Smad6 and two Smad7 (Smad7a and Smad7b) chick homologs and their expression and regulation in the developing limb. Smad6 and Smad7a are expressed in dynamic patterns reflecting the domains of BMP gene expression in the limb. Activation and inhibition of the BMP signaling pathway in limb mesenchyme indicates that negative Smad gene expression is regulated, at least in part, by BMP family signals.The inhibitory or negative Smads, Smad6 and Smad7, block TGFβ superfamily signals of both the BMP and TGFβ classes by antagonizing the intracellular signal transduction machinery. We report the cloning of one  Smad6 and Smad7), antagonize and block TGFβ superfamily signaling Bone morphogenetic proteins (BMP) are members of the transforming growth factor-β (TGFβ) ligand superfamily. BMPs have diverse essential roles during limb development which include establishment of the apical ridge and zone of polarizing activity, as well as in the regulation of cell death, chrondrogenesis, myogenesis, digit identity and fracture repair Smad6 expression in the developing chick limb have been reported Smad6 mRNA expression was also found to be upregulated in interdigital regions following Bmp5 protein application Smad7 expression or the dependence of negative Smad expression on BMP signaling have not been described. Thus while some information is available about Smad6 in the developing limb, how the negative Smad gene expression patterns and their expression levels are regulated, and how they might contribute to the dynamic control of BMP or TGFβ/Activin signaling during limb development remains to be determined.In previous studies limited descriptions of Smad6 and Smad7 from HH st12–15 whole chicken embryo and HH st20–24 chicken limb bud cDNA libraries. Multiple cDNAs encoding three distinct open reading frames were identified. Comparison with known Smad protein sequences indicates that one encodes a Smad6 homolog (Genbank Accession FJ417094), while the other two encode Smad7 homologs , and maps to a contig that is not associated with a particular chromosome within the sequenced chicken genome (Contig: NW_001472907.1). The Smad7a cDNA contains a partial open reading frame that is lacking only the four amino terminal amino acids as determined by comparison with other Smad7 proteins. This sequence maps to the predicted genomic Smad7 locus on the Z chromosome (XM_427238). Thus there are two chicken Smad7 loci: the cDNA we call Smad7a is equivalent to a genomic ‘Smad7’ locus on the Z chromosome. The sequence we call Smad7b is equivalent to a previously reported ‘Smad7’ cDNA, which is not encoded by the genomic Smad7 locus.Most vertebrate species have only one cSmad7a sequences with public databases identified two distinct genomic sequences with similarities to cSmad7a. Nucleotides 541–616 of cSmad7a are identical to sequences in an unassigned 1363 nucleotide genomic contig (Contig: NW_001482773.1), while cSmad7a nucleotides 615–1235 and genomic DNA sequences at the ‘Smad7’ locus have only one mismatch. These data suggest the cDNA spans at least two genomic fragments. Analysis of the conceptual protein sequences is consistent with this idea: The cSmad7a ORF encodes a protein of at least 384 amino acids, while the predicted genomic cSmad7 protein sequence is 222 amino acids long. cSmad7a aa207–384 (the translation of nt615–1235) and genomic cSmad7 aa45–222 are identical, consistent with the nucleotide alignments. Protein BLAST analysis of aa1–44 of genomic cSmad7 does not identify any protein other than genomic cSmad7, while similar analysis of cSmad7a aa1–206 or the unassigned genomic contig (NW_001482773.1) identifies numerous Smad7 homologs. These data imply that the first 45 aa predicted by the conceptual translation of genomic cSmad7 are incorrect, and that the unassigned contig belongs in the genomic sequence in this region.BLAST analysis of cSmad7a extend 5′ only to cSmad7a nucleotide 541, there is likely at least one additional sequence missing from the genomic sequence. The cSmad7a and cSmad7b cDNAs show multiple differences scattered throughout their sequences, and are most closely conserved with sequences at different chromosomal locations. Thus cSmad7a and cSmad7b are encoded by different genes, and are not splice or allelic variants. To the best of our knowledge, the chicken is the first example of an amniote species with two Smad7 genes.As cSmad7a is similar to cSmad7b through their most amino terminal amino acids, and genomic regions of identity to cSmad6 expression by whole mount in situ hybridization from HH stages 18–34, focusing on the developing limbs. At HH st18 expression is observed in the fore and hindlimb buds in small anterior and posterior mesenchymal domains . These data are consistent with, and extend, previously described cSmad6 expression patterns cSmad7a and cSmad7b expression by whole mount in situ hybridization from HH stages 17–34, again focusing primarily on expression in the developing limb bud. cSmad7a is expressed at HH st19 in the lateral mesoderm extending from a small mesenchymal domain in the posterior forelimb to the anterior hindlimb . We could not detect significant expression of cSmad7b in the developing limbs until HH st29/30 when expression was observed in interdigital mesenchyme and between the radius and ulna .We examined al ridge . From HHal ridge . From HHal ridge . Expressand ulna . cSmad7b HH st32 . cSmad7bcSmad6 and cSmad7a. We focused on cSmad7a rather than cSmad7b, as cSmad7a is expressed in the limb from HH st 19 through at least HH st32. Taken together the Bmp2, Bmp4 and Bmp7 expression domains bear a remarkable similarity to those of cSmad6 and cSmad7a  or applied recombinant BMP protein directly to limb tissue cSmad6 and cSmad7a expression was assessed up to 72 hours later. Using a virus-specific probe to monitor the extent of infection, we observed extensive but incomplete staining by 24 hours post-infection, and that by 48 hours post-infection had spread throughout the limb mesenchyme (data not shown). In the majority of cases both cSmad6 and cSmad7a were upregulated in their normal expression domains after 24 hr and were also ectopically induced in limb territories such as the central and proximal mesenchyme  or the BMP signaling antagonist noggin DNBMPR Ib virus infection down regulates, but does not completely abolish both cSmad6 and cSmad7a expression  st12–15 chicken embryo Smad6 and Smad7 genes Chicken Gallus gallus Genome Build 2.1. Sequences with the following Genbank accession numbers were used to generate the phylogenetic tree in Smad6 and Smad7a and Smad7b gene assignments were made based on similarities of the predicted protein sequences with published family members and sequences submitted to Genbank. Accession number for cSmad6: FJ417094; cSmad7a: FJ417093; cSmad7b: FJ417092.Overlapping clones for each gene were sequenced using standard dye termination chemistry. DNA and protein sequences were compared to the non-redundant GenBank databases and published Smad sequences using both NCBI Blast cSmad6, Clone 6, 1.7 kb; cSmad7a, Clone 10, 2.7 kb; cSmad7b Clone 33, 1.0 kb.Fertile White Leghorn chicken eggs  were incubated at 38°C in a humidified, forced air incubator and embryos collected at appropriate developmental stages. Experimental manipulations were performed on HH st15 to HH st21 right limb buds; the left limb bud served as a control. Embryos were harvested through 72 hr postmanipulation and were fixed overnight in 4% paraformaldehyde. Embryos were processed for non-radioactive whole-mount or section in situ hybridization (20 µm cryosections) and photographed as described noggin, RCAS BP(A)CABMPR Ia, RCAS BP(A)DNBMPR Ib and RCAS BP(A)CABMPR Ib viruses were described previously and caused phenotypic defects consistent with previously published reports 8 infectious units/ml The RCAS BP(A)<1?twb=.35w?>For limb infections, virus was injected into HH st17–21 wing buds. Injections were targeted such that either the entire limb or specific subregions were infected with virus. Targeting was monitored by nonradioactive whole mount in situ hybridization using either a pan-retroviral or an insert-specific in situ probe Heparin acrylic beads were soaked in 0.01 mg/ml recombinant human BMP2 or BMP7 protein for 1–2 hours. A tungsten needle was used to cut a small slit in the posterior proximal mesenchyme of HH st17–20 limbs, and a bead implanted into the slit. Embryos were incubated for approximately 15 hours, fixed in 4% paraformaldehyde and processed for in situ hybridization analysis.


