Cyclin-dependent kinase (CDK) 4/6 inhibition has been demonstrated to improve progression-free survival (PFS) in patients with human epidermal growth factor receptor 2 (HER2−), hormone receptor positive (HR+) in advanced breast cancer . PalbociCDK4/6 inhibitors have been approved by the US Food and Drug Administration (FDA) for initial endocrine therapy in postmenopausal women with metastatic or advanced HR+/HER2− breast cancer in combination with an aromatase inhibitor and for the treatment of endocrine therapy-resistant HR+/HER2− advanced or metastatic breast cancer in combination with Fulvesterant (a selective estrogen receptor degrader) . DespiteAnnals of Oncology Condorelli et al. [RB1 gene was noted through plasma ctDNA analyses at the point of disease progression. In the first patient a frameshift event involving exon 8 of RB1 was observed that was predicted to result in a non-functioning truncated version of the protein. This event was not observed through NGS analysis of a liver biopsy acquired before CDK4/6 inhibition. In the second patient of the case-series four RB1 alterations were noted at progression on palbociclib that were not detectable before initiation of therapy. The variant with the highest allele frequency in plasma at progression (Chr13(GRCh37): g.48937094G>A) has been previously shown in lung cancer to result in loss of the Rb protein region responsible for the binding of Rb to E2F-transcription factor complexes [Circulating tumour DNA (ctDNA) describes molecules of cell-free DNA circulating in plasma that originate from a patient’s tumour. ctDNA analyses by next-generation sequencing are demonstrating translational utility within clinical contexts ranging from non-invasive screening , trackini et al.  leverageomplexes . The finRB1 alterations potentially being selected at disease progression following intervention with CDK4/6 inhibitors in patients with metastatic breast cancer. These observations build on a previous invivo investigation of CDK4/6 inhibitor resistance using patient-derived tumour xenograft models that suggested Rb1 inactivation as a resistance mechanism to chronic CDK4/6 inhibition [This study is of interest for the following reasons. Firstly, it provides observational evidence of deleterious hibition . SecondlRB1 alterations and ctDNA analyses demonstrating acquired RB1 alterations—patient 1 received everolimus and exemstane before palbociclib and patient 2 received capecitabine and paclitaxel following ribociclib. Therefore, we cannot be certain that the acquisition of Rb1 alterations solely associate with selective pressure induced by CDK4/6 inhibition. Advancing the findings reported in this case-series will require a larger cohort to determine the incidence of Rb1 alterations as resistance mechanisms in patients with metastatic breast cancer on CDK4/6 inhibitors. Furthermore, more frequent ctDNA monitoring is necessary to follow the dynamics by which RB1 alterations emerge and ascertain the association of their emergence with disease progression.There are however caveats to consider regarding this case-series. The number of patients described within the manuscript is small and there is no indication as to the frequency by which Rb1 alterations are detected at progression on CDK4/6 inhibition in this patient population. Additionally, patients 1 and 3 in the case-series were treated with two lines of therapy in between the biopsies showing lack of RB1 and therefore could conceivably underlie an immune predatory selection pressure toward selection of Rb1 altered populations whilst undergoing treatment with CDK4/6 inhibitors. The fact that CDK4/6 inhibition has recently been shown to increase PD-L1 expression in mouse models of breast cancer provides a clear rationale for anti-PD1 treatment as a combination therapy with CDK4/6 inhibition before the emergence of Rb1 loss of function [Given this work, it is notable that CDK4/6 inhibition has recently been associated with increasing tumour cell antigen presentation through a mechanism involving downregulation of Rb1-E2F induced DNA methyltransferase 1 (DNMT1) activity, increased expression of endogenous retroviral elements and type III interferon production . This refunction .


CYP2A6 has been identified as the main gene that codifies the enzyme that metabolizes nicotine. Many alleles have been identified after the discovery of CYP2A6, suggesting a wide interethnic variability and a diverse smoking behavior of the allele carrying individuals. The main purpose of this review is to update and highlight the effects of the CYP2A6 gene variability related to tobacco consumption reported from diverse human populations. The review further aims to consider CYP2A6 in future studies as a possible genetic marker for the prevention and treatment of nicotine addiction. Therefore, we analyzed several population studies and their importance at addressing and characterizing a population using specific parameters. Our efforts may contribute to a personalized system for detecting, preventing and treating populations at a higher risk of smoking to avoid diseases related to tobacco consumption.Tobacco consumption has become a major public health issue, which has motivated studies to identify and understand the biological processes involved in the smoking behavior for prevention and smoking cessation treatments.  Tobacco consumption has become an epidemic affecting more than 1,000 million people worldwide and is considered the main cause of avoidable death causing approximately 6 million premature deaths each year . Thus, Among cigarette compounds, nicotine is responsible for causing dependence by stimulating the smoker and allowing the other compounds to access the body, causing chronic harmful effects and tobacco-related diseases.CYP2A6 gene plays an important role. Its protein product (by the same name) is the principal enzyme responsible for nicotine metabolism to cotinine and other sub-products in the human body. However, more than 45 alleles have been discovered suggesting wide interindividual and interethnic variety.Tobacco consumption is caused by environmental, psychosocial and genetic factors . PrevioCYP2A6 related to tobacco consumption reported in diverse human populations. Moreover, we aimed to consider these polymorphisms in future studies as possible genetic markers for the prevention and treatment of nicotine addiction.The main purpose of this review is to update and highlight the effects of the genetic polymorphisms of The CYP2A6 enzyme belongs to the enzyme superfamily known as the cytochrome P450 system (CYP450), also classified in the drug metabolizing enzymes group. These enzymes are found in the endoplasmic reticulum of the cells of some tissues in the body, particularly in the liver. Moreover, they are phase I enzymes responsible for metabolizing more than 80 % of drugs such as xenobiotics and endogen products in the body  that release neurotransmitters in the brain and cause a pleasant sensation in the smoker. The nicotine availability in the body is mediated by biological factors, mainly those related to its metabolism. Smokers tend to consume the same amount of nicotine each day to acquire the desired effects by modulating their smoking behavior to adjust nicotine availability for the purpose of regulating nicotine levels in the body .It has been reported that CYP2A6 is the main enzyme involved in the nicotine oxidation to cotinine. CYP2A6 catalyzes approximately 80 % (55-92 %) of this reaction via C-oxidation in addition to other metabolic pathways for nicotine and its metabolites  and are inactivated and removed from the body  making their genes ideal candidates for altering smoking behavior .CYP2A6 gene has a 6 kb extension length, and it is composed of 9 exons, which encode for a 494 amino-acid product  are also present. The CYP2A subfamily cluster includes the CYP2A6, CYP2A7, and CYP2A13 genes and other pseudogenes . It is al., 2001).Human CYP-Allele Nomenclature Database (HCAND)  was created in 1999 for the identification and characterization of CYP2A6 alleles (and other CYP450 genes). This database consists of a committee for unifying and assigning nomenclature for the already discovered alleles and alleles to be discovered in the future . To date, 42 well-characterized alleles and some haplotypes that are uncharacterized  have beCYP2A6*1A . CYP2A6al., 2000). In vital., 2009; Wang etal., 2009; Yoshidaal., 2009). Howeveal., 2000; Mwenifual., 2000, 2008; Nak; NakCYP2al., 2002; Rao et al., 2002; Schoedeal., 2002; Xu et aal., 2002); Atlantal., 2002; Sorianoal., 2002)) and Fral., 2002)) also hal., 2002)), Northal., 2002)) and Caal., 2002)) had a al., 2002)) and Swal., 2002)) also ral., 2002) and Tatal., 2002). Africaal., 2002; Nakajimal., 2002)), Africal., 2002; Nakajimal., 2002)), Ghanaal., 2002)), Ethioal., 2002)). Amerial., 2002)) have aal., 2002)) and moal., 2002)), Ecuadal., 2002)), Chileal., 2002)) and Hial., 2002)). On thal., 2002)) of thiCYP2A6*5 is found in a higher frequency in Asian populations, East and Southeast Asian populations such as the Vietnamese had the highest frequency ) comparal., 2009; Nurfadhal., 2009; Oscarsoal., 2009; Schoedeal., 2009), Koreanal., 2009; Kwon etal., 2009; Nakajimal., 2009; Yoshidaal., 2009) and Malal., 2009) populatal., 2009, 2006; Nak; NakCYP2al., 2006, 2004; Nak; NakCYP2al., 2006; Schoedeal., 2006)), Spanial., 2006)) and Caal., 2006)). Then,al., 2006), but inal., 2006; Schoedeal., 2006)) while al., 2006; Koontz al., 2006; Nakajimal., 2006; Oscarsoal., 2006; Schoedeal., 2006) did notal., 2006; Schoedeal., 2006)). In Afal., 2006; Schoedeal., 2006) and othal., 2006; Koontz al., 2006; Mwenifual., 2006; Nakajimal., 2006).CYP2A6*13 has been reported in Japanese ) and Koal., 2002)). It waal., 2002; Nakajimal., 2002).CYP2A6*14 has a higher frequency in Caucasian ) than ial., 2005; Nakajimal., 2005)), but wal., 2005; Nakajimal., 2005).CYP2A6*15 has been reported at a minimum frequency in Japanese ) and Koal., 2002)), but nal., 2002; Mwenifual., 2002; Nakajimal., 2002).CYP2A6*16 has been reported in Caucasian (0.3-3.6 %) and African (0-1.7 %) populations , but noal., 2002; Nakajimal., 2002).CYP2A6*17 has a frequency of approximately 7.3-10.5 % in African populations , but noal., 2004; Fukami al., 2004; Nakajimal., 2004) populatCYP2A6*18 has a higher frequency range in Caucasian ) than ial., 