TYK2 is recognized as a susceptibility gene for some inflammatory and autoimmune diseases (AID). Seven TYK2 variants have been associated with AIDs in Europeans, and establishing their causality remains challenging. Previous work showed that a protective variant (P1104A) is hypomorphic and also a risk allele for mycobacterial infection. Here, we have studied two AID-associated common TYK2 variants: rs12720270 located in intron 7 and rs2304256, a non-synonymous variant in exon 8 that causes a valine to phenylalanine substitution . We found that this amino acid substitution does not alter TYK2 expression, catalytic activity or ability to relay signaling in EBV-B cell lines or in reconstituted TYK2-null cells. Based on in silico predictions that these variants may impact splicing of exon 8, we: i) analyzed TYK2 transcripts in genotyped EBV-B cells and in CRISPR/Cas9-edited cells, ii) measured splicing using minigene assays, and iii) performed eQTL (expression quantitative trait locus) analysis of TYK2 transcripts in primary monocytes and whole blood cells. Our results reveal that the two variants promote the inclusion of exon 8, which, we demonstrate, is essential for TYK2 binding to cognate receptors. In addition and in line with GTEx (Genetic Tissue Expression) data, our eQTL results show that rs2304256 mildly enhances TYK2 expression in whole blood. In all, these findings suggest that these TYK2 variants are not neutral but instead have a potential impact in AID.TYK2 belongs to the JAK protein tyrosine kinase family and mediates signaling of numerous antiviral and immunoregulatory cytokines  in immune and non-immune cells. After many years of genetic association studies,  TYK2 locus an impact on susceptibility to systemic lupus erythematosus (SLE) and other autoimmune diseases (AID). The identification has been replicated in a number of recent analyses, and TYK2 is now recognized as a susceptibility gene in a variety of inflammatory and autoimmune diseases, including type I diabetes (T1D), psoriasis and multiple sclerosis , of which more than 500 cause non-synonymous  changes. Seven TYK2 variants have been associated with AIDs in European cohorts and for most the minor allele is protective  analysis of TYK2 in monocytes and whole blood from genotyped individuals, our results revealed an impact of these variants on splicing of a small exon which is essential for TYK2 binding to cytokine receptors.Here, we present a study of two additional disease-associated variants, which are in LD  (Ensembl/1000 Genomes). Rs12720270 has been associated to protection from SLE in UK families and in Finnish population , 12. Rs2TYK2 exon 8 and causes a valine to phenylalanine substitution   predicted that the C  to A (minor/derived) nucleotide change at rs2304256 could destroy a putative exonic splicing enhancer (ESE) regulated by the SR protein SF2/ASF, generating a new ESE recognized by the SRp55 protein or breaking a silencer motif. In parallel, we also studied rs12720270, which is located in intron 7, 36 nt upstream of the intron 7/exon 8 boundary  to A (minor/derived) nucleotide substitution could break a potential branch point and alter pre-mRNA splicing.Rs2304256 is a non-synonymous variant (C > A) that maps in l362Phe) . Valine l362Phe)  and, accl362Phe) . All linl362Phe) . Comparal362Phe) . To streK2-K930R . As showK2-K930R . In closboundary . The in TYK2. For this, we set up a RT-PCR assay with primers mapping in exon 7 and exon 9 of TYK2 and used as template cDNA from 30 EBV-B cell lines  present in the vector. The 520 nt product contained only exon 7. The 210 nt product contained the 5’ exon (SD) and 3’ exon (SA) of the pI-12 vector. In comparing the profile of transcripts expressed from the four minigenes, it appeared that, for each of the two SNPs, the presence of the minor allele A led to a higher level of a transcript retaining exon 8 . Using the pI-12 splicing vector, we generated a minigene reporter construct covering 1.2 kb of s2304256 , top. Ths2304256 , bottom.g exon 8 . Moreove293T cells are homozygous for the rs2304256 major allele. Using the CRISPR/Cas9 genome editing technology, we obtained clones homozygous for the minor A allele. RNAs from four edited clones (AA) were analyzed