ACVR2) is commonly mutated in microsatellite unstable (MSI) colon cancers, leading to protein loss, signaling disruption, and larger tumors. Here, we examined activin signaling disruption in microsatellite stable (MSS) colon cancers.Activin receptor 2  was evaluated for ACVR2 and ACVR1, and ACVR2 promoter methylation by methylation-specific PCR and bisulfite sequencing and chromosomal instability (CIN) phenotype via fluorescent LOH analysis of 3 duplicate markers. ACVR2 promoter methylation and ACVR2 expression were assessed in colon cancer cell lines via qPCR and IP-Western blots. Re-expression of ACVR2 after demethylation with 5-aza-2′-deoxycytidine (5-Aza) was determined. An additional 26 MSS colon cancers were assessed for ACVR2 loss and its mechanism, and ACVR2 loss in all tested cancers correlated with clinicopathological criteria.Fifty-one population-based MSS colon cancers were assessed for ACVR1, ACVR2 and pSMAD2 protein. Consensus mutation-prone portions of ACVR2 mutations were detected. Loss of ACVR2 expression was associated with LOH at ACVR2 (p<0.001) and ACVR2 promoter hypermethylation (p<0.05). ACVR2 LOH, but not promoter hypermethylation, correlated with CIN status. In colon cancer cell lines with fully methylated ACVR2 promoter, loss of ACVR2 mRNA and protein expression was restored with 5-Aza treatment. Loss of ACVR2 was associated with an increase in primary colon cancer volume (p<0.05).Of 51 MSS colon tumors, 7(14%) lost ACVR2, 2 (4%) ACVR1, and 5(10%) pSMAD2 expression. No somatic ACVR2 promoter hypermethylation, revealing distinct mechanisms for ACVR2 inactivation in both MSI and MSS subtypes of colon cancer.Only a small percentage of MSS colon cancers lose expression of activin signaling members. ACVR2 loss occurs through LOH and  Restoration of activin signaling and growth suppression occur in response to ACVR2 complementation in ACVR2-mutant colon cancer cells ACVR2 mutations in MSI-H colon cancers in conjunction with loss of ACVR2 protein expression TGFBR2 loss found in MSS colon cancers ACVR2 and distinct ACVR2 promoter methylation, but not genetic mutation. In colon cancer cell lines, mechanisms for ACVR2 loss also segregate according to microsatellite status, with MSI-H cell lines showing ACVR2 polyadenine tract mutation and MSS colon cancer cells demonstrating promoter hypermethylation. Thus we show that disruption of activin signaling occurs in MSI and MSS colon cancers by distinct mechanisms, revealing activin signaling as an important target in the two most common genomic subtypes of colon cancer.In this study, we explored activin signaling pathway disruption and possible mechanisms in primary MSS colon cancer specimens and colon cancer cells. We found that loss of ACVR2 expression occurs in a subset of MSS tumors, which is often associated with retained pSMAD2, the next downstream effector of both TGFβ and activin signaling. Unlike that of TGFBR2, ACVR2 loss in MSS tumors occurs through a combination of LOH at ACVR2 polyadenine tracts Our previous data suggested at least partial loss of ACVR2 protein expression in a subset of primary MSS colon cancer specimens despite wild type , All specimens with loss of at least one activin receptor pathway component revealed expression of both total SMAD2 and TGFBR2 , and 3/7 (43%) of MSS tumors with loss of ACVR2 protein expression also showed LOH , a common genomic mechanism of tumor suppressor inactivation in MSS colon cancers, we assayed for LOH at the owed LOH Figure 2ACVR2 (akin to corresponding mutations within TGFBR2 in MSS tumors)8 tract in ACVR2 lies in the kinase domain of this receptor, and frameshift mutations as well as other mutations in the kinase domain abolish the phosphorylating capacity of ACVR2. However, we found no tumor specific mutations corresponding with loss of ACVR2 protein expression. These data suggest that other inactivating mechanisms play a role in the loss of ACVR2 expression.We then sequenced the coding microsatellite as well as 3 separate hot spots in the kinase domain of ACVR2 promoter was hypermethylated in colon cancer tissue as compared to normal and if so, whether a specific methylation pattern of ACVR2 correlated with ACVR2 protein loss in primary MSS colon cancer specimens. We initially divided the ACVR2 promoter into three regions, region 1 (+142 to −603), region 2 (−607 to −958), and region 3 (−958 to −1484). Our bisulfite sequencing results showed that there was no methylation in region 1 and minimal methylation in region 2, but the 46 CpG dinucleotides between to −958 to −1484 nucleotides relative to the transcription start site (region 3)   within region 3 of the in vitro, we assessed mechanisms of ACVR2 loss in colon cancer cells lines based on microsatellite instability. We tested 3 MSI-H and 3 MSS colon cancer cell lines for: 1) mutation in the coding polyadenine tract of exon 10 of ACVR2, 2) ACVR2 promoter hypermethylation, 3) quantitative RT-PCR of ACVR2 mRNA, and 4) ACVR2 protein expression by immunoprecipitation. As previously reported ACVR2 (A8 to A7) in the MSI-H colon cancer cell line HCT116 causes loss of ACVR2 protein . HT29 cells revealed a distinct ACVR2 promoter hypermethylation pattern  phenotype correlated with hylation , suggestTaken together, these data suggest that the clinico-pathologic effects of ACVR2 protein loss may be similar in both MSI and MSS colorectal cancers despite differing underlying mechanisms of loss, implicating ACVR2 loss as an important step in colon carcinogenesis.ACVR2 in one of its polyadenine tracts ACVR2 expression is lost via a combination of LOH and epigenetic silencing of the ACVR2 promoter. These findings underscore the importance of abrogated activin signaling in colon tumorigenesis, as its disruption occurs in both MSI and MSS subtypes of colon cancer by differing distinct mechanisms.Disruption of activin signaling is common in MSI-H colon cancer cell lines through mutation of ACVR2 and TGFBR2 mutations in primary MSI colon tumors In MSI-H colon cancers, both TGFβ and activin are abrogated due to frameshift mutations in the type II receptor ACVR2 mutations may occur early in tumorigenesis and are associated with increased local growth ACVR2 mutations in MSI colon cancer may be similar to that of TGFBR2 in which the frameshift mutations occur in high grade dysplasia at the interface to malignancy It appears that in MSI colon cancers We show that in MSS colon cancers at least three members of the activin signaling cascade, ACVR2, ACVR1, and pSMAD2 are disrupted. A significant subset of colon tumors displayed a decrease in phosphorylated SMAD with intact ACVR2 and TGFBR2, indicating a separate primary event downstream of the primary receptors. One case of loss of ACVR1, ACVR2 and pSMAD2 was identified, which was TGFBR2 staining positive . This coACVR2 locus in 6% of MSS colon tumors, increasing to 50% in tumors with loss of ACVR2 protein expression. This overall rate is slightly lower than the frequency of ACVR2 LOH reported previously in a cohort with unknown ACVR2 status We detected LOH at the TGFBR2 kinase domain inactivation Our mutational analysis focused on hotspots akin to those implicated in inactivation of the ACVR2 promoter hypermethylation as a mechanism, and contrasts with the mechanisms of ACVR2 loss in the MSI colon cancer cells ACVR2 expression is between nucleotides −1297 to −958. Among genes that are targets for epigenetic silencing, hMLH1 is the best studied for methylation, and it has been proposed that the promoter region adjacent to the transcription start site (TSS) is critical for transcriptional silencing of this gene hMLH1 promoter region data is often used as a paradigm, and it is believed that for most genes, analysis of a similar promoter region is critical to correlate DNA methylation findings with loss of gene expression. In this study, we have analyzed >1500 bp proximal to the TSS, and found good correlation between ACVR2 promoter methylation (in regions −958 to −1297) and loss of protein expression by IHC. Our data suggests that instead of mere proximity to the TSS, differential access to methylation co-activators or repressors in a promoter determines gene silencing, and for ACVR2 such a region may occur upwards of 900 bp from the TSS.This is the first report describing ACVR2 loss in MSS colon cancers and ACVR2 was associated with the CIN phenotype, while ACVR2 hypermethylation correlated with the CIMP phenotype. While these categorizations allow attribution of loss of ACVR2 expression to promoter hypermethylation and/or chromosomal instability as a mechanism in MSS cancers, alternative mechanisms such as histone modification and/or microRNAs may be at play, particularly in the LOH negative/methylation negative cancers. Our data however, show a significant correlation between loss of ACVR2 expression and LOH/epigenetic silencing. Thus we provide evidence for the existence of either chromosomal instability or epigenetic modification of ACVR2 in colon cancer and identify a cell model for epigenetic silencing of ACVR2. The full clinical impact of this data will require further confirmation in future studies with larger patient samples.We are mindful that the total number of MSS patients with ACVR2 loss that were investigated in this study is not large, underscoring that this is a relatively infrequent event ACVR2 loss include LOH at ACVR2 and ACVR2 promoter hypermethylation between nucleotides −1297 and −958, which are associated with CIN and CIMP phenotypes, respectively. Loss of ACVR2 is associated with increased tumor size. Therefore, activin signaling can be inactivated by distinctive mechanisms in MSI and MSS colon cancers, suggesting the importance of this pathway in controlling colonocyte growth.In conclusion, loss of ACVR2, ACVR1 and pSMAD2 expression occurs in a subset of MSS tumors, and the evidence supports that this results in abrogation of the normal growth suppressive activity of activin signaling. Decreased pSMAD2 is commonly associated with wild type ACVR2 and ACVR1. Mechanisms for This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Institutional Review Board of the University of North Carolina hospitals. All patients provided written informed consent for the collection of samples as part of the under IRB approval as part of the North Carolina Colorectal Cancer Study (NCCCS) see below. Analysis as part of this study was completely de-identified and no identifying information was available to the PI, making this study exempt from a separate consent.Sporadic colon tumors were prospectively collected