2005; Nakajimal., 2005)), but hal., 2005; Nakajimal., 2005).CYP2A6*19 has been reported in a low frequency in Korean ) and Jaal., 2013; Nakajimal., 2013) populatCYP2A6*20 is present in African population at a frequency range between 1-1.7 % . This aal., 2005; Nakajimal., 2005).CYP2A6*21 has a higher frequency in Caucasian (0.5-2.3 %)  than inal., 2006; Nakajimal., 2006)). It haal., 2006) populatCYP2A6*22 has been reported at frequency less than 0.3 %  in Caucal., 2005) populatCYP2A6*23 has a frequency range among 1-2 %  in AfriCYP2A6*24 has a frequency range among 0.7-1.3 % in African populations , but itCYP2A6*25-*28 has only been reported in African populations, while CYP2A6*26-*27 has a frequency range of 0.7-0.9 %  and CYPal., 2009; Mwenifual., 2009).CYP2A6*31, *34 and *38 have not been studied in any population.CYP2A6*35 has been reported at a frequency between 2.5-2.9 %  in Afrial., 2010) populatCYP2A6*36 and *37 has been reported in Taiwanese populations at a frequency of 0.3 %, but not in African, Caucasian, Chinese nor Japanese , *40 (0.2 %), *41 (1.2 %), *42 (0.2 %), *43 (0.2 %), *44 (0.2 %) and *45 (0.6 %) have only been reported in the African population .CYP2A6*1X2, has been found at higher frequency in Asian populations as Asian ), Indiaal., 2006)), Chineal., 2006; Schoedeal., 2006; Xu et aal., 2006), Koreanal., 2006; Nakajimal., 2006) and Malal., 2006); the Hial., 2006) has a hal., 2006); Caucasal., 2006; Fukami al., 2006; Nakajimal., 2006; Rao et al., 2006; Schoedeal., 2006; Xu et aal., 2006), Spanisal., 2006), Swedisal., 2006), Serbiaal., 2006), North al., 2006) and Canal., 2006). In Afral., 2006)) and Etal., 2006; Nakajimal., 2006; Schoedeal., 2006).Most studies address the majority of a country's population as a general population. However, a few studies focus on more specific population classifications as ethnic and regional groups. In the Asian population, it was studied among Tottori, Shimane, Ehime and Fukuoka people of the respective districts of Yonago, Izumo, Matsuyama and Kurume located in Japan . In ChiCYP2A6 gene, which is highly polymorphic. This variability is due to changes in DNA, which have generated different responses to nicotine and are reflected in the individual smoking behaviors along with other factors. It has been reported that this variability has been generated and distributed over a long time in different human populations, showing well defined ethnic patterns. Although association studies between carriers of certain variants and different smoking behaviors have been numerous with plausible results, population studies reporting frequencies of these variants are few. General population studies exhibit most reliable information than association studies with a variable frequency, because the population requirements are usually more specific, creating a population bias. However, it would be advisable to address this type of methodology in higher risk populations of smoking and those where policies to control smoking are less efficient, and where more smoking-related diseases are reported in the population. Additionally, the population must be characterized by more specific requirements, such as including the ancestry informative markers and avoiding "self-reporting" as the unique classification criteria. While there is a Human CYP-Allele Nomenclature Database in which the genetic findings of CYP2A6 are unified, it is necessary to supplement it with updated data as per the population distribution. All of this could contribute to a personalized system that could detect, prevent and treat populations at risk of smoking, and in consequence, avoid tobacco consumption related diseases. The enzyme responsible for metabolizing nicotine is mostly encoded by the


Finding additional functional targets for combination therapy could improve the outcome for melanoma patients. In a spontaneous metastasis xenograft model of human melanoma a shRNA mediated knockdown of L1CAM more than sevenfold reduced the number of lung metastases after the induction of subcutaneous tumors for two human melanoma cell lines . Whole genome expression arrays of the initially L1CAM high MeWo subcutaneous tumors revealed unchanged or downregulated genes involved in epithelial to mesenchymal transition (EMT) except an upregulation of Jagged 1, indicating a compensatory change in Notch signaling especially as Jagged 1 expression showed an increase in MeWo L1CAM metastases and Jagged 1 was expressed in metastases of the initially L1CAM low MV3 cells as well. Expression of 17 genes showed concordant regulation for L1CAM knockdown tumors of both cell lines. The changes in gene expression indicated changes in the EMT network of the melanoma cells and an increase in p53/p21 and p38 activity contributing to the reduced metastatic potential of the L1CAM knockdowns. Taken together, these data make L1CAM a highly interesting therapeutic target to prevent further metastatic spread in melanoma patients. Malignant melanoma is an extremely dangerous disease with high mortality rates due to the aggressive metastatic potential of melanoma cells. Although the development of new therapies for patients with already metastasized melanoma over the last few years resulted in prolonged survival, for a considerable number of patients these new therapies still do not achieve stable remission for more than a few months  . To. To19]. Stained slides were scanned by a Mirax microscope  and the Panoramic Viewer software  was used to take images.Lung metastases were quantified as previously described . BrieflyAlu sequences was performed as previously described [DNA and RNA extraction from murine blood and tumors as well as quantification of human melanoma cells by quantitative real-time polymerase chain reaction (qRT-PCR) for human escribed .Continuous variables are displayed using boxplots.In all models the interaction of cell line (MeWo vs. MV3) and group (Luc vs. L1 kd) was tested using a likelihood-ratio test. In case of a significant interaction the group comparison was presented separately for each cell line and in case of insignificance the interaction was excluded from the model and an overall group comparison was presented.In vitro outcomes  were analyzed using linear models. Outcomes of the xenograft model were survival, tumor weight, metastases, and number of circulating tumor cells . Survival of mice was defined as time until mice were euthanized when the tumor ulcerated or reached an estimated mass exceeding 10% of the respective animal’s weight. As no censoring was existent it was analyzed using a linear model which was moreover adjusted for the type of death (ulcerated vs. not ulcerated). Tumor weight was analyzed in an analogous model. Number of metastases was analyzed and evaluated/quantified using a negative binomial regression which was adjusted for survival time and tumor weight and included furthermore CTCs as interesting variable. CTCs as outcome variable were logarithmized and analyzed using a linear model, which was adjusted for survival time and tumor weight.Parameter estimates are presented together with 95% confidence intervals (95%-CI). A two-tailed p<0.05 was considered to be statistically significant. All analyses were conducted using Stata 14.1 .Total RNA was extracted from the frozen tumor samples using the miRNeasy Mini Kit  as recommended by the manufacturer. RNA concentrations were determined by the Nanodrop 1000 spectrophotometer . RNA quality was checked using the Experion RNA StdSens Analysis Kit and Experion platform . For all samples RQI value  was greater than 8.Microarray expression analysis was performed using GeneChip® Human Transcriptome Array 2.0  as recommended by the manufacturer. Data processing was carried out using Signal Space Transformation–Robust Multi-Array Analysis (SST-RMA) method in Affymetrix Expression Console (version 1.4.1.46). Dispersion analysis (ANOVA) was applied to the logarithmic expression values using Affymetrix Transcriptome Analysis Console (version 3.0.0.466). For p-values correction we used the Benjamini-Hochberg method . AnalysiThe newly generated sublines were analyzed for L1CAM expression by flow cytometry.Fig 1A).Sublines with more than 85% (MeWo) and 75% (MV3) reduced L1CAM expression were pooled and used for all further experiments (designated MeWo and MV3 L1 kd). MeWo Luc and MV3 Luc cells with unchanged L1CAM expression, but with puromycin resistance were used as controls .In an XTT assay, MeWo L1 kd cells showed a significant increase in proliferation  compared with the MeWo Luc controls with unchanged surface L1CAM. For MV3 L1 kd there was a decrease in proliferation  in comparison to MV3 Luc controls .We found no significant overall influence of L1CAM knockdown on the cells’ ability for Fig 1D).In a colony forming assay in matrigel, a significant increase of colony numbers for MeWo L1 kd cells in comparison to Luc controls was observed  while there was no significant difference in colony numbers between L1 kd cells and Luc controls for MV3 .In laminar flow assays measuring adherence, tethering and rolling on activated endothelial cells (HUVEC), the number of events increased significantly for MeWo L1 kd cells  while it decreased for MV3 L1 kd cells  or no effect at all (invasion) for both human melanoma cell lines used. These in vitro results do not reflect the in vivo results from the following xenograft model (see below).Summarized, knockdown of L1CAM All animals developed subcutaneous tumors at the injection site and were euthanized when the tumor ulcerated or reached an estimated mass exceeding 10% of the respective animal’s weight. The time span between injections of melanoma cells until euthanization will subsequently be referred to as survival time.Fig 2A).L1CAM knockdown did not significantly change the animals’ survival time in MeWo and MV3 .Within the MV3 cell line the size of the L1CAM knockdown tumors was significantly greater  in comparison to the MV3 control tumors. The size of the MeWo L1CAM knockdown tumors was not significantly different from the control tumors . Metastasis  was 7.26 times higher in the MeWo and MV3 Luc group in comparison to the L1CAM knockdown group . Survival time also showed a significant influence on lung metastasis. With each day of survival, metastatic spread increased by 6.83% . Tumor weight and number of CTCs showed no influence on the number