LTBP1 and LTBP4 had two regions initiating different transcripts. Most of the family members were expressed in a range of mesenchymal and other cell types, often associated with use of alternative promoters or transcription start sites within a promoter in different cell types. FBN3 was the lowest expressed gene, and was found only in embryonic and fetal tissues. The different promoters for one gene were more similar to each other in expression than to promoters of the other family members. Notably expression of all 22 LTBP2 promoters was tightly correlated and quite distinct from all other family members. We located candidate enhancer regions likely to be involved in expression of the genes. Each gene was associated with a unique subset of transcription factors across multiple promoters although several motifs including MAZ, SP1, GTF2I and KLF4 showed overrepresentation across the gene family. FBN1 and FBN2, which had similar expression patterns, were regulated by different transcription factors. This study highlights the role of alternative transcription start sites in regulating the tissue specificity of closely related genes and suggests that this important class of extracellular matrix proteins is subject to subtle regulatory variations that explain the differential roles of members of this gene family.The fibrillins and latent transforming growth factor binding proteins (LTBPs) form a superfamily of extracellular matrix (ECM) proteins characterized by the presence of a unique domain, the 8-cysteine transforming growth factor beta (TGFβ) binding domain. These proteins are involved in the structure of the extracellular matrix and controlling the bioavailability of TGFβ family members. Genes encoding these proteins show differential expression in mesenchymal cell types which synthesize the extracellular matrix. We have investigated the promoter regions of the seven gene family members using the FANTOM5 CAGE database for human. While the protein and nucleotide sequences show considerable sequence similarity, the promoter regions were quite diverse. Most genes had a single predominant transcription start site region but  •We examine expression, promoter use and enhancers for the fibrillin/LTBP gene family.•Promoter switching was observed for most family members.•Multiple enhancers were identified for all family members.•Family members overlapped in tissue specificity with some unique expression patterns.•A degree of redundancy among family members is possible. In rats and mice the FBN3 gene appears to have degenerated and does not produce a functional mRNA FBN3 is likely to be active since transcripts have been detected (data from http://www.ensembl.org). There are a variable number of annotated LTBP genes across species, from two in fish to four in mammals: LTBP1, LTBP2, LTBP3 and LTBP4. It is possible that one or more of the gene family members take over the role of FBN3 in rats and mice.In vertebrates, including eutherian, marsupial and monotreme mammals, birds, reptiles and fish, fibrillins are encoded by three genes, Fbn1 mRNA is ubiquitous in mesenchymal cell types Fbn2 appears more restricted in expression . A similar pattern was reported for human FBN2FBN3 expression is restricted to embryonic/fetal tissues http://biogps.org; Expression of fibrillin/LTBP family members is principally found in cells and tissues of mesenchymal origin. In mouse, Consistent with their function in mesenchymal cell types, mutations in members of this gene superfamily result in phenotypes that primarily affect connective tissue types (reviewed in FBN1FBN2 (unpublished). However those data included few tissues showing expression of FBN3 in humans. The current FANTOM5 project involves many more tissue and cell types likely to express members of the fibrillin and LTBP families 154700; FBN1 OMIM: 134797), congenital contractural arachnodactyly , primary congenital glaucoma  and cutis laxa with severe systemic abnormalities  The FANTOM  projects co-ordinated by the RIKEN Institute in Japan have provided extensive information on gene expression in human and mouse and allowed the identification and characterization of a large number of gene promoter regions http://fantom.gsc.riken.jp/5/.This work is part of the FANTOM5 project. Data downloads, genomic tools and co-published manuscripts have been summarized at 22.1Five human cell lines and a primary adult derived mesenchymal stem cell culture were used to analyze expression patterns of fibrillin/LTBP gene family members. SAOS-2 (sarcoma osteogenic 2) is a hypotriploid line derived from the osteosarcoma of an 11-year-old Caucasian female and has been in culture since 1973 2.2http://www.amsbio.com/rna-bee.aspx). RNA was quantified using a Nanodrop spectrophotometer and cDNA was synthesized using MMLV reverse transcriptase  and an annealing temperature of 60 °C http://www.roche-applied-science.com, GAPDH was used as the reference gene . Quantitative PCR was performed in triplicate using LightCycler 480 SYBR Green 1 Master Mix (Roche) on the LightCycler 480 machine (Roche). Products were quantified using software supplied by the manufacturer.Expression of fibrillin/LTBP family members was initially examined in cell lines using quantitative reverse transcriptase PCR from RNA extracted from the cell lines described above. In addition RNA was extracted from cell pellets from Day 2 of embryoid body formation from two human ES cell lines, H1 http://www.amsbio.com/rna-bee.aspx). RNA was quantified using a Nanodrop spectrophotometer and cDNA was synthesized using MMLV reverse transcriptase  and an annealing temperature of 60 °C http://www.roche-applied-science.com, Supplementary Table 1). Human GAPDH was used as the reference gene . Quantitative PCR was performed in triplicate using LightCycler 480 SYBR Green 1 Master Mix (Roche) on the LightCycler 480 machine (Roche). Products were quantified using software supplied by the manufacturer.Expression of fibrillin/LTBP family members was initially examined in cell lines using quantitative reverse transcriptase PCR from RNA extracted from the cell lines described above. In addition RNA was extracted from cell pellets from Day 2 of embryoid body formation from two human ES cell lines, H1 The presence of the encoded proteins was ascertained using immunofluorescence with a range of antibodies. 20,000 NHDF cells per well were seeded into an eight well chamber slide (NUNC). After 7 days, the cells were fixed with 95% ethanol and 5% acetic acid for 20 min and washed with PBS. The samples were blocked with 1% BSA in PBS for 1.5 h prior to addition of a 1:100 dilution of the primary antibody and incubated over night at 4 °C. Following a wash with PBS, the secondary antibody was added at 1:1000 dilution, for 1 h at room temperature. The cells were then washed with PBS, and mounted with ProGold + DAPI (Invitrogen). Samples were viewed using Zeiss LSM 710 confocal microscope and analyzed with ZEN software at standard settings (Zeiss). Details of antibodies used are supplied in The presence of the encoded proteins was ascertained using immunofluorescence with a range of antibodies. 20,000 NHDF cells per well were seeded into an eight well chamber slide (NUNC). After 7 days, the cells were fixed with 95% ethanol and 5% acetic acid for 20 min and washed with PBS. The samples were blocked with 1% BSA in PBS for 1.5 h prior to addition of a 1:100 dilution of the primary antibody and incubated over night at 4 °C. Following a wash with PBS, the secondary antibody was added at 1:1000 dilution, for 1 h at room temperature. The cells were then washed with PBS, and mounted with ProGold + DAPI (Invitrogen). Samples were viewed using Zeiss LSM 710 confocal microscope and analyzed with ZEN software at standard settings (Zeiss). Details of antibodies used are supplied in Supplementary Table 1.2.3http://fantom.gsc.riken.jp/zenbu/). Composite promoters are those where two or more robust peaks occur within 100 bp of each other − 5The tissues and cells used for the FANTOM5 analysis of transcription initiation sites have been described in a parallel publication 2.4Express3DAll 81 DPI-detected robust promoters for the seven genes were clustered using Biolayout The parallel paper 2.5We downloaded the whole-genome alignment of the human genome with 45 other vertebrate genomes from the UCSC Genome Browser database 33.1We used quantitative reverse transcriptase PCR (qRT-PCR) to examine expression of gene family members in cell lines including H1 and RH1 embryonic stem cells, ADMSC mesenchymal stem cells from adipose tissue, SAOS2 and MG63 osteosarcoma cell lines and NHDF fibroblasts, . The resFBN1 expression was consistently high in ADMSC, NHDF and MG63 cell types but minimal in early ES cells (FBN2 was highly expressed early in embryoid body formation from H1 and RH1 embryonic stem cells and in SAOS2 and NHDF cells (FBN3 was present in both ES cell lines (H1 and RH1), with no expression in the remaining cell types  and 21 (LTBP2) robust promoters peaks associated with members of the gene family. FBN1 along with regulatory elements detected by the ENCODE project To assess further the tissue distribution and regulation of gene family members, we used FANTOM5 CAGE-based data for promoter expression. There were between 1 (FBN3) and 21 (LTBP2) robust promoters peaks associated with members of the gene family. FBN1 along with regulatory elements detected by the ENCODE project To assess further the tissue distribution and regulation of gene family members, we used FANTOM5 CAGE-based data for promoter expression. There were between 1  and this was attributable to LTBP1 , LTBP2  and FBN2 .Almost all the promoters were broad promoters in CpG islands and there were no TATA box promoters . The maj21 = 13.0; p = 0.0003) and this was attributable to LTBP1 , LTBP2  and FBN2 .Almost all the promoters were broad promoters in CpG islands and there were no TATA box promoters . The majority of robust peaks were within 100 bp of another peak, forming 18 composite promoters FBN1FBN1 CAGE tags localized to the main composite promoter region containing p1@FBN1 (Exon A), but p11@FBN1 is probably associated with the rarely used Exon B of previous reports FBN2 , were associated with the short transcript ENST00000507835.1. Two promoters were identified in the 3′ noncoding region of the LTBP2 gene . They may be associated with a short overlapping transcript from LOC730019.There were known transcripts  starting in the region of the majority of the detected promoters, and most known alternative transcripts were supported by this analysis . NotablyFBN1FBN1 CAGE tags localized to the main composite promoter region containing p1@FBN1 (Exon A), but p11@FBN1 is probably associated with the rarely used Exon B of previous reports FBN2 , were associated with the short transcript ENST00000507835.1. Two promoters were identified in the 3′ noncoding region of the LTBP2 gene . They may be associated with a short overlapping transcript from LOC730019.There were known transcripts  starting in the region of the majority of the detected promoters, and most known alternative transcripts were supported by this analysis (Supplementary Table 2). Notably, promoters for the three alternative first exons of FBN2 transcript ENST00000508053.1 which has an additional six 5′ non-coding exons , nor