under IRB approval as part of the North Carolina Colorectal Cancer Study (NCCCS), a population-based, case-control study comprising 503 patients The MSI colon cancer cells lines HCT116, SW48, DLD1, HCA7, HCT15, LoVo, LS174T, SNU175, SNU407, SW48, and RKO, as well as the MSS colon cancer cell lines CaCo2, HT29, SNU81, SNU503, SW480, and T84 were maintained in Iscove's Modified Dulbecco's Medium  and FET were maintained in F12/Dulbeco's Modified Eagles Medium at conditions previously described DNA from the formalin-fixed, paraffin-embedded material was extracted following microdissection using the Takara DEXPAT kit . Genomic DNA from cells lines was obtained using QIAamp DNA mini kit . RNA from cell lines was obtained using the TRIzol reagent .Rabbit anti-TyrGly mACVR2 (482–494) , with its target epitope in the C-terminus region of ACVR2 (beyond the resulting truncation from frameshift in exon 10) was used for immunohistochemistry as previously described Immunohistochemistry was performed as described previously ACVR2 and ACVR1 loci was performed as previously described ACVR2ACVR1 (D2S2686) (see Table S1).Assessment of LOH at the Table S1) was used to determine LOH at chromosome 5q, 17q and 18q. PCR amplifications were performed on genomic DNA templates from both tumor and corresponding normal tissues. The amplified fluorescent PCR products were electrophoresed on an ABI PRISM 3100 Avant Genetic analyzer and analyzed by GeneMapper fragment analysis software (Applied Biosystems). When comparing the signal intensities of individual markers in tumor DNA with that of the corresponding normal DNA, a reduction of at least 40% in the signal intensity was considered indicative of LOH.Analysis for CIN was performed as previously described DNA from matched colon cancer tissues and corresponding normal mucosa from CRC patients as well as DNA from colon cancer cell lines underwent bisulfite modification as described previously ACVR2 gene based upon mapping analysis using CpG island search program (http://www.uscnorris.com/cpgislands/cpg.cgi). Accordingly, we designed multiple primer sets for MSP and bisulfite sequencing that spanned the entire ACVR2 promoter region, including part of exon 1, to determine methylation density across all CpG dinucleotides with methylation specific primers (see Table S1). Bisulfite sequencing was initially performed on colon cancer cell lines as it allowed amplification of larger amplicons, and helped determine critical regions of promoter methylation that were subsequently PCR amplified in the clinical samples.For methylation analysis, we first identified the tentative CpG islands in the promoter region of the ACVR2TGFBR28 microsatellites of ACVR2 in exon 10 Table S1) and followed by sequencing as previously described ACVR2 were amplified from genomic DNA extracted from select ACVR2 expressing and non-expressing colon cancer cell lines using specific primers (see Table S1) and subjected to sequencing using the DNA Sequencing Shared Resource, UCSD Cancer Center. Any new mutations were to be deposited to GenBank.Three hotspots in conserved regions of the kinase domain of ACVR2 in colon cancer cells, we performed a quantitative polymerase-chain reaction. Briefly, all 17 colon cancer cell lines were grown to 60% confluence. RNA was extracted using a Trizol-based protocol. The concentration of RNA dissolved in DEPC-treated water was assessed using a Beckman-Coulter DU640B spectrophotometer . We performed RT-PCR using an oligo dT for incubation at 37°C to generate cDNA as previously described Table S1) Templates from each cell line were prepared in triplicate per target gene as 10 µL reactions . Templates were plated on fast optical 96-well plate  and spun for 2 minutes at 2000 rpm. Results were observed and analyzed on the StepOnePlus Real-Time PCR System . GAPDH mRNA amplification was performed in parallel  to obtain a normalized ACVR2-GAPDH after the relative expression of each gene was calculated using standard curves.To detect the amplification of To detect presence of ACVR2 in the colon cancer cell lines, we performed immunoprecipitation with ACVR2 followed by Western blotting as previously described Analysis was performed with the help of the UCSD Moores Cancer Center Biostatistics Core (K.M.) applying Fisher's exact test and Student's t-test with a p value of <0.05 indicating statistical significance. Further, Clopper-Pearson exact confidence interval was used to determine the 95% confidence interval.Table S1Specific primers used in LOH analysis, ACVR2 genotyping as well as ACVR2 promoter bisulfite sequencing. F denotes forward primer, R denotes reverse primer; U denotes unmethylated and M methylated.(0.09 MB DOC)Click here for additional data file.


The etiology of Kawasaki Disease (KD) is enigmatic, although an infectious cause is suspected. Polymorphisms in CC chemokine receptor 5 (CCR5) and/or its potent ligand CCL3L1 influence KD susceptibility in US, European and Korean populations. However, the influence of these variations on KD susceptibility, coronary artery lesions (CAL) and response to intravenous immunoglobulin (IVIG) in Japanese children, who have the highest incidence of KD, is unknown.CCL3L1 gene-containing duplication and CCR2-CCR5 haplotypes in 133 Japanese KD cases [33 with CAL and 25 with resistance to IVIG] and 312 Japanese controls without a history of KD. We observed that the deviation from the population average of four CCL3L1 copies  was associated with an increased risk of KD and IVIG resistance (adjusted odds ratio (OR)  = 2.25, p = 0.004 and OR = 6.26, p = 0.089, respectively). Heterozygosity for the CCR5 HHF*2 haplotype was associated with a reduced risk of both IVIG resistance  and CAL development .We used unconditional logistic regression analyses to determine the associations of the copy number of the CCL3L1-CCR5 axis may play an important role in KD pathogenesis. In addition to clinical and laboratory parameters, genetic markers may also predict risk of CAL and resistance to IVIG.The  Kawasaki disease (KD) is an acute, self-limiting systemic vasculitis of infants and children According to a current paradigm, KD is thought to be triggered by an infectious agent that elicits an inflammatory response directed at cardiovascular tissues in genetically susceptible hosts CCR5CCL3L1-gene containing segmental duplication CCR5 and CCL3L1 affect susceptibility to KD in parent-child trios from the United States There is evidence to suggest that recruitment of inflammatory cells in KD may be mediated through CC chemokine receptor 5 (CCR5) CCR5 genotypes and CCL3L1 copy number in different populations CCR5 haplotypes and CCL3L1 copy number influence KD susceptibility and two disease-related outcomes: development of CAL and IVIG resistance.However, there is significant variation in the prevalence of KD as well as the frequency of This study was approved by the institutional review boards of Yamaguchi and Kurume University Hospitals in Japan and the University of California San Diego and the University of Texas Health Science Center in San Antonio in the U.S. and written informed consent was given by the parents of all KD subjects and controls.We conducted an unmatched case-control study of 133 cases of KD and 312 controls collected between January 2002 and April 2005. The KD patients were recruited from three sites: the Department of Pediatrics, Yamaguchi University Hospital; Oita Children's Hospital; and Kurume University Hospitals, Japan. All patients met the Japanese criteria for the diagnosis of KD CCR5 polymorphisms are described elsewhere CCR5 were categorized into haplotypes as described previously and were designated as CCR5 human haplogroups A (HHA), HHB, HHC, HHD, HHE, HHF*1, HHF*2, HHG*1, and HHG*2 CCR5 haplotypes that bear the CCR5-Δ32 or CCR2-64I polymorphisms are designated as the CCR5 HHG*2 and HHF*2 haplotypes, respectively CCL3L1 gene-containing segmental duplication was estimated as described previously CCL3L1 copy number captures three separate CCL3L genes  as described previously The methods for genotyping CCR5 haplotypes was estimated using the PowerMarker software CCR5 haplotypes and CCL3L1 copy number with KD-related outcomes. The median number of CCL3L1 copies in the study population was 4 and for this reason the study subjects were trichotomized into those possessing <4, 4 and >4 CCL3L1 copies. In these regression analyses, we included CCR5 haplotypes  and CCL3L1 copy number (less than 4 and greater than 4) in the same regression model. To determine whether CCL3L1 gene copy number modified the KD-influencing effects of CCR5 haplotypes, we used the Mantel-Haenszel test of homogeneity. We used Stata 10.0  for the statistical analysis.Allele frequency and Hardy-Weinberg equilibrium for all the Among the cases there were 55 (41.35%) females and 78 (58.65%) males whereas in the control group there were 190 (60.90%) females and 122 (39.10%) males. KD patients with available echocardiographic data were categorized into 2 groups according to the presence of CAL. There were 33 (27.5%) and 87 (72.5%) patients with and without CAL, respectively. Mean age of disease onset was 43.5 months (range 2–270 months). Of the 95 cases who were treated with IVIG within the first 10 days of onset of fever, 25 (26.32%) were resistant to treatment.CCR5 haplotype was CCR5 HHC, followed by HHF*2 and HHE (CCR5-Δ32 mutation is very rare. The CCR5 locus was in Hardy-Weinberg equilibrium (Exact P = 0.9808 in controls and 0.5624 in cases). The median CCL3L1 copy number in both cases and controls was four  and >4 CCL3L1 copies  was associated with an increased risk of developing KD  and >4 CCL3L1 copies  remained associated with a significantly higher risk of developing KD. Thus, departure from the population average of 4 CCL3L1 copies  was associated with a significantly increased risk of KD before  and after adjustment for gender .To determine whether CCR5 haplotypes had a significant association with the risk of KD. In previous studies, we found that the copy number of CCL3L1 modified the SLE-, Kawasaki disease-, and HIV-1-disease-influencing effects of CCR5 haplotypes . Evaluation of the association for the outcome of CAL revealed that possession of the CCR2-64I-bearing CCR5 HHF*2 haplotype was associated with a reduced risk of developing CAL which trended towards statistical significance . However, we did not observe a significant association between CCL3L1 copy number and the risk of developing CAL.In our cohort, of the 25 subjects who were resistant to IVIG, 18 (72%) developed CAL. By