by RT-PCR as described above. The PCR product lacking exon 8 (550 nt) was undetectable in three edited clones and barely detectable in the fourth clone , top. ThTYK2 transcript (TYK2-204) lacks exon 8 (ENST00000525220). Both TYK2-204 and the small transcript detected in EBV-B cells retain the correct ORF and may give rise to a protein isoform missing the exon 8-encoded segment. We did not succeed in detecting such protein probably owing to its low expression level. Yet, the alternate transcript—and protein—may be more abundantly expressed in specific cell types and/or under particular conditions. On this basis, we undertook functional studies of a TYK2-ΔE8 protein. Our reference TYK2 cDNA was deleted of the exon 8 sequence . TYK2-WT and TYK2-ΔE8 expression vectors were stably transfected in TYK2-null cells and clones expressing similar TYK2 levels were chosen. We first compared the catalytic activity of WT and ΔE8. TYK2 was immunoprecipitated and subjected to in vitro kinase assay. As shown in The results described above indicated that rs2304256 and rs12720270 influence pre-mRNA processing at the level of exon 8. Interestingly, one annotated in vitro pull-down assay the ability of the N-ter portions  to interact in vitro with the cytoplasmic region of IFNAR1 , 10 mM MgCl2 and with or without 30 μM ATP at 30°C for 5 min in a total volume of 30 μl. The reaction was terminated by boiling in Laemmli buffer. Half of the sample was loaded for SDS-PAGE, transferred to a nitrocellulose membrane, and phosphorylated products were analyzed by western blotting with activation loop phospho-specific Abs. After stripping, membranes were reblotted with anti-TYK2 mAb, revealed using ECL detection reagents .Cells were lysed in 50 mM Tris (pH 6.8), 0.5% Nonidet P-40, 200 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM sodium vanadate, 1 mM sodium fluoride,10 mM PMSF, cocktail of antiproteases (Roche Applied Science). TYK2 and JAK1 were immunoprecipitated from 2 mg lysate using affinity-purified anti-VSV-G polyclonal Abs . Immunocomplexes were washed three times in buffer 1 , once in buffer 2 (50 mM Tris [pH 6.8] and 200 mM NaCl), and once in kinase buffer  with 0.5% bovine serum albumin and protease inhibitors for 60 min at 4°C. Beads were pelleted and washed three times in binding buffer. Bound proteins were eluted and boiled in 20 μl of Laemmli buffer, separated by SDS-PAGE, and analyzed by immunoblotting with the appropriate antibody.Histidine-tagged proteins were expressed in bacteria, purified on Ni-NTA agarose beads according to the manufacturer’s protocol (Qiagen), eluted, and dialyzed against 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10% glycerol, and 2mM EDTA, 1mM dithiothreitol (DTT). Proteins were concentrated by Vivaspin concentrator (Vivascience) and stored at 80°C. GST fusion proteins were affinity-purified on glutathione-Sepharose . For TYK2 expression in primary monocytes derived from 200 genotyped healthy male individuals of self-reported African and European ancestry are from the EvoImmunoPop project [TYK2 mRNA levels were measured in whole blood of healthy donors from the Milieu Intérieur cohort by the NanoString hybridization-based multiplex assay [TYK2 probe is complementary to the exon 3/exon 4 junction: GGGCCTGAGCATCGAAGAGGGCAAAGAGATTGAAGCAAGGAGGAGTGATACCAACTTTATGTGCAATGTGGATGCAGACTTCCTCAGCTGTCAGCGATGA. eQTL analyses were performed with the linear mixed model implemented in GenABEL R package [RNA-Sequencing (RNA-Seq) data on  project , and wer project , 39 at f project , based o project , and a p project . For the project  to extra project , includiex assay . The Milex assay , 68. The package .S1 Table(PDF)Click here for additional data file.S1 FigPartial exon 7 and exon 9 sequences are boxed. The central exon 8 encodes the 66 aa segment. Red arrowhead points to Val362.(PDF)Click here for additional data file.S2 Figet al, Nat. Struct. Mol. Biol. 2014. 21:443). Shown is the secondary structure of the TYK2 FERM-SH2 domain in complex with a short peptide of the IFNAR1 cytoplasmic portion (yellow). SH2 domain in light blue. The FERM comprises three lobes . Helices displayed as cylinders, strands displayed as block arrows, and loops displayed as lines. Arrows point to the start (5’) and the end (3’) of the exon 8-encoded segment, spanning from the β3-β4 loop to the β7 strand of the F3 lobe. Valine 362 is located in the unstructured β3-β4 loop and is tentatively indicated by an arrow.Taken and adapted from Supplem. S3 Fig. in Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