were there promoters for two long transcripts (AY203940 and AK022050AY203940AK022050) which overlap FBN3. Similarly there was no evidence for a promoter for the longest reported LTBP4 transcript, NM_003573 (LTBP4 A. —ENST00000545697 and ENST00000204005).Some published transcripts were not supported by CAGE tags. For example, there was no support at the robust level for a promoter for the long FBN2 transcript ENST00000508053.1 which has an additional six 5′ non-coding exons , nor were there promoters for two long transcripts (AY203940 and AK022050AY203940AK022050) which overlap FBN3. Similarly there was no evidence for a promoter for the longest reported LTBP4 transcript, NM_003573 .Some published transcripts were not supported by CAGE tags. For example, there was no support at the robust level for a promoter for the long LTBP1, alternate transcripts with a long or short 5′ UTR have been reported (LTBP1 D.). However, there was no CAGE support for the transcript with the shorter UTR (ENST00000354476.3). Instead, a small TSS cluster was detected midway between the main (long) start site about 200 bp upstream of the annotated beginning of the putative shorter transcript, which may indicate that the shorter transcript is incomplete. Similarly a number of identified TSS in LTBP3 did not correlate with known transcripts .Transcription start sites were also detected where there was no identified transcript. For instance, at the 5′ end of LTBP1, alternate transcripts with a long or short 5′ UTR have been reported . However, there was no CAGE support for the transcript with the shorter UTR (ENST00000354476.3). Instead, a small TSS cluster was detected midway between the main (long) start site about 200 bp upstream of the annotated beginning of the putative shorter transcript, which may indicate that the shorter transcript is incomplete. Similarly a number of identified TSS in LTBP3 did not correlate with known transcripts .Transcription start sites were also detected where there was no identified transcript. For instance, at the 5′ end of FBN1 promoter region contained a characteristic pyrimidine (T-rich) stretch, about 70 nucleotides upstream of the transcription start site associated with p1@FBN1 FBN2 promoter was in a C-rich region but the promoter region of the alternative transcript (p14@FBN2 and p18@FBN2) was not in a CpG island and was AT rich. The FBN3 promoter was also pyrimidine rich but shared no sequence homology with the other genes. The region around p1@LTBP1 contained alternating strings of purines and of pyrimidines.Although most promoters were located in CpG islands, there was little conservation of sequence around the promoters. Many of the promoter regions themselves were pyrimidine-rich (on the coding strand). The coding strand of the main FBN2 A. —red box compared with B.), which is almost exclusively expressed in testis, a tissue type not represented in the cell lines examined in the ENCODE Project.The major promoters were all supported by regulatory elements detected in the ENCODE project FBN2 A. —red box compared with B.), which is almost exclusively expressed in testis, a tissue type not represented in the cell lines examined in the ENCODE Project.The major promoters were all supported by regulatory elements detected in the ENCODE project 3.3LTBP1 . Promoters within a compound promoter were associated with the same predicted enhancer(s)  . All proions see , while aLTBP1 . Promoters within a compound promoter were associated with the same predicted enhancer(s) . This enhancer was not correlated with the other FBN2 promoters. p12@LTBP2 was uniquely associated with a bidirectional expression peak 50 kb upstream (Supplementary Table 3). In contrast, p10@LTBP3, p12@LTBP3 and p21@LTBP3 located within the second and third exons of the major LTBP3 transcript were correlated with one of the same predicted enhancers as the major promoter region containing p1@LTBP3. For LTBP4, each promoter tended to be associated with a different enhancer.p7@FBN1 and p25@FBN1, initiating the alternative first exon, Exon C 3.4The promoters all showed the characteristics of tissue restricted expression, as defined in the parallel paper The promoters all showed the characteristics of tissue restricted expression, as defined in the parallel paper http://fantom.gsc.riken.jp/5/sstar/ and To investigate this tissue restricted expression further, we used a number of ontologies http://fantom.gsc.riken.jp/5/sstar/ and Supplementary Table 4). Promoters in one composite cluster within a gene tended to be expressed in tissues associated with the same ontology terms    was highly expressed in adult heart samples and also expressed in tissue and cells types of mesenchymal origin. This composite promoter was associated with mesenchyme ontology terms including fibroblast but also with many muscle related terms , the high expression levels in cardiovascular cell types also led to a preponderance of terms associated with the vasculature   . Similaral cells . p2@LTBPyoblast) . The maied terms . Althougar cord) .FBN3 were strongly expressed in cells associated with ontology terms indicating mesenchymal origin  (Supplementary Table 4). Similar ontology terms were found for the main promoters of FBN2, which was expressed in a range of cells from fetal and embryonic tissues, fibroblasts, osteoblasts, placenta, hair follicle and lens epithelial cells  (Supplementary Table 4). The main composite promoter region of LTBP1  was highly expressed in adult heart samples and also expressed in tissue and cells types of mesenchymal origin. This composite promoter was associated with mesenchyme ontology terms including fibroblast but also with many muscle related terms (Supplementary Table 4). Although the promoter regions identified for LTBP2 were associated with some generic mesenchymal ontology terms (such as fibroblast and myoblast), the high expression levels in cardiovascular cell types also led to a preponderance of terms associated with the vasculature  (Supplementary Table 4).In general, the major promoters of all gene family members other than al cells . p2@LTBPpresumptive neural plate, neural tube, neural rod, structure with developmental contribution from neural crest, future spinal cord)  but also with terms associated with other organs . p7@LTBP2 demonstrated significant expression levels in both hematopoietic and epithelial samples and was enriched for terms reflection this expression pattern   . p3@FBN2pression . p2@LTBPal cell) . The higtron 25,  displayeve organ .presumptive neural plate, neural tube, neural rod, structure with developmental contribution from neural crest, future spinal cord) (Supplementary Table 4). p3@FBN2 peaked in hematopoietic cell types, and was associated with ontology terms such as classical monocyte as well as terms reflecting mesenchyme/mesoderm expression (Supplementary Table 4). p2@LTBP2 was also expressed in some hematopoietic cells while p3@LTBP4 was associated with lymphocytes of the immune system  but also with terms associated with other organs . p7@LTBP2 demonstrated significant expression levels in both hematopoietic and epithelial samples and was enriched for terms reflecting this expression pattern  (Supplementary Table 4). The high expression promoter of LTBP4, p1@LTBP4, was enriched in ontology terms reflecting an endodermal origin. p14 @FBN2 and p18@FBN2, located in intron 25,  displayed highest expression in testis and both promoters were associated with ontology terms such as testis, male reproductive organ, gonad and external genitalia/reproductive organ (Supplementary Table 4).A number of promoters were expressed in tissues of other origins. Thus p7@FBN1 and p1@LTBP3 were enriched in ontology terms reflecting development of the nervous system   l. Promotanscript  were higus layer .FBN3, p2@FBN3, was almost exclusively expressed in cells of fetal and embryonic origin. This expression pattern was associated with ontology terms such as embryonic stem cell, neuronal stem cell, neuroectodermal cell as well as some terms relating to pigmentation  (Supplementary Table 4l). Promoters of LTBP1 leading to the longer transcript  were highly expressed in embryonic and extraembryonic cell/tissue types including chorionic and amniotic membrane cells, fetal heart, epithelial and endothelial tissue samples. Within this region, p3@LTBP1 had the highest activity, and transcription from this region was found primarily in fetal heart. It was most strongly associated with ontology terms such as extraembryonic cell/structure, mesenchyme, compound organ and membranous layer (Supplementary Table 4).Some promoters were associated with early development. The sole robust promoter for LTBP1 promoter containing p1@LTBP1 showed strong trimethylation of H3K4 in human lung fibroblasts (NHLF), while the composite promoter containing p3@LTBP1 was strongly trimethylated in most of the cell lines, except embryonic stem cells and a human lymphoblastoid cell line (Gm12878) (LTBP1 B.).Variability of promoter use in different cell types was supported by the H3K4Me3 track from the ENCODE data , where dLTBP1 promoter containing p1@LTBP1 showed strong trimethylation of H3K4 in human lung fibroblasts (NHLF), while the composite promoter containing p3@LTBP1 was strongly trimethylated in most of the cell lines, except embryonic stem cells and a human lymphoblastoid cell line (Gm12878) .Variability of promoter use in different cell types was supported by the H3K4Me3 track from the ENCODE data , where different colored peaks (representing trimethylation levels in different cell types) can be seen for different promoters. For example, the composite 3.5Express3D. Biolayout Express3D employs a statistical approach to look at transcript-to-transcript similarities in expression pattern across the samples analyzed, by calculation of a Pearson pairwise correlation matrix LTBP2 promoters formed a separate cluster (Cluster 1) with no edges to promoters for any of the other genes, even at this low correlation level. This indicates that the LTBP2 promoters are more similar in expression pattern to each other than to any other promoters. FBN2 promoters formed two separate clusters (Clusters 3 and 11) and LTBP1 promoters were also in two clusters (Clusters 4 and 8) suggesting two patterns of expression for these genes. In contrast, at this low level of correlation LTBP3 and LTBP4 promoters were frequently in the same cluster (Clusters 5 and 7), or associated with the single FBN3 promoter (Cluster 6), showing that these promoters share some similarity of expression pattern. Some LTBP3 and LTBP4 promoters formed separate distinct clusters (Cluster 12 and Clusters 9 and 10 respectively). One LTBP1 and six LTBP3 promoters also clustered with the FBN1 promoters (Cluster 2).To explore further the similarities and differences in promoter expression across the gene family, we used RLE-normalized tag counts Express3D. Biolayout Express3D employs a statistical approach to look at transcript-to-transcript similarities in expression pattern across the samples analyzed, by calculation of a Pearson pairwise correlation matrix LTBP2 promoters formed a separate cluster (Cluster 1) with no edges to promoters for any of the other genes, even at this low correlation level. This indicates that the LTBP2 promoters are more similar