contrast, of the 68 who responded to IVIG treatment, only 5 (7.3%) developed CAL. This association between IVIG resistance and CAL development was highly significant . We also found that possession of <4 CCL3L1 copies was significantly associated with an increased risk of IVIG resistance . Although possession of >4 CCL3L1 copies was also associated with an increased risk of IVIG resistance   while possession of CCR5 HHF*2 haplotype was associated with a salutary IVIG response . Departure from the population average of 4 copies  was associated with a higher risk of IVIG resistance .We next determined whether ll model , possess= 0.146) , possessCCR5 HHF*2 haplotype was associated with a reduced risk of IVIG resistance as well as development of CAL, we next examined whether these associations were due to homozygosity and/or heterozygosity of the HHF*2 haplotype. We observed that heterozygosity but not homozygosity for HHF*2 was associated with a reduced risk of both CAL  and IVIG resistance .Because we observed that the CCL3L1 associates with susceptibility to KD and IVIG response whereas the CCR2-64I-containing CCR5-HHF*2 haplotype is associated with a reduced risk of both CAL development and IVIG resistance. Our finding that deviation from the average CCL3L1 copy number  found in the Japanese population is associated with increased risk of KD is noteworthy because we have previously found that deviation from median copy number of CCL3L1 is also associated with an increased risk of systemic lupus erythematosus (SLE) FCGR3B was associated with increased susceptibility to SLE and primary Sjogren's syndrome Our results suggest that in Japanese children, copy number variation of the segmental duplication bearing CCL3L1-containing segmental duplication in our study population was associated with increased KD susceptibility as well as an increased risk of IVIG failure is unknown. As noted, CCL3L1 is the most potent CCR5 ligand and CCR5 ligands are associated with pro-inflammatory effects CCL3L1-containing segmental duplication that is higher than the population average is associated with increased leukocyte chemoattraction CCL3L1 transcript (data not shown). In this light, it is conceivable that subjects bearing higher CCL3L1-containing segmental duplications may express higher levels of chemokines following antigenic stimulus that in turn may increase the risk of developing KD and possibly, IVIG resistance. In addition to causing an immunologic blockade of Fc receptor and inducing further antibody production, IVIG therapy is also known to play an important role in down-regulation of the cytokine/chemokine levels CCL3L1 gene copy numbers the currently used dose of IVIG may be insufficient to induce the desired degree of down-regulation of chemokines leading to IVIG resistance. The latter along with increased CCL3L1 associated inflammation may provide a partial explanation as to why we observed a trend for possession of a high CCL3L1 copy number and reduced clinical responsiveness to IVIG.The precise mechanistic basis by which deviation from the average copy number of the CCL3L1 copy number is associated with reduced CCL3-CCL3L1 chemokine expression levels CCL3L1-containing segmental duplication, respectively, may explain why all subjects do not respond to a single dose of IVIG and require additional treatments. This hypothesis is supported by the fact that greater than half of IVIG-resistant patients who receive an additional dose of IVIG become afebrile CCL3L1 gene copy associates with KD and IVIG non-responsiveness are currently lacking and require validation.On the other hand, a low CCR5 polymorphisms in KD susceptibility has been investigated previously. Significant attention has focused on the widely recognized 32-bp deletion (Δ32) mutation present in the coding region of CCR5 that is found mainly in populations of European ancestry. We reported previously that there was an inverse relationship between the global distribution of Δ32 allele and the incidence of KD CCR5-Δ32 allele across generations CCR5-Δ32-bearing HHG*2 haploype were modified by CCL3L1 copy number CCR5-Δ32 allele was lower in cases (6.5%) compared to controls (10.7%).The role of CCR5-Δ32-bearing HHG*2 haplotype is rarely found in Asian populations. The results of two prior studies in subjects with KD of European ancestry CCR5 locus also associate with susceptibility to KD. However, in the present study of Japanese subjects, we did not find an association between CCR5 haplotypes and KD susceptibility. By contrast, we did find an association of CCR5 haplotypes with KD outcomes and IVIG-resistance.The CCR5-HHG*2 haplotype was associated with not only reduced KD susceptibility, but also a lower risk of CAL CCR5 HHF*2 haplotype which bears the CCR2-64I polyrmorphism is associated with a reduced the risk of IVIG-resistance and CAL formation. Whether this association suggests an involvement of CCR2, a receptor critically involved in monocyte trafficking and activation, in KD pathogenesis and therapy responses is unclear because the CCR2-64I polymorphism is in linkage disequilibrium with promoter polymorphisms in CCR5CCR5 HHF*2 haplotype with KD-related outcomes may relate either directly or indirectly to inflammation.Early coronary lesions demonstrate marked infiltration of neutrophils ITPKC gene was associated with response to IVIG in US KD children. The results of the present study extend the notion that host genetic factors may influence IVIG resistance. IVIG has been shown to be effective across a range of autoimmune, inflammatory and infectious conditions, as well as for post-infectious complications Many demographic and laboratory factors such as patient age, white blood cell count, and plasma levels of aspartate amino transferase and C-reactive protein have been identified as risk factors for IVIG resistance CCL3L1 and CCR5 in KD CCR5-Δ32 polymorphism which has been intensively scrutinized in persons of European descent, the CCR2-64I polymorphism has also been associated with variable susceptibility to multiple diseases as well. Interestingly, while the CCR5-Δ32 polymorphism is rare among persons of Asian descent, the CCR2-64I-containing HHF*2 haplotype is very common, and has been associated with salutary effects (reduced risk) among persons of Japanese descent for several diseases with immunologic underpinnings including multiple sclerosis CCR2-64I-containing HHF*2 haplotype was associated with beneficial effects for both of these outcomes. Another limitation of our study is that we had to consider CAL outcomes as a dichotomous variable as the clinical centers in Japan did not uniformly use Z scores to characterize the dimension of the lumen of the coronary arteries Although a limitation of our study is the small sample size, our results are concordant with previous data suggesting a role for variations in Notwithstanding these limitations, our findings underscore that genetic determinants influence not only inter-individual differences in KD susceptibility but also inter-subject variation in cardiac complications and treatment response. In conjunction with previous studies that have focused on the relationship between variations in CCR5 and its ligands with KD


This is followed by logarithmic growth from 4 to 8 days with a doubling time of 18 hours, always similar in different animals. Eventually after 8 days and after about 100 million cells an asymptote is reached which is due to a fistula-like communication between blood-stream and peritoneal cavity. This terminal phase (9-10 days) is rapidly followed by death of the mouse.Reliable tumour cell counts can only be achieved between 4 and 8 days.


It is universally acknowledged that genome segment 4 of group A rotavirus, the major etiologic agent of severe diarrhea in infants and neonatal farm animals, encodes outer capsid neutralization and protective antigen VP4.To determine which genome segment of three group A equine rotavirus strains  with P[12] specificity encodes the VP4, we analyzed dsRNAs of strains H-2, FI-14 and FI-23 as well as their reassortants by polyacrylamide gel electrophoresis (PAGE) at varying concentrations of acrylamide. The relative position of the VP4 gene of the three equine P[12] strains varied (either genome segment 3 or 4) depending upon the concentration of acrylamide. The VP4 gene bearing P[3], P[4], P[6], P[7], P[8] or P[18] specificity did not exhibit this phenomenon when the PAGE running conditions were varied.The concentration of acrylamide in a PAGE gel affected VP4 gene coding assignment of equine rotavirus strains bearing P[12] specificity. Diarrheal disease is one of the principal causes of morbidity and mortality among young children in the developing world. Infectious diarrhea of neonatal animals is also one of the most common and economically devastating conditions encountered in the animal agriculture industry. Among an array of infectious agents including bacteria, viruses and parasites, group A rotaviruses are the single most important etiologic agents of diarrhea in infants and young children worldwide and in addition, they are the most commonly identified viral cause of diarrhea in neonatal food animals -4. In 19Reoviridae family, consists of eleven segments of double-stranded RNA numbered 1-11 according to their order of migration in polyacrylamide gels, segment 1 being the slowest and segment 11 the fastest  specificity, however, we did not find any predicted structures that were different between the equine VP4 sequences and the others (data not shown). In addition, we examined the VP4 sequences of selected rotavirus strains to look for potential changes in the equine VP4 sequence that might induce some sort of "pairing" of the ends of the molecule, however, we did not find any good candidate sequences.The relative position of the VP4 gene of three equine P[12] strains  varied (either genome segment 3 or 4) depending upon the concentration of acrylamide. The VP4 gene bearing P[3], P[4], P[6], P[7], P[8] or P[18] did not exhibit this phenomenon when the PAGE running conditions were varied. Caution needs to be exercised when PAGE analyses are used for VP4 gene coding assignment of rotaviruses.Table The standard phenol-chloroform method or TRIzol method was employed to extract rotavirus genomic dsRNA as previously reported ,29. AnalThe authors declare that they have no competing interests.All authors read and approved the final manuscript.LML, XW and ENO carried out the PAGE analyses. YH participated in the design of the study and drafted the manuscript.


