KRAS mutations are major factors involved in initiation and maintenance of pancreatic tumors. The impact of different mutations on patient survival has not been clearly defined. We screened tumors from 171 pancreatic cancer patients for mutations in KRAS and CDKN2A genes. Mutations in KRAS were detected in 134 tumors, with 131 in codon 12 and only 3 in codon 61. The GGT>GAT (G12D) was the most frequent mutation and was present in 60% (80/134). Deletions and mutations in CDKN2A were detected in 43 tumors. Analysis showed that KRAS mutations were associated with reduced patient survival in both malignant exocrine and ductal adenocarcinomas (PDAC). Patients with PDACs that had KRAS mutations showed a median survival of 17 months compared to 30 months for those without mutations (log-rank P = 0.07) with a multivariate hazard ratio (HR) of 2.19 (95%CI 1.09–4.42). The patients with G12D mutation showed a median survival of 16 months (log-rank-test P = 0.03) and an associated multivariate HR 2.42 (95%CI 1.14–2.67). Although, the association of survival in PDAC patients with CDKN2A aberrations in tumors was not statistically significant, the sub-group of patients with concomitant KRAS mutations and CDKN2A alterations in tumors were associated with a median survival of 13.5 months compared to 22 months without mutation (log-rank-test P = 0.02) and a corresponding HR of 3.07 (95%CI 1.33–7.10). Our results are indicative of an association between mutational status and survival in PDAC patients, which if confirmed in subsequent studies can have potential clinical application. Pancreatic ductal adenocarcinoma (PDAC) is the most fatal form of pancreatic malignancy with a 5 year survival of less than 4% KRAS being the most frequently mutated oncogene in pancreatic cancer with a reported frequency ranging between 20 and 100%, it has not been so far utilized in categorization of tumors for clinical purposes KRAS mutations in resected pancreatic cancers with prognosis Over the years somatic mutations have been shown to be legitimate targets for anti-cancer drugs because of casual relationship with tumor formation and maintenance KRAS mutations in pancreatic cancer were based on relatively small tumor numbers that lacked statistical power to determine association with the disease outcome. In order to address the issue of frequency of KRAS mutation in pancreatic cancer and impact of those mutations on disease outcome, we have in this study included a series of fully characterized 171 pancreatic tumors with complete patient data.Most of the earlier reports on The 163 patients with malignant tumors in this study comprised the following: i) 143 ductal adenocarcinomas that also included 5 adenosquamous and 4 anaplastic undifferentiated variants, ii) 16 rare carcinomas that were comprised of 2 acinar cell carcinomas, 2 (microcystic) tubulo-papillary carcinomas, 9 intraductal papillary mucinous neoplasm , 2 solid pseudopapillary neoplasms (Frantz tumors) and 1 cystadenocarcinoma, and iii) 4 papillary (ampulla of Vater) carcinomas. The non-malignant group was composed of 4 benign lesions in the form of serous cystic adenomas (SCA) and premalignant lesions in the form of 1 mucinous cystic neoplasm (MCN) and 3 non-invasive IPMN . All patKRAS gene was standardized using DNA from cell lines with known KRAS mutation. The sensitivity of SSCP, determined by titration experiments, showed that point mutations in tumor samples up to 5% tumor content were detectable. This provided confidence that our inclusion of tumor samples, only if those had at least 10% tumor content (n = 171), would more than adequately enable the detection of mutations. Another criterion applied for mutation detection was reproducibility. Mutations were scored only when band shifts were reproducible in at least two independent experiments. Repeat experiments using SSCP followed by DNA sequencing were used for confirmation and identification of mutations (KRAS mutations in a random sub-set (n = 6) analyzed blindly in the reference laboratory of the Institute of Pathology, University Hospital of Heidelberg.Mutation detection for utations . We alsoKRAS gene, we detected 134 mutations in 171 tumors (78%), with 131 mutations in exon 2 and 3 mutations in exon 3 , 3 at codon 83 , followed by solitary tumors with mutations at codon 58 , codon 129 , codon 130  and one tumor had 2 base pair insertion of GG at codon 78 (CTC>CGGTC). Deletions at the 9p21 locus were detected with varying frequency with 17–20% in the CDKN2A (p16INK4a) and 26–28% within the promoter associated with exon 1β of p14ARF transcript.A total of 43 tumors (25%) showed aberrations in the KRAS mutations tended to shorten survival of patients in general  and in all studied sub-categories (except tumor stage T4), however without reaching statistical significance  of 1.87 . The corresponding HR was 1.55  was poorest with median survival time of 13 months compared to 30 months for patients without any mutations in either KRAS or CDKN2A  showed a similar association with KRAS and CDKN2A genes in pancreatic cancer are well documented; however, their influence on disease outcome in patients with exocrine pancreatic tumors has remained unclear. In this study, we observed that KRAS mutation frequency in pancreatic tumors was consistent with ours and other previous European studies that were based on 70–100 tumors and reported a mutation frequency of 72–83% KRAS mutations in pancreatic cancer are believed to be the early events in neoplastic transformation. The hypothesis is supported by mice models based on conditional endogenous expression of the mutant KRAS. Those mouse models were developed with an assumption that KRAS mutation is an essential and early somatic genetic alteration in PDAC progression. Similar observations were reported for KRAS mutations in human acinar-ductal metaplasia (ADM) lesions of pancreas KRAS mutations existed only in the lesions associated with PanIN; the isolated ADM lesions were devoid of any KRAS mutation, with possible involvement of two distinct mechanisms, with and without KRAS mutations KRAS mutation may not be essential for human PDAC progression and other low frequency gene mutations could trigger alternate pathways. The pancreatic genome sequencing of 20,661 genes from 24 tumors identified other low frequency gene mutations KRAS, CDKN2A, TP53 and SMAD4 genes in about 30% of pancreatic tumors Mutations in KRAS gene are part of the conserved ‘G-domain’ (residues 1–165) required for signal transduction. Tumor malignancy depends not on the presence of a KRAS mutation but on the molecular configuration and constituent mutation type KRAS mutation in pancreatic tumors was associated with reduced survival time. Further analysis showed that the association was significant only for G12D sub-type of KRAS mutation. Lack of statistical power owing to low frequency of other mutation types likely precluded observation of the effect on survival. While our data being concordant with the paradigm of distribution of KRAS mutations, we clearly showed that patients, in particular, those harboring G12D mutation in tumors were at a 2-fold increased risk of death compared to those without any KRAS mutation. In an experimental study, the human cell lines with KRAS mutations were classified into KRAS dependent and independent. The ‘classical’ PDAC categorized as KRAS dependent were shown to be potentially amenable to the directed therapy KRAS has been established as a predictor of resistance to the treatment The codons 12, 13 and 61 of KRAS mutations could also be due to varying abilities to alter the RAS protein. KRAS mutations at codon 12, in general, have been shown to increase resistance to apoptosis and activate AKT/protein kinase B pathway KRAS G12D mutant was shown not only to initiate neoplastic lesions but was also involved in tumor maintenance KRAS is reported to promote widespread colonic epithelia hyperplasia and neoplasia KRAS mutation subtypes and survival. Our previous report on paraffin embedded tumors did not show any association between the presence of KRAS mutations and patient survival; however, there was difference in survival between the patients with different mutation types KRAS mutational status as predictive of survival was also reported in an earlier trial study of Gemctabine and Erlotinib therapy in patients with advanced pancreatic cancer KRAS mutations in the surgically negative resected margins have also been shown to be associated with clinical cancer recurrence, aggressive tumor biology and poor survival KRAS mutations in retroperitoneal margins, in the patients with complete pancreatectomy also showed poor prognosis The association between pancreatic cancer patient survival and specific sub-types of CDKN2ACDKN2A in the present study was in agreement with that reported in the COSMIC database G12DKras and Ink4a/Arf showed enhanced progression of pre-malignant lesions to PDAC KRAS and CDKN2A aberrations were at 2.5-fold higher risk of death than patients without any alterations in the two genes. In a previous study it was shown that 1–2 mutations in pancreatic tumors showed a median survival of 23 months compared to 13 months in our present study KRAS mutation KRAS mutations together with loss of heterozygosity on different chromosomal positions The other gene that has been consistently reported to carry high frequency of somatic mutation in pancreatic cancers is KRAS are frequent but not universal in pancreatic tumors and the presence of KRAS mutations in general, and G12D transformation in particular, were indicative of association with poor survival. Our results also showed that concomitant occurrence of KRAS mutations and aberrations in CDKN2A resulted in a sub-group of patients with lowest survival. Our data from this study is suggestive for a case for the prognostic classification of pancreatic cancer patients based on mutational status of KRAS and CDKN2A. However, the results need independent confirmation in additional studies with definite statistical confidence.In conclusion, our results show that mutations in For all samples analyzed, written informed consent was obtained from the patients. The study was approved by the local ethics committee of the University of Heidelberg.Tumor tissues were collected from pancreatic cancer patients during surgery between January 2002 and September 2009, snap-frozen in liquid nitrogen directly after resection and subsequently stored at −80 °C. A total of 171 tumor tissues, that contained at least 10% tumor by H&E staining were analyzed in the present study. The clinical and histopathological characteristics of the patients are given in Three different tissue sections were selected randomly for hematoxylin and eosin (H&E) staining and histological validation. Slides were scanned with the ScanScope GL System  and visualized using the ImageScope Software. For each tissue sample, three pathologists evaluated independently the histology and percentage of normal, tumor and stroma cells . Only saKRAS mutations in codon 12, 13 and 61 were used as controls that included, A549 cell line with G12S (GGT>AGT) mutation; MiaPaCa, G12C (GGT>TGT); SW 1116, G12A (GGT>GCT); SW 620 G12V (GGT>GTT); LS-174, G12D (GGT>GAT); LoVo, G13D (GGC>GAC); HS 766T, Q61H (CAA>CAC). DNA samples from healthy controls were included as negative control.Frozen pancreatic tissue samples were individually cut into 20 µm thick slices with a cryotome Leica CM 1850 UV at −34 °C. The tissue slices were covered with liquid nitrogen and gently ground by three turns with a micropestle made of polypropylene  that fitted into 2 ml Eppendorf tubes. DNA from tissue slices and from cell lines was extracted using the AllPrep Isolation Kit . DNA from cell lines with known 2, 0.11 mM each dNTP, 1 µCi [α-32P] dCTP, 0.2 µM each gene specific primer  which contained 30 probes mapping chromosome 9p21 and 9p22, 13 reference probes and 9 internal controls. Reference probes were located in genomic regions with low frequency copy number changes. The hybridization and ligations were carried out as per instructions and fragment analysis was performed on an ABIPRISM 3130xl capillary sequencer. The data were visualized using peak scanner v1.0 software and the exported data was analyzed with Coffalyser software v8 . Calculation of signal ratios was carried out as described by Mistry et al. MLPA was used to detect homozygous deletions at the Of 171 tumors that were analyzed for mutations, 163 were malignant and 8 non-malignant tumors. Of the 163 patients with malignant tumors, survival data were available for 153 patients, of whom 150 patients had malignant tumors of pancreatic origin including ductal adenocarcinomas (n = 135) and rare carcinomas (n = 15). The rest (n = 3) were carcinoma of ampulla of Vater . The KapFigure S1Histomorphological examination of pancreatic tumor tissue sections with Hematoxylin and Eosin stains. Representative photomicrographs of three sections with low, medium and high tumor contents are shown.(TIF)Click here for additional data file.Figure S2Representative SSCP of KRAS codon 12 and codon 61 in pancreatic tumors. (A) The lanes 1–4 contain amplified fragments of exon 2 (codon 12) and lanes 5–6 contain amplified fragments of exon 3 (codon 61) from tumor DNA samples. The shifted bands seen in lane 1 contain GGT>GAT (G12D) mutation, lane 2 contains GGT>CGT (G12R), lane 3 contains GGT>GTT (G12V) mutation and lane 4 contains tumor DNA without mutation in exon 2. The shifted bands in lane 5 contain CAA>CAC (Q61H) mutation and lane 6 contains tumor DNA without mutation in exon 3. (B) Sequence analysis of a part of exon 2 of KRAS gene (coding strand) with GGT>GAT (G12D) mutation. (C) A part of exon 2 sequence showing GGT>CGT (G12R) mutation. (D) A part of exon 2 sequence showing GGT>GTT (G12V) mutation. (E) A part of the exon 2 showing wild type sequence at codon 12 and codon of KRAS. (F) A part of exon 3 sequence showing CAA>CAC (Q61H) mutation. (G) A part of the exon 3 showing the wild type sequence at codon 61 of KRAS.(TIF)Click here for additional data file.Figure S3Kaplan-Meier survival curves showing difference in overall survival in exocrine cancer patients with and without mutations. (A) Median survival of patients with KRAS mutations was 17 months against 30 months for patients without mutations in the gene. (B) Median survival of patients with KRAS codon 12 GGT>GAT (G12D) mutations was 16 months against 30 months for patients without any mutation in KRAS. (C) Median survival of patients with concomitant alterations in KRAS and CDKN2A genes was 13 months against 30 months for patients without any alterations in both KRAS and CDKN2A.(TIF)Click here for additional data file.Table S1Primer sequences and SSCP conditions for detection of mutations in the KRAS and CDKN2A genes.(DOC)Click here for additional data file.Table S2Mutation frequency by clinic pathology and effect on survival of pancreatic cancer patients.(DOC)Click here for additional data file.Table S3Clinico-pathological details and tumor mutational status of all pancreatic cancer patients.(DOC)Click here for additional data file.