The role of human papillomavirus 16 (HPV16) in human cancers is well established; however, to date there are no antiviral therapeutics that are available for combatting these cancers. To identify such targets, we must enhance the understanding of the viral life cycle. Innate immune genes (IIGs) are repressed by HPV16, and we have reported that this repression persists through to cancer. Reversal of this repression would boost the immune response to HPV16-positive tumors, an area that is becoming more important given the advances in immunotherapy. This report demonstrates that E6 and E7 synergistically repress IIG expression in the context of the entire HPV16 genome. Removal of either protein activates the expression of IIGs by HPV16. Therefore, gaining a precise understanding of how the viral oncogenes repress IIG expression represents an opportunity to reverse this repression and boost the immune response to HPV16 infections for therapeutic gain. per se can activate the DDR following infection, and this activation is a known inducer of IIG expression, which may induce cellular senescence. To combat this, E6 and E7 synergistically combine to manipulate the DDR and actively repress innate immune gene expression promoting cellular growth; neither protein by itself is able to do this.Human papillomaviruses (HPV) are causative agents in 5% of all cancers, including the majority of anogenital and oropharyngeal cancers. Downregulation of innate immune genes (IIGs) by HPV to promote the viral life cycle is well documented; E6 and E7 are known repressors of these genes. More recently, we demonstrated that E2 could also repress IIGs. These studies have been carried out in cells overexpressing the viral proteins, and to further investigate the role of individual viral proteins in this repression, we introduced stop codons into E6 and/or E7 in the entire HPV16 genome and generated N/Tert-1 cells stably maintaining the HPV16 genomes. We demonstrate that E6 or E7 individually is not sufficient to repress IIG expression in the context of the entire HPV16 genome; both are required for a synergistic repression. The DNA damage response (DDR) is activated by HPV16 irrespective of E6 and E7 expression, presumably due to viral replication; E1 is a known activator of the DDR. In addition, replication stress was apparent in HPV16-positive cells lacking E6 and E7, manifested by attenuated cellular growth and activation of replication stress genes. These studies led us to the following model. Viral replication IMPORTANCE The role of human papillomavirus 16 (HPV16) in human cancers is well established; however, to date there are no antiviral therapeutics that are available for combatting these cancers. To identify such targets, we must enhance the understanding of the viral life cycle. Innate immune genes (IIGs) are repressed by HPV16, and we have reported that this repression persists through to cancer. Reversal of this repression would boost the immune response to HPV16-positive tumors, an area that is becoming more important given the advances in immunotherapy. This report demonstrates that E6 and E7 synergistically repress IIG expression in the context of the entire HPV16 genome. Removal of either protein activates the expression of IIGs by HPV16. Therefore, gaining a precise understanding of how the viral oncogenes repress IIG expression represents an opportunity to reverse this repression and boost the immune response to HPV16 infections for therapeutic gain. Human papillomavirus (HPV) is the most common sexually transmitted infection in the United States, with an estimated 80% of sexually active adults acquiring an HPV infection in their lifetime . Of the Much has been discovered relating to the viral oncoproteins E6 and E7 driving cellular growth and promoting infection –9. Most –In addition to targeting cellular proteins, both E6 and E7 can regulate transcription from the host genome of