The proliferation index after phytohaemagglutinin (PHA) stimulation of isolated peripheral blood mononuclear cells was also investigated. Sixteen patients undergoing hypothermic  and normothermic  cardiopulmormry bypass (CPB) were enrolled in this study. As a control, we evaluated four patients undergoing thoracovascular operations without CPB. Blood samples were collected before CPB but after anaesthesia, every 30 min during CPB, at the end of CPB and 10 min after protamine administration. Both C3a and TGF-β1 increased significantly during CPB and after protamine administration in the hypothermic as well as the normothermic group. In the latter case the increase of C3a and TGF-β1, although more prominent, was not significantl higher than in the former group. Conversely, the proliferation, index of peripheral mononuclear cells had already decreased 30 min after CPB was started and remained depressed throughout the CPB time. These results suggest a possible role of C3a and TGF-β1 in the immunological changes occurring during extracorporeal circulation.The aim of this study was to evaluate plasma levels of two mediators with immunosuppressive properties, complement fraction C3a (C3a) and transforming growth factor-β


Pax6-/- ES cells died rapidly after neuronal differentiation in vitro.Embryonic stem (ES) cells can differentiate into all cell types and have been used extensively to study factors affecting neuronal differentiation. ES cells containing mutations in known genes have the potential to provide useful in vitro models for the study of gene function during neuronal differentiation. Recently, mouse ES cell lines lacking the neurogenic transcription factor Pax6 were reported; neurons derived from these Pax6-/- ES cells and the assessment of their ability to survive and differentiate both in vitro and in vivo. Neurons derived from our new Pax6-/- lines were viable and continued to elaborate processes in culture under conditions that resulted in the death of neurons derived from previously reported Pax6-/- ES cell lines. The new lines of Pax6-/-ES cells showed reduced neurogenic potential, mimicking the effects of loss of Pax6 in vivo. We used our new lines to generate Pax6-/- ↔ Pax6+/+ chimeras in which the mutant cells survived and displayed the same phenotypes as Pax6-/- cells in Pax6-/- ↔ Pax6+/+ chimeras made by embryo aggregation.Here we report the derivation of new lines of We suggest that loss of Pax6 from ES cells reduces their neurogenic capacity but does not necessarily result in the death of derived neurons. We offer these new lines as additional tools for those interested in the generation of chimeras and the analysis of in vitro ES cell models of Pax6 function during neuronal differentiation, embryonic and postnatal development. Pax6 cause failure of eye morphogenesis and severe abnormalities of brain development  that were Pax6+/- and homozygous for a reiterated β-globin repeat transgene  and disrupted to give individual clusters of between 1-5 cells. Cell clusters were then transferred to a fresh gelatinised well containing N2B27 media containing LIF (1000 U/ml) with the addition of BMP4 (10 ng/ml). Primary colonies of ES cells were visible after approximately 5 days in culture. ES cell lines were passaged at least twice in feeder-free conditions in BHK-21 Glasgow MEM  with 15% fetal bovine serum (FBS) and LIF (1000 U/ml). Cell lines were genotyped as described previously [ES cells that were either wild type  supplemented with N2 and B27  for 6 or 8 days  before fixation and processing.The neural differentiation protocol followed a previous description . BrieflyPax6+/- animals were isolated. Each cortex was kept separate, its cells were dissociated into separate wells and were allowed to grow for 7 days in DMEM:F12 1:1 culture media supplemented with 100 μg/ml transferrin, 25 μg/ml insulin, 20 nm progesterone, 30 nm sodium selenite, 60 μg/ml putrescine, 20 ng/ml EGF and bFGF . The genotypes of individual embryos were discovered by PCR analysis as described previously [The cortices of E11.5 embryos generated from timed matings of Antibodies used in this study were against β-III-tubulin , microtubule associated protein 2 , UK, Pax6 , glial fibrillary acid protein  and BrdU . Visualisation was achieved using Alexafluor-488 or Alexafluor-568  conjugated secondary antibodies and cell nuclei were counterstained using TOPRO3 . For counting, 3-8 areas were selected randomly per well. Apoptotic cells were identified by their dense chromatin condensation visible with TOPRO3 staining. Chromatin condensation was easily recognized, is a defining feature of apoptosis  and has For flow cytometry, cells were stained with propidium iodide to allow discrimination of single cells and analysis of DNA content. Staining reactions were carried out in duplicate. Cells were analyzed on a Beckman-Coulter XL flow cytometer with Expo32 software. 8,000-20,000 cells were analyzed per sample.Pax6-/- ES cells were dissociated using Tryple Express™ and plated in GMEM media containing LIF and FBS. Twenty-four hours later they were co-transfected using Fugene  with pCMV-Script plasmid containing the full length Pax6 cDNA [Sub-confluent ax6 cDNA  and plasPax6+/+ ES cells (wtMM1-9) and three lines of Pax6-/- ES cells (SeyD1-3) containing the Tg transgene [Pax6-/- ES cell lines displayed no abnormalities of growth during routine passage; two (SeyD1 and SeyD2) were analysed in detail.We established 9 lines of ransgene . Karyotyransgene . Pax6-/-Pax6-/- line SeyD1 was assessed by injection into wild type blastocysts to create chimeras. We produced ten Pax6-/- ↔ Pax6+/+ chimeras aged between embryonic day (E) 10.5 and postnatal day (P) 5 and ten control Pax6+/+ ↔ Pax6+/+ chimeras aged between E10.5 and E14.5. In controls, Tg+ cells derived from wtMM4 were distributed throughout eye and brain tissues, which exhibited no abnormal phenotypes. In Pax6-/- ↔ Pax6+/+ chimeras, Tg+Pax6-/- cells derived from SeyD1 showed abnormal distributions in eye and brain tissues, similar to those reported previously for Pax6-/- cells in Pax6-/- ↔ Pax6+/+ chimeras produced by aggregation of Pax6+/+ and Pax6-/- morulae [Pax6-/- cells derived from SeyD1 formed clusters in the subventricular zone of the embryonic cortex  from previously-derived genitors . In agreu et al.  of theirPax6-/- lines at Day 8 .Proportions of apoptotic cells on Day 6 and Day 8 after LIF withdrawal were low with no significant differences between the wild-type and the Pax6-/- ES cell lines SeyD1 and SeyD2 lines showed a significantly lower proportion of cells positive for β-III-tubulin and MAP2 (around 20-30%) in Day 14 cultures  population of cells by flow cytometry as described previously [Pax6+/+, 12.6% ± 2.10 s.e.m.; Pax6-/-, 7.8% ± 2.85, n = 3 cultures each). These data indicate that loss of Pax6 in primary neural progenitors does not increase cell death.To test whether primary neural progenitors derived from eviously . No signPax6-/- ES cells resembles that of previously derived Pax6-/- ES cells [Pax6-/- progenitors taken from mutant embryos [Pax6-/- mutant lines produced EBs containing proliferating progenitors in large proportions that were similar to those in EBs of wild type ES cells and of previously derived Pax6-/- ES cells. This agrees with a previous conclusion that the generation of early neurogenic progenitors from ES cells is not perturbed in the absence of Pax6 [Pax6-/- ES-derived cells in the dorsal telencephalon of chimeras. Thirdly, previous studies have shown that primary Pax6-/- progenitors from mutant cortex generate reduced proportions of neurons in culture [Pax6-/- ES cells in vitro. Together these data suggest that our novel Pax6-/- ES cell lines are mimicking the in vivo and in vitro characteristics of neuronal progenitors derived from primary tissue of the Pax6-/- embryo.In many respects, the in vitro development of our new ES cells  and that embryos . Firstly of Pax6 . Secondl of Pax6  showed t culture ; we founPax6-/- ES cell lines developed quite differently to Pax6-/- ES cell lines reported previously. Our Pax6-/- ES cell-derived neurons showed no signs of early failure of process development and the extensive cell death previously reported; on the contrary, they were continuing to develop ever more elaborate processes at an age when virtually all of the previously described cells had died [In one respect, however, our new had died .Pax6-/- ES cells, possible explanations for this difference in viability must centre of the nature of the ES cells themselves. One possibility is variation in genetic background. Our ES cells were derived from Pax6 mutant mice inbred on a 129Sv(Ola) background whereas those used by Nikoletopoulou et al. [Pax6-/- phenotype [Since the culture protocol we used is similar to that used to differentiate previously derived u et al.  were notPax6-/- cells derived from their mutant ES cells in mouse chimeras. They did, however, describe the introduction of Pax6-/- ES cell derived progenitors into chick telencephalon: some of the neurons derived from these cells survived in the chick telencephalon, suggesting that even in these cells the absence of Pax6 does not necessarily cause extensive cell death given an appropriate supporting environment. Therefore induction of Pax6 genotype-dependent neuronal cell death might depend on both the genetic background of the mutant cells and the environment in which they are placed. It seems quite conceivable that loss of Pax6 from ES cells might cause the rapid death of all derived cells with some genetic backgrounds in vitro but not in vivo, whereas loss of Pax6 from ES cells with other genetic backgrounds might generate cells that are viable both in vitro and in vivo, as is the case in our novel Pax6 mutant cell lines.Nikoletopoulou et al.  did not Pax6-/- ES cell derived neurons in culture reflects an increased incidence of cell death in the cerebral cortex of Pax6-/- mice, other studies did not report an increase in apoptotic cells in the Pax6-/- cortex in vivo or among Pax6-/- primary cells in culture [Pax6-/- neurons have been shown to survive for extended periods, even into postnatal life following transplantation into wild type embryos [Pax6 [Pax6-/- ES-derived neurons in chimeras also survived into postnatal life. Overall, most published studies point to the importance of Pax6 in the early regulation of neurogenesis [Although Nikoletopoulou et al.  suggeste culture ,9,30. Da embryos  and follos [Pax6 . In the ogenesis ,9,30-33,Pax6-/- ES cells that develop in ways predicted from previous work on Pax6-/- progenitors and neurons in vivo and in vitro. Neurons derived from our new lines show enhanced viability compared to neurons derived from previously studied Pax6-/- ES cell lines. Our new lines offer a good model for the study of Pax6 gene function in vivo and in vitro.In conclusion, we have successfully derived JQ and MM carried out the ES cell derivation and JQ, MM and TN performed the differentiation analysis. JQ, JM and DP conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