of metastases (p = 0.070 and p = 0.436 respectively).Metastatic spread of the human melanoma cells was exemplarily studied in the animals’ lungs. All control (Luc) metastases displayed high (MeWo) or low (MV3) L1CAM expression and even most L1 kd metastases still showed weak (MeWo) or very weak (MV3) L1CAM expression  in the bone marrow show a tendency towards the results for the animals’ lungs: In the MeWo luc group 33.3% of the animals were positve for DMCs, 23.8% were positive in the MeWo L1 kd group. For MV3, 55.6% were positive for DMCs in the Luc group and 22.2% in the L1 kd group.Melanoma cell numbers (determined by RT-qPCR) in the bone marrow of the animals were low: most of the samples showed no values above background, thus only samples with at least 1 melanoma cell per 10Fig 2D).Regarding influence factors on the number of CTCs we found the mass of the primary tumor to be significant: CTC numbers increased by the factor 3.33 per 1 g increase of the primaries . Survival time and L1CAM knockdown showed no significant influence on CTC numbers (p = 0.454 and p = 0.161 respectively)  genome expression analyses starting with the corresponding primary xenograft tumors of the initially L1CAM high cell line MeWo (MV3 microarray data see below).S1 Table). Analysis of this regulation profile using the Pathway Studio software suggests a central role of Tumor Growth Factor β  as many genes that have been reported to be directly or indirectly regulated by TGFβ display a respective up- or downregulation in the L1 kd tumors .Considering all genes with a significant  expression fold change of at least 1.51, in the xenograft tumors formed by the originally L1CAM high MeWo cells, the expression of 981 genes was up- and of 1000 (excluding L1CAM itself) downregulated  regulated genes were screened for EMT related genes:The phenotype of the L1CAM knockdown tumors appeared less mesenchymal than the control tumors’; e.g. significant reduction of N-cadherin (CDH2), Tyrosine-protein kinase receptor UFO (AXL), Matrix Metalloproteinase 2 (MMP2), TGFB1 and Secreted Protein Rich in Cysteine (SPARC) expression. An exception from this reduction of EMT related gene expression, however, was an increased expression of the NOTCH ligand JAG1 (Jagged 1) in the L1CAM knockdown tumors. As this gene was the sole exception among the EMT related genes, we chose JAG1 to exemplarily verify the array results. Interestingly, the increase in JAG1 expression was accompanied by a significant increase in NOTCH1 and 2 expressions, albeit only 1.40 and 1.41 fold, respectively, and a significant increase in NOTCH signaling activator Contactin 1 (CNTN1) expression. With CCNC (Cyclin-C), HIF1A (Hypoxia-inducible factor 1-α) and KAT2B (K acetyltransferase 2B) three other genes involved in NOTCH signaling are downregulated.Fig 5B).On protein level, a tendency towards increased Jagged 1  and NotcTable 1, analysis see Fig 6). Immunohistochemical stainings showed clear Jagged 1  and Notch 1 expression in the subcutaneous MV3 tumors and lung metastases, however, we did not see a tendency towards higher or lower expression in the L1 kd tumors or metastasis compared with the Luc controls which already displayed a higher Jagged 1 expression than the MeWo Luc tumors . This lack of difference for MV3 is in line with the results on RNA level as no significant difference in Jagged 1 and Notch was detected by analysis of the arrays as well.As L1CAM knockdown also resulted in a reduction in metastasis for the initially L1CAM low MV3 cells, an additional whole genome expression analysis of the corresponding MV3 primary xenograft tumors was performed . Genes wFig 6), up-regulation of RFWD2  in the L1 kd tumors should lead to an increase in p53/p21 pathway activity. This could be verified for the subcutaneous tumors of both cell lines using immunohistochemistry; however, there was a difference between the two cell lines: The L1CAM wildtype MeWo Luc tumors showed no p53 and low p21expression but the respective L1CAM knockdown tumors displayed clear p53 and p21 expression. In contrast to the MeWo Luc tumors, a pronounced p53 expression was found for the MV Luc tumors which did not change for in the L1 kd tumors. However, as for the MeWo tumors, p21 expression did increase in the MV3 L1 kd tumors . According to the presumption that tumor cells with increased p53/p21 pathway activity should display a marked reduction in their metastatic potential, the remaining metastasis in the lungs of the L1 kd animals indeed showed no pronounced change in p53/p21 expression compared with their Luc counterparts with unchanged L1CAM expression .Of the genes regulated in the L1 kd tumors of both the initially L1CAM high (MeWo) and L1CAM low (MV3) cell lines .The only up-regulated gene of the 17 –MAP2K6  indicated an increase in p38 phosphorylation in the L1 kd tumors. The subcutaneous tumors for both cell lines showed an extreme intra-tumoral heterogeneity in p38 phosphorylation ranging from areas with extremely high phospho-p38 to areas with no detectably phospho-p38 which made a reasonable analysis by immunohistochemistry impossible. The L1 kd metastases from both cell lines, however, displayed an increase in phospho-p38 compared with their Luc counterparts . An exception to this was the expression of the Notch ligand Jagged1 which showed an increase in expression. Genes with changed expression in the xenograft tumors after L1CAM knockdown for both melanoma cell lines indicate an increase in p53/p21 and also p38 activity which could be verified on protein level using immunohistochemistry. Both, reduced expression of EMT-network genes and increased p53/p21 and P38 activity probably contribute to the reduction of the metastatic potential of the human melanoma cells after L1CAM knockdown.in vitro experiments with the MeWo and MV3 Luc and L1CAM knockdown cells gave no clear indication how L1CAM influenced the metastatic behavior of the corresponding xenograft tumors in vivo  as both cell lines displayed different changes, e.g. in proliferation, after L1CAM knockdown. Melanoma cells are rather heterogenic in many aspects [in vitro changes in behavior differed between the two cell lines. We used an originally L1CAM high (MeWo) and an L1CAM low (MV3) cell line which might have been differently affected by L1CAM knockdown as their original dependence on L1CAM probably also differed. L1CAM knockdown led to a decrease in in vitro proliferation for MV3, but obviously even increased tumor growth in vivo. However, the in vitro proliferation assay is performed with cells in 2D culture with a standard cell culture medium. The conditions in this assay are thus quite different from the growth conditions in the animals and it appears not always to be very helpful for predicting the in vivo behavior of the cells. Considering the conflicting in vitro data we think that it is all the more remarkable that the influence of the L1CAM knockdown on the in vivo metastatic potential did not differ between the cell lines as the xenograft model of this study clearly demonstrated that L1CAM expression promotes melanoma metastasis. The unchanged CTC numbers in the xenograft indicate that the number of cells that left the primary tumor and entered the bloodstream (to become CTC) and were able to leave the vasculature to invade underlying tissue indeed did not differ between Luc and L1 kd cells. However, this number might not be directly correlated with the number of metastases as the L1CAM knockdown probably affects steps of the metastatic cascade following extravasation as the increase in p38 phosphorylation  indicates that the melanoma cells in the L1 kd metastases are more stressed than the Luc controls, but L1CAM expression  could be detected even in the metastases formed by the L1CAM knockdown cells  indicating that L1CAM is necessary for the outgrowth of metastases. Additionally, involvement of L1CAM in spreading on the abluminal surface of capillaries has been demonstrated in a xenograft model of brain metastasis with human breast and lung cancer cells [The performed ent ones) which mier cells .Fig 4). Interestingly, L1CAM is able to directly or indirectly regulate TGFB1 expression and not only vice versa.Although melanocytes reside in the stratum basale of the epidermis they are not epithelial cells themselves but are derived from the neuronal crest. During malignant transformation these non-epthelial cells do not undergo a classic EMT (as observed in carcinoma), however, many molecules involved in EMT do also play important roles in the malignant transformation of melanocytes although these roles are sometimes different from classic EMT . For exaFor L1CAM low melanoma, a partial compensation for the lack of L1CAM could be an overexpression of JAG1 (Jagged 1), modulating Notch signaling . UpregulFig 6), RASAL2 (Ras GTPase-activating protein nGAP) and MALAT2 (long non-coding RNA Metastasis Associated Lung Adenocarcinoma Transcript 2) have been implicated in EMT networking, with the role of RASAL2 still under discussion [) and p38  pathways could indeed be verified by immunhiostchemistry.Of the genes regulated in the L1 kd tumors of both the initially L1CAM high and L1CAM low cell lines  in subcutaneous (s.c.) tumors of human melanoma cells (MeWo and MV3) with unchanged L1CAM expression  and L1CAM knockdown . All scale bars: 100 μm. Stainings show ncreased p53 for MeWo L1 kd tumors only but increased p21 for MeWo and MV3 L1 kd tumors.(TIF)Click here for additional data file.S2 FigImmunohistochemical staining for p53 (left panels) or p21 (right panels) expression (red) in lung metastases of human melanoma cells (MeWo and MV3) with unchanged L1CAM expression  and L1CAM knockdown . All scale bars: 50 μm. Stainings show no change in p53 and p21 expression for MeWo and MV3 L1 kd cells.(TIF)Click here for additional data file.S3 FigImmunohistochemical staining for phospho-p38 (red) in lung metastases of human melanoma cells (MeWo and MV3) with unchanged L1CAM expression  and L1CAM knockdown (respective lower panels). All scale bars: 50 μm.(TIF)Click here for additional data file.S1 TableIncluded are only fold changes at least +/- 1.51; ANOVA and adjusted p-values <0.05.(DOCX)Click here for additional data file.S2 TableIncluded are only fold changes at least +/- 1.51; ANOVA and adjusted p-values <0.05.(DOCX)Click here for additional data file.