in expression pattern to each other than to any other promoters. FBN2 promoters formed two separate clusters (Clusters 3 and 11) and LTBP1 promoters were also in two clusters (Clusters 4 and 8) suggesting two patterns of expression for these genes. In contrast, at this low level of correlation LTBP3 and LTBP4 promoters were frequently in the same cluster (Clusters 5 and 7), or associated with the single FBN3 promoter (Cluster 6), showing that these promoters share some similarity of expression pattern. Some LTBP3 and LTBP4 promoters formed separate distinct clusters (Cluster 12 and Clusters 9 and 10 respectively). One LTBP1 and six LTBP3 promoters also clustered with the FBN1 promoters (Cluster 2).To explore further the similarities and differences in promoter expression across the gene family, we used RLE-normalized tag counts FBN3 promoter, p2@FBN3, was in this excluded group as were a promoter for FBN2 and LTBP1, seven for LTBP3 and two for LTBP4. Nine of these 11 were singleton promoters while two were within a composite promoter. The network of associations at P ≥ 0.5 is shown in LTBP2 promoters continued to cluster together (Cluster 1) while FBN1 (Clusters 2 and 12), FBN2 (Clusters 3 and 11) and LTBP1 (Clusters 4 and 6) formed two clusters each. p6@LTBP1 clustered with the majority of the FBN1 promoters, indicating an expression pattern more similar to FBN1 than to the other LTBP1 promoters. p7@FBN1 and p25@FBN1, initiating the alternative first exon, Exon C FBN1 promoters. LTBP3  and LTBP4  formed four clusters each. These results suggest that the most diverse expression patterns were found for promoters of LTBP3 and LTBP4, while LTBP2 had the most similar expression pattern across all promoters.At a Pearson correlation coefficient threshold of 0.5, 11 promoters were excluded from the analysis indicating that their expression pattern was sufficiently different from any other promoter that they did not correlate at this level. The sole LTBP2 promoters and single clusters for FBN1, LTPB1 and LTBP4 .Promoters for LTBP genes tended to fail to cluster or formed clusters with other promoters for the same gene, particularly in the primary cell analysis. p1@LTBP1, p2@LTBP1, p4@LTBP1 and p5@LTBP1 formed a small cluster when all samples were analyzed and p3@LTBP1, p7@LTBP1 and p8@LTBP1 formed a cluster when primary cells were analyzed (Supplementary Table 2). However some LTBP promoters clustered with promoters for mesenchymal genes and occasionally were found with promoters for other members of the fibrillin/LTBP gene family. 3.7FBN1 , the motifs for GTF2I, NKX3-1, TBX4-5 and TFAP4 regulated five promoters, while for FBN2 (6 promoters) HIF1A was found to regulate four promoters and TFDP1, KLF4, TFAP2B and TOPORS regulated three promoters. There were no significant regulatory edges for FBN3  for FBN3 l. HoweveFBN1 , the motifs for GTF2I, NKX3-1, TBX4-5 and TFAP4 regulated five promoters, while for FBN2 (6 promoters) HIF1A was found to regulate four promoters and TFDP1, KLF4, TFAP2B and TOPORS regulated three promoters. There were no significant regulatory edges for FBN3 (Supplementary Table 2). However, binding motifs for the transcription factors MAZ, PATZ1, RREB1, SP1 and TFAP4 were detected in the promoter region of FBN3.We used Motif Activity Response Analysis (MARA) LTBP1  and LTBP2 (regulated at 16 promoters) showed significant regulation by the MAZ motif in seven and eight promoters respectively. Five promoters of LTBP1 were also regulated by HIC1 and NFY while four promoters were regulated by PRDM1, RXR and SOX 17. LTBP2 was predicted to be regulated by KLF4 (at 10 promoters), SP1 (at eight promoters), GATA4 and TEAD (at five promoters) and XCPE1 (at four promoters) was associated with LTBP2. Regulation at seven LTBP3 promoters was detected, through motifs for GTF2I and SP1 (at six promoters), MED-1 (at five promoters) and TFAP2 and ZFP161 (at four promoters). Only two LTBP4 promoters were significantly associated with motifs, with TRAF2B found for both  and LTBP2 (regulated at 16 promoters) showed significant regulation by the MAZ motif in seven and eight promoters respectively. Five promoters of LTBP1 were also regulated by HIC1 and NFY while four promoters were regulated by PRDM1, RXR and SOX 17. LTBP2 was predicted to be regulated by KLF4 (at 10 promoters), SP1 (at eight promoters), GATA4 and TEAD (at five promoters) and XCPE1 (at four promoters) was associated with LTBP2. Regulation at seven LTBP3 promoters was detected, through motifs for GTF2I and SP1 (at six promoters), MED-1 (at five promoters) and TFAP2 and ZFP161 (at four promoters). Only two LTBP4 promoters were significantly associated with motifs, with TRAF2B found for both (Supplementary Table 2).4Members of the fibrillin/LTBP gene family appear to play two roles in the extracellular matrix. Firstly, they contribute structurally to the formation of the matrix. In particular, fibrillin-1 and fibrillin-2 are integral components of the 10 nm microfibrils which provide strength and elasticity to the matrix In this study we have examined gene expression and regulation of the seven fibrillin/LTBP family members using results from the FANTOM5 CAGE analysis, a genome wide analysis of transcription start sites and bidirectional putative enhancers FBN1 was highly expressed in a range of connective tissue cells, consistent with previously published transcription factor activity There were multiple examples of differential use of promoters in different tissue types (promoter switching) and this was frequently associated with differences in ENDOCE regulatory elements and distinctive predicted enhancer activity. The main promoter region of FBN1 was highly expressed in a range of connective tissue cells, consistent with previously published transcription factor activity There were multiple examples of differential use of promoters in different tissue types (promoter switching) and this was frequently associated with differences in ENDOCE regulatory elements and distinctive predicted enhancer activity. The main promoter region of FBN2, where p14@FBN2 and p18@FBN2 were expressed in reproductive cell types. The transcript associated with these promoters, ENST00000507835.1, covers coding exons 26 to 34 of the full length FBN2 and the polypeptide would consist of eight cbEGF domains with short N- and C-terminal flanking sequences (http://www.ensembl.org). Preliminary examination of the mouse CAGE data from FANTOM5 showed an equivalent promoter exclusively expressed in mouse testis, suggesting that this transcript has a real function, perhaps involved with sequestration of calcium. Further investigation of this novel transcript should reveal more about its role.Promoter switching with a change in tissue specificity was also seen for LTBP1 is documented to have at least two alternative transcription start regions (http//:www.ensembl.org) and this switch in promoters was confirmed in our analysis. One promoter region demonstrated high expression in early development and low expression in mesenchymal cells  while the alternate promoter region  was associated with mesenchyme-related ontology terms and expressed in heart. The major promoters p1@LTBP1 and p2@LTBP1 were associated with the same transcription factor motifs , while the alternative promoter region was associated with a distinct set of transcription factors motifs .Putative enhancers within 500 kb of the promoter regions were significantly associated with many of the promoters examined. The majority were downstream of the promoter region and some were as close 2 kb from the promoter. In general many promoters in one gene were correlated with the same predicted enhancers although there were a few examples of promoter-specific enhancers, associated with promoters of unique expression pattern.FBN2 showed similar expression patterns and ontology term enrichment to FBN1. However, FBN1 and FBN2 promoters did not cluster together in the co-expression analyses and there was little overlap of transcription factor motif activity between FBN1 and FBN2 promoters, suggesting that there were subtle differences in their expression patterns and regulation. In particular, FBN2 promoters tended to be associated with the epithelial and pluripotency transcription factor KLF4 FBN1 promoters were associated with mesenchymal transcription factors Cluster analysis of expression from the different promoters highlighted the differences between the gene family members. Members of a composite promoter often clustered together, but few promoters for different genes were found in the same cluster. For example, most promoters for LTBP2 A —blue box and C.), for example, showed significant potential to bind GATA4 , for example, showed significant potential to bind GATA4  or Ltbp4 (through p5@LTBP4). Preliminary analysis of FANTOM5 data for mouse indicates that the equivalent of p3@LTBP1 is strongly expressed in embryonic tissue, especially neuron derived neurospheres, while a region equivalent to p5@LTBP4 is expressed in embryonic stem cells , supporting this possibility.Because its expression is restricted to fetal and embryonic tissue types FBN1, FBN2, LTBP2, LTBP3 and LTBP4 have been associated with connective tissue disease in humans. Transgenic and natural mutation mouse models, available for Fbn1, Fbn2, Ltbp1, Ltbp2, Ltbp3 and Ltbp4 also show phenotypic abnormalities of connective tissue (reviewed in Mutations in human 5This study supports the differential roles of members of the fibrillin/LTBP gene family. We have shown the strong mesenchymal expression of most family members and most promoters and highlighted promoter and enhancer switching associated with changes in tissue specificity. We were unable to find evidence for a single “master mesenchyme regulator” but there was considerable overlap in the transcription factor activity associated with these genes. These data aid in better understanding the overall function and activity of the members of the fibrillin/LTBP family.Promoters of the fibrillin/LTBP family members.qPCR primers and antibodies.Promoter characteristics, expression levels, transcription factor associations and expression clusters.Enhancers of fibrillin/LTBP family members.Ontology terms associated with cell types expressing fibrillin/LTBP family member promoters.The following are the supplementary data related to this articlehttp://dx.doi.org/10.1016/j.ymgme.2013.12.006.Supplementary data to this article can be found online at The data in the paper will be freely available.http://fantom.gsc.riken.jp/5/. FANTOM5 enhancers can also be accessed from. http://enhancer.binf.ku.dk.The FANTOM5 atlas is accessible from MRD carried out bioinformatic and molecular genetic analysis, participated in the design of the study and helped to draft the manuscript. RA provided the enhancer analysis. JS and NB supported the analysis and visualization. JKB performed the initial clustering of all CAGE detected promoters. MdH carried out the MARA analysis. HK managed the data handling. ARRF was responsible for FANTOM5 management and concept. KMS conceived this study, participated in its design and coordination, carried out much of the analysis and interpretation, performed the clustering analysis of the promoters and drafted the manuscript. All authors read and approved the final manuscript.