There are relatively few articles addressing long-term follow-up in women with breast cancer at very young ages.BRCA1, BRCA2 and TP53, with 115 of 288 consecutive cases being tested. Kaplan–Meier curves were generated to assess overall survival, contralateral breast cancer and other second primaries.We have updated and extended our population-based analysis of breast cancer diagnosed at the age ⩽30 years in North-west England to include an extra 15 patients with mutation testing in P=0.05). Contralateral breast cancer rates were at a steady rate of 0.6 per 1000, although the rates in mutation carriers were ∼2 per 1000. Altogether, 16 BRCA1, 9 BRCA2 and 6 TP53 mutations have now been found among the 115 cases on whom DNA analysis has been performed. BRCAPRO accurately predicted the number of carriers for BRCA1 and BRCA2 and was sensitive and specific at the 10 and 20% threshold, respectively. However, BRCAPRO did not seem to give any weight to DCIS, which accounted for two BRCA1 carriers and three TP53 carriers and overpredicted mutations at the high end of the spectrum, with only 6 of 11 (54%) with a >90% probability having identifiable BRCA1/2 mutations.Survival analysis of all 288 patients showed poor overall survival, although this improved from a 15-year survival of only 46% in those diagnosed between 1980 and 1989 to 58% in those diagnosed between 1990 and 1997 (Rates of new primaries are predicted to some extent by mutation status. BRCAPRO is useful at determining those patients aged ⩽30 years to be tested. BRCA1, BRCA2 and TP53 account for a proportion of early-onset and familial breast cancer. These mutations confer a lifetime breast cancer risk of 43–85% (BRCA1/2 and TP53 mutations in families with breast and/or ovarian cancer or Li–Fraumeni (LFS) or LFS-like (LFL) syndrome  and data are lacking for TP53.Mutations in BRCA1/2 and TP53 mutations in early-onset breast cancer in earlier papers. These papers showed a predominance of BRCA1/2 mutations in familial aggregations of breast cancer, with penetrance estimates of breast cancer as high as 80–90% by 70 years of age. This paper presents a detailed analysis of survival, contralateral breast cancer and other tumour incidence in index cases aged ⩽30 years, both with and without BRCA1/2 or TP53 mutations. It also presents results of including an extended series of BRCA1/2 and TP53 index cases diagnosed with breast cancer at ⩽30 years of age.The 288 original patients were ascertained from a population-based series of consecutive breast cancers from the North Western Cancer Intelligence Service (NWCIS) diagnosed with breast cancer at or under 30 years of age between 1 January 1980 and 31 December 1997. Diagnoses were confirmed using hospital records and pathology reports. Those patients who were proven to have a histological diagnosis other than breast carcinoma were excluded. Patients were approached through their consultant for permission to be interviewed and, after informed consent, to provide blood for DNA testing. A detailed three-generation pedigree was obtained along with copies of hospital notes, where available. Family histories of malignancy were confirmed through the cancer registry, hospital notes or death certificates.Patients were classified as familial, LFS, LFL or non-familial groups on the basis of FH at initial diagnosis. Familial patients were defined as those with a FH of breast cancer <65 years of age or ovarian cancer at any age in first- or second-degree relatives at the time of the index cases’ primary breast cancer diagnosis. Both LFS and LFL were defined as in BRCA1/2 or TP53 were included for some analyses of contralateral and other tumour risks to enrich the inherited subtypes. Of these patients, 24 were diagnosed in the northwest after 31 December 1997.A further series of 84 patients diagnosed with breast cancer at or under 30 years of age who had tested positive for mutations in in situ (DCIS) included and excluded. A comparison between cases diagnosed before and after 1990 was also undertaken. Kaplan–Meier curves were derived for the occurrence of contralateral breast cancer after the initial diagnosis and for other cancer diagnoses following initial breast cancer diagnosis with the inclusion of mutation carriers from the additional data set. Kaplan–Meier curves for contralateral breast cancer from diagnosis were derived using the enriched data set with a confirmed FH or mutation: comparisons were made between no FH (sporadic): BRCA1; BRCA2; TP53; and FH but no mutation.Kaplan–Meier (KM) survival curves from diagnosis of breast cancer were derived from the original series alone, with ductal carcinoma BRCA1, BRCA2 and TP53 as described previously  or, in the absence of FISH, if the immunohistochemical score was 3+ .Pathology was taken from the original pathology report and NWCIS entry. All available tumours from patients who had donated blood samples for DNA analysis were reviewed by a single pathologist (FK). Oestrogen receptor (ER) , PR (PGR312 Novocastra) and HER2 (c-erbB2 CB11 Novocastra) immunohistochemistry was performed in cases in which tissue was available. Both ER and PR were scored using the Allred (Quick) score based on the assessment of both proportion and intensity of staining. Her-2 was scored according to standard protocol  from the University of Texas Southwestern Medical Center . Additional information about CancerGene is available at http://www.utsouthwestern.edu. The Manchester scoring system was used for comparison  were derived for survival and contralateral tumour incidence.Assessment of KM curves was undertaken by A total of 276 women were registered on the NWCIS with confirmed early-onset primary breast carcinoma diagnosed between 1 January 1980 and 31 December 1997 in the strict regional boundaries. At original ascertainment between 1993 and December 1997, 116 (42%) women were dead and 160 (58%) were alive. As of December 2008, 144 (52%) women were dead and 132 (48%) were alive. The age of diagnosis and grade of tumour did not significantly differ between the living and dead cases. The mean age at diagnosis was 28 years and 3 months (range 18 years 5 months to 30 years 11 months). Consultant permission to approach the patient was refused for 26 living patients (4 subsequently came forward unprompted). Of the remaining 135 cases, 102 consented to participate, 32 refused and 1 could not be traced. Blood samples were available from a further five deceased patients, and were tested after family consent. Further blood samples were obtained from an additional 8 of 12 women affected at the appropriate age and study period on the NWCIS, but outside the strict regional boundaries. As such, genetic status could be established for 115 women from the NWCIS who developed breast cancer at ⩽30 years of age (15 more than our previous report).In all, 46 patients (42%) had a significant FH, which was consistent with LFS or LFL in 6 cases. A further five women had a FH of breast cancer, which was not consistent with the original study (breast cancer in third-degree relatives or >65 years of age). The remaining 64 cases had no known FH of breast or ovarian cancer at the time of diagnosis and were classified as non-familial.BRCA1, BRCA2 and TP53 were identified in 31 women: 26 of 53 (49%) familial and 5 of 62 (8%) non-familial cases  diagnosed with breast cancer at ⩽30 years of age. BRCA2 mutations were detected in nine women (8%). Pathogenic TP53 mutations were found in 5 patients (5%) including 3 of 6 (50%) of the LFS/LFL subgroup. The updated analysis demonstrated an additional TP53 mutation: 659A>G in a family fulfilling LFS criteria, four further BRCA1 mutations  in four familial breast cancers and 1 BRCA2 mutation (1058C>A) out of the 15 additional samples obtained since our last report (BRCA1 mutation among sporadic cases increase above 10% (2 of 16). However, the overall detection rate in all triple-negative grade 3 cases including those with a FH was 10 out of 27 (37%).Pathogenic BRCA1 mutations diagnosed at ⩽30 years of age after 31 December 1979 and 19 with BRCA2 mutations with the same criteria were identified from our clinic mutation database. In addition, six TP53 carriers diagnosed at ⩽30 years of age were found. In all, 13 of the total 84 mutation carriers  have had risk-reducing contralateral mastectomy (RRM) representing 20% of the total. We are not aware of any RRMs being performed in the remaining patients.An additional 30 patients with in situ as their initial diagnosis. Of these cases, two had comedo DCIS and were shown to have a TP53 mutation. They subsequently died from a primary glioma and retroperitoneal sarcoma, respectively. Two patients died from subsequent ipsilateral invasive breast cancer and one from non-cancer-related event. Survival analysis from diagnosis in the whole data set is presented in P=0.05).Of the 288 cases in the population-based series, 18 had carcinoma TP53 carrier, 4 in BRCA1 carriers and 1 in BRCA2. In the enriched mutation carrier data set, there is a 2% annual risk of contralateral breast cancer up until 15 years, after which the number of patients is too small for any stable estimate , no ovarian cancers have occurred. Of 59 women with intact ovaries, 25 are now >40 years of age.The incidence of non-breast primary cancer was 4.2% at 25 years . SurprisP=0.05). In isolated sporadic unilateral cases, the only patient exceeding the 10% threshold (25.8%) with BRCAPRO was a Jewish woman who did not have a mutation. In 11 families with a predicted BRCA1/2 mutation status >90% (10.47 predicted), only 6 BRCA1/2 mutations were found (1 in BRCA2). Two of the TP53 carriers had a predicted BRCA1/2 score of >90% . The prediction of DCIS cases is also presented in BRCA1 carriers with DCIS reached the 10% combined threshold, it is clear that this was because of the very strong FH. Indeed, there seems to be no weighting for DCIS, as sporadic cases had a 0% rating for BRCA1 and even those with a FH were no more likely in BRCAPRO to be a BRCA1 carrier than an unaffected sibling.Performance of the BRCAPRO model and myriad tables can be seen in The myriad model did not perform well with poor sensitivity, especially at the 20% level. The Manchester score was partly developed using initial data from this series before the extended follow-up and addition of 15 women. Using the updated pathology-adjusted Manchester score .BRCA1/2 and TP53 mutations in this cohort , demonstrating the importance of accurately documenting a FH when estimating the likelihood of a mutation carrier. Few mutations were found in those women without a FH. These data would therefore not support the testing of BRCA1/2 in patients below the age of 31 years without a FH of breast cancer. In particular, even in sporadic cases with grade 3 cancers, the detection rate was only 5%. Among grade 3 triple-negative sporadic cases, the rate of mutation detection was only 12.5% (2 of 16). BRCA1 mutation by pathological grade and ER status at