Higher plants are sedentary organisms which inevitably endure a variety of environmental stresses throughout the life cycle. Abiotic stresses can be atmospheric like cold, heat and UV irradiation; or can also be edaphic like salinity, drought, and heavy metal toxicity  was observed in Zea mays during cold acclimation. This resulted in the global deacetylation at the lysine residues on the histone subunits H3 and H4  along with reduced DNA methylation and dimethylation at H3K9 (H3K9me2) in the unsilenced stretches  crucially phosphorylate histone H3-Thr 3 (H3T3ph) in the pericentromeric/knob regions in Arabidopsis to maintain heterochromatin organization and chromosome functions  and Galactinol Synthase 3 in Arabidopsis. H3K27me3 decrease permitted the de-repression of these cold responsive genes and ensured systemic acclimatization to low temperatures  and COR413 in Z. mays exposed to cold / DREB1s, CORs, Responsive to Dehydration genes (RDs), cold induced genes (KINs), and the cold induced TF, ICE1 during cold stress  chromatin to maintain FLC expression  and their target genes  and the 600 kDa polycomb complex . The MSI-like protein, MSI4/FVE negatively regulated Chorispora bungeana exposed to 4°C chilling and −4°C freezing stress. Rapid alterations in cytosine methylation occurred throughout the periods of chilling and freezing  , VRN2 and Vernalization Insensitive 3 (VIN3). Cold stress induces the expression of VIN3, a homeodomain finger containing gene encoding protein. VIN3 associates with an unidentified complex and triggers repressive H3K27me3 and H3K9me in the FLC chromatin  and VAL2 stably repress FLC during prolonged cold treatment  in the late phases of vernalization. High ColdAIR levels reduce H3K4me3 in FLC and facilitates flowering after vernalization  recruits epigenetic factors to regulate vernalization-related floral gene expression in winter annual accessions of FLC during vernalization is maintained as a cellular memory via the mitotic inheritance of the repressive histone marks in the chromatin  has verified the increase in gene activating H3K9me3 and decrease in gene suppressing H3K27me3 in VRN1 during vernalization  were observed in both the vernalized and non-vernalized seedlings. This depicts that unlike in VRN1, cold treatment does not modify the chromatin of these genes (Achrem et al., VRN2 and FT expression in the germinating caryopses but refrain from regulating VRN1 expression in the shoot apices (Li and Liu, Arabidopsis seedlings exposed to 30°C, directly after cold treatment exhibited low H3K27me3 at the FLC locus (Bouche et al., Trans-generational repression of Triticum monococcum where VRN1 initiates the regulatory cascade to down-regulate cold acclimation genes like CORs and CBFs (Galiba et al., Epigenetic regulation under cold stress is a manifestation of multiple crosstalks Figure  and planAR and SW suggested the title of the opinion article. AB designed the article, drafted the entire manuscript and arranged the references. AR incorporated all the necessary modifications. All the authors checked and confirmed the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The first sentence of the sixth paragraph in the Discussion section should have cited references 39–45, 56 instead of 39–45. The correct sentence should read: It can be therefore safely inferred that the copper metal composing the axe was extracted from Southern Tuscan ores and not from Alpine or Balkanic ores, despite the fact that copper deposits in both areas were certainly known and exploited during the Copper Age .The third sentence of the eighth paragraph in the Discussion section should have cited reference 42 instead of references 42–56. The correct sentence should read: Virtually all of the objects found south of the Alps  were made of Tuscan or southern Alpine copper , whereas all of the objects found north (Tyrol) [41] and east (Serbia) [42] of the Alps were produced from Balkanic copper, mainly from Serbia and Bulgaria.Fig 5. 2D projections of the available lead isotope data for Neolithic and Copper Age objects from Italy , Tyrol [41], and Serbia [42] (from the late 5thmillennium to the early 3rdmillennium BC). (a) 206Pb/204Pb vs 207Pb/204Pb plot; (b) 206Pb/204Pb vs 208Pb/204Pb plot.The Fig 5 legend should have cited reference 42 instead of 42–56. The correct Fig 5 legend is:


AKT3, HNRNPU and ZBTB18 are highly expressed in the brain and point mutations in these genes have been recently identified in children with neurodevelopmental phenotypes. In this study, we report the clinical and molecular data from 17 patients with 1q43q44 microdeletions, four with ZBTB18 mutations and seven with HNRNPU mutations, and review additional data from 37 previously published patients with 1q43q44 microdeletions. We compare clinical data of patients with 1q43q44 microdeletions with those of patients with point mutations in HNRNPU and ZBTB18 to assess the contribution of each gene as well as the possibility of epistasis between genes. Our study demonstrates that AKT3 haploinsufficiency is the main driver for microcephaly, whereas HNRNPU alteration mostly drives epilepsy and determines the degree of intellectual disability. ZBTB18 deletions or mutations are associated with variable corpus callosum anomalies with an incomplete penetrance. ZBTB18 may also contribute to microcephaly and HNRNPU to thin corpus callosum, but with a lower penetrance. Co-deletion of contiguous genes has additive effects. Our results confirm and refine the complex genotype-phenotype correlations existing in the 1qter microdeletion syndrome and define more precisely the neurodevelopmental phenotypes associated with genetic alterations of AKT3, ZBTB18 and HNRNPU in humans.Subtelomeric 1q43q44 microdeletions cause a syndrome associating intellectual disability, microcephaly, seizures and anomalies of the corpus callosum. Despite several previous studies assessing genotype-phenotype correlations, the contribution of genes located in this region to the specific features of this syndrome remains uncertain. Among those, three genes, The online version of this article (doi:10.1007/s00439-017-1772-0) contains supplementary material, which is available to authorized users. AKT3 is the main candidate for microcephaly, ZBTB18 for AnCC and HNRNPU for epilepsy  is associated with a complex neurological phenotype, including moderate to severe intellectual disability (ID), microcephaly, epilepsy and anomalies of the corpus callosum (AnCC). More than 40 patients with 1q43q44 microdeletions of variable sizes identified by chromosome microarray have been reported. Comparison of their clinical phenotypes has established some genotype-phenotype correlations and has identified three genes, preferentially expressed in the brain and located in a genomic region spanning 1.36 Mb , as the main genes contributing to the 1qter microdeletion phenotype: AKT3, ZBTB18 and HNRNPU have recently been identified by whole exome sequencing in patients with different neurodevelopmental phenotypes. AKT3 encodes a serine/threonine protein kinase involved in the mammalian target of rapamycin (mTOR) signaling pathway. Gain-of-function point mutations or microduplications leading to abnormal AKT3 and mTOR activation, most of which are limited to somatic brain populations, cause a spectrum of disorders characterized by cerebral hemisphere overgrowth such as hemimegalencephaly (HME), megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) and megalencephaly-capillary malformation (MCAP)  encodes a C2H2-type zinc finger transcription factor negatively controlling the expression of genes involved in neuronal development, including cell division of progenitor cells and survival of postmitotic cortical neurons    U, an abundant nucleoplasmic phosphoprotein able to bind pre-mRNA in vivo, possibly involved in pre-mRNA splicing  in humans or mice , Genematcher  . Clinical data were collected using a standardized questionnaire directly from the referring clinicians. Microcephaly was considered for patients with an occipitofrontal circumference (OFC) of at least −2.5 standard deviation (SD) below the mean. A radiologist, a neuropediatrician and a geneticist collegially ascertained brain MRI anomalies. AgCC designates the absence of one or all parts of the CC, DysCC is used for complete CC with an abnormal shape or abnormally small CC, ThCC is used for complete CC with insufficient thickness.We then collected clinical and molecular data of patients with We retrieved the probability of loss-of-function (LoF) intolerance (pLI) calculated by the Exome Aggregation Consortium (ExAC) and the haploinsufficiency score (HI) established by Huang et al.  for genehttp://genetics.bwh.harvard.edu/pph2) and SIFT (http://sift.bii.a-star.edu.sg).Missense variants were assessed in silico for possible pathogenicity using Alamut Visual 2.7 , PolyPhen-2 (https://genome-euro.ucsc.edu) to align microdeletions on a schematic representation of the 1q chromosome. Alignments were performed using different colors explained in the figure legends. Minimal critical regions were defined as the smallest deleted region of overlap found in at least 95%  or 85% (AnCC) of patients harboring a given phenotypic trait. Frequencies were compared using the Fisher’s exact test.We used UCSC  were predicted to be probably or possibly intolerant to haploinsufficiency among the 44 genes other than OR genes (Table S1). Among these genes, AKT3, ZBTB18 and HNRNPU are clearly those with the highest pLI scores and HI ranks as well as the highest expression in the brain (Table S1). We then decided to focus our study on these three genes that likely contribute to most clinical features of 1q43q44 microdeletion syndrome, as predicted from previous genotype-phenotype correlation studies. Specifically, 12 microdeletions encompassed AKT3, ZBTB18 and HNRNPU; six completely or partially included AKT3 but not ZBTB18 and HNRNPU; nine encompassed AKT3 and ZBTB18 but not HNRNPU, two ZBTB18 and HNRNPU but not AKT3, and 25 deleted HNRNPU but not AKT3 and ZBTB18 .To decipher the contribution of genes located in 1qter region to the corresponding microdeletion syndrome, we collected clinical data from 17 patients with 1q43-q44 microdeletion (Table S2) and compiled them with those of 37 previously reported patients fulfilling our criteria . Altogether, the 54 deletions span a 10 Mb region comprising 83 genes, 39 of which encode clustered olfactive receptors (OR). Seven genes ; 47/54 patients had available brain imaging: 20 had AnCC, including ten with agenesis , five with dysgenesis  and five with thin corpus callosum . Finally, 36/54 patients had epilepsy .AKT3 and ZBTB18, regardless of the presence of HNRNPU in the deletions . AKT3 was included in 24/26 deletions identified in individuals with microcephaly, whereas only 2/23 patients with microdeletions sparing AKT3 had microcephaly (p = 1.46E−9). The number of patients with microcephaly who had deletions including ZBTB18 (n = 21/22) and sparing ZBTB18 (5/27) was also significantly different (p = 4.25E−8). Four of the six deletions including coding sequences of AKT3 only and one of the two deletions encompassing ZBTB18 but not AKT3 were associated with microcephaly. The minimal critical region for microcephaly  mapped to a region encompassing the 5′ upstream region and the five first exons of AKT3  ZBTB18 haploinsufficiency may independently lead to microcephaly with a lower penetrance; and (3) co-deletion of AKT3 and ZBTB18, which are neighboring genes spaced from only ~200 Kb, may have an addictive effect, resulting in constant microcephaly.The alignments of microdeletions found in patients with a known OFC and comparison of their gene content showed that microcephaly was present in all 20 patients with deletions encompassing both AKT3 deletion was not significantly associated with AnCC. More precisely, 11/26 patients with AKT3 deletion versus 16/21 without AKT3 deletion had a normal CC (p = 0.06). Accordingly, all six patients with a microdeletion involving only AKT3 had a normal CC. The proportions of patients with AnCC who had deletions encompassing (17/32) or sparing HNRNPU (5/15) were also not significantly different (p = 0.23). In contrast, the number of patients with