Human infections were from a virus clade undergoing antigenic drift that showed resistance to adamantanes but sensitivity to neuraminidase inhibitors. An outbreak of highly pathogenic avian influenza A (H5N1) has recently spread to poultry in 9 Asian countries. H5N1 infections have caused >52 human deaths in Vietnam, Thailand, and Cambodia from January 2004 to April 2005. Genomic analyses of H5N1 isolates from birds and humans showed 2 distinct clades with a nonoverlapping geographic distribution. All the viral genes were of avian influenza origin, which indicates absence of reassortment with human influenza viruses. All human H5N1 isolates tested belonged to a single clade and were resistant to the adamantane drugs but sensitive to neuraminidase inhibitors. Most H5N1 isolates from humans were antigenically homogeneous and distinct from avian viruses circulating before the end of 2003. Some 2005 isolates showed evidence of antigenic drift. An updated nonpathogenic H5N1 reference virus, lacking the polybasic cleavage site in the hemagglutinin gene, was produced by reverse genetics in anticipation of the possible need to vaccinate humans. Highly pathogenic avian influenza viruses of the H5N1 subtype are circulating in eastern Asia with unprecedented epizootic and epidemic effects , neuraminidase (NA), and M2 are essential viral proteins targeted by host antibodies or antiviral drugs such as oseltamivir and rimantadine  What is the genetic diversity of H5N1 viruses involved in human infections? 2) Can the relationship between human and avian H5N1 isolates help explain the source of infection? 3) Do genetic changes correlate with enhanced viral transmissibility in humans? 4) How sensitive are H5N1 isolates to antiviral drugs? 5) What is the antigenic similarity between human H5N1 viruses and current candidate vaccines? and 6) Can candidate vaccine reference stocks be developed in time for an effective public health response?All work involving infectious H5N1 influenza was performed in government-approved biosafety level 3–enhanced containment facilities with experimental protocols in compliance with applicable federal statutes and institutional guidelines. Influenza A (H5N1) viruses isolated in Asia and A/Puerto Rico/8/34 (PR8) (H1N1) were propagated in embryonated chicken eggs or in Madin-Darby canine kidney (MDCK) cells. The African green monkey kidney Vero cell line was from a cell bank certified for human vaccine production.Viral RNA was extracted by using a commercial lysis solution and resin kit and amplified by reverse transcriptase–polymerase chain reaction with specific oligonucleotide primers. Nucleotide sequencing reactions were performed with a cycle sequencing kit and resolved on an ABI 3100 Genetic Analyzer . DNA sequence analysis was performed by using version 10 of the GCG sequence analysis package (Postinfection ferret antisera were prepared as previously described (50) values for oseltamivir and zanamivir were determined by using NA-Star substrate and Light Emission Accelerator IITM  as previously described  of influenza virus PR8 strain , South Mimms, United Kingdom; Saint Jude Children's Research Hospital (SJCRH), Memphis, Tennessee, USA; and Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA. For brevity, the reverse genetics derivation method described represents a consensus of the institutions; minor unpublished protocol details unique to each laboratory were not described and are available upon request. The VN/04x/PR8 reassortant virus was recovered in embryonated eggs and identified in the allantoic fluid by HA assay. The genetic and antigenic properties of the resulting reassortant virus were determined as described previously . These findings are consistent with the epidemiologic data that suggest that humans acquired their infections by direct or indirect contact with poultry or poultry products . The amino acid sequence of the M2 protein of clade 1 viruses as well as of HK/213/03 indicated a serine-to-asparagine substitution at residue 31 (S31N), known to confer resistance to adamantanes (including amantadine and rimantadine)  and have coevolved with the respective HA genes . No evidInfluenza vaccines whose HA are antigenically similar to circulating strains provide the highest level of protection from infection . Viruses,,,,Antigenic analysis of human isolates from 2005 provided evidence of antigenic drift among the most recently circulating H5N1 strains . For exaThe growing H5N1 epizootic in eastern Asia could expand the environmental load of virus and cause more infections in mammals  can be traced back to viruses isolated in 1997 in Hong Kong (clade 3) and from geese in China (goose/Guangdong/96) . The phyThe 2004–2005 H5N1 isolates are sensitive to 2 neuraminidase inhibitors that are recommended for prophylactic or therapeutic intervention against human infections with recent H5N1 strains. Rapidly testing potentially pandemic influenza viruses for their susceptibility to licensed drugs is essential to establish appropriate control measures.An effective H5N1 vaccine is a public health priority and the cornerstone for pandemic prevention and control. Reverse genetics approaches allow the rapid production of high-growth PR8 reassortant viruses by engineering a virus with a homologous HA gene lacking the polybasic amino acids associated with high virulence. These candidate H5N1 pandemic vaccine viruses have been made available to vaccine manufacturers to produce pilot lots for clinical trials and are available for possible large-scale manufacturing should the need arise.Genetic and antigenic analyses have shown that, compared to previous H5N1 isolates, 2004–2005 isolates share several amino acid changes that modulate antigenicity and perhaps other biological functions. Furthermore, our molecular analysis of the HA from isolates collected in 2005 suggests that several amino acids located near the receptor-binding site are undergoing change, some of which may affect antigenicity or transmissibility. For example, an isolate (VN/JP12-2/05) showed a change from serine to asparagine at position 223 of the HA1 (S223N) that may affect receptor-binding specificity (,The phylogenies of the 8 genomic segments from the clade 1 and 2 isolates from 2004–2005 showed that all genes are of avian origin. All H5N1 isolates from both clades belong to 1 of the genotypes recently circulating in Eastern and Southern Asia, e.g., genotypes V and Z (The currently circulating H5N1 viruses were reported to infect domestic or wild captive felids, such as tigers, feeding on infected bird carcasses, and the infected cats can transmit H5N1 to pen mates (


RB1 gene through diverse mechanisms including point mutations, nucleotide insertions, deletions, loss of heterozygosity and promoter hypermethylation. Recently, a novel mechanism of retinoblastoma initiation was proposed. Gallie and colleagues discovered that a small proportion of retinoblastomas lack RB1 mutations and had MYCN amplification [RB1 gene mutations and compared the mechanism of RB1 inactivation to the recurrent copy number variations in the retinoblastoma genome. In addition to the previously described focal amplification of MYCN and deletions in RB1 and BCOR, we also identifed recurrent focal amplification of OTX2, a transcription factor required for retinal photoreceptor development. We identifed 10 retinoblastomas in our cohort that lacked RB1 point mutations or indels. We performed whole genome sequencing on those 10 tumors and their corresponding germline DNA. In one of the tumors, the RB1 gene was unaltered, the MYCN gene was amplified and RB1 protein was expressed in the nuclei of the tumor cells. In addition, several tumors had complex patterns of structural variations and we identified 3 tumors with chromothripsis at the RB1 locus. This is the first report of chromothripsis as a mechanism for RB1 gene inactivation in cancer.Retinoblastoma is a rare childhood cancer of the developing retina. Most retinoblastomas initiate with biallelic inactivation of the  RB1) which is rate limiting for tumorigenesis [RB1 gene was cloned, researchers have focused on identifying genetic lesions in retinoblastoma that contribute to tumor progression following RB1 inactivation [DEK gene is within the 0.6 Mb minimal region of chromosome 6p22 that is gained in retinoblastoma and there is a significant increase (~2.5 fold) in gene expression in tumors with 6p22 gain (n=5) compared to those without 6p22 gain (n=2) [ with significantly elevated expression (1.7–2.2 fold increase) in tumors with 6p gains [Most retinoblastomas are believed to initiate with biallelic inactivation of the retinoblastoma susceptibility gene  lack RB1 mutations. The authors suggested that in those patients, MYCN amplification may be sufficient for retinoblastoma tumorigenesis [Another recurrent focal amplification found in ~9% of retinoblastomas is a region of the genome on chromosome 2p spanning the blastoma  but it iication ≥0 copies While aCGH and cytogenetic studies have contributed to the identification of recurrent chromosomal lesions in human retinoblastoma, higher-resolution platforms (i.e. SNP 6.0 arrays) may help to identify additional candidate oncogenes or tumor suppressor genes that contribute to retinoblastoma progression. However, such analyses could be complicated by chromosome instability because it may be difficult to distinguish driver mutations that contribute to retinoblastoma progression from those regions of the genome that are inherently unstable but do not directly contribute to tumorigenesis. Recent whole genome sequencing of 4 primary retinoblastomas and their matched germline DNA demonstrated that at least some retinoblastomas have relatively stable diploid genomes with few CNVs or somatic nucleotide variations (SNV). TherefoMYCN was the most commonly amplified gene in 8.5% (8/94) of tumors in our retinoblastoma cohort. We also identified focal amplification in OTX2 in 3% (3/94) retinoblastoma samples. In addition to recurrent deletions in RB1, the most common focal deletions were in BCOR in 4% (4/94) of our retinoblastomas. To characterize the relationship between MYCN amplification and RB1 gene inactivation, we analyzed the RB1 gene status in 46 retinoblastomas with sufficient DNA for custom capture Illumina sequencing. Ten of those tumors had no evidence of RB1 SNVs or indels in the coding region. We performed whole genome sequencing (WGS), RB1 immunohistochemistry and fluorescence in situ hybridization (FISH) using probes spanning the RB1 locus on all 10 tumors. One of the tumors had a wild type RB1 gene and expressed nuclear RB1 protein in virtually all the tumor cells. It also had MYCN amplification consistent with previously published data showing that 1.5% of retinoblastomas may initiate by this mechanism [RB1 gene and 3 tumors had focal chromothripsis on chromosome 13 spanning the RB1 locus. This provides a novel mechanism of retinoblastoma initiation and suggests that molecular assays to detect RB1 chromothripsis should be included in future analyses of this important tumor suppressor pathway.In this study, we performed SNP 6.0 analysis of 94 human retinoblastomas and their matched normal germline DNA. These data allowed us to more precisely define the boundaries of recurrent chromosomal gains and losses and to identify recurrent focal lesions in individual genes or small groups of genes. Previous aCGH and cytogenetic studies of human retinoblastoma have identified several recurrent whole chromosome gains and losses including chromosome 16 monosomy (18%) , 10, gaiMDM4 in 45% (43/94) on chromosome 1 has been described previously[In addition to whole chromosome gains