infected cells –12. Amon–11–17–In order to investigate the contribution of the individual viral proteins to IIG repression, we generated mutant HPV16 genomes that had stop codons in E6, E7, or both . These were transfected into the hTERT immortalized foreskin keratinocyte cell line N/Tert-1 to establish stable cell lines containing wild-type HPV16 and the assorted E6/E7 mutants. We have demonstrated that HPV16 represses IIGs in these cells , 31. The25–We introduced stop codons for E6 (residue C110 mutated to a T) or E7 (residue T584 mutated to an A) into the HPV16 genome and both together. We prepared stable pooled N/Tert-1 cell lines containing the wild-type and mutant HPV16 genomes. These were named N/Tert-1+HPV16, N/Tert-1+HPV16 E6 STOP, N/Tert-1+HPV16 E7 STOP, and N/Tert-1+HPV16 E6E7 STOP. The presence of HPV DNA in the cells was confirmed by PCR using E6, E2, and E5 primers . While tAs markers for E6 and E7 activity, the expression levels of p53 and pRb were investigated . Both p5Our previous work in N/Tert-1 cells demonstrates that there is downregulation of innate immune gene expression at various stages of the interferon signaling pathway by HPV16 , 31. FolWe looked at the expression of the ISGF3 components in the mutant cells . As prevTo determine whether the activation of STAT1 expression in the absence of E6 and E7 was due to an increase in interferon production, we monitored the RNA levels for interferon genes. There was no detectable expression of alpha or beta interferon (IFN-α or IFN-β) in any of the N/Tert-1 cells. However, there was an increase in IFN-κ RNA in the absence of both E6 and E7 expression, in comparison with parental cells , IFN-κ. In doing the above-described experiments, we noticed that the N/Tert-1+HPV16 E6E7 STOP cells grew slower than the other N/Tert-1 cell lines. To quantitate this, we carried out a growth curve analysis and demonstrated that this was indeed the case . The preIt is well established that HPV16 activates the DNA damage response (DDR) in keratinocytes and that individual overexpression of E6 or E7 can also activate the DDR 20–, 34, 35.25–These results suggested that the N/Tert-1 cells containing HPV16 were under replication stress irrespective of E6/E7 status of the viral genome. We next investigated the RNA expression pattern of stress response genes in the N/Tert-1 cells containing the HPV16 genomes . BRCA1, Several laboratories have demonstrated repression of the innate immune response by high-risk HPV in keratinocytes –16. It h–36–We next investigated the expression of interferon-stimulated genes (ISGs) in these cell lines and made several striking observations. First, HPV16 represses the expression of ISGs in N/Tert-1 cells, as we have previously reported , 31. SecThese results demonstrate that in the absence of E6 and E7, E2 cannot repress IIG expression alone. However, neither E6 nor E7 can repress these genes by themselves; therefore, there remains the possibility that E2 synergizes with E6 and E7 to repress IIGs in the context of the entire HPV16 genome. It is also possible that E2, E6, and E7 have roles to play in IIG repression during the different stages of the viral life cycle, and this possibility is under investigation.It is clear that both E6 and E7 are required to suppress innate immune gene expression in the context of the entire HPV16 genome. While both proteins can repress genes when overexpressed individually, abrogation of expression of either one results in a loss of innate immune gene repression  and 3. TThe next question we addressed was what could be responsible for the elevated innate immune gene expression in the absence of E6/E7? The innate immune response is activated by detection of cytoplasmic DNA via the cGAS-STING pathway, and cytoplasmic DNA can be detected in cells treated with DNA-damaging agents . HoweverAll together, the results presented here prompt us to propose the following model. Following infection, the viral genome copy number increases from 1 to around 50 copies per cell. This increase in copy number would by itself activate the DDR due to torsional stress on the viral DNA. This activation promotes recruitment of homologous recombination factors to the viral genome, a process required for HPV16 