ADH) gene clusters (ADH1B and ADH1C) and Aldehyde dehydrogenase (ALDH2)], one microsomal ethanol oxidizing enzyme cytochrome p450 (CYP2E1) and three oxidative stress response (OSR) genes  among 490 Bengali individuals (322 ALD and 168 control) from Eastern and North-Eastern India and validation was performed in a new cohort of 150 Bengali patients including 100 ALD and 50 advanced non-alcoholic steatohepatitis (NASH). Out of 14 genetic variants, carriage of 5 genotypes  were noted significantly higher among ALD patients while inter or intra group gene-gene interaction analysis revealed that addition of risk genotype of any OSR gene enhanced the possibility of ALD synergistically. Multiple logistic regression analysis showed independent association of rs2066701CC, rs4880TT and GSTM1 null genotype with ALD while lower frequencies of those genotypes in advanced NASH patients further confirmed their causal relation to ALD. Thus these findings suggest that the three variants of ADH1C, MnSOD and GSTM1 can be used to identify individuals who are at high risk to develop ALD and may be helpful in proper management of Indian alcoholics.Genetic susceptibility is an important modifier of clinical outcome and natural history of progression in Alcoholic liver disease (ALD). While the significance of ethnicity in this evolution is very clear, subtle inter-individual genetic variant(s) might be important and thus we investigated those in an Indian population. Fourteen markers were genotyped within two alcohol metabolism genes [Alcohol dehydrogenase ( Alcoholic liver disease (ALD) is one of the fast emerging common causes of chronic liver diseases across the globe. It is the clinical consequences of continuous alcohol over consumption  . AlthougADH) and aldehyde dehydrogenase (ALDH) (particularly ALDH2) in risk of development of ALD have been extensively investigated  at the –9 amino acid position of the N-terminal signal sequence , two for ADH1C genes [ADH1C*1 (Arg272Ile350) and ADH1C*2 ]. ADH1B*2 allele codes for a higher activity of the enzyme in ethanol oxidation with increased formation of acetaldehyde and ROS and is highly prevalent among neighbouring East-Asians such as Chinese, Japanese and Korean whereas it is least common among Indians [ADH1B polymorphism [ADH1C*1(rs698), which encodes for alcohol metabolism enzyme with high activity, has consistently found protective in alcoholic individuals in Chinese and Korean population [ADH1B (rs2066701) and two of ADH1C, rs1693425 (synonymous variants) and rs1789920 identified with significant association to the increased risk of ALD than ALC. Intronic variants may show association for being in linkage disequilibrium with a causal variant and also if situated at the beginning of an intron may result in alteration of the protein by affecting the splicing phenomenon of the nascent mRNA. rs1789920 which is present at the beginning of intron 2 of ADH1C may have an important role during the splicing phenomenon.Among the 7 human  Indians –26. A reH1C*1 Arg2Ile350 apulation , but foupulation , but faiALDH enzymes, ALDH2  is highly expressed in the liver and stomach and it has a very high affinity for alcohol detoxification by acetaldehyde oxidation [ALDH2 exhibits accelerated risk of progression of gastrointestinal cancers such as gastric cancer, esophageal cancer and colon cancer but its role in the liver, the major organ of alcohol metabolism, is still controversial [ALDH2 gene  but none of them showed sigificant association with ALD among “Bengalis” which is similar to the previous observation by Bhaskar et al with six ethnic populations from four linguistic groups in India [Out of nineteen putative functional genes that encodes for xidation ,27,28. Doversial –30. In tin India .CYP2E1 and generates acetaldehyde and free radicals to induce progression of liver diseases to cirrhosis. The 5’-flanking region of CYP2E1 gene is highly polymorphic at two non-coding loci, rs3813867 and rs2031920 [CYP2E1*5B is associated with higher transcription of CYP2E1. Significant association of this variant with ALD and other liver diseases has been documented in Oriental [Chronic alcohol intake induces the level and activity of s2031920 . These tOriental  and NortOriental  but not Oriental . These lGSTT1 and GSTM1) and superoxide dismutases. Consistent with the North Indian study [GSTM1 gene and coding variant of MnSOD (rs4880Ala) with lower enzyme activity were observed with significantly higher frequencies among ALD in this study. Association of deletion variant of GSTM1 with ALD has been observed in several studies [GSTM1 plays critical role in liver diseases as supported by our gene-gene interaction data where cumulative effect of this null variant has been observed with GSTT1 null genotype, rs4880 of MnSOD and rs2066701 of ADH1B. Again, Alanine to Valine change in the 9th position of mitochondrial target sequence of the single mitochondrial superoxide scavanger MnSOD which alters its amphiphilic helical structure crucial for transport and processing of mitochondrial protein varies among ethnic groups. Although a controversial data is available in the literature, a very few studies documented Valine as risk factor against a large data available for Alanine as effective variant which spontaneously dismutase superoxide to peroxide and oxygen [TNF-α mediated apoptosis whereas Valine variant inhibits entrance of MnSOD to inner membrane and enhances programmed cell death. Furthermore, our logistic regression analysis revealed significant association of null genotype of GSTM1 and rs4880TT of MnSOD with severity of liver diseases.The oxidative stress generated in alcoholics is neutralized by two important enzymes, glutathione-s-transfereases  are independently associated with the development of ALD among Bengali alcoholics. A genetic interaction amongst them also deserves importance in disease development. As two genotypes in oxidative stress response genes were also found in significantly higher frequencies among North Indian alcoholics, these genetic variants may serve as powerful predictor of ALD in advance and hence, these markers can be useful in the management of Indian alcoholics. Further stratification in a new cohort of patients and powerful complementary mechanistic studies to determine the physiological and pathophysiological role of those variants in liver disease development has been initiated which may help to improve diagnostic and therapeutic strategies for ALD patients.In conclusion, the current study confirms that the genetic variants in S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(a)Genotypic association of the Alcohol metabolism and oxidative stress related genes in Indian ALD patients. (b): Distribution of genotypes at different loci, which were either monomorphic or not significant in Bengali population of India.(DOC)Click here for additional data file.S4 Table(a)Interaction between rs2066701 of ADH1B and rs1693425 of ADH1C gene. (b): Interactions between anti-oxidative genes GSTT1, GSTM1 and rs4880 of MnSOD. (c): Inter gene interaction between ALD metabolism and oxidative stress related genes.(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file.


Two hand hygiene techniques are promoted internationally: the World Health Organisation’s 6 step and the Centre for Disease Control’s 3 step techniques; both of which may be considered to have suboptimum levels of empirical evidence for use with alcohol based hand rub (ABHR).The aim of the study was to to compare the effectiveness of the two techniques in clinical practice.A prospective parallel group randomised controlled trial (RCT) was conducted with 1:1 allocation of 6 step versus the 3 step ABHR hand hygiene technique in a clinical setting. The primary outcome was residual microbiological load. Secondary outcomes were hand surface coverage and duration. The participants were medical and nursing participants (n=120) in a large teaching hospital.The 6 step technique was statistically more effective at reducing the bacterial count 1900cfu/ml  to 380cfu/ml  than the 3 step 1200cfu/ml  to 750cfu/ml  (p=0.016) but even with direct observation by two researchers and use of an instruction card demonstrating the technique, compliance with the 6 step technique was only 65%, compared to 100% compliance with 3 step technique. Further those participants with 100% compliance with 6 step technique had a significantly greater log reduction in bacterial load with no additional time or difference in coverage compared to those with 65% compliance with 6 step technique (p=0.01).To our knowledge this is the first published RCT to demonstrate the 6 step technique is superior to the 3 step technique in reducing the residual bacterial load after hand hygiene using alcohol based hand rub in clinical practice. What remains unknown is whether the residual bacterial load after the 3 step technique is low enough to reduce risk of transmission from the hands and whether the 6 step technique can be adapted to enhance compliance in order to maximise reduction in residual bacterial load and reduce duration.None declared.


K13 mutations superimposed onto a long-standing background of Pfmdr1 amplification.The pivotal factor leading to the declining efficacy of the artemisinin-based combination on the Thailand–Myanmar border (mefloquine–artesunate) to a clinically unacceptable level is the increasing local prevalence of  Background. Deployment of mefloquine–artesunate (MAS3) on the Thailand–Myanmar border has led to a sustained reduction in falciparum malaria, although antimalarial efficacy has declined substantially in recent years. The role of Plasmodium falciparum K13 mutations (a marker of artemisinin resistance) in reducing treatment efficacy remains controversial.Methods. Between 2003 and 2013, we studied the efficacy of MAS3 in 1005 patients with uncomplicated P. falciparum malaria in relation to molecular markers of resistance.Results. Polymerase chain reaction (PCR)–adjusted cure rates declined from 100% in 2003 to 81.1% in 2013 as the proportions of isolates with multiple Pfmdr1 copies doubled from 32.4% to 64.7% and those with K13 mutations increased from 6.7% to 83.4%. K13 mutations conferring moderate artemisinin resistance (notably E252Q) predominated initially but were later overtaken by propeller mutations associated with slower parasite clearance (notably C580Y). Those infected with both multiple Pfmdr1 copy number and a K13 propeller mutation were 14 times more likely to fail treatment. The PCR-adjusted cure rate was 57.8%  compared with 97.8%  in patients with K13 wild type and Pfmdr1 single copy. K13 propeller mutation alone was a strong risk factor for recrudescence (P = .009). The combined population attributable fraction of recrudescence associated with K13 mutation and Pfmdr1 amplification was 82%.Conclusions. The increasing prevalence of K13 mutations was the decisive factor for the recent and rapid decline in efficacy of artemisinin-based combination (MAS3) on the Thailand–Myanmar border. Plasmodium falciparum malaria in 1985. However, resistance developed rapidly ) compared with admission isolates . In the recurrent isolates, more recrudescent isolates also had K13 mutant alleles compared with reinfections .Pfmdr1 copy numbers were measured in 726 (71.4%) admission isolates and 65 (34.8%) recurrent isolates (Supplementary Table 2). The proportion of infections caused by parasites with multiple (>1) Pfmdr1 copies on admission doubled from 32.4%  in 2003 to 64.7%  in 2013  compared with admission isolates 377/726 . Among these recurrent isolates, more recrudescent isolates also had multiple copies of Pfmdr1 compared with reinfections .s and 65 4.8% recuPfmdr1 gene because these relevant single-nucleotide polymorphisms (SNPs) are rare in Thailand [Pfmdr1 wild-type background [K13 were associated with single-copy Pfmdr1, while K13 mutations were associated with amplified Pfmdr1  .Our study did not characterize the single nucleotide polymorphisms of the Thailand  and meflckground , 28–30. K13 and Pfmdr1 was lower in recurrent infections because of significantly lower parasitemia (P = .0004) and thus much lower parasite DNA concentrations.The success rate for genotyping of P < .001, test for trend; Table K13 gene was the strongest risk factor for day 3 positivity, with later year of treatment, higher parasitemia, higher hematocrit, and fever on admission (but not Pfmdr1 amplification) as independent risk factors  were each significantly associated with day 3 positivity (Table Clearance data were available for 957 patients 95.2%). There was a significant increase in the proportion of patients who were parasitemic at day 3  recrudescences and 53 (28.5%) reinfections, with 2 indeterminate results, 1 amplification failure, and 13 missing samples. PCR-adjusted parasitological efficacy at day 42 remained above or close to 90% from 2003 to 2009 but declined sharply thereafter  along with year of recruitment and age  and lowest in patients with multiple copies of Pfmdr1 and any K13 propeller SNP . Cure rates declined markedly in recent years as the prevalence of K13 mutations rose .Cure rates were highest for infections with isolates with single-copy K13 and Pfmdr1 amplification were 69.0 and 41.9%, respectively. The PAF for the 2 factors in combination was 82%. Hence these 2 factors alone explain almost entirely the increased ACT treatment failure rate observed in this population.The PAFs  for Supplementary Materials . There was no association between the presence of K13 propeller mutations  or multiple copies of Pfmdr1  and gametocytemia, either at admission or during follow-up.The proportions of patients presenting with pre-treatment gametocytes or with post-treatment gametocytemia and the associated risk factors are shown in the P. falciparum malaria has had a remarkable therapeutic longevity and a dramatic impact on morbidity and mortality on the Thailand–Myanmar border. When artesunate was introduced in 1991, mefloquine was a failing drug, the incidence of falciparum malaria was rising, and most parasite isolates had multiple copies of Pfmdr1 associated with mefloquine resistance [Pfmdr1 copies were replaced by single copy–containing isolates [Pfmdr1 [P. falciparum parasites with K13 propeller mutations was the definitive event that led to the demise of MAS3. The temporal sequence of K13 selection is informative. The E252Q mutation , associated with moderate slowing of parasite clearance [K13 propeller mutations, conferring greater reductions in parasite clearance rates. The main propeller mutation was C580Y, a polymorphism common in Cambodia [K13 mutation is proportional to the degree of parasite clearance prolongation. It is possible that genetic changes outside the K13 locus have contributed to the emergence of artemisinin resistance and the selection of K13 mutations [MAS3 for the treatment of uncomplicated sistance . After disolates . High cuisolates , 32. In  [Pfmdr1 . The rislearance , predomiCambodia , 15. Theutations . It remaK13 mutations on treatment failure. K13 mutations in admission samples are associated with a failure rate of 21.5%. In combination with multiple copies of Pfmdr1, this rises to 42.2%. The sharp decline in MAS3 cure rates temporally corresponds with the emergence of K13 propeller mutant isolates on a long-standing genetic background of Pfmdr1 amplification. The proportion of treatment failures that can be attributed to K13 mutations and multiple copies of Pfmdr1  is 82%. Hence, these 2 factors alone explain the majority of increase in treatment failure observed. Other parasite genetic factors that our study was not designed to detect or changes in the demographics of the patient population may have contributed to the 18% of unexplained variation in treatment failure.The large body of data presented here strongly support the hypothesis that there is a substantial impact of K13 mutations and Pfmdr1 amplification have a multiplicative (rather than an additive) effect on risk of treatment failure. Synergy between these 2 resistance determinants may help to explain why failure rate declined precipitously in 2009—parasites carrying both markers only became common as K13 mutations rose in frequency. Second, in contrast to the 2 Cambodian studies, we observed multiple K13 mutations in this study. These data demonstrate significant heterogeneity in the impact of different K13 mutations on treatment failure. A mutation outside the K13 propeller (E252Q) increased treatment failure relative to wild-type parasites, but the 3 common propeller mutations had a much greater effect , which has a resistance mechanism that is similar to that of mefloquine, involving fication , 33, 34.despread , 35–38. P. falciparum malaria in our treatment centers on the Thailand–Myanmar border was changed from MAS3 to DHA–piperaquine, which is currently highly efficacious in this area but relies increasingly on the piperaquine component. The recent emergence of piperaquine resistance in Cambodia [P. falciparum from the area before the recent and substantial gains in malaria control are reversed.ACTs containing an alternative partner drug should provide relief in the short term. In 2012 the first-line treatment of uncomplicated Cambodia  and assoCambodia , 21, 40 Supplementary materials are available at http://cid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author.