and losses, several whole chromosome arms and/or large chromosomal regions (>3Mb) have been found to be gained or lost in retinoblastoma , 12. In reviously–15. The  that had statistically significant increase in expression and were more that 2-fold increased in tumors with a 6p gain  . We identified 4 genes on chromosome 6p 05) Fig. . RAB23 iand liver. HCG18 i    and one of them (UPEN-RB-31) had a focal RB1 deletion  so we performed RB1 immunohistochemistry on that sample  and normal adjacent retinal tissue as a positive control. The SJRB011 retinoblastoma had abundant nuclear RB1 protein in virtually all the tumor cells  of retinoblastomas with .5% of reion Fig. . Subsequple Fig. . We usedlls Fig. . To detesis Fig. .RB1, we performed custom capture and Illumina sequence analysis of all 27 RB1 exons in the 46 tumors with sufficient DNA from patients treated at St. Jude Children's Research Hospital  of the tumor and matched normal DNA. Using a paired-end-sequencing approach, we generated 3064 Gb of sequence data for the 10 pairs of samples (germline DNA and tumor DNA); 2793 Gb (91%) were successfully mapped to the reference genome   Table .RB1 gene even though there was evidence of a reduction in RB1 gene copy number and LOH spanning the RB1 locus  had a deletion  had evidence of chromothripsis on chr13 spanning the RB1 gene  had relatively high rates of chromosomal, regional and focal CNVs with more than 20 lesions per tumor. A larger subset (22%) had an intermediate rate with 10–20 lesions per tumor and the majority of tumors (60%) had few lesions (1–9) or none at all (7%). In addition to the previously reported recurrent focal losses of RB1 mutation and the number of lesions. A much larger study will be required to determine if there are more subtle associations between the clinicopathological features of retinoblastoma and the rate of CNVs.Previously, we performed whole genome sequencing on 4 primary retinoblastomas and their matched germline DNA, as well as an orthotopic xenograft derived from one of the primary tumors that was continuously passaged for 9 months before sequencing . The anaThe relatively higher rate of chromosomal, regional and focal lesions in a subset of tumors (30%) does not necessarily indicate that those retinoblastomas have unstable genomes. Chromosome instability is a dynamic process that cannot be accurately measured at a single time point because it involves the acquisition of sequential chromosomal lesions over time. It is important to distinguish between such dynamic processes that reflect the continuous accumulation of genetic lesions from more acute genomic events such as chromosomal “shattering” called chromothripsis , 25. To RB1 gene and the BCOR gene were the most common deletion events and amplification of the MYCN gene was the most common focal recurrent gain[OTX2 in 3% of retinoblastomas. OTX2 is a homeobox gene that is involved in photoreceptor and retinal pigment epithelium development[OTX2 plays a role in modulating the photoreceptor differentiation program in retinoblastoma in future studies.The overall low rate of CNVs was consistent with the paucity of focal recurrent lesions in genes. As reported previously, we found that inactivation of the rent gain. Here wevelopment, 26–28. velopment, 29 and  we found to be altered by more than 2-fold and significantly associated with the copy number alteration. The RAB23 gene is upregulated in retinoblastomas with 6p gain and there is evidence that RAB23 may potentiate Hedgehog signaling in cancer [COX4I1 gene is downregulted in tumors with 16q loss and previous studies have shown that this gene is downregulated in skin cancer [For the most common large chromosomal lesions  we integrated our data on copy number changes with the gene expression data from the same tumors. While there was an overall trend of subtle changes in expression for the genes on those altered regions, only 5 genes n cancer –16. Alson cancer . The othDEK, NUP153, E2F3 and TTRAP[DEK, NUP153, E2F3 and TTRAP and additional studies will be required to determine the functional significance of elevated gene expression in human retinoblastoma.The genes that have been previously found to show changes in gene expression that correlate with gain of 6p were MDM4 is found on a region of chromosome 1 that is gained in 45% of retinoblastomas but the gene and protein are increased in virtually all retinoblastomas. Therefore, the lack of a statistically significant association between copy number and gene expression does not necessarily mean that the gene is dispensable for tumorigenesis.One important caveat of our analysis is the possibility that genes involved in tumor initiation and/or progression may be altered in retinoblastoma irrespective of copy number changes. For example, MYCN, BCOR, or OTX2 because we did not have enough samples with both copy number data and gene expression data. A much larger study will be required to determine if those tumors have distinct gene expression signatures and if there is any association between the focal genetic lesion and the expression of those genes.We were not able to perform detailed integrated analysis of gene expression and copy number changes for the less frequent focal lesions such as MYCN amplification and RB1 inactivation to determine if a subset of retinoblastomas can be driven by a single oncogenic lesion (MYCN amplification) without RB1 loss [MYCN in retinoblastoma tumorigenesis was first suggested in 1984 when Lee and colleagues described retinoblastomas with MYCN amplifications [MYCN amplification is excluding all possible mechanisms of RB1 gene inactivation including SNVs, indels, LOH, deletions, translocations and promoter hypermethylation. In our cohort, we identified 10 retinoblastomas that had no evidence of RB1 gene inactivation by sequencing the 27 exons of the gene. Whole genome sequencing of those 10 tumors and their matched normal germline DNA showed that at least 5 had mutations. One had an indel that was missed due to poor coverage of that exon in our custom capture sequencing analysis. One had a deletion and LOH that was also missed in our targeted sequence analysis. Remarkably, 3 of the tumors had complex structural variations that occurred focally on chromosome 13 spanning the RB1 gene locus. These lesions had all the features of chromothripsis and had breakpoints in the RB1 gene leading to loss of protein expression. This is an important discovery because it is the first report of focal chromothripsis as a mechanism of RB1 gene inactivation in cancer. Moreover, it suggests that chromothripsis can initiate tumorigenisis by inactivating a tumor suppressor gene. This type of lesion would be missed by conventional RB1 mutational analysis and this is why it has gone undetected since the RB1 gene was first cloned in 1986 [RB1 sequence because all of the exons are intact in a sample with chromothripsis spanning RB1. Copy number and LOH analysis may also show distributions that are difficult to distinguish from the germline reference because both alleles are present and copy number changes are focal and subtle. Our data suggest that a significant proportion of retinoblastomas (3/10) thought to have wild type RB1 may actually have gene inactivation by chromothripsis. Whole genome sequencing combined with break-apart FISH analysis of the RB1 locus and RB1 IHC can be used to identify this unique subset of retinoblastoma tumors.One of the goals of our study was to explore the relationship between ications . However in 1986 . SpecifiRB1 gene . Among those, only SJRB011 had MYCN amplification and nuclear expression of RB1 protein. The other 3 had reduced RB1 protein expression and all 3 lacked MYCN amplification. There were no known cancer genes mutated in those 3 tumor samples. It is possible that those tumor samples had biallelic promoter hypermethylation and this caused the reduction in protein expression. Unfortunately, we did not have sufficient DNA to perform methylation analysis. It is also possible that there is a novel oncogenic drive that is altered in those tumors that has not yet been characterized and this in turn leads to downregulation of the RB1 protein. A much larger analysis will be required explore the spectrum of genomic lesions that contribute to retinoblastoma initiation in the absence of RB1 gene mutation.It is important to emphasize that in our cohort of 10 retinoblastomas that we analyzed by WGS, FISH and IHC there were 5 tumors that had at least 1 intact Details for tumors samples acquired at St. Jude Children's Research Hospital have been previously described . In addiDetails for the SNP6.0 arrays have been previously described . The SNPRB1 mutation analysis was performed as described previously [RB1, including the flanking intronic regions, and the promoter region were amplified by the polymerase chain reaction (PCR) and Sanger sequenced. Large deletions and rearrangements were detected by quantitative-real time PCR assays designed to measure the copy number of each individual exon of RB1, including the promoter and 3 ' untranslated regions. Quantitative-real time PCR was performed using a StepOne Plus instrument (Applied Biosystems). Experimental plate setup and analysis was performed in StepOne software v 2.1 (Applied Biosystems). A master mix was prepared for each assay following the manufacturer's instructions. The data was analyzed by the comparative Ct method. The Ct values were then compared with the endogenous control (RNase P) and the reference sample to calculate a ΔΔCt value. Copy number (CN) was then calculated using the formula: CN = 2*2−ΔΔCt.eviously . All 27 RB1 promoter of tumor samples was analyzed using the Methyl-Profiler™ DNA Methylation qPCR assays following the manufacturer's instructions, with an assay for RB1 gene .Methylation state of the MYCN copy number was detected by a Taqman copy number assay  using quantitative-real time PCR as described above.OTX2 copy number was detected by quantitative real-time PCR using Sybr Green (Applied Biosystems 4472942) and analyzed by Eppendorf Mastercycler ep Realplex2 system  following manual instruction. DNA was isolated from primary tumor and blood as indicated above. Whole genome amplified DNA was used for samples acquired at St. Jude Children's Research Hospital [OTX2 at chr. 14:56339578 and CTNNA3 designed to SNP_A- 2245588 (rs2105702) at chrl0:67396520 as an internal diploid reference based on SNP6.0 data analysis. Primers were designed using Primer3 [Hospital . Copy nu Primer3 , 39 for OTX2: forward — AACAGGGCTGGTAAAGAG; reverse - GAGTAGTGCCACTCAGCACA. CTNNA3: forward- CAGGTAGGCCAACAAGTCC; reverse — AAGGTACCTGCCATGTGAATA.RB1 gene in these tumors. Purified BAC DNA from two RB1 3-prime clones (RPH-115122 and RP11-90K7) were labeled with a green-dUTP  by nick translation, and one RB1 5-prime clone (RP11-795F23) was labeled with a red-dUTP . In normal cells that contain normal RB1 gene this probe set produces very tightly linked red and green signals since the probes are separated by only 80kb. This assay was specifically designed to detect any disruption of the RB1 gene occurring between introns 6 and 16. One hundred interphase nuclei from each tumor were scored for the presence of either normal RB1 genes  or disrupted or deleted RB1 genes . Details for FISH protocol have previously been described [The Cancer Center Core Cytogenetic Laboratory received 10 formalin fixed paraffin embedded (FFPE) retinoblastoma tissue specimens in order to determine by FISH if there is a disruption of the Details of IHC protocol have previously been described . IHC wasWhole genome sequencing and exome capture validation has previously been described .