replication . The actMutations to introduce T584A (E6 STOP), C110T (E7 STOP), or both C110T and T584A (E6E7 STOP) into HPV16 in the peGFP-N1HPV16 plasmid were carried out by Genscript. Briefly, the target area was released from the rest of the plasmid by EcoNI and NotI restriction digestion, base changes were introduced and amplified via PCR, and then the target area was religated into the plasmid backbone. Successful mutagenesis was confirmed by sequencing. These mutations resulted in (i) an early stop codon in the E6 gene, (ii) an early stop codon in the E7 gene, or (iii) an early stop codon in both the E6 and E7 genes .2–95% air atmosphere and passaged every 3 to 4 days. Cells containing wild-type or mutant HPV16 were grown in the same medium but also containing 150 μg/ml G418 (Thermo Fisher Scientific). All cells were routinely checked for mycoplasma contamination.Cell lines containing the HPV16 genomes were generated using N/Tert-1 cells as previously described , 31. The5 cells per dish and grown to 80% confluence . Cells were then harvested by trypsinization and stained with trypan blue, and viable cells were counted. A total of 3 × 105 cells per dish were replated, and this was repeated three times in total.To measure cell growth, cells were seeded in triplicate onto 10-cm dishes at a density of 3 × 106 cells were plated onto 100-mm plates, trypsinized and pelleted after 24 h, and washed twice with phosphate-buffered saline (PBS).For downstream protein and nucleic acid analysis, 1 × 10RNA was isolated using the SV total RNA isolation system (Promega) by following the manufacturer’s instructions, including the DNase treatment step. Two micrograms of RNA was reverse transcribed into cDNA using a high-capacity reverse transcription kit (Applied Biosystems). cDNA and relevant primers were added to PowerUp SYBR green master mix (Applied Biosystems), and real-time PCR was performed using a 7500 fast real-time PCR system. Primer sequences  were as follows: HPV16 E2 F, ATGGAGACTCTTTGCCAACG; HPV16 E2 R, TCATATAGACATAAATCCAG; HPV16 E6 F, TTGAACCGAAACCGGTTAGT; HPV16 E6 R, GCATAAATCCCGAAAAGCAA; HPV16 E5 F, CACAACATTACTGGCGTGCT; HPV16 E5 R, ACCTAAACGCAGAGGCTGCT; GAPDH F, GGAGCGAGATCCCTCCAAAAT; GAPDH R, GGCTGTTGTCATACTTCTCATGG; IRF9 F, GCCCTACAAGGTGTATCAGTTG; IRF9 R, TGCTGTCGCTTTGATGGTACT; STAT1 F, CAGCTTGACTCAAAATTCCTGGA; STAT1 R, TGAAGATTACGCTTGCTTTTCCT; STAT2 F, CCAGCTTTACTCGCACAGC; STAT2 R, AGCCTTGGAATCATCACTCCC; IFN-k F, GTGGCTTGAGATCCTTATGGGT; IFN-k R, CAGATTTTGCCAGGTGACTCTT; IFIT1 F, AGAAGCAGGCAATCACAGAAAA; IFIT1 R, CTGAAACCGACCATAGTGGAAAT; MX1 F, GGTGGTCCCCAGTAATGTGG; MX1 R, CGTCAAGATTCCGATGGTCCT; CHK1 F, ATATGAAGCGTGCCGTAGACT; CHK1 R, TGCCTATGTCTGGCTCTATTCTG; CHK2 F, TCTCGGGAGTCGGATGTTGAG; CHK2 R, CCTGAGTGGACACTGTCTCTAA; ATR F, GGCCAAAGGCAGTTGTATTGA; ATR R, GTGAGTACCCCAAAAATAGCAGG; BRCA1 F, TTGTTACAAATCACCCCTCAAGG; BRCA1 R, CCCTGATACTTTTCTGGATGCC; BRCA2 F, ACAAGCAACCCAAGTGTCAAT; BRCA2 R, TGAAGCTACCTCCAAAACTGTG; RAD50 F, TACTGGAGATTTCCCTCCTGG; RAD50 R, AGACTGACCTTTTCACCATGC; OAS1 F, TGTCCAAGGTGGTAAAGGGTG; OAS1 R, CCGGCGATTTAACTGATCCTG.DNA was extracted from monolayer-grown cells by lysis in HIRT lysis buffer . Cell extracts were incubated with 50 g/ml RNase A, and with proteinase K sequentially to remove residual RNA and proteins, followed by phenol-chloroform extraction. DNA was resuspended in Tris-EDTA (TE) buffer and quantitated by spectrophotometry. Following dilution, 10 ng of DNA and relevant primers were added to PowerUp SYBR green master mix (Applied Biosystems) and real-time PCR was performed using a 7500 fast real-time PCR system, using SYBR green reagent. Primer sequences were as follows: HPV16 E2 F, 5′-ATGGAGACTCTTTGCCAACG-3′; HPV16 E2 R, 5′-TCATATAGACATAAATCCAG-3′; HPV16 E6 F, 5′-TTGAACCGAAACCGGTTAGT-3′; HPV16 E6 R, 5′-GCATAAATCCCGAAAAGCAA-3′; HPV16 E5 F, 5′-CACAACATTACTGGCGTGCT-3′; HPV16 E5 R, 5′-ACCTAAACGCAGAGGCTGCT-3′.6 cells were lysed in 50 μl NP-40 lysis buffer  supplemented with protease inhibitor  and phosphatase inhibitor cocktail (Sigma). The cell and lysis buffer mixture was incubated on ice for 20 min and centrifuged for 20 min at 184,000 relative centrifugal force (rcf) at 4°C, and the supernatant was collected. Protein levels were determined utilizing the