various ages in women not selected by FH. These data were also from an unselected series. Women aged between 20 and 29 years with grade 3 ER-negative breast cancers had a 35% chance of a BRCA1 mutation, with a similarly high risk for women aged 30–34 years at 26.5%. Only after the age of 34 years did the risks fall below 10%  was highlighted in 1990 . This moTP53 mutation. One patient with a renal carcinoma and a de novo TP53 mutation went on to develop a sarcoma behind the remaining kidney that was subjected to regular screening radiation from intravenous urograms. An oesophageal cancer also occurred in a BRCA2 carrier. However, surprisingly, no ovarian cancers occurred in BRCA1/2 carriers, even though 21 women have lived beyond 40 years of age without an oophorectomy. Population studies have indicated that the subsequent ovarian cancer risk is higher if an individual is diagnosed with breast cancer at a young age, particularly if there is a FH of ovarian cancer (BRCA1/2 mutation rather than another mechanism increasing ovarian cancer risk. The data from our study do not suggest a higher ovarian cancer risk in women with known mutations with early-onset breast cancer, compared with mutation carriers with later-onset disease.The diagnosis of a second primary tumour should prompt the clinician to consider a BRCA1/2 mutations even in early-onset breast cancer cases without a FH are low. An adopted sporadic case would need to be diagnosed at the age of ⩽24 years to breach the threshold using BRCAPRO. Nevertheless, BRCAPRO does seem to overestimate the BRCA1/2 probabilities in bilateral cases. It is also interesting to note the handling of DCIS. Although both BRCA1 carriers with DCIS were identified using the 10% combined threshold, they were as likely to be a carrier as an unaffected sibling. It seems that no weight is given to DCIS to increase either the BRCA1 or BRCA2 probability of either the individual or the family in BRCAPRO. Ductal carcinoma in situ at the age of ⩽30 years is clearly an important diagnosis, as 45% of the 11 cases had a pathogenic mutation, with DCIS being a particular marker for a TP53 mutation. The myriad tables (TP53 carriers had scores above 94%, yet their FHs were consistent with LFS. We have estimated that a combined score of 40+ using the Manchester score suggests a very high probability of a BRCA1/2 mutation (This study assessed the performance of mutation prediction models in this population-based data set. We chose to assess the models at last follow-up to determine how the models dealt with new contralateral disease. Direct comparisons with the Manchester scoring system are inappropriate, as this system was partly developed from the original data set at the time of diagnosis. Another model, BOADICEA, also used this data set in development (The strengths of this study are that it is population based and has obtained DNA samples on the great majority of living cases. Overall death rates and second primary rates are secure because of the cancer intelligence service.This study does have some limitations. The pathology reports on patients diagnosed before 1990 had little information with regard to grade and do not have information regarding hormone receptor status. It was not possible to determine reliable survival data on the basis of tumour grade as a result of this deficiency. However, in the group that submitted a blood sample, a detailed pathological review was possible in most cases.BRCA1, BRCA2 and TP53 mutations in women aged ⩽30 years with a FH. Among sporadic patients, mutations are generally in those with grade 3 triple-negative tumours. Rates of further primary tumours other than contralateral breast cancer are not high, except in TP53 carriers, and very young-onset BRCA1/2 carriers do not seem to be at enhanced risk of ovarian cancer compared with other BRCA1/2 carriers. Contralateral breast cancer rates seem stable at ∼0.6% annually in all women and at 2–3% in mutation carriers.This analysis has shown an improvement in survival for women with very early-onset breast cancer in more recent years. There are high rates of


We investigated K-ras mutations in cell samples microdissected by laser capture microscopy at multiple sites from lung tissue sections representing tumour tissue and matched histologically normal tissue obtained from 48 lung cancer patients. K-ras mutations were detected in cell samples from 10 of 38 (26.3%) lung adenocarcinomas and in none of the histologically normal or tumour cell samples taken from 10 lung squamous cell carcinomas. Of the K-ras mutation-positive adenocarcinomas, in 4 cases a mutation was found in only the tumour tissue, in 1 case a mutation was found only in the histologically normal tissue, and in 5 cases mutations were found in both the tumour tissue and histologically normal tissue. Among these 5 cases, 2 had identical mutations in both the tumour tissue and histologically normal tissue, 2 had 1 mutation in the tumour tissue and 2 mutations in the histologically normal tissue, 1 of which was identical to the mutation found in the tumour, and 1 case had 2 codon 12 mutations in tumour tissue and 2 mutations, in codons 9 and 11, in histologically normal tissue. These results showed that K-ras mutations are frequent in histologically normal cells taken from outside lung adenocarcinomas and suggest that some of these mutations may represent early events which could pave the way of lung carcinogenesis. © 2001 Cancer Research Campaign http://www.bjcancer.comMutations in the K-


P<0.01). Female sex, increased age, elevated alanine transaminase and urea and early grade 1 PPE were significant independent prognostic factors for the development of CTC grade 2 or worse stomatitis (P<0.01). Early CTC grade 1 diarrhoea predicted CTC grade 2 or worse diarrhoea (P<0.01). Older, female patients with good performance status and impaired liver and renal function who develop early grade 1 PPE alone or in combination with diarrhoea are at highest risk of subsequently developing grade 2 or worse PPE or stomatitis during treatment with PVI 5FU. Reduction of infused 5FU dose should be considered for these patients. Such an approach could both reduce severe toxicity owing to chemotherapy and minimise treatment delays, and should be evaluated prospectively.This study used a prospectively managed clinical database in order to identify 1470 patients with gastrointestinal cancers receiving protracted venous infusion (PVI) fluorouracil (5FU). It aimed to determine the time course of toxicity due to PVI 5FU and to analyse factors predicting toxicity. The initial development of stomatitis occurred more rapidly than diarrhoea or palmar plantar erythema (PPE). The percentage of patients with National Cancer Institute Common Toxicity Criteria (CTC) grade 2 or worse PPE peaked at 9% between weeks 8 and 17, whereas this peak occurred earlier for stomatitis and diarrhoea. The development of CTC grade 1 toxicity in the first 28 days after commencement of chemotherapy was classified as early grade 1 toxicity. Multivariate Cox regression analysis showed that female sex, better performance status, elevated bilirubin, early grade 1 PPE and early grade 1 diarrhoea were independent prognostic factors for the development of CTC grade 2 or worse PPE ( Fluorouracil (5FU)-based chemotherapy is used for the adjuvant and palliative treatment of a variety of different tumour types, particularly for gastrointestinal cancers. A recent meta-analysis demonstrated that the efficacy of 5FU was dependent on its schedule of administration. Infused schedules of 5FU resulted in superior response rates with a small but statistically significant prolongation of survival in advanced colorectal cancer compared with bolus schedules . In addiThe development of chemotherapy-induced toxicity has an adverse impact on quality of life and in the most severe cases may cause death during treatment. The management of toxicities owing to chemotherapy are generally made on an empirical basis only after the development of dose-limiting side effects and generally involve interruption of chemotherapy and dose reduction in order to prevent future episodes. This study aimed to analyse the principal dose-limiting toxicities of infused 5FU therapy observed in a large cohort of patients treated at a single centre. It aimed to identify clinical, laboratory and temporal factors, which could be combined in order to identify those patients who were at greatest risk of toxicity.The Gastrointestinal Unit (GI unit) at the Royal Marsden Hospital (RMH) has maintained a prospective database of all patients receiving chemotherapy. This database contains details of baseline demographic data, haematological and biochemical parameters, chemotherapy administration, tumour response to chemotherapy and survival and toxicity data graded according to the National Cancer Institute Common Toxicity Criteria version 1  7 mg/m2 q 6 weekly. MMC is associated with a mild and predominantly haematological toxicity profile; consequently in patients receiving MMC with PVI 5FU, the toxicities of diarrhoea, PPE and stomatitis were considered to most likely result from 5FU  5FU 300 mg mTreatment with PVI 5FU with or without MMC was continued up to 24 weeks, unless patients developed progressive disease or excessive toxicity.−2 dose reduction, grade 3 toxicities to 100 mg m−2 dose reduction and grade 4 toxicities to 150 mg m−2 dose reduction. Therefore, for this analysis dose-limiting toxicities were defined as grade ⩾2 toxicities as this severity of toxicity would usually result in a dose reduction.Nonhaematological toxicities developing during PVI 5FU therapy were managed uniformly as follows. Grade 1 stomatitis and diarrhoea were treated with oral sucralfate and codeine phosphate, respectively, and grade 1 PPE was treated with oral pyridoxine. No dose reduction or delay of 5FU was made for grade 1 toxicities. Grade 2 or worse nonhaematological toxicities led to cessation of 5FU treatment until resolution of toxicity followed by a dose reduction of 5FU except for PPE developing for the first time after 10 weeks of therapy, where treatment was initially recommended at the original starting dose. Otherwise, grade 2 toxicities led to a 50 mg mP-values of less than 0.01 were considered significant.Time to the development of toxicity was calculated using the inverted methods of Between January 1994 and January 2001, 1470 patients with gastrointestinal cancer received treatment using infused 5FU at the Royal Marsden Hospital (RMH). The baseline demographics of these patients are shown in Figure 1A-3AThe time course of grade 2 or worse toxicities was studied in further detail, as this severity usually required modification of chemotherapy dose. The proportion of patients with grade 2 or worse diarrhoea, PPE and stomatitis was therefore determined during each week of chemotherapy. These results depict both initial as well as subsequent episodes of grade 2 or worse toxicities for all patients who were continuing to