Amplification of chromosome 3q is the most common genomic aberration and this region harbours genes having role as novel targets for therapeutics. There is no standard definition on how to score and report 3q amplification. False versus true 3q chromosomal amplification in squamous cell lung carcinoma may have tremendous impact on trials involving drugs which target DNA zones mapping on 3q. Forty squamous lung carcinomas were analyzed by FISH to assess chromosome 3q amplification. aCGH was performed as gold-standard to avoid false positive amplifications. Three clustered patterns of fluorescent signals were observed. Eight cases out of 40 (20%) showed ≥8 3q signals. Twenty out of 40 (50%) showed from 3 to 7 signals. The remaining showed two fluorescent signals (30%). When corrected by whole chromosome 3 signals, only cases with ≥8 signals maintained a LSI 3q/CEP3 ratio >2. Only the cases showing 3q amplification by aCGH (+3q25.3−3q27.3) showed ≥8 fluorescent signals at FISH evidencing a 3q/3 ratio >2. The remaining cases showed flat genomic portrait at aCGH on chromosome 3. We concluded that: 1) absolute copy number of 3q chromosomal region may harbour false positive interpretation of 3q amplification in squamous cell carcinoma; 2) a case results truly “amplified for chromosome 3q” when showing ≥8 fluorescent 3q signals; 3) trials involving drugs targeting loci on chromosome 3q in squamous lung carcinoma therapy have to consider  Squamous cell cancer accounts for about thirty per cent of lung cancer The interphase in situ hybridization technique is becoming a routinely available standard molecular assessment requested to reference Pathology Labs, due i.e. to the value of the Her-2 gene in breast, gastric cancers, 1p/19q in oligodendrogliomas or EGFR and ALK genes in lung adenocarcinoma In the actual study we sought to evaluate the subtypes of genotypic abnormalities of the entire chromosome 3 and the distal locus specific 3q that maps the SOX-2 and PI3CA genes in a serie of squamous lung carcinoma Finally, we inizialize a scoring for the assessment of 3q amplification, in order to provide a tool to support the clinical laboratories either for the diagnostic goal either for selection to clinical trials encountering inhibitors targeting the 3q region such as anti- PI3CA or –SOX2 targeted drugs.We used tissue samples from human participants. All tissue blocks have been previously declaired to be available for the purposes of the actual study by the Istitutional Review Board (study conducted according to the principles expressed in the Declaration of Helsinki). Our institutional review board and the ethics committee approved the original human work that produced the tissue samples . All processing in obtaining the material has been performed after a written informed consent.Full name Ethic/Institutional Review Board: Prof. Marco Chilosi, Prof. Aldo Scarpa, Prof. Francesco Calabrò, Prof. Guido Martignoni, Nucleo Ricerca&Innovazione.Fourty squamous lung carcinomas were recruited from the LUNG VERONA DATABASE files, Italy. All samples per tumour were reviewed by a pathologist expert in the field (MC) and an appropriate sample containing at least 90% neoplastic cells has been selected for the interphase cytogenetic and molecular studies.Immunophenotyping was performed by using Δn-p63 , CK5 , CK7 , TTF-1 , Napsin A , and SOX-2 .Interphase cytogenetic fluorescence in situ hybridization analysis was performed using commercially available telomeric specific probe  mapping on chromosome 3q telomere  and a centromeric  probe mapping on 3p11-q11. FISH procedure was developed according to previous reports from our group 5 µm sections were cut from paraffin-embedded blocks. The procedure of FISH has been performed as detailed in previous manuscript Centromeric probes for chromosomes 3 and locus specific probes for 3q  were used. Each probe was diluted 1∶10 in tDenHyb2 buffer . Ten microliters of diluted probe were applied to each slide and cover slips were placed over the slides. Denaturation was achieved by incubating the slides at 80°C for 10 minutes in a humidified box; then hybridization was done at 37°C for 16 hours. The cover slips were then removed and the slides were immersed at room temperature in 0.5 XSSC for 2 minutes and in 2 XSSC for 2 minutes. The slides were air dried and counterstained with 10 µl DAPI/Antifade .The slides were examined using an Olympus BX61 (Germany) with appropiate filters for SpectrumOrange , SpectrumGreen , and the UV Filter for the DAPI nuclear counterstain. The signals were recorded with a CCD camera .Fluorescent in situ signals were evaluated on carcinomatous and normal pulmonary adjacent parenchyma.Fluorescent 3q signals was initially scored per each case. These cases were corrected per the score of chromosome 3 signals (LSI 3q/CEP3 >2 “amplified”). From 60 to 250 neoplastic nuclei were assessed per tumours, similarly per adjacent normal parenchyma.Genomic DNA was isolated using QIAamp DNA mini kit  and quantified on the NanoDrop Spectrophotometer . The quantities and qualities of the DNA allowed us to continue with the analyses. As reference was used DNA from pooled peripheral blood leucocytes of healthy males. With Agilent Human 44K array format containing around 44 000 oligonucleotide probes, covering coding and noncoding genome region , we screened for copy number alteration 6 tumours . Briefly, 1.5 µg of tumor and reference DNA were digested, labeled and hybridized according with the Agilent Protocols (version 4). The digested DNAs were labeled by random priming with Cy3-dUTP (reference DNA) and Cy5-dUTP (patient DNA) by use of the Agilent Labelling Kit, after which the labelled DNAs were purified. The resolution of the fragments analyzed varied from several Kb to <1 Mb depending upon the case selected.The array images obtained after scanning (high resolution Agilent scanner) were processed with the Feature Extraction software (version 10.5), and the output data files were analyzed with the Agilent Genomic Workbench. To identify copy number alterations we used Aberration Detection Method 2 (ADM-2) algorithm and to exclude the small variances in the data we set up a custom aberration filter identifying an alterations in copy number if minimum of 8 probes gained or lost to be present and with minimum absolute average log ratio for region to be 0.5. The region with small copy number variations were excluded by comparing and visualizing the copy number variant regions tool of the Genomic workbench software.All carcinomas showed pure squamous morphology ; 11 caseImmunophenotipically p63 and SOX2 were expressed in all cases (100%) . The whole aberrations span about 27.5 Mb. Specifically, findings from oligonucleotide probes spanning the SOX2 and PI3CA zones revealed more relevant gains for the PA3CA in respect to the SOX2 zones, although significant differences were not observed.The remained cases had normal profile. Relevant genomic abnormalities were observed in three cases (+5p).When matching cases analyzied by both FISH and aCGH, only those two cases showing 3q amplification by aCGH scored ≥8 fluorescent at FISH and evidenced a 3q/CEP3 ratio >2. The remaining four cases showed a flat genomic profile at aCGH with focus on the 3q and centromeric regions.false versus true 3q chromosomal amplification in squamous cell lung carcinoma merit consideration at light of emerging trials involving anti-PI3CA and SOX-2 targeted drugs.In our study we observed that 1) absolute copy number of 3q chromosomal region may harbour false positive interpretation of 3q amplification in squamous cell carcinoma; 3) correction for chromosome 3 is important to identify true 3q amplification; 4) we propose to consider a case “amplified for chromosome 3q” when scoring ≥8 fluorescent signals; 5) Amplification of chromosome 3q has been described in squamous neoplastic transformation from different sites In our study we highlighted the findings of three distinctive clustered cytogenetic patterns and propose that correction for chromosome 3 is critical for the determination of true 3q amplification status. Today, there is no definitive consensus about the optimal scoring system for assessing 3q amplification. Due to the fact that the 3q region maps genes, such as SOX2 and PI3CA genes, that may have potential role as novel targets for therapeutics, we believe that the need in proposing guidelines for appropriate evaluation of the 3q region status is important and may have impact to different clinical levels. The absolute 3q copy number or the alternative 3q/CEP3 ratio has to be evaluated appropriately, when the predictivenesss to drug efficacy is going to be determined. In our serie eight cases displayed true amplification  when corrected by chromosome 3. We validated these findings after wide genomic aCGH analysis. False positive 3q amplification is present when absolute copy number of 3q signals ranges from 3 to 7 per nuclei. Wide genomic molecular profiling such as the array comparative genomic hybridization technique may help in appropriate definition of new cut-offs and validate standard ISH results and interpretation. aCGH may distinguish gains of a chromosome due to polyploidy or genetic instability versus true chromosomal amplification.The chromosomal region spanning the 3q encounters many interesting genes At routinely laboratory level, fluorescence in situ hybridization (FISH) on formalin-fixed, paraffin-embedded tissue is an increasingly useful technique in the detection of many diagnostic chromosomal abnormalities in different oncological fields Tonon et al. observed and highlighted different degree of gains of chromosomes/genes in term of magnitude such as focal copy number alterations, such as five genes gained in lung carcinomas and others recurrent copy number alterations including high-amplitude amplifications The PI3K gene also set in the 3q chromosomal region. There are ample genetic and laboratory studies that suggest the PI3K–Akt pathway is vital to the growth and survival of cancer cells In our study we did not observe relevant differences in term of magnitude of genome gains when analyzing findings from oligonucleotide probes spanning the SOX2 and PI3CA chromosomal regions.versus true 3q chromosomal amplification in squamous cell lung carcinoma are observed and its impact to trials involving anti-PI3K and -SOX2 targeted drugs is a direct consequence. Correction for chromosome 3 is critical for the determination of true 3q amplification status: the definition of a detailed scoring system and a consensus is mandatory.The clinical impact of the multifaceted genotypic abnormalities, observed in the actual study, merits further investigation. We evidenced that absolute enumeration of chromosomal copies is challenging and the absence of analytical standardisation may limit pre-clinical and clinical studies focusing on the predictiveness of biomarkers mapping on the 3q region. False