AnCC was significantly higher in cases of deletions containing ZBTB18 (17/22) compared with deletions sparing this gene . However, these proportions reached statistical significance when considering AgCC instead of AnCC (p = 0.01). These results suggest that (1) the main driver for AnCC, and more particularly AgCC, in the 1q region is ZBTB18, although with incomplete penetrance, (2) HNRNPU haploinsufficiency can be associated with ThCC, and (3) deletion of both ZBTB18 and HNRNPU, which are 792 kb distant, has an additive effect, resulting in more penetrant AnCC phenotype and more frequent AgCC.Considering all types of AnCC, the alignment of deletions revealed that, contrary to microcephaly, HNRNPU and only one had a deletion sparing HNRNPU  or spared (n = 25/27) and the absence of seizures in patients with deletion restricted to this gene confirmed that AKT3 was not involved in epilepsy. The difference in the number of epileptic patients with or without ZBTB18 deletion was also not significant . These results suggest that the loss of one HNRNPU allele is the primary cause of epilepsy.Deletions identified in epileptic patients showed a shift toward the telomeric extremity of the 1q region . Of the 36 patients with epilepsy, 35 had a deletion including HNRNPU mutations, six had constitutive de novo mutations and one has a mosaic frameshift mutation . Two of them are located in zinc finger domains in which missense mutations tend to cluster  inherited from his father who was reported to have an intelligence within the normal range , (2) 13 with a deletion including HNRNPU but not AKT3 and ZBTB18 walked at a mean age of 32.6 months , and (3) two had a large deletion encompassing the three genes and walked at a mean age of 6.5 years (range 5–7 years). Among non-ambulatory patients, one carried a HNRNPU deletion and the three others had a deletion of the three genes. These data suggest that the loss of one HNRNPU allele has a more deleterious effect on walking acquisition than the loss of ATK3/ZBTB18 genes but larger deletions including all three genes have even more severe consequences. Walking abilities in patients with HNRNPU point mutations were similar to those with deletions of the gene. Patients with ZBTB18 mutations globally walked later than those with deletions including AKT3 and ZBTB18 although this should be confirmed on larger patient series.Since formal evaluations of cognitive functioning were unavailable for most other patients, we used the postural and language milestones to evaluate their developmental level. Walking abilities were available for 22 patients with 1q43q44 deletions older than 2 years. Four of them were unable to walk and 18 walked independently at a mean age of 35 months (median age 24 months). Among ambulatory patients, (1) three had microdeletions encompassing n = 5) or full (n = 1) sentences. None of the four patients with a deletion of all three genes made sentences, whereas 2/2 patients with deletions including AKT3 and ZBTB18 but not HNRNPU, and 4/10 patients with HNRNPU deletions did. Thus, patients with HNRNPU point mutations globally had more severe speech impairments than patients with deletions including HNRNPU, who had more variable language abilities. Yet, language abilities were more preserved in patients with ZBTB18 point mutations and individuals with deletions including ZBTB18 but sparing HNRNPU, since 6/7 could speak sentences.Language abilities were available for 15 patients with 1q43q44 deletions older than 4 years of our series only. Three patients did not speak any word, six had acquired a few words, six were able to speak short  in individuals with missense mutations leading to increased mTOR signaling that are usually—but not always—limited to mosaic brain tissues  three deletions sparing AKT3 (one encompassing ZBTB18 and HNRNPU and two HNRNPU but not ZBTB18) were also associated with microcephaly, and (2) all patients with deletion comprising AKT3 but extending to and including ZBTB18 and/or HNRNPU had microcephaly. The OFC is known for nine patients with ZBTB18 mutations [from the literature (Table S6) and our series] and three have microcephaly. This observation, combined with the microcephaly phenotype described in Zbtb18−/− mice, suggests that microcephaly can also be associated with ZBTB18 mutations and deletions with a lower penetrance. In contrast, data from patients with HNRNPU mutations  AKT3 haploinsufficiency is sufficient to cause microcephaly with high but incomplete penetrance, (2) the heterozygous loss of ZBTB18 may cause microcephaly with a lower penetrance, and (3) other regions located more distally (including ZBTB18) may contribute to microcephaly in addition to AKT3 deletion.The preponderant role of on that:  three deZBTB18 to be the main candidate gene for AnCC in the 1q43q44 deletion syndrome  three patients carrying HNRNPU deletions sparing ZBTB18 and two with HNRNPU point mutations had ThCC (one had DysCC), and (3) patients with ZBTB18 point mutations had either partial AgCC, DysCC or ThCC. Thus, ThCC is the main category of AnCC observed when HNRNPU is deleted and is possibly related to insufficient myelination of crossing axons rather than indicating malformation of the CC. We conclude that ZBTB18 haploinsufficiency predisposes to different types of AnCC, particularly partial AgCC, while HNRNPU anomalies are more specifically associated with ThCC. Furthermore, AgCC is significantly more frequent in patients with microdeletions comprising ZBTB18 extending towards the telomeric end of the 1q region, i.e., encompassing both ZBTB18 and HNRNPU, compared to those encompassing ZBTB18 but sparing HNRNPU. This suggests that the loss of genetic determinant(s) in 3′ of the ZBTB18 coding sequence or that the co-deletion of ZBTB18 and HNRNPU has an additive effect resulting in AgCC. Moreover, the AgCC observed in patients with point mutations or small deletions altering mainly ZBTB18 is partial and characterized by a small splenium with the absence of beak  no patient with 1q43q44 deletion sparing HNRNPU and COX20. COX20 encodes a protein contributing to the assembly of mitochondrial cytochrome C oxidase and has been involved in a recessive disease with healthy heterozygous carriers  epilepsy is present in 90% of patients with deletions comprising HNRNPU and absent in 14/15 patients with deletions sparing HNRNPU, and (2) all patients with constitutive HNRNPU mutations have epilepsy. The only epileptic patient with a deletion sparing HNRNPU had a deletion encompassing ZBTB18. Given that 3/11 patients with ZBTB18 mutations, including two of our series, had seizures; epilepsy may also be a minor phenotypic trait in some patients with ZBTB18 haploinsufficiency. The pro-epileptogenic effect of ZBTB18 alteration could be masked by the strong penetrance of HNRNPU-related epilepsy in patients with loss of both genes.Two thirds of patients with 1q43q44 deletions have epilepsy. The minimal critical region for epilepsy included HNRNPU point mutations from both our series and the literature (n = 9) was 13.5 months (median 8.5 months), versus 12.3 months (median 12 months) in individuals with deletions encompassing HNRNPU but sparing ZBTB18 (n = 13) and 12.9 months (median 12 months) in patients with deletions encompassing both HNRNPU and ZBTB18 (n = 7). These observations suggest that the age at seizure onset is independent of the size of the deletion, and that the loss of one HNRNPU allele is the strongest factor determining the age at seizure onset.The mean age at seizure onset in patients with HNRNPU mutations or deletions. Tonic–clonic seizures and atypical absences are the most frequently reported seizure types. Seizures occur with variable frequencies, are frequently triggered by fever at the onset of the disease and are pharmacoresistant in some patients. Diffuse or focal slow-waves or a slow background activity are recurrently reported on EEG recordings together with various epileptiform features.To date, available data did not reveal specific epileptic features in patients with AKT3 gene  and 5/8 of them aged 3–37 years were not able to speak sentences or to associate several words. Thus, the impression of a relatively preserved development in patients with ZBTB18 mutations from our series should probably be attenuated. These differences are apparently unrelated to the nature (missense versus truncating) of the mutation but likely to the small sample sizes. An overview of acquired developmental milestones in these series of patients shows that those with HNRNPU deletions or mutations have a globally more severe postural and speech delay that those with AKT3/ZBTB18 deletions and those with ZBTB18 mutations. Because the most severe group of patients have deletions encompassing HNRNPU, AKT3 and ZBTB18, co-deletions of these three genes could have an additive detrimental effect on neurodevelopment.Patients with AKT3 is the main gene driving microcephaly, ZBTB18 defect is responsible for AnCC and HNRNPU is the main gene accounting for epilepsy. These correlations can be summarized as follows: (1) AKT3 deletion causes microcephaly with incomplete penetrance but ZBTB18 and HNRNPU deletions may also be involved with a weaker effect; (2) epilepsy and the loss of one HNRNPU allele are strongly associated; and (3) AgCC, is dependent on the loss of ZBTB18 allele but is also influenced by the alteration of neighboring genes. Neurodevelopmental impairment in patients with ZBTB18 LoF is more variable and less severe than that with HNRNPU LoF. Additional studies are required to investigate factors controlling this phenotypic variability in more details, including in particular the possibility of modifiers variants located on the trans allele.In conclusion, the complete neurodevelopmental phenotype of the 1q43q44 microdeletion syndrome is the consequence of the deletion of three main genes spanning 1.36 Mb. Our data confirm that Figure S1. Alignment of all 1q43-q44 microdeletions included in this study. L1, L2, etc (literature) and D1, D2, etc  in the left margin refer to patients’ identities used in the manuscript (JPEG 531 kb)Figure S2. Alignment of 1q43q44 deletions found in patients with microcephaly, anomalies of the corpus callosum and epilepsy. A. Alignment of deletions found in patients with (red bars) and without (blue bars) microcephaly showed a shift of microcephaly-associated deletions towards the centromere, i.e. encompassing AKT3 . B. Alignment of deletions according to the “CC status”  did not easily suggest the involvement of ZBTB18 . C. Deletions found in patients with (red bars) and without (blue bars) epilepsy were shifted towards the telomeric end of the regions, suggesting the involvement of HNRNPU  (JPEG 2965 kb)Figure S3. Alignment of 1q43q44 deletions found in patients with normal and abnormal corpus callosum. These alignments show the different categories of AnCC  in patients with deletions of different sizes. They show that i) deletions comprising ZBTB18 and AKT3 but not HNRNPU (A) are associated with all types of AnCC, ii) deletions comprising HNRNPU but not ZBTB18 and AKT3 (C) are not associated with AgCC, iii) most AgCC are observed in patients with large deletions including ZBTB18 and mostly extending to the telomere (B). (JPEG 767 kb)Figure S4. Orthologous ZBTB18 protein alignments in the regions surrounding the three affected amino acids altered by missense mutation reported in this study  (TIFF 502 kb)Table S1. Probability of haploinsufficiency intolerance (pLI) calculated by the Exome Aggregation Consortium (ExAC) and haploinsufficiency score (HI) for genes of the 1q43q44 region comprised between genomic positions 239,990,618 to 249,208 (PDF 240 kb)Table S2. Genetic and clinical data from the 17 patients with 1q43q44 deletions (PDF 169 kb)Table S3. Molecular and clinical data from 37 patients with 1q43q44 deletions in the literature (XLSX 29 kb)Table S4. Additional clinical data from the six patients with HNRNPU mutations (PDF 21 kb)Table S5. Clinical and molecular data from patients with HNRNPU mutations in the literature (PDF 113 kb)Table S6. Clinical and molecular data from patients with ZBTB18 mutations in the literature (XLSX 14 kb)Below is the link to the electronic supplementary material.