Bio-Rad protein estimation assay. Equal amounts of protein were boiled in 2× Laemmli sample buffer (Bio-Rad). Samples were then loaded into a Novex 4-to-12% gradient Tris-glycine gel (Invitrogen), run at 100 V for approximately 2 h, and then transferred onto nitrocellulose membranes (Bio-Rad) at 30 V overnight using the wet blot method. Membranes were blocked in Odyssey blocking buffer (diluted 1:1 with PBS) at room temperature for 6 h and probed with relevant antibody diluted in Odyssey blocking buffer overnight at 4°C. Membranes were then washed with PBS supplemented with 0.1% Tween (PBS-Tween) before probing with the corresponding Odyssey secondary antibody (goat anti-mouse IRdye800cw or goat anti-rabbit IRdye680cw) diluted 1:10,000 for 1 h at 4°C. Membranes were washed in PBS-Tween before infrared scanning using the Odyssey CLx Li-Cor imaging system. The antibodies to the following proteins were used for Western blot analysis at 1:1,000 dilutions in Odyssey blocking buffer, diluted 1:1 with PBS: IFIT1 (D2X9Z), MX1 (D3W7I), and IRF9 (D8G7H) from Cell Signaling Technology and STAT1 (sc-346), STAT2 (sc-1668), pSTAT1 Tyr 701 (sc-135648), pRb (SC-102), p53 (sc-47698), and GAPDH (sc-47724) from Santa Cruz Biotechnology.A total of 1 × 10For immunoprecipitation of E7, 0.5 mg of total protein (extracted as described above) was incubated overnight with E7 antibody  at 4°C with rotation in a total volume of 500 μl. This was then incubated for 8 h with prewashed protein A-Sepharose 4B fast flow beads (Sigma). The lysate-bead mixture was rotated at 4°C for 6 h, and the beads were washed five more times with lysis buffer to remove nonspecific protein binding. The beads were then boiled in 4× Laemmli sample buffer (Bio-Rad), and the supernatant of this mixture was loaded into a Novex 4-to-12% gradient Tris-glycine gel (Invitrogen) and treated as a Western blot, as described above.Cells were grown on coverslips to 70% confluence, fixed with formaldehyde, and permeabilized with NP-40. Cells were incubated with the primary antibody for 1 h , diluted in 10% normal goat serum. Coverslips were washed three times in PBS-Tween (0.1% Tween), and immune complexes were visualized using Alexa 488- or Alexa 595-labeled anti-species-specific antibody conjugates (Molecular Probes). Cellular DNA was stained by inclusion of 4′,6-diamidino-2-phenylindole  in the penultimate wash. Microscopy was performed at the VCU Microscopy Facility. Immunofluorescence was observed using an LSM 700 laser scanning microscope and ZEN 2011 software (Carl Zeiss). Images were assembled in Adobe Photoshop CS 6.0.t test.The standard error was calculated from at least three independent experiments, and significance was determined using Student's


The Han population of the province is approximately 59.15 million [D19S433, D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX, Penta E, TH01, D12S391, D2S1338, and FGA). The Investigator HDplex kit  contains the amelogenin locus, one CODIS (D18S51), and 11 Non-CODIS loci . Both kits contain D18S51 and D12S391, which can be used for sample concordance.The Han population accounts for the majority of the Chinese population. Hunan is a large province located at 108°47′–114°15′ E and 30°08′–24°38′ N in Southern China, specifically in the area in the middle reaches of Yangtze River. The province has a total area of 218 400 kmGenomic DNA was extracted from the bloodstain samples by using the Chelex-100 protocol according to the description by Walsh et al. . DNA conhttp://genepop.curtin.edu.au). The allele frequency distributions among groups were compared using Arlequin version 3.5 software package [Genotype frequencies at each locus, power of discrimination (PD), power of exclusion (PE), probability of matching (PM), polymorphism information content (PIC), observed heterozygosity (Ho) and Hardy–Weinberg equilibrium (HWE) were evaluated with modified PowerStat (version 1.2) program . Linkage package . Pairwis package . Neighbo package .The currSE33 locus and the least number of 6 alleles at the TPOX locus. Genotyping results should be combined with the standard DNA allelic ladder to avoid genotyping errors because of the complex internal sequence structure of the SE33 locus [P-value for