receive chemotherapy. The denominator at each time point was the number of patients receiving chemotherapy at that time. The percentage of patients with grade 2 or worse diarrhoea peaked at about 4.5% between weeks 5 and 8. Approximately 30% of patients with grade 2 or worse diarrhoea also had another grade 2 or worse toxicity . The perP<0.001) and stomatitis . This result suggested that the time at which grade 1 toxicity initially developed could be used to identify patients who were at higher risk of subsequently developing a more severe toxicity.While the development of more severe toxicities led to a dose delay or dose reduction, the development of individual grade 1 toxicities alone did not mandate any dose modification. The risk of developing a worse grade toxicity after the development of an initial grade 1 toxicity was analysed in more detail, in order to assess whether the time of onset of grade 1 toxicity had any prognostic significance for the subsequent development of more severe toxicity. Univariate Cox regression analysis demonstrated that each extra day after commencing chemotherapy before the development of a grade 1 toxicity resulted in a lower risk of subsequently developing a grade 2 or worse toxicity in the case of PPE (hazard ratio (HR) 0.99, 95% confidence interval (CI) 0.985–0.994; The median time until the first development of any grade 1 toxicity was 28 days. Patients with early grade 1 toxicities were therefore defined as those patients who developed grade 1 but not grade 2 or worse diarrhoea, PPE or stomatitis before completing 28 days of chemotherapy. This time point also predates the times at which the largest proportions of patients have grade 2 or worse toxicities see .P<0.001). A total of 19.9% of patients with early grade 1 diarrhoea subsequently developed grade 2 or worse diarrhoea compared with 11.9% of patients without early grade 1 diarrhoea (P=0.003). Totally, 19.5% of patients with early grade 1 stomatitis subsequently developed grade 2 or worse stomatitis compared with 11.4% of patients without early grade 1 stomatitis (P=0.003).In all, 55.1% of patients with early grade 1 PPE subsequently developed grade 2 or worse PPE compared with 27.3% of patients without early grade 1 PPE  and renal function (urea⩾4.8 mmol/l−1) who develop early grade 1 PPE alone or in combination with diarrhoea could be considered for early dose reduction, at the time of development of early toxicity, as this group of patients is at the highest risk of grade 2 or worse PPE and stomatitis.The multivariate Cox regression analysis allows identification of subgroups of patients at higher risk of toxicity with PVI 5FU. Based on the multivariate Cox regression analysis, older, female patients with good performance status, impaired liver function . Severe 5FU toxicity may occur in patients with partial or complete DPD deficiency and these individuals show marked impairment of 5FU clearance (On this basis, measurement of DPD levels has been suggested as a possible method to identify patients at increased risk of 5FU toxicity. However, there are limitations with such an approach. Although the majority of 5FU is metabolised by the liver, assessment of DPD levels is typically based on the levels of the enzyme expressed in peripheral blood mononuclear cells because of their easier accessibility. Unfortunately, assessment of peripheral blood DPD levels correlates only weakly with 5FU clearance and is a poor predictor of 5FU toxicity (An alternative approach to predict and ultimately prevent severe 5FU toxicity is pharmacokinetic monitoring. Patients with DPD deficiency may demonstrate a markedly altered pharmacokinetic profile with significant reduction in the plasma levels of 5FU metabolites. The impaired clearance of 5FU is thought to contribute to its toxicity in these patients. Pharmacokinetic evaluation provides a more direct information about whole-body 5FU catabolism, and small studies suggest that with infused 5FU, higher plasma levels of 5FU correlate with increased toxicity (This study provides a strong empirical basis for the use of information about the temporal development of toxicity in combination with baseline clinical factors in order to assess the risk of toxicity. While it appears logical that patients who develop early grade 1 toxicity would be at higher risk of developing a subsequent more severe toxicity, the extent of the risk and the possible implications for patient management have never previously been evaluated. Thus, the data suggest that older female patients with good performance status and impaired liver function who develop early grade 1 PPE or the combination of early grade 1 PPE and diarrhoea would be at a higher risk of grade 2 or worse stomatitis and PPE. Such patients could receive a reduction in the dose of 5FU at the time of development of early grade 1 toxicity, which could prevent the development of more severe toxicity altogether. This approach, if successful, would be likely to achieve significant benefit in improvements in quality of life as well as minimise treatment interruption due to toxicity.However, in order to evaluate rigorously such an approach, it will be important to validate the models generated in this study prospectively. It will also be important to study the use of early dose reduction for selected patients at high risk of toxicity prospectively. This should involve assessment of response, progression-free survival, overall survival and quality of life in addition to toxicity outcomes in order to determine whether efficacy and palliative benefit is affected using such an approach.An increased understanding of factors associated with toxicity due to chemotherapy may potentially allow early identification of patients at risk of severe toxicity. This study suggests that evaluation of the temporal pattern of toxicity can assist in this process. Similar approaches using data derived from large patient cohorts treated with other chemotherapy agents may allow identification of prognostic factors, including temporal factors associated with toxicity due to other drugs, which could prove useful for clinical management.


From December 2003 through January 2004, the Phnom Tamao Wildlife Rescue Centre, Cambodia, was affected by the highly pathogenic influenza virus (H5N1). Birds from 26 species died. Influenza virus subtype H5N1 was detected in 6 of 7 species tested. Cats from 5 of 7 species were probably infected; none died. On January 24, 2004, the first confirmed outbreak of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in Cambodia was reported to the Office International des Epizooties . The center is divided into 3 main sections that cover 37 ha. Birds were kept in sections S1–1, S1–2, and S2, and the cats were in all sections . The infSpilornis cheela), was reported on December 15, 2003, in S2 (Nycticebus sp.), did not become ill. None of the 27 animal keepers, who were 20–50 years of age, were reported to have gotten sick.The first case, in a crested serpent eagle . The cats were sick for 5–7 days and exhibited anorexia and lethargy but no respiratory illness.>96.5%) to HPAIV (H5N1) strain H5 sequences from Vietnam and Thailand in 2004 (data not shown). All belonged to the H5 clade 1 (>97.12% identity) and to 2004 Vietnamese and Thai N1 sequences (>96%) (data not shown). The HA and NA sequences of the isolates were deposited in GenBank .Laboratory investigations of the organs from 8 birds sampled in December 2003 were performed . For thoRetrospective investigation of the villages surrounding the PTWRC and Phnom Penh showed that chickens from 2 flocks in which deaths had been reported in mid-December had been provided to the PTWRC, either for the restaurants or for the captive animal feeding. Furthermore, at the time of the outbreak, many wild crows were found dead in the forest surrounding the PTWRC.The 4 cat serum samples, each from a different species, were positive for HPAIV (H5N1) with serum neutralization test . None ofThe sources of introduction of HPAIV (H5N1) within the PTWRC were probably multiple: virus-infected chicken bought to feed the carnivorous species, infected live chickens brought to restaurants near S2 , and contact between infected wild and captive birds. The introduction through infected chickens is supported by the absence of an outbreak at the PTWRC after the feeding of chickens to carnivorous species was discontinued; however, deaths in domestic poultry continued in the area. In addition, almost all carnivorous bird species in S2 died  as did most species usually fed chicken meat in captivity . Diet was also the origin of the outbreak among tigers and leopards in Thailand (Corvus macrorynchos) among the 5 species of Passeriformes in the aviaries showed clinical signs and later was confirmed by RT-PCR to be positive for HPAIV (H5N1). This outbreak confirms that Falconiformes and Strigiformes are sensitive to HPAIV (H5N1) infection and disease (–) and shows that numerous species of these orders can be affected by HPAIV (H5N1) (Anas poecilorhyncha), did not show any clinical signs. Heterogeneity in the susceptibility of wild ducks to HPAIV (H5N1), including asymptomatic infection, has been demonstrated  infection at PTWRC ranged from severe illness and death to complete absence of clinical signs, as described (V (H5N1) . PsittacCatopuma temminckii) and the clouded leopard (Neofelis nebulosa) broadens the host range of the virus among mammals.The serologic evidence of influenza virus (H5N1) infection in 4 species of wild cats is in agreement with previous infection in Thailand  infection. It also confirms the broadening range of susceptible species that may be specific to this clade 1 virus.


KIP2, IGF2 and H19 genes was studied by quantitative RT-PCR in 24 paired samples of urothelial carcinomas and morphologically normal mucosa obtained by cystectomy, and in bladder carcinoma cell lines. The most frequent alteration in tumour tissue was decreased expression of KIP2 identified in 9/24 (37%) specimens. Decreased IGF2 and H19 mRNA levels were found in five (21%) and three (13%) tumours, respectively. One tumour each overexpressed IGF2 and H19. Loss of H19 expression was only found associated with loss of KIP2 expression, whereas decreased expression of IGF2 mRNA occurred independently. Almost all bladder carcinoma cell lines showed significant changes in the expression of at least one gene with diminished expression of KIP2 mRNA as the most frequent alteration. IGF2 mRNA levels were diminished in several lines, but increased in others. The KIP2 gene could be an important target of genetic and epigenetic alterations in bladder cancer affecting the maternal chromosome 11p15.5. However, reminiscent of the situation in Wilms’ tumours, expression of the IGF2 gene on the paternal chromosome can also be disturbed in bladder cancers. © 2000 Cancer Research CampaignTo identify targets of genetic and epigenetic alterations on chromosome 11p15.5 in human bladder cancer, expression of the imprinted