PDE4B subtype selective inhibitors are known to reduce the dose limiting adverse effect associated with non-selective PDE4B inhibitors. This makes the development of PDE4B subtype selective inhibitors a desirable research goal. To achieve this goal, ligand based pharmacophore modeling approach is employed. Separate pharmacophore hypotheses for PDE4B and PDE4D inhibitors were generated using HypoGen algorithm and 106 PDE4 inhibitors from literature having thiopyrano  Pyrimidines, 2-arylpyrimidines, and triazines skeleton. Suitable training and test sets were created using the molecules as per the guidelines available for HypoGen program. Training set was used for hypothesis development while test set was used for validation purpose. Fisher validation was also used to test the significance of the developed hypothesis. The validated pharmacophore hypotheses for PDE4B and PDE4D inhibitors were used in sequential virtual screening of zinc database of drug like molecules to identify selective PDE4B inhibitors. The hits were screened for their estimated activity and fit value. The top hit was subjected to docking into the active sites of PDE4B and PDE4D to confirm its selectivity for PDE4B. The hits are proposed to be evaluated further using  Most of the mortality related to these inflammatory disorders occurs in low and low-middle income countries , a predominant family of phosphodiesterase (PDE) enzymes expressed in immune and inflammatory cells, includes three subtypes PDE4A, PDE4B and PDE4D. Inhibition of PDE4 has been shown to suppress a diverse spectrum of inflammatory responses  in-vivo -5. More  in-vivo , 6, 7, a in-vivo -10.in-vitro and in-vivo. Investigation in ferrets also showed significantly less emesis with this compound compared with the non-selective PDE4 inhibitor cilomilast  and C-terminal domain CR3 (i.e Leu in PDE4D vs Gln in PDE4B)  , 17. Then PDE4B) -18.The availability of PDE4B and PDE4D inhibition data for recently reported PDE4 inhibitors allows the development of pharmacophore models of PDE4B and PDE4D inhibitors -21. PharData set50 of the above series were taken from the original papers. Numbers used in original papers were used to denote molecules belonging to triazine series while numbers used in original papers for molecules belonging to 2-arylpyrimidine and thiopyranoPyrimidine series were suffixed with a and b respectively.Selective PDE4B inhibitors belonging to thiopyrano Pyrimidines,2 arylpyrimidines and triazines class reported recently, along-with their PDE4B and PDE4D inhibitory activities, were used for the present study  19-21).-21.19-213D QSAR pharmacophore modelingPharmacophore generationPharmacophore modeling is the most widely used method for identification of essential structural features required for biological activity. In the present study, HypoGen algorithm was applied to build the 3D QSAR pharmacophore models for both PDE4B and PDE4D inhibitors using DS V2.0 software  . 50) between 3.0 nM and 18755 nM were selected as training set, which was used to engender the hypotheses. The training set selected complies with the requirements specified in the literature. To validate the hypothesis, the test set was prepared using the specified requirements. Test set contains 24 molecules having wide range of activity values. Sketch function of DS was used to sketch the two-dimensional (2D) chemical structures of all molecules which were later converted into 3D structures. Maximum of 250 conformations were generated for each molecule using the best conformation model generation method based on CHARMm force field and Poling algorithm , HBD (hydrogen bond donor), and H (hydrophobic), features were selected based on the training set molecules. The number of features allowed in the model were kept in the range 0-5. The ‘Uncertainty’ values for all the 75 molecules in the training set were set to 2.0, and the default values for other parameters were kept constant. Subsequently, pharmacophore models were computed and the 10 top scoring hypotheses for both PDE4B and PDE4D inhibition were selected for further study. The qualities of the hypotheses were reliant on the fixed cost, null cost, and total cost values .Assessment of pharmacophore quality Quality of the developed pharmacophore was assessed using three different methods. Initially, cross validation was performed by the Fischer’s randomization test. Secondly, the prediction of the activity values of the test set was performed. The correlation between the experimental and predicted activities was used to assess predictive ability of the model. All queries were addressed using the ligand pharmacophore mapping protocol. Virtual Screening50 less than 20 nM were selected and subsequently subjected to screening using the validated pharmacophore model Hypo1D in the same manner as in the previous step. The hit compounds were chosen that showed Hypo1D estimated fit value less than 4. The validated pharmacophore model (Hypo1B and Hypo1D) of PDE4B and PDE4D inhibitors was used as a query in a sequential manner to search the zinc database. Zinc is a comprehensive database of small molecules containing a total of 17,900,742 drug like molecules . In the i.e. 34b, were docked into the active sites of PDE4B (PDB ID: 4NW7) and PDE4D (PDB ID: 1Y2B). First the protein structures were prepared using the automatic protein preparation module of DS V2.0 software using the default parameters. The structures of the identified hit as well as the standard molecule (34b) were prepared using the prepare ligand module of DS V2.0 software. Docking of the prepared ligands into the active site of the prepared structures of PDE4B and PDE4B was carried out using CDOCKER program available in DS V2.0 software  with default parameters  for PDE4B inhibition consist of three HBA which established the highest cost difference (143.378), best correlation coefficient (0.9571), maximum fit value (5.8678) and lowest root mean square (RMS) of 1.86 . The resTop scoring model (Hypo1D) for PDE4D inhibition consists of two HBA and three H with highest cost difference (164.419), best correlation coefficient (0.9563), maximum fit value (8.1515), and lowest root mean square (RMS) of 1.66 . As in t50 values.The experimental and predicted activities by Hypo1B and Hypo1D for 75 training set molecules are shown in dFit value indicates how well the features in the pharmacophore overlap the chemical features in the molecule. Fit value = weight x [max] where SSE = (D/T)2, D = displacement of the feature from the center of the location constraints and T = the radius of the location constraint sphere for the feature (tolerance).eDifference between the predicted and experimental values. ‘+’ indicates that the predicted IC50 is higher than the experimental IC50; ‘-’ indicates that the predicted IC50 is lower than the experimental IC50; a value of 0 indicates that the predicted IC50 is equal to the experimental IC50.3 and hydrophobic groups like halogen atoms in the aromatic ring (Ar) to orient properly for interaction with CR3 will show significant selectivity for PDE4B as compared to PDE4D. This is consistent with the findings described previously in the original papers in which these compounds have been reported  for the test set given by Hypo1B was 0.8579  while thVirtual ScreeningZinc, a comprehensive database of small drug like molecules was used for the sequential virtual screening using the pharmacophore models. Screening of zinc database using the validated pharmacophore model (Hypo1B) of PDE4B inhibitors retrieved a set of 6015 hits, mapping to the pharmacophore model Hypo1B. The hits comprised of some compounds structurally similar to that of the existing PDE4B inhibitors, and some novel scaffolds. 50 less than 20 nM were selected and subsequently subjected to screening using the validated pharmacophore model Hypo1D. 5 hit compounds that showed Hypo1 PDE4D estimated fit value less than 4 were identified (The 397 hit compounds showing Hypo1B estimated ICentified . Among tentified . ZINC091The results of docking studies of ZINC09157416 and 34b with PDE4B and PDE4D further confirmed the selectivity of ZINC09157416 for PDE4B over PDE4D .Ligand-based pharmacophore models for a diverse class of PDE4B and PDE4D inhibitors were developed. The best pharmacophore models Hypo1B and Hypo1D were validated using different methods to evaluate their predictive power over the diverse test set compounds. Hydrogen bond acceptors were identified to be mainly responsible for PDE4B inhibition while both hydrogen bond acceptors as well as hydrophobic groups were found to be responsible for PDE4D inhibition. The highly predictive pharmacophore hypotheses were further used in sequential virtual screening for identification of selective PDE4B inhibitors. Zinc drug like database was used in virtual screening. The hits from the virtual screening were filtered based on the estimated activity and fit value. Five molecules with different backbones were identified as final hits. The most selective hit molecule ZINC09157416 exhibited better selectivity for PDE4B than the standard compound 34b in the docking studies. The activity of the hit compound has not been reported in the literature as we explored by PubChem and SciFinder Scholar search tools. Thus, the sequential virtual screening strategy using 3D QSAR pharmacophores for PDE4B and PDE4D inhibitors proved to be an effective strategy to identify novel selective PDE4B inhibitors.