In such cells, as well as in yeast, the selective TORC1 inhibitor rapamycin blocks this activation in contrast to Hsp90 inhibitors which potently activate Hsf1. Potentially therefore rapamycin could prevent the Hsf1 activation that frequently compromises the efficiency of Hsp90 inhibitor cancer drugs. Little synergy was found between the effects of rapamycin and the Hsp90 inhibitor radicicol on yeast growth. However certain rapamycin resistance mutations sensitised yeast to Hsp90 inhibitor treatment and an Hsp90 mutation that overactivates Hsf1 sensitised cells to rapamycin. Rapamycin inhibition of the yeast Hsf1 was abolished by this Hsp90 mutation, as well as with the loss of Ppt1, the Hsp90-interacting protein phosphatase that is the ortholog of mammalian PP5. Unexpectedly Hsf1 activation was found to have a requirement for the rapamycin binding immunophilin FKBP12 even in the absence of rapamycin, while TORC1 “bypass” strains revealed that the rapamycin inhibition of yeast Hsf1 is not exerted through two of the major downstream targets of TORC1, the protein phosphatase regulator Tap42 and the protein kinase Sch9 – the latter the ortholog of human S6 protein kinase 1.In human cells TORC1 mTOR (Significance: A problem with most of the Hsp90 inhibitor drugs now in cancer clinic trials is that they potently activate Hsf1. This leads to an induction of heat shock proteins, many of which have a “pro-survival” role in that they help to protect cells from apopotosis. As the activation of Hsf1 requires TORC1, inhibitors of mTOR kinase could potentially block this activation of Hsf1 and be of value when used in combination drug therapies with Hsp90 inhibitors. However many of the mechanistic details of the TORC1 regulation of Hsf1, as well as the interplay between cellular resistances to rapamycin and to Hsp90 inhibitors, still remain to be resolved. Heat shock protein 90 (Hsp90) provides a molecular chaperone function essential for the conformational maturation, activation and maintenance of proteins essential for sustaining all of the hallmarks of cancer. As such it is now a prime target for drug development, with several Hsp90 inhibitors currently in cancer clinic trials , 2. Inhitarget of rapamycin) protein kinase , wh, wh11], In this study we found little synergy between the inhibitory effects of rapamycin and the Hsp90 inhibitor radicicol on the growth of yeast, irrespective of whether the cells possessed a stress-inducible form of Hsf1 Fig. . Such fitor1Δ, fpr1Δ; Fig. The novelty of this work is that it has provided the first demonstration that Hsp90 inhibitor resistance can be influenced by mutations causing resistance to rapamycin Fig.  and, conTOR1.1, TOR2.1 and fpr1Δ rapamycin resistance mutations sensitise cells to Hsp90 inhibitor treatment  and measurement of levels of β-galactosidase expression were as previously described [fpr1Δ containing this HSE-LacZ vector was transformed with additional YCplac111-based LEU2 vectors bearing either the wild type or F43Y mutant forms of the FPR1 gene under native promoter control, then grown on SD minus uracil and leucine prior to measurements of their HSE-LacZ expression.Yeast strains used in this study are listed in Table escribed . Culture5 cells/ml), this dilution being used to set up radicicol and rapamycin or radicicol and caffeine dose response matrixes  in 96-well microtiter plates. After 46h growth at 28°C the cells were re-suspended by agitation, their absorbance determined at 595 nm with a Multiskan Ascent platereader with correction for the background from the corresponding medium, and the data quantitatively displayed with color using the program Java Treeview 1.1.3 (http://jtreeview.sourceforge.net/).For drug sensitivity assays, overnight YPD cultures were either serially diluted, then pinned onto YPD agar containing the indicated antibiotic and the plates incubated as indicated in the figure legends; or they were inoculated into complete SD medium (1×10


Antibodies 14F7 and the corresponding anti-idiotype 1E10  were mistakenly conflated in this review. 14F7 reacts with (Neu5Gc)GM3, was recently humanized . The firThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The authors would like to clarify that some of the blots previously depicted in The correct blots for The authors have provided new versions of Figs The text in the Results sections titled “The effects of acute Herceptin treatment on HER3 and PKB phosphorylation” and “The discordant effects of Herceptin on ERK and PKB pathways” has been edited to accommodate the removal of panel E in The corrected text, Figs We found that the downregulation of HER2 receptors was detectable after Herceptin treatment in SKBR3 and BT474 cells, and was associated with an increase in HER2 phosphorylation . Lee-HoeWe analysed the effects of Herceptin on the downstream signalling pathways in HER2 positive breast cancer cells. It was found that the effect of acute Herceptin treatment on phosphorylation of PKB and ERK1/2 was not concordant Figs , in contAcute Herceptin exposure increased EGFR/HER2 and HER2/HER4 dimerisation, correlating with an increase in ERK phosphorylation . In contTherefore, Herceptin treatment decreased PKB phosphorylation due to a decrease in HER3 phosphorylation induced by HER2 downregulation. However, one hour of Herceptin treatment increased ERK phosphorylation as a result of ligand-dependent EGFR and HER4 activation.We found that although Herceptin downregulated HER2 receptors, the remaining cells had persistent and increased HER2 phosphorylation in both SKBR3 and BT474 cells . Since HWe postulated that the increased dimerisation of EGFR, HER3 and HER4 with HER2 was due to activation by their respective ligands. We proceeded to assess the levels of endogenous ligands, using heregulin (ligand for HER3 and HER4) and betacellulin (ligand for EGFR and HER4) as examples. Herceptin treated cells were lysed and endogenous ligand levels were detected using ELISA. We found that Herceptin induced a statistically significant upregulation of heregulin and betacellulin (p = 0.0152 and p = 0.0286 respectively) after 1 hour of Herceptin treatment compared to untreated cells in both SKBR3 and BT474 cells (data on BT474 cells are shown in Thus, Herceptin increased the dimerisation of EGFR, HER3 and HER4 with HER2 as a result of the activation by their ligands.


Systemic onset Juvenile Idiopathic Arthritis (SoJIA) is a subset of juvenile idiopathic arthritis (JIA) that describes patients with intermittent fever, arthritis and skin rash with complex genetic trait in children less than 16 years old. The etiology of disease has not yet been identified; but because of high level of IL-1β in the blood of patients; and relation between its complications, and clinical signs of disease; the only theory is mutation in factors which are involved in the control of production and secretion of this proinflammatory cytokine, like IL-1Ra, IL-1 type II decoy receptor proteins and soluble IL-1R accessory protein (IRAP).The aim of this study was to identify the association between MEFV mutations in exons 2 and 10, and SoJIA disease in 30 Iranian patients.Thirty Iranian children with SoJIA disease and 30 healthy control, were screened for MEFV mutations in exons 2 and 10 by sequencing. All patients were diagnosed according to the classification criteria of EULAR for SoJIA. The control group consisted of 30 healthy individuals  who did not have any history of immune system disorders and other diseases with known genetic or hereditary predisposition.Statistical analysis showed that the MEFV mutations in the patients and control groups were significantly different (p<0.01). The mutations were detected in 17 of 30 patients (56.6%). The respective figure for the control subjects was 1 of 30 (3.3%). Leading mutation was R202Q mutation which detected in 13 patients ( 6 homozygote and 7 heterozygote). Following eight patients had M694V mutation ( 2 homozygote and 6 heterozygote). Three patients had E148Q mutation in heterozygote form.MEFV mutations may be an important cause of high level of IL-1β in SoJIA patients. It shows that SoJIA is better to be classified as auto-inflammatory disorder.None declared.