FOXC1 transcription factor gene or duplications of the 6p25 region is associated with a spectrum of ocular abnormalities including ASD. However, mapping data and phenotype analysis of human deletions suggests that an additional locus for this condition may be present in the same chromosomal region as FOXC1. DHPLC screening of ENU mutagenised mouse archival tissue revealed five novel mouse Foxf2 mutations. Re-derivation of one of these (the Foxf2W174R mouse lineage) resulted in heterozygote mice that exhibited thinning of the iris stroma, hyperplasia of the trabecular meshwork, small or absent Schlemm's canal and a reduction in the iridocorneal angle. Homozygous E18.5 mice showed absence of ciliary body projections, demonstrating a critical role for Foxf2 in the developing eye. These data provide evidence that the Foxf2 gene, separated from Foxc1 by less than 70 kb of genomic sequence (250 kb in human DNA), may explain human abnormalities in some cases of ASD where FOXC1 has been excluded genetically.Anterior segment dysgenesis (ASD) is characterised by an abnormal migration of neural crest cells or an aberrant differentiation of the mesenchymal cells during the formation of the eye's anterior segment. These abnormalities result in multiple tissue defects affecting the iris, cornea and drainage structures of the iridocorneal angle including the ciliary body, trabecular meshwork and Schlemm's canal. In some cases, abnormal ASD development leads to glaucoma, which is usually associated with increased intraocular pressure. Haploinsufficiency through mutation or chromosomal deletion of the human  Anterior segment dysgenesis covers a spectrum of disorders affecting the iris, cornea, trabecular meshwork and Schlemm's canal of the eye, which can result in abnormal aqueous humor drainage from the eye leading to raised intraocular pressure and glaucoma FOXC1, PITX2, PITX3, FOXE3, PAX6, MAF, CYP1B1 and LMX1B. Mutations in the FOXC1 gene FOXC1 can all cause iridogoniodysgenesis; as can mutations in the PITX2/RIEG1 gene FOXC1 mutations have a milder average prognosis for glaucoma development than do patients with any one of the known PITX2 mutations Anterior segment dysgenesis (ASD) phenotypes are inherited as autosomal dominant traits with variable expressivity and incomplete penetrance, pointing to a complex etiology FOXC1 may also be involved in the underlying etiology of iridogoniodysgenesis and other eye abnormalities associated with glaucoma. For example, deletion of 6p24-p25 proximal to the FOXC1 locus causes anterior segment abnormalities FOXC1 as the causative gene FOXC1 but may have been monosomic for FOXF2 Genetic evidence suggests that other genes near Foxf2 gene could be involved in anterior segment development and dysgenesis we took advantage of an ENU mutagenised DNA archive Foxf2 mutant lineages. We describe the genetic analysis of an identified Foxf2 mutation and the phenotypic features of the affected animals. These analyses suggest Foxf2 is essential for normal anterior segment development, and that the FOXF2 gene should be considered as an additional candidate for anterior segment dysgenesis in humans.To investigate whether the nearby Foxf2 genomic DNA .The eyes from ten wildtype ) and a lwildtype . The canFoxf2W174R heterozygous mice (16/18) revealed no substantial damage to the optic nerve, retina or cornea (To investigate the effect of the W174R mutation in older mice (6 months), the retina, cornea and optic nerve of heterozygous mice were examined to determine if there was any apparent glaucomatous damage. Histological analysis of 18 mice showed a range of anterior segment defects as previously seen in younger mice. In addition two mice appeared to have bulging eyes that can be associated with raised intraocular pressure. However, on histological investigation there appeared to be extraneous amorphous tissue between the retina and the lens. Histological analysis in the majority of r cornea . In two r cornea . In miceFoxf2W174R embryos displayed iridocorneal defects, E18.5 embryos were examined by histology. Evagination of tissue from the anterior optic cup begins at E14 to form the future iris and ciliary body. At E18.5 finger-like projections of tissue forming the ciliary body processes are clearly visible in wildtype littermates . Previous reports of cytogenetic abnormalities are consistent with the notion that the eye is exquisitely sensitive to both reduced and increased dosage in this chromosomal region. Although FOXC1Foxf2W174R individuals could be the result of a mutation within a Foxc1 regulatory region. However, other evidence to support the involvement of Foxf2 in anterior segment dysgenesis, including the patterning of its ocular expression Foxc1 coding mutations; in combination with the observed physical phenotype - all contribute towards a greatly strengthened candidacy of Foxf2.Data from the characterisation of a 200 kb deletion located 1.2 Mb upstream of Foxf2 caused palate malformations and an abnormal tongue Foxf2 knockout mice subsequently revealed megacolon, colorectal muscle hypoplasia and agangliosis Foxf2W174R mutation. Foxf2 is expressed in the absence of its closest paralogue (Foxf1) in the CNS, ear, and limb buds as well as the eye Foxf2-associated phenotypes.Previous studies have shown that targeted deletion of Foxf2 knockouts Foxf2 expression in the periocular mesenchyme of the developing eye at about E12.5 in situ hybridisation established that there was continued Foxf2 expression from E13 through to adult stages Foxf2 expression at E17 were observed in the developing ciliary body and choroid. These data support the abnormal morphological finding in the developing ciliary body in homozygous Foxf2W174R embryos.The effect on eye development was not examined in previous analyses of Foxf2W174R is a hypomorphic allele, However it is also possible that the differences are due to genetic background and that the mutation causes a complete loss of function. Molecular modelling of FOXC1 in a previous study revealed that a tryptophan residue (Trp152) - the direct homologue of Trp174 in Foxf2, is one of nine critical intramolecular interaction residues that maintain structural integrity of the forkhead winged helix structure Foxf2 would lead to protein instability. Another example of an unstable forkhead transcription factor with a mutation in the DNA binding region is the I87M variant of FOXC1Foxf2W174R mutation and would be consistent with the hypothesis that haploinsufficiency plays a key role in the pathogenesis of Fox associated anterior segment anomalies.The difference in phenotype that was identified between targeted knockout and homozygous missense mutation could suggest that Foxf2W174R mice are variable in eyes from different individuals, recapitulating the variable expressivity observed in human patients with ASD. Schlemm's canal was often smaller than typically seen in wild-type eyes and trabecular meshwork was either missing or was underdeveloped, suggesting abnormal migration of mesenchymal cells into the iridocorneal angle. The ciliary body malformations may affect aqueous humor production and secretion of antioxidant proteins into the aqueous humor FoxF2W174R mice are very reminiscent of those seen in mice that are heterozygous for Foxc1 or Foxc2 mutations Foxc1, Foxc2 and Foxf2 are all expressed in the developing periocular mesenchyme, this suggests that this tissue is particularly sensitive to gene dosage The ocular abnormalities found in FOXE3 gene affect lens development and can be inherited as either an autosomal dominant or recessive trait FOXC2 gene cause lymphedema-distichiasis syndrome FOXC1FOXL2 mutations cause blepharophimosis-ptosis-epicanthus inversus syndrome  Foxg1 , Foxd1Foxn4 Foxf2 gene can be added to this growing list.Despite the high level of conservation in their DNA binding domain, forkhead transcription factors are an extraordinarily diverse group of genes with roles as varied as development, homeostasis, stress response and cell cycle control FOXC1/FOXF2/FOXQ1) in which FOXC1 is separated from FOXF2 by less than 250 kb of genomic DNA, and FOXQ1 is 470 kb proximal of FOXC1. Because duplication and deletions of this region in human disease often contain more than one of these genes, confirmation of pathogenicity has relied on specific mutations in animal models. Although gene knockouts FOXC1 deletions or mutations, no model carrying an additional functional copy of FOXC1 has been developed to explore gain-of function effects seen in interstitial gene duplication events. Since our data provides evidence that Foxf2 in mice is also critically involved in anterior segment development, then duplications or deletions containing both FOXF2 and FOXC1 in patients may contribute to the phenotype. This is supported by clinical observations where interstitial duplication of FOXC1 alone causes an iris hypoplasia phenotype, whereas duplications containing both genes  cause microcornea and ptosis, without iris hypoplasia The 6p25 region contains a forkhead cluster .PCR amplification products from pooled DNA that exhibited evidence of heteroduplexes in their DHPLC profiles, were individually PCR-amplified and screened by DHPLC. The single DNA heteroduplex that was identified was sequenced on both strands to determine the mutation. PCR products were purified using a QIAquick PCR purification kit (Qiagen) and sequencing was carried out using BigDye 3.1 terminator chemistry on an ABI prism 377 DNA sequencer. Sequences were aligned and compared with consensus data obtained from the mouse genome database  and BsrI (wildtype locus) restriction digestion of the exon1c PCR product to distinguish Foxf2W174R heterozygotes from homozgotes and wildtypes. Because the C3H mice carry a Pde6brd1retinal mutation affecting the eye, the identified Foxf2W174R mice were outcrossed to C57BL/6 mice for 2 generations. To exclude rd1 carriers, genotyping was performed with the following two primers F: 5′-ACCTGAGCTCACAGAAAGGC-3′ and R: 5′-GCTTCTAGCTGGGCAAAGTG-3′ as described previously DdeI restriction digest (which cuts the rd1Pde6b mutant locus) and SnabI (wildtype locus) thus allowing differentiation between rd1Pde6b heterozygotes, homozygotes and wildtypes. All subsequent analyses were carried out on mice with only the Foxf2 mutation. Primers for sequencing the Foxc1 gene are in Recovery of the mutant mouse lineage was achieved by Eyes were enucleated and placed in 50% Karnovsky's fixative for 45 minutes. Eyes were then washed 3×30 min in PBS, dehydrated through a graded ethanol series  and then embedded in paraffin wax. Whole eye sections cut sagitally to a thickness of 5 µm were counterstained with hematoxylin and eosin. Retinal histology was imaged using a digital camera mounted on an Olympus 1×71 microscope.


There was an error in the text in the "Cell dissociation and culture" section of Materials and Methods. The correct text is: "The chopped tissues were then transferred into 50 ml tube and incubated with collagenase II (5mg/ml) in 37°C shaker for 60 min."


