReferences given in S3 Table following manuscript numbering.(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


PAX3/7 fusions and alterations in cell cycle regulators. In contrast, mostly showing embryonal histology, both E1 and E2 subtypes are characterized by high frequency of CN alterations and/or allelic imbalances, FGFR4/RAS/AKT pathway mutations and PTEN mutations/methylation and in E2, also by p53 inactivation. Despite the better prognosis of embryonal RMS, patients in the E2 are likely to have a poor prognosis. Our results highlight the close relationships of the methylation status and gene mutations with the biological behaviour in RMS.Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood. Here we studied 60 RMSs using whole-exome/-transcriptome sequencing, copy number (CN) and DNA methylome analyses to unravel the genetic/epigenetic basis of RMS. On the basis of methylation patterns, RMS is clustered into four distinct subtypes, which exhibits remarkable correlation with mutation/CN profiles, histological phenotypes and clinical behaviours. A1 and A2 subtypes, especially A1, largely correspond to alveolar histology with frequent  Rhabdomyosarcoma is a common childhood soft-tissue cancer. Here Seki and Nishimura analyse the exome, transcriptome, copy number and DNA methylome of 60 sarcomas and identify distinct methylation subgroups associated with genetic and clinical features. PAX3– or PAX7–FOXO1 fusion genes, whereas ERMS commonly harbours loss of heterozygosity at 11p15.5 and gains of chromosomes 2, 8, and 12 in varying combinations3FGFR4/RAS/AKT pathway, FBXW7, CTNNB1 and BCOR667Rhabdomyosarcoma (RMS), a highly aggressive soft-tissue sarcoma, typically affects children, and it is classified into two major subtypes having alveolar (ARMS) and embryonal (ERMS) histologies1n=2) and metastatic (n=1) samples (http://genomon.hgc.jp/exome/en/index.html), 604 in 531 genes (88%) were validated by deep sequencing  and FGFR4/RAS/AKT pathway genes , which were predominantly detected in ERMS tumoursFGFR4/RAS/AKT pathway mutations, RAS pathway genes were mutated in 15 cases, in which all mutations in NRAS/KRAS/HRAS (n=10) and PTPN11 (n=2) occurred in hot-spot amino acids involved in gene activation, whereas three of four NF1 mutations were frameshift indels resulting in premature truncation of the protein. Five of six FGFR4 mutations affected highly conserved amino acids within the kinase domain of which four were previously reported activating mutationsPIK3CA, which have previously been reported in other cancers, are thought to be activating in nature1213BCOR and ARID1A. All BCOR mutations and four of six ARID1A mutations were frameshift or nonsense mutations, suggesting the importance of inactivation of these genes in the pathogenesis of RMS.Among the 531 mutated genes, only 18 were recurrently mutated , which nand PTEN . Thus, tand PTEN . Overalland PTEN . The mosFGFR4/RAS/AKT signalling  and cell cycle regulation  and STAT6 (12q13.3) (PAX3/7 (n=6) and FOXO1 (n=6) were frequently accompanied by local amplifications15A number of genes and pathways were also recurrently affected by CN alterations and thought to be implicated in deregulated 12q13.3) . As prevPAX3–FOXO1 fusion found in two cases of ARMS. The NSD1–ZNF346 fusion that was previously identified in an ERMS cell lineNSD1–ZNF346 fusion detected in our study was out-of-frame and thus functional significance of this fusion transcript is still elusive.Fusion transcripts were investigated by RNA sequencing in eight cases of RMS, including five ERMS and three ARMS cases . In totan=160; PAX3–FOXO1 or PAX7–FOXO1 fusions were grouped into the A1/A2 clusters, although the separation between A1 and A2 did not coincide with the presence or absence of fusions , whereas only two of 28 (7.1%) in A1/A2 tumours showed PTEN hypermethylation . DNA methylation profiling based on unsupervised hierarchical clustering identified four unique clusters having distinct methylation signatures , and the fusions . In our  fusions . Among thylation . SubsequP=0.045; . GATA4 eA1 to A2 . Althoughttp://www.ingenuity.com/) using probe lists of E1/E2 versus A1/A2, E1 versus E2 and A1 versus A2 . Compared with the A1/A2 clusters, the E1/E2 clusters were characterized by high frequencies of multiple chromosomal CN changes