During the proton irradiation of thorium targets, large amounts of 103Ru are generated through proton induced fission. The development of a two part chemical separation process to isolate 103Ru in high yield and purity from a proton irradiated thorium matrix on an analytical scale is described herein. The first part employed an anion exchange column to remove cationic actinide/lanthanide impurities along with the majority of the transition metal fission products. Secondly, an extraction chromatographic column utilizing diglycolamide functional groups was used to decontaminate 103Ru from the remaining impurities. This method resulted in a final radiochemical yield of 83 ± 5% of 103Ru with a purity of 99.9%. Additionally, measured nuclear reaction cross sections for the formation of 103Ru and 106Ru via the 232Th103,106Ru reactions are reported within.Ruthenium-103 is the parent isotope of  This decay gives rise to the emission of low-energy Auger/Coster-Kronig electrons (2.3 electrons/decay) . The beam profile was assessed following the experiment by the activation of thin stainless steel plates whose dimensions significantly exceeded those used for the thorium foils. The steel plates were exposed to Gafchromic® film following the end of irradiation in order to map the beam profile, which was determined to have been quantitatively incident on the desired targets in both experiments.Thin thorium foils were irradiated in two separate experiments at the Los Alamos Neutron Science Center (LANSCE) at LANL using incident proton energies of 100 and 200 MeV as described previously . In eachA 10 g thorium metal target was irradiated at the Isotope Production Facility (IPF), Los Alamos National Laboratory . The target was encapsulated in Inconel cladding and placed into the high energy “A” slot  of the IPF target assembly. IPF targetry and 4π water cooling were identical to the design as described previously , 19. The225Ac. The target was dissolved in 200 mL 10 M HCl and 0.1 mL of 2M HF with heating (80–90°C) for approximately 2 hours. A 0.1 mL aliquot of the dissolved target was diluted to 5.1 mL with 0.1M HNO3. This solution was then used as a stock solution for radiotracers that represent radionuclides previously identified in the target [103Ru, eight 5 mL fractions of 10 M HNO3 were added to the column and collected (fractions 8–15). All fractions were analyzed by HPGe spectroscopy using the characteristic γ-rays as identified in 225Ac purification process as described previously [The irradiated 10 g thorium target was shipped to Oak Ridge National Laboratory (ORNL) for recovery of e target . For theeviously , 14.103Ru eluted in fractions 8–15. Contaminants present included 95Zr, 95Nb, 233Pa, 230Pa, 117mSn and 124,125Sb. These fractions were brought to near dryness and reconstituted in 10 mL 10 M HCl. This solution was then passed through a column containing 1 mL DGA  equilibrated with 10 M HCl. The eluent was collected (fraction 16) and the column was washed with an additional 20 mL 10 M HCl (fraction 17). All separation experiments were performed in triplicate.A second column was developed to remove contaminants from the 103Ru, and 106Ru are plotted in Measured excitation functions of 225Ac, Ra, Ba, lanthanides and bulk thorium along with the majority of 95Zr passed through the anion column in the loading fraction and 10 mL 10 M HCl wash (fractions 1–3). Ruthenium is most strongly retained on the anion exchange resin using 1 M HCl [95Nb and 123mTe along with approximately 45% of the Pa. The loading and washing of the column resulted in 103Ru losses of 8–15%. Thirty milliliters of 10 M HNO3 resulted in the elution of 85 ± 5% of 103Ru with a radiochemical purity of 82%. The main impurities present in this fraction consisted of 117mSn and 125,126Sb with trace amounts of 230,233Pa, 95Nb, and 95Zr. The cationic species such as 3 solutions and several papers discuss fission product behavior in these systems [103Ru from the DGA column was 98 ± 1% resulting in a final 103Ru recovery of 83 ± 5% with a radiochemical purity of > 99.9%. Ruthenium speciation is a complicated subject with respect to its separation in acid based systems and is likely responsible for the high variability in the loss of 103Ru from the anion column (8–15%) [103Ru is an ancillary activity with respect to 225Ac recovery. A flow diagram of the whole process is shown in According to a paper published by Pourmand et al. , Nb, Zr, systems , 27–32.  (8–15%) . Ideally117mSn and 123mTe have the same identifying γ-ray lines without a viable secondary gamma, a spike of 121mTe was added to the sample to help deconvolute the separation of 123mTe and 117mSn. This information led to the determination that 123mTe was present in the 1 M HCl fractions and 117mSn was present in the 10 M HNO3 fractions. This elemental distribution is further corroborated by prior reports that tellurium is eluted from anion columns in 1 M HCl while tin is retained strongly, and that tin elutes with 103Ru in 10 M HNO3 [As both 0 M HNO3 , 35.106Ru. However as 106Ru decays to 106gRh (29.9 s), 103mRh obtained from a generator would be isotopically pure five minutes after elution. The predicted experimental yield calculated from measured cross sections of 103Ru, with anticipated full scale 225Ac production, is (~ 3 Ci (111 GBq)) at end of bombardment. This would significantly increase the current supply of 103Ru for medical research needs.Ruthenium-103 obtained from this method contains the isotopic impurity 103Ru/103mRh generator system. Solvent extraction generators have been designed employing a carbon tetrachloride extraction, however given the toxicity associated with CCl4 this method is not amenable for biomedical applications [103mRh with minimal breakthrough of 103Ru. This would preferably entail the use of mineral acids that can be readily removed from the product such as HCl or HNO3.Future work needs to be performed in order to determine suitable conditions for a ications , 36. A s103Ru that is produced concurrently with 225Ac. This method results in a final 103Ru chemical recovery yield of 83 ± 5% with a radiochemical purity of > 99.9%. The measurement of energy dependent cross sections for the proton induced fission production of 103Ru and 106Ru at proton energies less than 200 MeV on 232Th targets predict thick target yields of ~111 GBq. This process can be implemented with the existing 225Ac recovery flow sheet at minimal impact to the 225Ac process. Additionally, future work to develop a robust 103Ru/103mRh radionuclide generator is essential to the success of 103mRh for auger therapy.A method was obtained for the recovery and purification of


COL4A3, COL4A4 or COL4A5 but in half of families a gene is not identified. We investigated a Cypriot family with autosomal dominant microscopic haematuria with renal failure and kidney cysts.Hereditary microscopic haematuria often segregates with mutations of We used genome-wide linkage analysis, whole exome sequencing and cosegregation analyses.COL4A1. This mutation predicts truncation of the protein with disruption of the C-terminal part of the NC1 domain. We confirmed its presence in 20 family members, 17 with confirmed haematuria, 5 of whom also had stage 4 or 5 chronic kidney disease. Eleven family members exhibited kidney cysts (55% of those with the mutation), but muscle cramps or cerebral aneurysms were not observed and serum creatine kinase was normal in all individuals tested.We identified a novel frameshift mutation, c.4611_4612insG:p.T1537fs, in exon 49 of COL4A1 that encode the CB3 [IV] segment of the triple helical domain (exons 24 and 25) are associated with HANAC syndrome . Missense mutations of COL4A1 that disrupt the NC1 domain are associated with antenatal cerebral haemorrhage and porencephaly, but not kidney disease. Our findings extend the spectrum of COL4A1 mutations linked with renal disease and demonstrate that the highly conserved C-terminal part of the NC1 domain of the α1 chain of type IV collagen is important in the integrity of glomerular basement membrane in humans.Missense mutations of  Isolated microscopic haematuria is a significant risk factor for kidney failure in later life  and can COL4A3, COL4A4 or COL4A5 genes that encode type IV collagen, and the clinical features overlap with the carrier state for autosomal recessive or X-linked Alport syndrome , three requiring renal replacement therapy.COL4A3 and COL4A4) were sequenced in the proband by amplification of each exon and Sanger sequencing. This did not reveal any rare or likely pathogenic variants. A genome-wide linkage study was performed using DNA from 12 affected family members. This excluded linkage with loci containing the genes COL4A3, COL4A4, COL4A5, MYH9 and CFHR5 (LOD < −2 at each locus), but did demonstrate linkage (LOD = 3) with loci on chromosomes 6 and 13 that include the COL4A1 gene on chromosome 13  and predicted a change in amino acid sequence of a protein  were identified. Of these, 644 were heterozygous and 270 were novel (i.e. not reported in dbSNP). Two rare variants, both heterozygous, occurred within the linked loci. One variant predicted a missense mutation p.A101T in TMEM14C (NM_016462) on chromosome 6. This gene has not previously been associated with kidney disease and the variant is predicted to be benign, with SIFT and Polyphen scores of 0.42 and 0.023, respectively. The other variant was in the COL4A1 gene and encodes a novel frameshift mutation c.4611_4612insG; p.T1537fs (transcript NM_00185). This was considered a good candidate for causing disease since other mutations in this gene are associated with HANAC syndrome, in which haematuria, kidney cysts and renal impairment are features.Genes associated with autosomal dominant thin basement membrane nephropathy  exhibited renal impairment, haematuria, proteinuria and kidney cysts  and individual II-6. No rare or likely pathogenic mutations of these genes were identified in either of these individuals. We conclude that the COL4A1 mutation is responsible for the familial renal disease in this kindred. We went on to examine whole exome sequencing data from four other families in which glomerular basement membrane abnormalities and microscopic haematuria segregated as a dominant trait in the absence of a COL4A3, COL4A4 or COL4A5 mutation but did not find any additional likely pathogenic variants in COL4A1.To determine whether an additional mutation might be segregating in other family members we went on to sequence Renal failure was unpredictable, with creatinine rising above the normal range in some affected members after the age of 40 years. End-stage renal failure ranged from 63 to 70 years in three of nine siblings in the first generation. In those affected, one or more kidney cysts were detectable by ultrasound from the age of 40 years in 12 family members. Hypertension was variable, but could be present from 35 years of age. No family members reported a history of muscle cramps or cerebral aneurysms, and serum creatine kinase was normal in all 22 family members tested. Some family members had evidence of vascular disease  and 3 had an elevated NAG:creatinine ratio .Little or no proteinuria  was found until the eGFR was <50 mL/min/1.73 mA kidney biopsy was performed in one family member at age 47 years to investigate familial nephropathy and reduced eGFR. Light microscopic examination was normal with the exception of minimal global glomerulosclerosis (1 of 25 glomeruli) and <1% tubular atrophy. Immunostains for type IV collagen were not performed, but stains for immunoglobulins and complement were negative. Electron microscopy demonstrated areas of thinning of glomerular basement membranes (down to 93.5 nm) and subtle lamellation of tubular basement membranes  that have a more restricted distribution of expression and are less broadly conserved across species [Type IV collagen is an important constituent of the extracellular matrix and forms a complex meshwork that provides structural integrity to basement membranes . Individ species .COL4A1 are associated with neurological, vascular and renal disorders. Previously described phenotypes cluster into primarily neurological disease, in which porencephaly (the occurrence of fluid filled cavities in the brain) and cerebral vasculopathy or haemorrhage cause significant neurological damage early in life [COL4A1-associated diseases are incompletely understood. However, it is unlikely that haplo-insufficiency is a major contributor in most cases: previously described pathogenic mutations in humans are almost all missense variants and do not include early nonsense mutations or heterozygous whole gene deletions; only a single collagenous-domain frameshift mutation that reduced transcript levels has been reported to date [Col4A1 allele do not display any detectable phenotype [COL4A1, encoding a region of the protein that contains multiple potential integrin-binding sites, and it has been suggested that disruption of normal interaction between type IV collagen and integrins might be responsible for HANAC syndrome [COL4A1-associated diseases.Pathogenic mutations of  in life , and HAN in life . The mec to date . Moreovehenotype . Mutatiosyndrome , 24. ThiCOL4A1 are exceedingly rare, with only one example observed (encoding p.P438fs) in >120 000 alleles tested in the ExAC project and a single pathogenic allele (p.G696fs) reported previously in the medical literature [211), which is essential for the sulfilimine bond cross-linking adjacent trimers to form hexameric type IV collagen [Frameshift variants of terature . The p.Tcollagen . It alsoSupplementary Video S1). Alternatively, since it is known that α1 type IV chains are expressed in podocytes and present in glomeruli of healthy adults [COL4A1 is considered less likely for the reasons stated above. A homozygous truncating mutation within the C-terminal NC1 domain of COL4A4 has previously been reported in association with autosomal recessive Alport syndrome, but the phenotype of the obligate carrier parents was not documented in this report [One possible mechanism whereby loss of this part of the NC1 domain could cause disease includes disruption of sulfilimine bond(s) by inclusion of one or more defective α1 chain missing the C-terminal part of its NC1 domain within an α1.α1.α2 type IV collagen heterotrimer (See related article by Savige. A further genetic cause of thin basement membrane nephropathy.