Pcp4 chromatin and regulates Pcp4 expression in the second arch. Hoxa2 is also sufficient to induce Pcp4 expression in anterior first arch cells, which are Pcp4-negative.Branchial arches are externally visible tissue bands in the head region of all vertebrate embryos. Although initially formed from similar components, each arch will give rise to different head and neck structures. In a screen designed to characterize the molecular control of branchial arch identity in mouse, we identified Pcp4 as a second branchial arch-specific molecular signature. We further show that the transcription factor Hoxa2 binds to  Hoxa2 and Hoxb2; the first arch does not express any Hox genes. Hoxa2 specifies second branchial arch (IIBA) identity: the second arch follows a first arch fate in the absence of Hoxa2, and IIBA-skeletal derivatives are replaced by typical first branchial arch (IBA) skeletal elements in a mirror image configuration Branchial arches are transient, repetitive structures of the vertebrate embryo, in which cells of the cranial neural crest and mesoderm are encapsulated by epithelia. There are five to six pairs of arches in amniotes, labeled according to their position along the embryo antero-posterior axis; the first corresponds to the most anterior arch (lying below the forebrain) and the sixth to the most posterior (close to the developing heart). Each of the arches shares a similar architecture and ability to form skeletal elements, their associated muscles, blood supply and nerves, but has its own molecular identity and contributes to diverse head and neck structures. Early development of the branchial arches is instructed by signaling molecules and transcription factors. Dlx transcription factors regulate proximo-distal patterning within each branchial arch in vitroPcp4-null mice Pcp4 is a small calmodulin (CaM)-binding protein. It affects the rates of association and dissociation of Ca (2+) from CaM and can attenuate the activity of a number of CaM-dependent enzymes, including CaM kinase II Hoxa2, the main determinant of second arch fate, in first arch cells and analyzed changes in global expression. By intersecting the data obtained with related data sets, we uncover novel genes regulated by Hoxa2. In addition, we identify the gene encoding for Pcp4 as highly differentially expressed in first and second arch cells. We show that, while anterior first arch cells are Pcp4-negative, Pcp4 is enriched in second arch cells, and that Pcp4 expression is directly controlled by the transcription factor Hoxa2 in vivo.To understand the molecular control of inter-branchial arch identity, we forced expression of Hoxa2 in Hox-negative IBA cells , distributed in 36 upregulated and 23 downregulated genes  Hoxa2 loss of function mutant IIBA Hoxa2 gain of function and downregulated in Hoxa2 loss of function. This group, which contains genes activated by Hoxa2, is frequently associated with Hoxa2 bound regions (Hoxa2 loss of function mutant (repressed by Hoxa2) are not affected by Hoxa2 overexpression and are seldom associated to Hoxa2 binding events, suggesting these changes may be indirect effects of Hoxa2 absence , which may potentially compensate Hoxa2 loss of function and mask changes in the expression of Hoxa2 targets. Gain of function of Hoxa2 in IBA cells provides therefore a complementary system to identify Hoxa2 direct targets in branchial arches mesenchymal cells. Finally, few genes displayed changes in expression with the same sign in Hoxa2 gain and loss of function  and IIBA cells and instructs a second arch developmental fate  [2], [3]BA cells . Cells ied genes . We idened genes . We inteHoxa2, with low to undetectable level of expression in GFP-positive first arch cells . Interestingly, expression of Pcp4 was also highly different when comparing expression profiles of cells isolated from the first and the second arch . Since Hoxa2 expression is sufficient to induce Pcp4 expression in IBA cells, we asked whether IIBA cells, which are Hoxa2-positive, endogenously express Pcp4. We analyzed the expression of Pcp4 in branchial arch cells using immunofluorescence: Pcp4 was visualized only in IIBA cells, and was not present in cells derived from the IBA  and Hoxa2 mutant (−/−). We found strong Pcp4 expression in wild type IIBA, but no expression in Hoxa2 mutant IIBA  IIBA; b) Hoxa2 is sufficient to induce Pcp4 expression in IBA mesenchymal cells; and c) Hoxa2 is required for expression of Pcp4 in the IIBA, indicate that Hoxa2 is a main regulator of Pcp4 in embryonic development. Additional experiments will be required to understand the function of Pcp4 in the development of the second branchial arch. Local modulation of calcium signal transduction is a key determinant of beak shape Pcp4 by Hoxa2, leading to its expression in the second arch, and the ability of Pcp4 to modulate Ca (2+) signaling suggest that acquiring IIBA identity may partly rely on a differential ability of IIBA cells to control Ca (2+) levels.Taken together, the observations that a) Hoxa2 is strongly bound to Hoxa2 mutant mice were described previously 2. Ecotropic-Phoenix cells were transfected with Fugene and pMYs-IRES-GFP  or pMYs-Hoxa2-IRES-GFP (containing Hoxa2 in front of the IRES), and branchial arch cells were infected using supernatants from Ecotropic-Phoenix packaging cells, supplemented with polybrene at 2 µg ml/1 final concentration. After 72 hours cells were sorted by retroviral gene expression of fluorescent proteins using flow cytometry. Animal experiments were carried out under ASPA 1986.RNA was extracted from sorted cells using Trizol (Life Technologies). Labeled targets were generated from total RNA obtained in two independent experiments, using the 3′ IVT Express Kit and hybridized to GeneChip Mouse Genome 430 2.0 arrays Branchial arch cells were isolated as described above and grown on 8-well culture slides  for 24 hours, fixed and incubated with Pcp4 antibody (Sigma) diluted 1∶200 and visualized with AlexaFluor 488 goat anti-rabbit (Invitrogen).Pcp4 gene ChIP was performed as described +/−Hoxa2 intercrosses were dissected out and snap-frozen in dry ice. After genotyping the embryos, pools were made with the wild type and −/−Hoxa2 branchial arches and total RNA was extracted using Trizol. RT-PCR was performed as described Pcp4 F 5′-ATGAGTGAGAGACAAGTGCC’-3; Pcp4R 5′-CTAGGACTGTGATCCTGCCT-3′, respectively. Whole mount in situ hybridization was performed as described Hoxa2Pcp4 probe, amplified from IIBA cDNA using the primers above.Second branchial arches of E11.5 embryos from Figure S1Differential Pcp4 expression in the first and second branchial arch. Semiquantitative RT-PCR on cDNA isolated from first (I) and second (II) arch. Pcp4 is detected in IIBA and not IBA. Gapdh is a positive control gene; H2O is the negative control, using both Pcp4 and Gapdh primers.(JPG)Click here for additional data file.Table S1Top regulated genes in Hoxa2 gain of function in IBA cells. Genes are ranked using fold changes. The following cut-offs were applied to the probe sets: average fold difference ≥2.5; P value ≤0.05.(XLS)Click here for additional data file.Table S2Microarray data expressed as fold change and p values for the Hoxa2 loss of function (LOF) and Hoxa2 gain of function (GOF) microarray experiments. Affymetrix probesets are only included if they have a fold change > = 1.75 in either of these experiments. Additional information shows if a gene passes combined filter thresholds of + or − 1.75; 1 indicates yes and 0 indicates no. Also indicated is whether the gene is located close to a Hoxa2-bound region in ChIP-seq binding site (where “close” is defined as being one of the two closest genes to a ChIP-seq peak).(XLS)Click here for additional data file.


There are different risk factors or determinants of AKI, and some are related to cardiopulmonary bypass (CPB). In this study, we explored the association between metabolic parameters (oxygen delivery (DO2 and VCO2 levels of each patient were monitored during CPB. Outcome variables were related to kidney function (peak postoperative serum creatinine increase and AKI stage 1 or 2). The experimental hypothesis was that nadir DO2 values and nadir DO2/VCO2 ratios during CPB would be independent predictors of AKI. Multivariable logistic regression models were built to detect the independent predictors of AKI and any kind of kidney function damage.We conducted a retrospective analysis of prospectively collected data at two different institutions. The study population included 359 adult patients. The DO2 level < 262 mL/minute/m2 and a nadir DO2/VCO2 ratio < 5.3 were independently associated with AKI within a model including EuroSCORE and CPB duration. Patients with nadir DO2 levels and nadir DO2/VCO2 ratios below the identified cutoff values during CPB had a significantly higher rate of AKI stage 2 . The negative predictive power of both variables exceeded 90%. The most accurate predictor of AKI stage 2 postoperative status was the nadir DO2 level.A nadir DO2 level during CPB is independently associated with postoperative AKI. The measurement of VCO2-related variables does not add accuracy to the AKI prediction. Since DO2 during CPB is a modifiable factor (through pump flow adjustments), this study generates the hypothesis that goal-directed perfusion management aimed at maintaining the DO2 level above the identified critical value might limit the incidence of postoperative AKI.The nadir DO The ear2) .2 level of 272 mL/minute/m2 during CPB was independently associated with acute renal failure and peak postoperative serum creatinine levels [2 levels < 260 mL/minute/m2 during CPB were associated with increased lactate formation [2 levels and an increased CO2 production (VCO2) during CPB, with critical values settled at a VCO2 > 60 mL/minute/m2 and a DO2/VCO2 ratio < 5.0 [In 2005, in a retrospective series, we demonstrated that a lowest (nadir) DOe levels . Subsequormation  and thatio < 5.0 .2 during CPB falls below a critical value (in the range of 260 to 270 mL/minute/m2), organ dysoxia may be triggered, with consequent tissue acidosis leading to increased VCO2, and that this mechanism may be a determinant of impaired postoperative renal function.These data generate the hypothesis that when the DO2 and VCO2 is still not the standard of care during CPB. The quality indicators used for CPB management are wide-ranging and are mostly 'stand alone' parameters. There is little or no evidence in the literature to suggest that goal-directed perfusion pressure using current quality indicators [2 during CPB is an independent determinant of postoperative AKI is still based on a single-centre observation.Despite this information, routine measurement of DOdicators  influenc2 and VCO2 during CPB might be independently associated with postoperative AKI.In the present dual-centre large series of patients where these values were routinely monitored during CPB, we investigated the hypothesis that DO2 and VCO2 measurement were introduced as part of standard monitoring during CPB in 2009. The primary end point of this study was the determination whether the nadir DO2 value and DO2/VCO2 ratio and the VCO2 peak value during CPB are independently associated with postoperative renal function impairment, which we defined as the peak postoperative serum creatinine value and the presence of AKI according to the AKI Network (AKIN) criteria [2- and VCO2-related variables with general outcome measurements .In this retrospective analysis of prospectively collected data collected at two institutions  DOcriteria . BrieflyThe study was approved by the local ethics committee and the institutional review boards of the two participating institutions , and the need for written informed consent from the patients was waived.Three hundred fifty-nine patients  who had surgery between September 2009 and June 2010 were admitted into the study. Entry criteria were patient age > 18 years undergoing CPB and expected CPB duration > 90 minutes. Exclusion criteria were the presence of chronic renal failure being treated with dialysis and patients who had undergone heart or heart and lung transplantation.The patients were consecutively enrolled at both Institutions. Since only one dedicated software system was available for data collection at each Institution, some patients were not included in cases where two operations were being performed simultaneously on two suitable candidates for enrolment. This criterion accounted for a missing cases rate of 23%. The patients included in the present series were not included in the previous studies dealing with the same experimental hypothesis.2 (< 260 mL/minute/m2) was calculated as 35%. The study was powered to detect a double AKI stage 2 rate in patients with low DO2. Considering the unequal distribution