Computational cardiac modelling has been established as a valuable tool for simulating electrophysiology and electromechanics of the heart , with pr2). The entire left ventricle was covered in both systole and diastole with a total of 12 slices with a spatial resolution of 2.2×2.2x6mm3.Data from one healthy volunteer was acquired using a dual-phase dual-slice stimulated echo acquisition mode (STEAM) method  on a 1.53 and 18ms, respectively. Diastolic and systolic diffusion tensors were mapped into a prolate spheroidal coordinate system [To correct diffusion tensors for material strain, additional 3D tagging data were acquired and incorporated into diffusion tensor calculation  with spae system  for 3D dData across the entire left ventricle were successfully acquired in both systole and diastole. Figure The data presented here provides a comprehensive set of information on microstructure and motion and may serve as realistic input for computational modeling projects.This work is supported by UK EPSRC.


A mutation in the abnormal limb mutant 5 (ALI5) mouse in the region coding for the hydrophobic ridge loop 3 (HRL3) of the phospholipaseCγ2 (PLCγ-2) gene, corresponding to human PLCγ-2 exon 27, leads to acute and chronic inflammation and granulomatosis. For that reason, we screened exons 11, 12 and 13 coding for the hydrophobic ridge loop 1 and 2  and exon 27 of the PLCγ-2 protein by single strand conformation polymorphism (SSCP), sequencing and PCR/ restriction fragment length polymorphism (RFLP) analyses. In addition, we screened indirectly for disease association via 4 microsatellites with pooled DNA in the PLCγ-2 gene.Wegener Granulomatosis (WG) is a multifactorial disease of yet unknown aetiology characterized by granulomata of the respiratory tract and systemic necrotizing vasculitis. Analyses of candidate genes revealed several associations, Although a few polymorphisms in these distinct exons were observed, significant differences in allele frequencies were not identified between WG patients and respective controls. In addition, the microsatellite analyses did not reveal a significant difference between our patient and control cohort.PLCγ-2 gene in the pathogenesis of WG in our case-control study.This report does not reveal any hints for an involvement of the  Staphylococcus aureus is a risk factor for disease exacerbation in WG ]]StaphyloPLCγ-2, was proposed on the basis of a novel animal model system. A mutation in the abnormal limb mutant 5 (ALI5) mouse + status Pooling of DNA was performed as reported before . PatientIn this study 3 intragenic microsatellites as well as one in the immediate vicinity of the gene were included  and 50 ng DNA.For PCR we used the 'tailed primer PCR' as described before . For ampElectrophoreses were run using ABI standard protocols. Raw data were analyzed by the Genotyper software (ABI) producing a marker-specific allele image profile )7]). AIPStatistics for comparisons of allele frequencies of patients and controls was performed as described before [ and 7]]]7]]. CasExons 11, 12, 13 and 27 were analysed by the SSCP method. PCR was performed using oligonucleotides reported before (, exon 27PJ and SW carried out the molecular genetic studies, performed the statistical analysis and drafted the manuscript. PY participated in study design and helped to draft the manuscript. EC and WLG provided the samples and performed diagnostics of the patient group. JTE conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


TGFBR1*6A, a common hypomorphic variant of the type I Transforming Growth Factor Beta receptor, is emerging as a tumor susceptibility allele that predisposes to the development of breast, colon and ovarian cancer. The association with prostate cancer has not yet been explored. A total of 907 cases and controls from New York City were genotyped to test the hypothesis that TGFBR1*6A may contribute to the development of prostate cancer. TGFBR1*6A allelic frequency among cases (0.086) was slightly higher than among controls (0.080) but the differences in TGFBR1*6A genotype distribution between cases and controls did not reach statistical significance (p = 0.67). Our data suggest that TGFBR1*6A does not contribute to the development of prostate cancer.Prostate cancer is the most commonly diagnosed cancer in men and one of the leading causes of cancer deaths. There is strong genetic evidence indicating that a large proportion of prostate cancers are caused by heritable factors but the search for prostate cancer susceptibility genes has thus far remained elusive.  TGFBR1*6A is a common variant of the type I TGF-ß receptor, TGFBR1 [TGFBR1*6A (*6A) has a deletion of three GCG triplets coding for alanine within a nine alanine (9A) repeat sequence of TGFBR1 (*9A) exon 1, resulting in a six alanine (6A) repeat sequence. The 9-bp deletion that differentiates *6A from *9A is located within the predicted signal sequence cleavage region. In vitro studies have demonstrated that TGFBR1*6A responds less effectively than TGFBR1 to TGF-ß growth inhibitory signals [TGFBR1*6A heterozygotes and homozygotes among patients with a diagnosis of cancer as compared with the general population led us to postulate that TGFBR1*6A might act as a tumor susceptibility allele [TGFBR1*6A carriers may have an increased risk of breast, colon and ovarian cancer [TGFBR1*6A may contribute to the development of prostate cancer, we conducted a case control study of patients with biopsy verified prostate cancer cases and geographically and ethnic-status matched controls.Transforming Growth Factor Beta (TGF-ß) is one of the most potent inhibitor of cell growth . Almost , TGFBR1 . TGFBR1* signals ,5. The an cancer . To testTGFBR1*6A. The mean age of cases was significantly higher than controls (p < 0.01) but there were no differences in ethnic status between the two groups. There were 59 TGFBR1*6A heterozygotes and three TGFBR1*6A homozygotes among cases, 62 TGFBR1*6A heterozygotes and 1 TGFBR1*6A homozygote among controls. TGFBR1*6A allelic frequency among cases (0.086) was slightly higher than among controls (0.080) but the differences in TGFBR1*6A genotype distribution between cases and controls did not reach statistical significance (p = 0.67)  but was not significant after adjustment for race and age strata within groups  (Table TGFBR1*6A was significant (p = 0.01).A total of 907 cases and controls were genotyped for BRCA2 gene have an increased prostate cancer risk [BRCA2 mutations in the general population, it is unlikely to account for a significant proportion of prostate cancer cases. Approximately 14% of the general population carries at least one copy of TGFBR1*6A, which makes it the most common candidate tumor susceptibility allele reported to date. While there is growing evidence that TGFBR1*6A predisposes to the development of breast, colon and ovarian cancer, our data do not suggest that it predisposes to the development of prostate cancer. We have previously shown that TGFBR1*6A homozygotes have an O.R of 2.69 and 2.02 for ovarian and colon cancer, respectively. The present study has the power to detect an O.R. for prostate cancer of 1.70 or higher and therefore rules out a major association between TGFBR1*6A and prostate cancer. However, it does not exclude a smaller O.R., which might have clinical relevance given the high TGFBR1*6A allelic frequency in the general population. It is possible that age differences in cases and controls affected the allele frequencies observed. If the TGFBR1*6A allele predisposes to a lethal malignancy such as prostate cancer, however, its frequency could be higher, not lower, in a younger cohort. Thus, the younger mean age of controls could result in a bias toward the null hypothesis, resulting in a stronger association than that observed. The intriguing findings of a high TGFBR1*6A allelic frequency among prostate cancer cases diagnosed before the age of 55 have to be cautiously interpreted given the fact that this group only included 46 patients. We have previously shown that TGFBR1*6A is not associated with an increased risk of bladder cancer [TGFBR1*6A and prostate cancer is at best very weak but further studies are needed to formally exclude an association with early onset prostate cancer.Prostate cancer is the most common cancer and the second most common cause of cancer death among U.S. men . A similcer risk . HoweverDNA was extracted from lymphocytes of blood specimens from 465 consecutive individuals diagnosed with adenocarcinoma of the prostate who received care at the outpatient urology clinic at Memorial Sloan-Kettering Cancer Center from April 2000 to September 2002. The blood samples were collected following completion of diagnostic studies. They were unselected for age or family history. Clinical and pathological records were reviewed to confirm the diagnosis of prostate cancer in all subjects. Once pathological diagnosis of prostate cancer was confirmed, the age of diagnosis was recorded, and all other identifying links were destroyed. The study design and anonymization method were approved by the Memorial Sloan-Kettering Cancer Center Institutional Review Board.A population of 465 healthy male controls aged 20 to 87 years with well-defined ethnic background who had donated blood for various reasons  constituted the control group. Controls were matched to the cases on ethnicity and were from the same geographic locations as the prostate cancer cases. None of the controls had any personal history of cancer at the time of blood donation. This was ascertained by a questionnaire completed by each control. Exact age information was not available for 205 controls since it was not collected prospectively but the age range (20 to 40) was known. All personal identifiers were permanently removed from both cases and controls.® GC rich kit . The PCR reaction mixture included 20 ng of genomic DNA in a 10-µL reaction volume and the following concentration of other reagents: primers (0.25 µM each), 1X GC genomic PCR reaction buffer, 1.625 mM Mg2+, 0.2 mM dNTPs and 0.16 µL of Advantage-GC genomic polymerase mix. Polymerase chain reaction cycling conditions consisted of an initial denaturation period of 3 minutes at 94°C, then 35 cycles of denaturation for 30 seconds at 94°C and annealing/extension for 2 minutes at 72°C, followed by a final extension step of 5 minutes at 72°C. Quality controls were run on a 2% agarose gel. The ABI Prism 310 Genetic Analyzer  was used for data acquisition. A peak at 115 base pairs corresponded to TGFBR1 allele, whereas a peak at 107 base pairs corresponded to the TGFBR1*6A variant. The rare equivocal results were confirmed by cloning of the PCR product followed by automated sequencing. Samples were read by two independent investigators unaware of the case /control status. Ten percent of samples were randomly selected and run for quality assurance. Concordance rate was 100%.DNA was extracted by standard technique using the Qiagen DNA extraction kit. The PCR primers used were 5'-CCA CAG GCG GTG GCG GCG CGA TG-3' in the forward direction and 5'-CGT CGC CCC CGG GAF CAG CGC CGC-3' in the reverse direction. A standard solution was prepared using the Clontech AdvantageTGFBR1 genotypes, age, and ethnicity were compared between cases and controls using Fisher's exact tests. To test the hypothesis that the hypomorphic TGFBR1*6A gene is related to an increased prostate cancer risk, adjusted odds ratios of prostate cancer were estimated using both conditional and unconditional logistic regression models. Both models were run since the matched controls of cases with missing genotypes had to be excluded in the conditional models but could be included in the unconditional models. Adjusted odds ratios of prostate cancer were estimated comparing carriers of TGFBR1*6A versus non-carriers under dominant models. Potential confounders such as age (in four strata) and ethnicity were controlled in the analysis. Whether the effects of TGFBR1*6A on prostate cancer differ by age was evaluated by stratified analysis and tests for multiplicative interaction. A small p value indicates that interaction of age and gene is statistically significant on the multiplicative level. For the unconditional models, sensitivity analysis was conducted to evaluate the impact of the fact that the exact age of some controls with age 20–40 years is unknown (N = 126). With 442 cases and 465 controls, the power to detect an OR of 1.7 and 2 in the present study was 0.86 and 0.98, respectively, based on a two-tailed test at the 0.05 significance level.Distributions of All authors made substantial contributions to this paper, including conceiving of the ideas, discussion and writing. All authors read and approved the final manuscript.