NOD2 contributes to the innate immune response and to the homeostasis of the intestinal mucosa. In response to its bacterial ligand, NOD2 interacts with RICK and activates the NF-κB and MAPK pathways, inducing gene transcription and synthesis of proteins required to initiate a balanced immune response. Mutations in NOD2 have been associated with an increased risk of Crohn’s Disease (CD), a disabling inflammatory bowel disease (IBD). Because NOD2 signaling plays a key role in CD, it is important to further characterize the network of protein interacting with NOD2. Using yeast two hybrid (Y2H) screens, we identified new NOD2 interacting proteins (NIP). The primary interaction was confirmed by coimmunoprecipitation and/or bioluminescence resonance energy transfer (BRET) experiments for 11 of these proteins . These proteins are involved in diverse functions, including endosomal sorting complexes required for transport (ESCRT), cytoskeletal architecture and signaling regulation. Additionally, we show that the interaction of 8 NIPs is compromised with the 3 main CD associated NOD2 mutants . Furthermore, to determine whether these NOD2 protein partners could be encoded by IBD susceptibility genes, a transmission disequilibrium test (TDT) was performed on 101 single nucleotide polymorphisms (SNPs) and the main corresponding haplotypes in genes coding for 15 NIPs using a set of 343 IBD families with 556 patients. Overall this work did not increase the number of IBD susceptibility genes but extends the NOD2 protein interaction network and suggests that NOD2 interactome and signaling depend upon the NOD2 mutation profile in CD. Toll Like Receptors (TLR) and Nod-Like Receptors (NLR) are major receptors of the innate immune system . These pNOD2 is the main CD susceptibility gene. Up to 50% of Caucasian CD patients carry mutations in NOD2 . Fi. FiNOD2 assessed  Tables. assessed .Several clones coding for RICK protein were identified in the colon or lung library, validating the Y2H screening procedure, since RICK is an undisputed NIP. All RICK positive clones contained the C-terminus CARD domain of RICK, a region known to be involved in a heterotypic interaction with the N-terminus CARD domains of NOD2 . ImportaThe NIP candidate CHMP4b was found several times only in the colon library . As CHMPAs a result of Y2H screens, 14 preys were selected to further investigate their interaction with NOD2. Several proteins like RICK, CHMP5, TRIM41, SDCCAG3, VIM, and ANKHD1 were chosen because they were identified independently in the colon and the lung cDNA library. The other selected proteins  included mostly NIP candidates isolated a minimum of 3 times.Co-immunoprecipitation experiments were performed to confirm Y2H interactions. The full length (or near full length) cDNAs coding for the 14 selected NIPs including RICK were cloned in a plasmid to express EGFP fused proteins. Each EGFP-tagged NIPs was co-expressed with Myc tagged NOD2 protein in HEK293T cells by transient transfection. The cells were then lysed in a “stringent” buffer containing 1% TX100 and subjected to immunoprecipitation with anti-myc (to immunoprecipitate myc-NOD2) or anti-GFP antibodies (to immunoprecipitate each EGFP-NIP) and immunoprecipitated proteins were analysed by Western blot using anti-myc and anti-GFP antibodies. RICK and NOD2 were used as positive controls . Six putThe Bioluminescent Resonance Energy Transfer (BRET) approach reveals direct interaction between proteins in the cellular context without cell lysis . FollowiIn order to understand the impact of NOD2 activation on the NIP-NOD2 interaction, BRET experiments in live cells were also carried out with the addition of MDP following transfection. Among all the proteins investigated, only the CHMP5/NOD2 BRET profile appeared clearly modified by the addition of MDP. In that case, an overnight incubation with MDP induced a decrease in BRETmax of the NOD2/CHMP5 interaction, indicating either a conformational change or dissociation of the protein complex after MDP activation . This reAn interaction score was calculated as an attempt to classify the NIPs characterized in this study. This score (arbitrary unit) integrates, the number of hits in Y2H screens, the “strength” of the co-immunoprecipitation and the profile of the BRET saturation curves and MDP response . In summTo evaluate the functional role of each NIP on the NOD2 dependent NF-κB signaling, reporter assays were carried out in HEK293T cells transfected with plasmid encoding luciferase under the control of NF-κB response elements. As reported elsewhere, overexpressing NOD2 in these cells stimulates NF-κB and a MDP incubation further increased NF-κB activation . The celNod2 is constitutively expressed by the THP-1 monocyte cell line. The expression of Nod2 and 10 NIP genes was studied by qRT-PCR arrays upon MDP, LPS or LPS + MDP stimulation . Nod2, VThe interaction between 8 NIPs and the three main CD associated NOD2 mutants were tested by Y2H. By this analysis , we obseNOD2 mutation.Genetic analyses were performed for genes encoding 15 NIPs with a set of 101 Single Nucleotide Polymorphisms (SNP) chosen to cover the vast majority of gene haplotypes with a frequency higher than 0.05, as estimated on the Hapmap website . StudiesDOCK7, GOLGB1, IKBIP, PRR16, and VIM  was tested for each SNP independently and for all possible haplotypes with a combination of 2 or 3 of the genotyped SNPs in the different family groups. Among the 15 genes, we found a weak distortion of transmission for haplotypes in 5 NIPs: M Tables  and 5.DOCK7, a positive TDT was seen for rs17381383 in the group of CD only families . This marker tags a haplotype with a frequency of 0.142 in the Hapmap CEU panel and 0.15 in our cohort  and for another haplotype in the whole group of CD families (T: 24 U: 9 p = 0.0090)  were found in the IBD family cohort most often with haplotypes containing of the intronic SNP rs13161840. Similarly, distortions of transmission were observed in the NOD2 mutated CD families with haplotypes containing the minor frequency allele of rs300970. However, the p values of all these TDT did not remain significant after applying the Bonferoni correction for multitesting.For r cohort . For GOL 0.0090) . A nomin 0.0090) . PRR16 iVIM is defined by one haplotypic block and 8 haplotypes. The 4 genotyped SNPs were available defining only 3 different haplotypic groups in the 8 haplotypic group defining VIM in HapMap CEU panel. A distortion of transmission was observed for an haplotype with a frequency of 0.557 in NOD2 WT CD families . These novel protein partners extend the NOD2 interactome and provide new leads about mechanisms of NOD2 function, regulation, and subcellular localization. The use of two independent methods to confirm Y2H interactions allowed validation of a total of eleven NOD2 interacting candidates. Importantly, some interactions could not be seen by co-immunoprecipitation, but were positive in BRET experiments, suggesting that the intracellular milieu and/or the subcellular localization can be important for some specific interactions to occur.Based on a generic NF-κB reporter assay, combined with the use of siRNA, none of the NIPs isolated here, except RICK, appear to be major regulators of the NOD2 dependent NF-κB pathway at least in HEK293T cells. These data are however consistent with a recent publication reporting the identification by genome wide RNAi screen in HEK293 cells of new positive and negative regulators of the NOD2 dependent NF-κB pathway . Of noteThe expression of several NIPs is upregulated by MDP and LPS in THP-1 cells suggesting that these proteins may play a role as feedback control to amplify or down regulate the NF-κB signaling pathway. Alternatively some NIPs may be involved in other specific NOD2 functions including autophagy induction, Interleukin production (IL-8 or IL1β) or in the subcellular localization and traffic of NOD2 during signaling.Importantly, our data indicate that the 3 main NOD2 mutants associated with CD, R702W, G908R and 1007fs  interact differentially with 8 NIPs and this could have functional consequences in term of downstream signaling. Future studies will help define whether these alterations (loss or gain) of interactions between NIPs and NOD2 CD associated mutants could contribute to the pathophysiology and severity of CD.In The NIPs identified in this study are possible candidate genes for susceptibility to CD or UC, another IBD condition. We thus analysed the main NIPs in a cohort of more than 300 European IBD families. This cohort may appear limited when considering the very large cohorts used in GWAS to date, however the type of analysis is quite different and good power can be attained with a modest number of inferred meioses from the family data.NOD2 mutations. Unfortunately, this result was not reproduced in the second family sample. Similarly, a weak association has been reported by Stevens et al. between VIM polymorphisms and CD but without firm validation [RICK, a well-known NIP [Among all the tested NIPs, we observed a positive association with some haplotypes of VIM in the CD family group carrying no lidation , especialidation . We thusnown NIP  was likenown NIP .C10orf67 and SDCCAG3 have been proposed as putative genes associated with CD [C10orf67 was found following a genome scan to isolate genes associated with sarcoidosis and CD. The SDCCAG3 locus is in a region where a SNP associated with CD was found in the vicinity of another IBD gene candidate, CARD9. However, none of the SNPs tested here for SDCCAG3 and C10orf67 showed any association. For SDCCAG3 and C10orf67, we also genotyped the two SNPs previously found to be associated rs10870077 and rs1398024 respectively and we were unable to replicate the previous observation . It is nevertheless very striking that the C10orf67 locus was identified in our study on NOD2 partners and in an independent study of CD genetics based on totally different screening procedure [C10orf67 and C10orf115. Surprisingly the cDNAs sequences of our Y2H positives clones were actually chimeric between the C10orf67 and C10orf115 loci. However, the full cDNA that was used in this study for validation experiments spans only sequences of the C10orf67 gene. Further biochemical studies are thus required to determine the exact nature of protein(s) encoded by transcripts arising from this 10p13 region and whether they interact with NOD2 and could possibly contribute to CD susceptibility. In summary, we report here a set of new NOD2 protein partners that connect into the NOD2 protein network  are shown in parallel with the Nomarski field (left). The white scale bar represents 10μm.(PDF)Click here for additional data file.S1 TableGrowing yeast colonies were tested and scored by 3 phenotypic assays .(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.