of patients with low and high DO2, an α value of 0.05 and a β value of 0.20, the following sample size was calculated: (1) 195 patients with high DO2 (65%) comprising 18 patients with AKI (9%) and (2) 105 patients with low DO2 (35%) comprising 18 patients with AKI (17%). The total patient population included in the study was therefore between 300 and 400 patients to account for possible lack of data during the study period.Power analysis was performed before retrospective data collection. On the basis of historical data from the two institutions, the postoperative AKI stage 2 rate was calculated as 12%. The ratio of patients with low DOThe following data were recorded for all patients.1. Preoperative data: Patient demographics, baseline serum creatinine level (mg/dL), estimated creatinine clearance (measured using the Cockcroft-Gault equation), left ventricular ejection fraction (%), comorbidities , baseline haemoglobin (Hb) value (g/dL), logistic EuroSCORE and acute renal failure risk according to a validated risk score .2 (mL/minute/m2), peak VCO2 (mL/minute/m2) and nadir DO2/VCO2 ratio during CPB, peak serum lactate during CPB and number of packed red cell (PRC) units transfused during CPB.2. Perioperative data: Reoperation, type of operation, CPB duration, nadir body temperature (°C) during CPB, nadir haematocrit (HCT) level (%) (measured at the start of the CPB operation and every 20 minutes thereafter), nadir DO3. Postoperative data: Peak serum creatinine level (mg/dL), peak postoperative change in creatinine level (percentage change from baseline), estimated lowest creatinine clearance, AKI stage defined according to the AKIN criteria, number of PRC units transfused and days spent in the ICU and postoperatively in the hospital.The number of PRC units transfused was considered as a potential risk factor for renal function impairment. Since transfusions may be the consequence rather than the cause of renal dysfunction, we considered the number of PRC units transfused before the onset of the renal dysfunction (perioperatively or soon after surgery).All patients were treated with moderately hypothermic CPB (27°C to 36°C). The CPB circuit consisted of an Avant oxygenator , an HVR Evo hard-shell reservoir (Sorin Group) in London and a closed CVR 1200 soft-shell reservoir (Sorin Group) in Gent, Belgium. The hardware consisted of a Stöckert S5 heart-lung machine (Sorin Group) and a Stöckert Heater Cooler System 3T (Sorin Group).2 at the outset of CPB and subsequently adjusted according to the patient's core temperature . A Hb value less than 6 to 7 g/dL during CPB was considered the trigger point for PRC transfusion. All patients received tranexamic acid according to the routine protocol of each institution, and no aprotinin was used.Priming solutions consisted of 1,400 mL of Hartmann's solution and 10,000 IU of heparin (London site) and 1,200 mL of gelatine , 15% mannitol (200 mL) and 2,500 IU of heparin (Gent site). No hydroxyethyl starch was used for priming the circuit or as additional fluid during CPB. Cardioplegic solution was a 4:1 ratio of cold blood cardioplegia (St. Thomas's solution) at an induction dose of 1,000 mL, with subsequent 200-mL doses every 20 minutes of (London site) and 800- to 1000-mL single-dose Saint Thomas II crystalloid cardioplegia with additional doses if considered necessary by the surgeon (Gent site). Pump flow was settled at 2.4 to 2.8 L/minute/m2- and VCO2-related measurements were performed using a dedicated software system provided by Dideco (Sorin Group). Data were collected at 10-minute intervals during CPB. Data required to calculate DO2 and VCO2 were arterial Hb (g/dL), pump flow (L/minute/m2), arterial O2 saturation, gas flow into the oxygenator (L/minute) and expiratory CO2 tension (mmHg) measured at the site of the oxygenator exhaled gas port with a Datex-Ohmeda capnograph ), or with a ventilator-integrated capnograph.DO2 and VCO2 were described in a previous article [2 was calculated using the following equation:All blood gas data were corrected for temperature by using standard equations. The equations used to determine DO article . Briefly2 content was calculated as follows:where arterial O2 content (mL/100 mL) = Hb (mg/dL) × 1.34 × Hb saturation (%) + 0.003 × O2 tension (mmHg).Arterial O2 was calculated using the following equation:VCOGas volume and flow are expressed as dry standard temperature and pressure. Adequate corrections according to body temperature and pressure saturated conditions were applied.2 level was defined as the lowest DO2 value registered for at least two consecutive measurements, the peak VCO2 value was defined as the highest VCO2 level registered for at least two consecutive measurements and the nadir DO2/VCO2 ratio was defined as the lowest value registered for at least the two consecutive measurements. The normal DO2 and VCO2 values in healthy, awake subjects are usually around 500 mL/minute/m2 and 100 mL/minute/m2, respectively. Under moderate hypothermia, anaesthesia and haemodilution during CPB, these values are always reduced to unspecified values.The nadir DO2, nadir DO2/VCO2 ratio and peak VCO2 during CPB were tested for association with kidney function variables using bivariate linear regression analysis (for peak postoperative serum creatinine) or bivariate logistic regression analysis (for AKI stage 1 or 2 and any kind of AKI). Other possible confounders were tested for associations with kidney function variables using the same technique. The linearity assumption was checked by exploring the decile-based distribution of the AKI stage 2 rate with spline curves. For the independent predictors of AKI, adequate cutoff values were identified using a receiver operating characteristic (ROC) curve, and the area under the curve (AUC) was used to determine the accuracy of the model. The cutoff values were chosen according to Youden's index ). For each cutoff value, the sensitivity, specificity and negative and positive predictive values were measured.Data are expressed as means ± standard deviation  or as frequencies and percentages . Nadir DOP < 0.1) with AKI stage 2 or any kind of AKI were entered into multivariable stepwise forward logistic regression models for definition of the independent predictors of kidney function impairment to generate odds ratios with 95% confidence intervals. To avoid overfitting of the models, only one independent variable per 10 events was entered into the model. To avoid multicollinearity, variables affected by mathematical coupling were separately entered into different models. In cases of intercorrelation, the best single independent variable was chosen. For all the statistical tests, a P value < 0.05 was considered significant. Data analysis was performed using SPSS 13.0 statistical software .All the factors associated (N = 359), we identified 31 patients (8.6%) with AKI stage 1 and 44 patients (12.2%) with AKI stage 2, for a total of 75 patients (21%) with any kind of AKI. Within the AKI stage 2 group, five patients (1.4%) with AKI stage 3 were included.The demographics, preoperative, operative and postoperative details of the patient population are shown in Table 2, peak VCO2 and nadir DO2/VCO2 ratio during CPB were all associated with renal outcomes and were therefore admitted into the following multivariable analyses. The other perioperative variables were not significantly associated with renal outcomes.The variables listed in Table 2 levels and nadir DO2/VCO2 ratios with postoperative AKI stage 2, we explored the rate of AKI stage 2 outcomes according to the decile distribution of the two independent variables. The graphical analysis of the AKI stage 2 rate according to nadir DO2 level and DO2/VCO2 ratio during CPB , this value was included in the analysis , with slightly lower values for the nadir DO2/VCO2 ratio (0.62) and nadir the HCT level (0.61). The best cutoff values according to the Youden's index were set at a nadir DO2 < 262 mL/minute/m2, a nadir DO2/VCO2 ratio < 5.3 and a nadir HCT < 23.5%.The best AUC was identified for the nadir DO2 value and nadir DO2/VCO2 ratio were separately analysed because of mathematical coupling between the two variables. The various factors identified in the univariate analysis were tested for intercorrelation. There was a significant intercorrelation between centres and EuroSCORE. Centre 1 included patients at significantly (P = 0.001) higher risk for postoperative AKI (EuroSCORE 13.4 ± 17) than centre 2 (EuroSCORE 4.1 ± 2.1). We therefore included the EuroSCORE and not the centre in the multivariable models. Some of the other univariate predictors of AKI are included in the EuroSCORE  and were not included in the model to avoid multicollinearity.The multivariable analysis was performed with AKI stage 2 rate and the rate of any kind of AKI as dependent variables. For each model, nadir DO2 < 262 mL/minute/m2 and nadir DO2/VCO2 ratio < 5.3. After correction for the other explanatory variables, both a nadir DO2 < 262 mL/minute/m2 and a nadir DO2/VCO2 ratio < 5.3 during CPB remained independently associated with AKI stage 2 rate  higher rate of postoperative AKI stage 2 (23.2% vs. 7.4%), with a negative predictive value of 92.5%. The patient group with a nadir DO2/VCO2 ratio < 5.3 during CPB had a significantly (P = 0.001) higher rate of AKI stage 2 (20% vs. 8%), with a negative predictive value of 92%. The patient group with a nadir HCT < 23.5% had a significantly (P = 0.014) higher rate of AKI stage 2 (18.5% vs. 9.4%).The cutoff values identified for the nadir DO2 Figure . The pat2, peak VCO2 and nadir DO2/VCO2 ratio during CPB) with the ICU and postoperative hospital stays using linear regression analysis. We found significant  negative associations between ICU length of stay and postoperative hospital length of stay with nadir DO2 during CPB. Conversely, there was no association of ICU length of stay and postoperative hospital length of stay with the peak VCO2 value and the nadir DO2/VCO2 ratio  and DO2/VCO2 ratio (5.3), and for both these values the negative predictive power exceeds 90%. However, the measurement of CO2-related variables did not increase the accuracy of the DO2-based predictive model. In clinical terms, our data generate the hypothesis that by maintaining the DO2 at a level > 262 mL/minute/m2 during CPB, the likelihood of experiencing a postoperative AKI might be decreased.In the present study, we investigated the role of potentially modifiable factors related to CPB surgery in determining postoperative AKI. Our results demonstrate, in a relatively large series of patients treated at different sites, that a low DO2 as the lowest acceptable DO2 during CPB is associated with significant differences in terms of ICU and postoperative hospital lengths of stay .Moreover, the cutoff value of 262 mL/minute/m2 < 272 mL/minute/m2 during CPB have been associated with an increased rate of acute renal failure [2 have been associated with increased lactate formation [This study confirms some of the critical values previously identified in other studies ,12,13. V failure , and val2 level, O2 consumption cannot be maintained using aerobic energy production, and that to provide energy to the cells, the anaerobic mechanism is activated [2. This mechanism was identified for kidney tissue in 1966 [2 level is around 300 mL/minute/m2. In our study, we identified and confirmed that under anaesthesia and moderate hypothermia, values as low as 262 mL/minute/m2 can be sustained, but below this value kidney function starts to decline.It is well known that below a critical DOctivated ,18. As a in 1966 , the cri2 -related variables did not increase the accuracy of AKI prediction in our series. VCO2 may increase even under normal aerobic conditions, and during CPB this invariably happens during the rewarming phase as a result of increased metabolic needs. Theoretically, if coupled with the DO2 measurement (DO2/VCO2 ratio), the measurement of VCO2 should offer additional advantages for detecting dysoxia, as suggested by other authors who demonstrated that CO2-related variables, in combination with O2-related variables, are good indicators of critical hypoperfusion [2-related variables was detected.The measurement of VCOerfusion ,22. Unde2 supply during CPB might lead to a hypoxic insult to the kidney, as previously hypothesized by other authors who demonstrated the relationship between a low Hb value during CPB and bad renal outcomes [The results of our study, linking postoperative AKI to a condition of 'dysoxia' during CPB, strengthen the hypothesis that a condition of inadequate Ooutcomes -11.2. As such, they have already been identified as independent predictors of postoperative AKI [2 had a marginally higher accuracy in predicting postoperative AKI, as suggested in a previous