This series of homologous polyhedra is found in endohedral clusters of the group 14 atoms such as the endohedral germanium cluster anions [M@Ge10]3−  and [Ru@Ge12]3− The next members of this series have been predicted to be the lowest energy structures of the endohedral silicon clusters Cr@Si14 and M@Si16 . The largest members of this series correspond to the smallest fullerene polyhedra found in the endohedral fullerenes M@C28 . The duals of the oblate (flattened) ellipsoidal deltahedra found in the dirhenaboranes Cp*2Re2Bn−2Hn−2  are prolate (elongated) trivalent polyhedra as exemplified experimentally by the germanium cluster [Co2@Ge16]4− containing an endohedral Co2 unit.The duals of the most spherical  Similarly bare group 15 element vertices  vertices as well as Co(CO)3 and (η5-C5H5)Ni vertices are valence isoelectronic and isolobal with CH vertices. The duals of these most spherical deltahedra represent a series of most spherical trivalent polyhedra in which the degree 3 vertices correspond to the triangular faces of the most spherical deltahedra. This paper surveys the wide range of cluster structures ranging from molecular endohedral germanium clusters to the smallest endohedral fullerenes based on trivalent polyhedra that are duals of the deltahedra found in polyhedral borane and metallaborane chemistry.The fundamental structural units for polyhedral boranes, including the Briangles . 1,2,3],2,3nHn2−closo deltahedra have the following features:(1)All faces are triangles—hence their designation as deltahedra relating to the shape of the Greek letter delta (∆). This feature maximizes the number of edges for a given number of vertices and thus maximizes the connectivity between the vertices.(2)The vertices are as nearly similar as possible thereby providing the best approximation to a sphere rather than a prolate or oblate ellipsoid.The most spherical closo deltahedra can be classified into the following three types:(3)hO) 6-vertex regular octahedron with exclusively degree 4 vertices and go as far as the Dd4 10-vertex bicapped square antiprism with two degree 4 vertices and eight degree 5 vertices. These deltahedra are found in the borane dianions BnHn2− and the isoelectronic carboranes CBn−1Hn− and C2Bn−2Hn 3−  a copper atom is encapsulated by a closo 9-vertex tricapped trigonal prismatic tin or lead cluster [closo 10-vertex Dh4 bicapped square antiprism encapsulating a metal atom was found to be the structural motif for the anionic indium cluster Zn@In108− found in the intermetallic [8In10Zn as well as in the lead clusters M@Pb102− found in [K]2[M@Pb10] . [Cv3 tetracapped trigonal prism in the M@Ga1010− clusters found in the K10Ga10M intermetallics  [104+ in Bi14PdBr16 (=[Pd@Bi10][BiBr4]4), [12]2− contains a platinum atom encapsulated in a closo regular icosahedron of lead atoms. 4),  have alld atoms. 10 pentagonal prism in the anion Co@Ge103− of [K]4[Co@Ge10][Co2]•toluene [103− of [K]3[Fe@Ge10] [dT 28-vertex polyhedron found in the endohedral fullerenes M@C28 . [In view of these considerations the discovery of an outer Ge•toluene  and in tFe@Ge10]  was a ma Th, U).  In this oblatocloso dirhenaboranes Cp*2Re2Bn−2Hn−2   . 3[Ru@Ge12]·4py [8As4]3−, also found in a [K]+ salt of more complicated stoichiometry [7Bi7]4−  tetraanions ·4py ,25 and ihiometry . The larraanions  and is pic beams . The 16-ructures ,30,31.n fullerenes having only pentagonal and hexagonal faces. Even with carbon vertices, such polyhedra become large enough to encapsulate metal atoms. Thus the 28-vertex tetratruncated tetracapped tetrahedron  6− with an outer Si20 regular (hI) dodecahedron (n2 for n = 4) counting one skeletal electron for each group 14 element vertex and all of the valence electrons of the endohedral atom. This relates to the spherical aromaticity [The M@Cgeometry  and Pu@Cgeometry   have central oblate (flattened) ellipsoidal Re2Bn−2 deltahedra ]4[Co2@Ge16]·en where one of the two isolated and crystallographically characterized isomers has a structure based on a prolate trivalent polyhedron with two hexagonal faces, four pentagonal faces, and four tetragonal faces  ellipsoidal deltahedron such as those found in the hypoelectronic dirhenaboranes leads to a prolate (elongated) trivalent polyhedron. The elongated shapes and relatively large internal volumes of such prolate trivalent polyhedra make them particularly suitable to incorporate a pair of interstitial metal atoms. This is seen experimentally in the .,23.2@Ge1closo borane deltahedra having from 6 to 16 vertices form a series of homologous spherical trivalent polyhedra having even numbers of vertices from 8 to 28. This series of homologous polyhedra is found in endohedral clusters of the group 14 atoms. Thus the smallest members of this series are found in the endohedral germanium cluster anions [M@Ge10]3−  and [Ru@Ge12]3− with Dh5 pentagonal prism and Dd2 bisdisphenoid dual geometries, respectively. The next members of this series have been predicted to be the lowest energy structures of the endohedral silicon clusters Cr@Si14 and M@Si16  with Dh3 tritruncated trigonal bipyramid and Dd4 bitruncated tetragonal trapezohedron geometries, respectively. The largest members of this series correspond to the smallest fullerene polyhedra. The last member of this series, namely the dT tetratruncated tetracapped tetrahedron dual to the 16-vertex Frank-Kasper deltahedron, is found in the endohedral fullerenes M@C28 , which are the smallest fullerene polyhedra known experimentally. Thus the homologous series of most spherical trivalent polyhedra represent a transition from molecular endohedral clusters of the heavier group 14 elements to the smallest fullerene derivatives. In addition, the duals of the oblate (flattened) ellipsoidal deltahedra found in the dirhenaboranes Cp*2Re2Bn−2Hn−2  are prolate (elongated) trivalent polyhedra as exemplified experimentally by the germanium cluster [Co2@Ge16]4− with an endohedral Co2 unit.The duals of the most spherical


Due to a Production error the references were numbered incorrectly. Reference 14, "Singapore", was split into two references (14 and 15). So, reference 16 "Taiwan", should be renumbered as "15", reference 17, "Thailand", should be renumbered as "16", and so on through the rest of the references .


Mutations in GJB2 gene are the leading cause of deafness in autosomal recessive inheritance, and the 35delG mutation is the most common in many ethnic groups. Besides the 35delG mutation in homozygosis, the mutation is also found in compound heterozygosis, coupled with other mutations in genes GJB2 and GJB6.To determine the prevalence of 35delG/GJB2 and del (GJB6-D13S1830) mutations in patients with sensorineural hearing impairment in residents from the Espirito Santo state, Brazil.77 unrelated individuals with moderate to profound sensorineural hearing loss were evaluated. The 35delG mutation was studied by PCR / RFLP; and the del (GJB6-D13S1830) mutation was screened by the technique of multiplex PCR.88.3% had normal genotype for the studied mutations, 1.3% were compound heterozygotes, 3.9% homozygotic for the 35delG mutation, 6.5% heterozygotic for 35delG/GJB2. The frequency of 35delG/GJB2 and del (D13S1830/GJB6) alleles in the sample was 7.8% and 0.65%, respectively.The data confirmed the existence of the mutations studied in cases of sensorineural hearing loss in a population from Espírito Santo / Brazil. These findings reinforce the importance of genetic diagnosis, which can provide early treatment for children and genetic counseling for the affected families. Deafness is the most frequent sensory deficit in human beings; its reported incidence worldwide ranges from 1:300 to 1:1,000 children.13,Connexins are transmembrane proteins that form cell surface cylindrical hexameric structures which bind to other connexin hexamers in adjacent cells to form intercellular communication channels.,Mutations in three connexin encoding genes, GJB2 (Cx 26), GJB6 (Cx 30), and GJB3 (Cx 31) have been found to cause hearing loss.,,13,The 35delG mutation accounts for most of the mutant alleles (60-85%) in Caucasians, among the DFNB-causing GJB2 gene mutations described so far.The 35delG mutation is a deletion of a guanine base in a sequence of six guanines that extend from nucleotide positions 30 to 35 on the GJB2 gene encoding exon, resulting in a stop codon. This deletion causes the polypeptide to be synthesized incompletely; it contains 12 amino acids, rather than the usual 226 amino acids.Analyses of the GJB2 gene in patients with autosomal recessive inherited deafness, especially because of the 35delG mutation, have shown that about 10 to 50% presented only one mutant allele.22Environmental factors cause about 80% of hearing loss cases in Brazil; the remaining 20% supposedly are of inherited causes.,Estimates of the prevalence of 35delG heterozygotes in several European countries range from 2 to 4% of the normal hearing population.Finding the prevalence of mutations in Brazil may help implement expedited molecular diagnosis tests to inform physicians about therapy, along with genetic counseling for family members of patients. It is therefore essential to learn about the genetic diversity of deafness to design improved proposals for the molecular diagnosis of population groups.The purpose of this study was to estimate the prevalence of the 35delG and D13S1830 mutations of the GJB2 and GJB6 genes in a sample of patients from Espirito Santo (Southeast Brazil), with bilateral prelingual non-syndromic sensorineural hearing loss of unknown causes.A cross-sectional cohort study included 77 unrelated hearing loss patients, of which 38 were male and 39 were female. The age of patients ranged from 1 to 52 years. All resided in the state of Espirito Santo.Patients came from auditory oral schools and hearing loss patient support centers in several regions of Espirito Santo; a clinical history to identify the onset of hearing loss, the presence of other cases in their families, to confirm non-syndromic deafness, and to exclude ambient causes, such as prenatal infection  approved this study (protocol no. 121/2006). A commercial DNA-extraction kit (Puregene DNA Purification Kit - Gentra Systems) was used to extract genomic DNA according to the manufacturer's instructions.A primer pair (F: 5 TCT TTT CCA GAG CAA ACC GC 3 and R: 5 GCT GGT GGA GTG TTT GTT CAC ACC CGC 3) was used to amplify the genomic region at 65°C annealing temperature for analyzing the 35delG mutation on the GJB2 gene. The resulting 89 bp PCR product was digested with the BstNI restriction enzyme. In the absence of deletions, the enzyme yields two fragments (69 bp and 20 bp); if the deletion is present, the enzyme does not cut the DNA (89 bp). The digested fragments were analyzed in 8% polyacrylamide gel electrophoresis.,The del (GJB6-D13S1830) mutation was tracked using the PCR multiplex technique with three primers  at 62°C annealing temperature.We originally evaluated 308 subjects with hearing loss; 77 of these had no environmental causes for their clinical picture, and were included in the study (25% of cases).The 77 