including gains of chromosomes 2, 8 or 12 (P=5.2 × 10−4). In addition, the mutation frequency of the FGFR4/RAS/AKT pathway was much higher in the E1/E2 clusters than in the A1/A2 clusters (P=4.6 × 10−4), and these mutations were predominantly observed in the E2 cluster . Furthermore, E2 tumours had significantly frequent TP53 mutations (45.5%) compared with E1 tumours . In contrast, mutations and CN alterations affecting cell cycle regulators, such as MYCN, CDK4 and CDKN2A/B, except for MDM2, were particularly enriched in the A1/A2 clusters compared with the E1/E2 clusters (P=0.016), suggesting that these genes may have driver roles in A1/A2 tumours.Next, we analysed the genetic and clinical characteristics of each methylation subgroup. E1 tumours showed a lower mutation rate  than E2 tumours , although not statistically significant  significantly affected the overall survival of E1/E2 patients (P=2.9 × 10−3). However, the impact of the E1/E2 classification on overall survival was more prominent than that of the TP53 mutations  for which intensification or novel therapeutic approaches need to be considered. We also found an extremely high frequency of epigenetic silencing of This study cohort comprised 66 tumours from 60 cases with RMS  were available in 16 cases, which were subjected to whole-exome sequencing. In three ARMS cases, samples from both primary and recurrent or metastatic tumours were available, which were analysed by whole-exome sequencing. Detailed information on subjects and samples is provided in In 19 samples from 16 cases, whole-exome capture libraries were constructed from tumour and matched normal DNA using Agilent SureSelect Human All Exon 50 Mb, V4 or V5 (Agilent Technologies) according to the manufacturer's protocol. Enriched exome libraries were sequenced with the Hiseq 2,000 platform (Illumina). For identifying candidates for somatic mutations from exome-sequencing data, the EBcall In eight tumour samples from our RMS which high-quality RNA  was available, RNA sequencing was performed using Hiseq 2,000. RNA sequencing libraries were prepared using the Truseq RNA Sample Preparation kit (Illumina). Candidates of fusion transcripts were identified using Genomon-fusion. All candidates of gene fusion, which represented more than two paired-reads were subjected to confirm by RT–PCR-based Sanger sequencing. The primer sets used for RT–PCR analysis were listed in NotI-tagged primers. Pooled PCR amplicons were digested with NotI, followed by ligation with T4 DNA ligase. Ligated PCR amplicons were sonicated into fragments of an average size of 200 bp using a Covaris sonicator. After being enriched and multiplexed, the prepared libraries were subjected to deep sequencing using Illumina Hiseq 2,000 or Miseq with a 100-bp or 150-bp pair-end reads option. Both tumour and germline DNA were examined to confirm somatic variations. Selective variants of which PCR primers could be constructed by Primer 328To validate putative genomic variants detected in whole-exome sequencing, targeted deep sequencing was performedTP53, BCOR, ARID1A, GAB1, ROBO1, PTEN and KRAS), and the associated genes , PCR-based deep sequencing of the candidate genes was performed in 66 samples using Hiseq 2000 or Miseq (Illumina). NotI-tagged primers  according to the manufacturer's protocol. Beta-values were converted to eijk is an observation error, aik is the methylation pattern depending on sample i, and bjk is the methylation pattern depending on probe location j, respectively. The parameters aik, i=1,…,n and bjk, j=1,…,pk are estimated by Bayesian principal component analysisaik, i=1,…,n are chosen so that they are proportional to the average beta value of pk locations of gene k. The z-score to see if aik is significantly larger or smaller than those of the normal skeletal muscles is then calculated by:where where T, we can declare the methylation status of gene k for sample i to be an aberration compared with that of normal skeletal muscles if |zik|>T. Here identifying aberrations can be thought of as a multiple-testing problem where we are testing the following hypothesis for each gene within each sample:For a given threshold T can be calculated byAn estimator of the false discovery rateT is then chosen so that The threshold Quantitative RT–PCR for PTEN was performed