study [Hb content and HCT level are physiological variables that concur in the definition of the DOtive AKI -11. In ous study , but the2 supply. Under normal physiologic conditions, peritubular capillaries are nourished by efferent glomerular arteries [2 (pO2) levels as low as 25 mmHg [2. In vitro studies have demonstrated that some highly susceptible areas of the kidney are prone to ischemic injury in cases of even slight reductions in renal DO2 [Kidney physiology and function are particularly dependent on Oarteries , which carteries . This efenal DO2 .2 content in cases of acute anaemia is usually compensated by reduced blood viscosity with increased blood flow in the microcirculation and by a compensatory increase in cardiac output. This last mechanism may be impaired during CPB, where pump flow is usually adjusted on the basis of the patient's body surface area and temperature, not the Hb value.Reduced O2 belongs to the field of the modifiable risk factors; and (3) the negative predictive value of the identified cutoff value is very high, suggesting the potential clinical usefulness of these values within a preemptive strategy of renal protection.Our study highlights some concepts related to postoperative AKI prevention in cardiac surgery: (1) CPB is one of the major determinants of AKI, and, in our analysis, both CPB duration and the metabolic variables measured during CPB were independent predictors of AKI; (2) opposite to other determinants of postoperative AKI, DO2. Historically, it has been difficult to deploy CPB goal-directed strategies because of the limitations of parameters such as cardiac index, mean arterial pressure, venous saturation, arterial blood temperature, minimum Hb, partial pressure of CO2 and pO2.Goal-directed strategies are key to improving artificial heart and lung support during CPB. This is especially true, given our more complex population with more comorbidities than those in other studies. An effective goal-directed strategy should be based on proven, clinically significant quality indicators such as DO2 may limit the risk of postoperative AKI. To maintain an acceptable DO2, the pump flow should be adjusted according to the Hb value of the perfusate, not only according to the patient's body surface area and temperature. In other words, the pump should be used to adjust the cardiac output exactly, as it happens as a compensatory physiological mechanism during acute, severe anaemia.The take-away message from our results is that the maintenance of adequate DO2 are likely to occur. The study is not powered to detect other organs' responses to low DO2. Finally, this study was not a randomized, controlled trial comparing a specific strategy (targeted at a DO2 > 262 mL/minute/m2) with conventional CPB management.Our study has limitations. It lacked patients treated under hypothermic conditions < 27°C, when different limits of critical DO2. This concept should be tested for its clinical effects on kidney function and the function of other organs in an adequately large, randomized, controlled trial.The concept of goal-directed CPB perfusion merits further investigations that are based on clinically significant quality indicators such as DO2 < 262 mL/minute/m2 during CPB is independently associated with AKI stage 2 following cardiac operations.• A nadir DO• Other determinants of AKI are patient age, the presence of comorbidities, the type of operation  and CPB duration.2 level is significantly associated with prolonged ICU and postoperative hospital lengths of stay.• Nadir DO2 during CPB (by reducing haemodilution and maintaining high pump flow) should be tested as a possible preemptive strategy for postoperative AKI in a randomized, controlled trial.• Goal-directed perfusion aimed at preserving DO2: oxygen delivery; Hb: haemoglobin; PRC: packed red cells; ROC: receiver operating characteristic; VCO2: carbon dioxide production.AKI: acute kidney injury; AUC: area under the curve; CPB: cardiopulmonary bypass; DO2 and VCO2 monitoring during CPB and received honoraria from Medtronic Inc. and Sorin Group for speaking at congresses and symposia. No financial or nonfinancial competing interests exist for any of the other authors.MR is the owner of a patent for DOMR designed the study, performed the statistical analysis and prepared the manuscript. FDS and MB were responsible for data acquisition and interpretation. JM was responsible for data acquisition and interpretation and drafted the manuscript. GVN was responsible for data acquisition and manuscript preparation. TA performed the statistical analysis and designed the study. All authors read and approved the final manuscript.


Sse1 is a conserved protein that is a noncanonical member of the Hsp70 protein superfamily. Sse1 influences the cellular response to heat stress and has also been implicated in playing a role in the propagation of prions in yeast. Sse1 can seemingly exert its effects in vivo through direct or indirect actions by influencing the nucleotide exchange activity of canonical cytosolic Hsp70s. Using a genetic screen based on the inability to propagate the yeast [PSI+] prion, we have identified 13 new Sse1 mutants that are predicted to alter chaperone function through a variety of different mechanisms. Not only are these new Sse1 mutants altered in the ability to propagate and cure yeast prions but also to varying degrees in the ability to grow at elevated temperatures. The expression levels of chaperone proteins known to influence yeast prion propagation are unaltered in the Sse1 mutants, suggesting that the observed phenotypic effects are caused by direct functional alterations in these mutants. Mapping the location of the mutants onto the Sse1 crystal structure suggests that more than one functional alteration in Sse1 may result in changes in prion propagation and ability to function at elevated temperatures. All Sse1 mutants isolated provide essential functions in the cell under normal growth conditions, further demonstrating that essential chaperone functions in vivo can to some degree at least be detached from those related to propagation of prions. Our results suggest that Sse1 can influence prion propagation through a variety of different mechanisms.The yeast Hsp110 chaperone  Saccharomyces cerevisiae by the Sse1 and Sse2 proteins. SSE1 and SSE2 constitute an essential gene pair in yeast  and are noncanonical members of the Hsp70 superfamily  result in similar phenotypic defects, supporting the hypothesis that Sse1 is an evolutionary vestige of Hsp70  for Hsp70. Sse1 does reflect that of canonical Hsp70s. It consists of a N-terminal nucleotide-binding domain (NBD), a β-sandwich domain (SBD-β) and a three helical bundle domain (3HBD or SBD-α) toward the C-terminus. The Sse1 protein has a compact structure with tight interactions between the NBD and substrate-binding domain (SBD). Unlike Hsp70, the Sse1 SBD-α does not form a lid over its binding pocket but instead interacts with the flank of the Sse1 NBD  was amplified from a cDNA clone purchased from Origene . All plasmids constructed in this study were verified by sequencing.Strains and plasmids used and constructed in this study are listed and described in PSI+] was carried out as described  and SUQ5 causes efficient translation read through of the ochre mutation in the ade2-1 allele. Non-suppressed ade2-1 mutants are Ade- and are red when grown on medium containing limiting amounts of adenine due to the accumulation of a pigmented substrate of Ade2. Partial suppression of ade2-1 by [PSI+] allows growth without adenine and eliminates the pigmentation . Cells were transformed with wild-type (WT) or mutant SSE1 alleles and transformants were selected on medium lacking leucine. At this stage all cells (at least 100) were scored for color phenotype on the basis of being white, red or sectored.Effects on  prion  to others having minor effects on color phenotype . As expected, all Sse1 mutants that could not propagate [PSI+] could not grow on medium lacking adenine .Applying the plasmid shuffle technique as described in +] prion , Table 3 adenine . However adenine . The rea of Sse1 . DeletioFes1 and Sse1 have been shown to be NEFs for cytosolic Hsp70s , cells were placed onto YPD to assess color and –ADE –HIS medium to assess the ability to grow on medium lacking adenine. Although the color phenotype on YPD for Sse1 WT or mutant cells harboring the vector or overexpressing FES1 is consistent with presence of Sse1 alone (compare FES1 YPD panels), the ability of some CMY02 Sse1 mutant cells to grow on medium lacking adenine is influenced greatly by the absence of histidine (compare FES1 –ADE panels). Only G616D appears altered in color on YPD by the presence of FES1 overexpression. However, this color change does not correlate with a significant increased ability to grow on –ADE medium  chaperone family expression levels in all the Sse1 mutants. Sse1. Only the P37L mutant appeared to have slightly increased levels of Hsp104 and Ssa, but taking into account previous findings these are unlikely to be the cause of any prion or temperature-related phenotypes . We theSse1 mutants are unable to grow at 39°. One possible explanation for this phenotype is that such Sse1 mutants are unstable at this temperature. We therefore used Western blotting to assess the stability of Sse1 mutants following exposure to 39° for 1 hr and found no difference in stability between any Sse1 mutants compared to wild-type protein (data not shown).As shown in Sse1 protein alone and in complex with cytosolic Hsp70 has been determined . Of the nine mutants identified within the NBD four are predicted to affect ATP binding , three to alter interaction with cytosolic Hsp70 , and three remain unclear  (File S1). The four mutants isolated in the SBD domain are predicted to alter either Sse1 interaction with cytosolic Hsp70 , substrate binding (S440L), or protein−protein interactions (E504K)  , File S1Figure S1 shows an alignment of Sse1 and Sse2. Although these proteins share 76% identity, Sse2 is unable to compensate for Sse1 in terms of [PSI+] prion propagation or growth at higher temperatures  could propagate [PSI+] in the G600 background. We found that both Fes1 and HSPH1 were unable to complement essential Sse1/2 functions in the CMY02 strain . This new role for Hsp110 proteins is conserved across species and provides the first clearly identified protein disaggregation machinery in mammalian cells  in modulating the histidine and/or adenine biosynthetic pathways. Both pathways are part of the “super-pathway of histidine, purine and pyrimidine biosynthesis” (Saccharomyces Genome Database) and converge on production of the biosynthetic intermediate aminoimidazole carboxamide ribonucleotide, accumulation of which can be toxic to the cell. If Sse1 is involved in modulating this super-pathway then our mutants may be affected in the ability to synthesize either histidine or adenine (or both) and toxic intermediates on this pathway may also be caused to accumulate. The addition of histidine or adenine to growth medium would have the effect of switching off these pathways and therefore suppressing any impaired growth phenotype due to the accumulation of toxic intermediates.Phenotypic analysis of the eratures , Table 3 complex , Table 3henotype . CurrentPSI+] propagation and also heat shock we were surprised to discover that all the Sse1 mutants were unable to efficiently cure the [URE3] prion. In a previous study, Sse1G223D mutant  was unable to cure [URE3] whereas Sse1K69M (can bind ATP but defective in hydrolysis) efficiently cured [URE3]. Thus, it seemed that efficient Sse1 NEF activity is required to cure [URE3]. Our data suggest that this may be an oversimplification. The clear phenotypic differences observed for the Sse1 mutants in respect of [PSI+] propagation and heat shock cannot be explained by a single unifying change in Sse1 function in all mutants. This suggestion is also supported by the location of the mutations on the Sse1 structure. Therefore it appears that a variety of mechanisms that alter Sse1 function can alter the ability to cure [URE3]. However, it should be noted that the ability to cure [URE3] could be influenced by the prion variant that is present in the cells. The [URE3] variants present in the SB34 strain and strains used by Given the variation in the effects of mutants upon . When this residue is mutated to create Sse2Q504E [PSI+] can be propagated albeit not to the same extent as Sse1  was shown to be unable to complement heat shock phenotypes of a sse1 deletion strain constructed in the W303 background  family is well characterized in its ability to modulate prion propagation and how this function can be distinct from roles in the heat shock response (Sse1.The isolation of a set of new