subjects with idiopathic deafness underwent molecular screening for the 35delG mutation in the GJB2 gene and the del (D13S1830) mutation in the GJB6 gene. The 35delG mutation was found in homozygosis in three patients (3.9% of cases), which established the etiology in these subjects. The 35delG mutation was found in heterozygosis in five subjects (6.5% of cases), but the presence of a single allele did not explain the cause of deafness in these patients. Normal and heterozygotic patients for the 35delG mutation were also investigated for the del (GJB6-D13S1830) mutation; one among 74 subjects had this mutation (1.35% dos cases). The heterozygotic patient for the del (D13S1830/GJB6) mutation was also heterozygotic for the 35delG/GJB2 mutation, which defined the double genetic etiology for this patient. These mutations were not found in 68 patients; other related mutations to Cx26 and Cx30 may be associated with the clinical pictures of these patients. The frequency of the 35delG/GJB2 mutant allele in the sample was 7.8%; the frequency of the del (D13S1830/ GJB6) mutant allele was 0.65%.The genetic heterogeneity of non-syndromic hearing loss makes its molecular diagnosis more complicated, given the number of mutations that have been described in dearness-related genes; additionally, the predominance of each varies significantly in different populations.,,The 35delG mutation in the GJB2 gene is the main cause of genetic deafness in Caucasian populations; it may be found in homozygosis or compound heterozygosis (with other mutations in the GJB2 and GBJ6 genes).,,,,,The frequency of the 35delG/GJB2 mutant alleles may vary in different regions of the world, as follows: United States of America (1.0%); Australia (1.0%); Austria (1.7%); Turkey (1.8%); Portugal (2.2%); Spain (2.5%), France (2.7%), and Italy (2-4%).,,Because of widespread racial mixing in Brazil, the 35delG/GJB2 mutation is not rare. Three studies in the state of Sao Paulo have revealed mutant allele frequencies ranging from 0.97% to 2.24%.A survey in 10 cities in different regions of Brazil revealed the following frequencies for 35delG mutation: North region (2.1%); Southeast region (1.5%); South region (1.2%), and Northeast region (0.8%); these differences were not significant.,,40In the present study, the 35delG/GJB2 mutation and the del (D13S1830/GJB6) mutation were investigated in patients with idiopathic deafness in the state of Espirito Santo, Brazil. Among 77 subjects, nine unrelated individuals had the 35delG/GJB2 mutation; of these, one had the mutation in compound heterozygosis with the D13S1830 mutation in the GJB6 gene. The frequencies of the 35delG/GJB2 and del (GJB6-D13S1830) mutant alleles in the sample were respectively 7.8% and 0.65%. Our data concur with other studies done in the Brazilian population , Table 3Although molecular analysis of hearing loss is not frequent in developing countries, it is essential to investigate GJB2 and GJB6 gene mutations for public health and genetic counseling purposes. The proportion of hearing loss patients because of genetic causes tends to increase as a result of investments and improvements in healthcare systems in developing countries such as Brazil. Establishing the prevalence and the types of mutations that cause non-syndromic hearing loss in Brazil, as was done in this study, may help implement simple and specific models to detect the main mutations causing genetic deafness in this country. The molecular diagnosis leads to accurate genetic counseling for family members and makes it possible to provide early rehabilitation for the children in affected families.Our data confirmed the presence of the 35delG mutation in the GJB2 gene in cases of non-syndromic bilateral moderate to profound sensorineural hearing loss in Espirito Santo, Brazil, a result which concurs with other published findings. The del (GJB6-D13S1830) mutation was found in compound heterozygosis with the 35delG/ GJB2 mutation in one patient. These findings underline the importance of a genetic diagnosis that may clarify the etiology and provide early treatment for children and genetic counseling for their family members.


TET2 and canonical ASXL1 (c.1934dupG) mutations. The number of cases is small, but the variant allele fraction (VAF) sums of the TET2 mutations, as well as the persistence of TET2 mutations in a case of relapsed BPDCN, suggest an ancestral/founder nature of TET2 clones in the cases. Further literature review shows a high frequency of biallelic TET2 mutations in reported cases of BPDCN.Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is recurrently mutated in epigenetic pathway genes. We studied the myeloid‐related genetic mutations in a cohort of five patients with BPDCN and one paired relapse case at our institution and identified a high frequency of biallelic  Our study was aimed to identify genetic mutations in our BPDCN cohort, and compare our findings to published BPDCN cohorts. Our work identified a high frequency of biallelic TET2 mutations and canonical ASXL1 mutations with cooccurrence in a subset of cases.Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive type of hematologic malignancy derived from plasmacytoid dendritic cell precursor cells. Next‐generation sequencing (NGS) techniques have expanded the understanding of the genetics of BPDCN. Epigenetic pathway genes  and 3 (60%) patients, respectively. Two (40%) patients had both TET2 and ASXL1 mutations. TET2 and IDH2 mutations were mutually exclusive, a pattern consistent with other myeloid neoplasms.Genomic DNA extracted from involved bone marrow or skin biopsies was amplified and subjected to mutation analysis. Cases 1 and 2 were sequenced using the Oncomine Myeloid Research Assay of 40 genes and analyzed with Thermo Fisher Scientific Ion Reporter . Cases 3 through 6 were sequenced using the institutional Myeloid Neoplasm Next‐Generation Sequencing panel of 53 genes on Illumina platform . All variants identified were in genes included in both panels. The limit of detection for the reported variants was set at 5% VAF. Among the newly diagnosed BPDCN cases [TET2 mutations spread across the coding exons of the gene  to 5‐hydroxymethylcytosine (5‐hmC) and promotes DNA demethylation. TET2 mutations are generally loss of function variants (frameshift and nonsense) that could be monoallelic or biallelic and occur throughout the length of the gene. Biallelic TET2 mutations have been reported in 10%–30% of MDS and AML patients and approximately 30% of CMML patients [TET2 mutations were founder lesions in 72% of CMML cases in one study [TET2 mutations likely result in a relatively more competitive advantage over single TET2 mutations [TET2 mutant allele dosage has potential theranostic impact for myeloid neoplasms. MDS patients with 1 or more TET2 mutations showed response to hypomethylating agents such as azacitidine (AZA) in some studies [TET2 mutations is associated with increased response to hypomethylating agents [patients . The Bia studies . Higher TET2 loss‐of‐function was a shared ancestral genetic event in some reported cases before divergent clonal evolution occurred in each neoplasm [TET2 mutations. The type and frequency of TET2 mutations in BPDCN appear similar to those observed in other myeloid neoplasms [TET2 mutations appears significantly higher in BPDCN compared to other myeloid malignancies  variant, with the VAFs ranging from 24% to 28%. ASXL1 encodes a polycomb repressive complex protein with a vital role in chromatin regulation. ASXL1 mutations are commonly frameshift or nonsense, and most are located within or upstream of the catalytic domain, which causes C‐terminal truncation of the protein. The ASXL1 G646fs*12 mutation is a hotspot mutation and commonly referred to as the “canonical” ASXL1 mutation. The ASXL1 G646fs*12 mutation accounts for approximately half of all ASXL1 mutations in AML [ASXL1 mutations in reported BPDCN cases . This study also underwent review in the Case Comprehensive Cancer Center Protocol Review and Monitoring Committee (PRMC) due to the inclusion of cancer patients, to further ensure ethical retrospective review of medical records. All study personnel were certified as having up to date relevant training in research ethics, compliance and safety.Following detailed review of the study, the requirement for patient consent was waived by the IRB due to the minimal risk to the subjects and historical nature of the data.Supplemental InformationClick here for additional data file.


Dear Editor,1. Several distinct functional modules have been characterized for the 1.4-MDa SAGA complex, including a histone acetyltransferase (HAT) module and a histone deubiquitinase (DUB) module that establish histone modification signatures for transcription activation, the largest subunit TRRAP that directly interacts with transcription activators, the core module that serves as a structural scaffold, and the spliceosome U2 snRNP factors SF3B3 and SF3B52  complex, human SAGA (abbreviated as hSAGA) complex is a well-established transcription coactivator that regulates the transcription of numerous genesHere we report the cryo-EM structure of endogenous hSAGA coactivator complex purified from HEK293 cells at an overall resolution of 3.7 Å  is vacant and potentially available for the association of TBP  of the yeast SAGA complex can recruit the U2 snRNP ATPase Prp5 to the promoter region, and consequently mediating a balance between transcription and pre-spliceosome assembly10. Accordingly, we presume that the incorporation of SF3B3 and SF3B5 into hSAGA complex might play a similar role in the coordination of transcription initiation and pre-mRNA splicing. Further studies will be required to examine the specific roles of SF3B3 and SF3B5 within the context of hSAGA complex by generating mutations that selectively disrupt the attachment of SF3B3 and SF3B5 to TAF6L.SF3B3 and SF3B5 usually serve as the structural components of the SF3B complex in the spliceosome U2 snRNP. Structural superposition with the SF3B complex revealed that TAF6L occupies the surface where the HEAT repeat domain of SF3B1 interacts with SF3B3 and SF3B5, suggesting that the incorporation of SF3B3 and SF3B5 into the two complexes is mutually exclusive (Supplementary Fig. 2, further indicating the possible functional differences between human and yeast SAGA in multiple physiological activities. Future work on the structural mechanism for the regulation of hSAGA on transcription factors and its substrate nucleosomes will help understand how the unique configuration of hSAGA contributes to its functions. Our studies also elucidate the structural basis for the association of the splicing factors SF3B3 and SF3B5 with hSAGA complex and will promote functional investigations on the roles of the splicing factors in hSAGA complex.In summary, our cryo-EM analysis reveals that human SAGA complex adopts an overall architecture distinct from yeast SAGA complex due to the different organization of TRRAP relative to the core module (Supplementary Fig. Supplementary InformationSupplementary Table S3Supplementary Table S4