in 19 samples from which enough RNA was available. RNA (200 ng) extracted from the fresh-frozen tumours were subjected to reverse transcription using the SuperScript VILO MasterMix (Invitrogen) according to the manufacturer's protocol. Quantitative mRNA expression levels were measured using the QuantiTect SYBR Green PCR kit  with an iCycler iQ real-time PCR detection system (Bio-Rad). The optional thermal-cycling condition was as follows: 40 cycles of a two-step PCR  after the initial denaturation (95 °C for 10 min). For the purpose of normalization, relative expression levels were calculated by dividing the expression level of the respective gene by that of GAPDHPTEN methylation, bisulfite sequencing using nested primers was performed. Genomic DNA (500 ng) was bisulfite-modified using the EpiTect Plus DNA Bisulfite Kit (Qiagen) according to the manufacturer's instructions. Primer sequences for PCR amplicons are listed in To confirm NotI-tagged primes as described above. For data analysis, the same method of targeted deep sequencing was used, with modified mapping to bisulfite converted reference genome. Methylated cytosine was calculated as variant allele frequency. Validated probes were listed in To validate the result of DNA methylation array, targeted bisulfite deep sequencing was performed. Five samples among each cluster were randomly selected for validation. As positive controls, two samples of replicated normal DNA were also analysed by bisulfite sequencing. Genomic DNA  was bisulfite modified using the EpiTect Plus DNA Bisulfite Kit (Qiagen) according to the manufacturer's instructions. Among extracted 266 probes , we selected probes to validate, which recurrently detected in three or more. Finally, 160 probes were subjected to target deep sequencing. Bisulfite PCR was performed in 22 samples (20 tumours and two controls) with http://www.ingenuity.com/) was performed for extracted genes listed in Ingenuity pathway analysis (Nsp or CytoScan HD (Affymetrix) according to the manufacturer's protocol. Details regarding the use of these platforms are listed in 35DNA extracted from RMS samples was subjected to SNP array analysis using Affymetrix GeneChip 250K PAX3/7–FOXO1 fusion gene was examined by RT–PCR followed by Sanger sequencing in 34 tumours for which RNA or cDNA was available. The primers and the PCR condition are listed in The status of the t-test.Fisher's exact test was used to evaluate the differences in chromosomal CN changes between ARMS and ERMS or the correlation between CN changes and prognosis in each subgroup. The numbers of mutations or CN changes were compared by Accession codes: Whole-exome sequence data and the methylation microarray data has been deposited in the European Nucleotide Archive, hosted by the European Bioinformatics Institute, under the accession code EGAS00001000884. The SNP array data have been deposited in the Gene Expression Omnibus under accession number GSE41263 and GSE63891.How to cite this article: Seki, M. et al. Integrated genetic and epigenetic analysis defines novel molecular subgroups in rhabdomyosarcoma. Nat. Commun. 6:7557 doi: 10.1038/ncomms8557 (2015).Supplementary Figures 1-10Patient characteristics of RMS cases analyzed by next generation sequencing.List of detected mutations by whole exome sequencing.Patient characteristics of validation cohort.List of detected mutations by targeted deep sequencing.Significant gain regions detected by GISTIC analysis.Significant loss regions detected by GISTIC analysis.Validated fusion transcripts detected in RNA sequencing.Differentially methylated probes between cluster E1/E2 and A1/A2.Differentially methylated probes between cluster E1 and E2.Differentially methylated probes between cluster A1 and A2.Pathway analysis for gene list of E1/E2 vs. A1/A2.Functions annotation analysis for gene list of E1/E2 vs. A1/A2.Pathway analysis for gene list of E1 vs. E2.Functions annotation analysis for gene list of E1 vs. E2.Pathway analysis for gene list of A1 vs. A2.Functions annotation analysis for gene list of A1 vs. A2.Primer sets for targeted deep sequencing.Primer sets for nested PCR of bisulfite sequencing and RT-PCR in PTEN.Selected probes for validation of methylation array and primer sequences for bisulfite targeted deep sequencing.Primer sequences for RT-PCR.