Hardy–Weinberg testing did not significantly deviate from HWE after Bonferroni correction (P = 0.05/29 = 0.0017), only D21S2055 (P = 0.000 2) and D7S1517 (P = 0.0012) had minor departures from HWE because of errors in random sampling. Among the 29 STR loci, the SE33 locus was the most informative marker, with a PD value of 0.9930 and a PIC value of 0.9500, whereas the TPOX locus presented the lowest forensic efficiency, with a PD value of 0.7590 and a PIC value of 0.5200. The Ho ranged from 0.5930 (TPOX) to 0.9240 (Penta E). The combined PM and combined PE for the 29 STR loci were 1.34 × 10−36 and 0.999 999 999 998, respectively. Pairwise LD tests demonstrated that only 18 pairs (Table S2) of the loci remained in LD for 406 pairwise comparisons after Bonferroni correction (P = 0.05/406 = 0.00012). These significant pairs in LD may be attributed to random sampling errors because the pairs are located on different autosomal chromosomes or chromosome arms.The allele frequencies and population genetics parameters of the 29 STR loci are summarized in Table S1. We detected 403 alleles across the 29 STR loci, with the largest number of 50 variants at the P-value) in the Investigator HDplex kit between the Hunan Han population and previously published data of other ethnic populations at the same loci. Evident differences (P < 0.05) were found between the Hunan Han and the Dutch [D3S1744, P = 0.0723) [D3S1744, P = 0.1267 and D8S1132, P = 0.2776) [D2S1360, D3S1744, D10S2325, SE33 and D12S391 with the Hebei Han [D4S2366, D8S1132, SE33 and D12S391 with the Sichuan Han [D3S1744, D10S2325, SE33 and D12S391 with the Shanghai Han [Table S3 shows the locus-by-locus population differences   and Swedhuan Han ; and at ghai Han . Table SD7S820, Penta E and TH01), Huai'an Han [Penta E), Yangzhou Han [D19S433, D3S1358 and TH01) and Taizhou Han [D16S539, Penta D and TH01) populations. Table S6 and Figure S2 present the genetic distances and phylogenetic tree between the Hunan Han and other populations, respectively. The minimum genetic distance and close genetic relationship were detected between the Hunan Han and Guangdong Han (0.0040) populations. The largest distance was found between the Hunan Han and Liaoning Manchu (0.0618) populations.Our results for 19 STR loci in the Goldeneye™ 20A kit compared with other populations are also summarized. Table S5 shows that the distribution of alleles for the Hunan Han versus Suzhou Han , Henan Hi'an Han  (except zhou Han  (except zhou Han  (except In summary, data suggested that the use of 29 STR loci could provide highly informative polymorphic data for forensic identification and establish a database for the Hunan Han group in China. The data also demonstrated that the distribution of allele frequencies varied in different areas or even in the same area for different subpopulations. Close genetic distance and genetic relationship were noted among regions separated by small distances because of their geographic proximity and high gene mobility.This paper strictly follows all the guidelines for publication of population data as required by this target journal.


TP53, encoding p53, is the most frequently mutated gene in human cancers. p53 is a transcription factor that suppresses tumors by regulating myriad genes critical for diverse cellular outcomes including growth arrest and death. This study addresses the mechanism by which iASPP, a p53 partner, influences p53 target gene selection. Using next-generation sequencing, we found genes coregulated by iASPP and p53, and characterized their DNA sequence signatures. Our crystal structure of iASPP and p53 reveals that iASPP displaces a loop of p53 that recognizes DNA signatures. iASPP inhibits p53 through a protein surface distinct from other characterized p53 cellular partners but overlapping that targeted by the viral oncoprotein human papillomavirus E6. These findings open prospects for designing p53-targeting anticancer agents. The most frequently mutated protein in human cancer is p53, a transcription factor (TF) that regulates myriad genes instrumental in diverse cellular outcomes including growth arrest and cell death. Cell context-dependent p53 modulation is critical for this life-or-death balance, yet remains incompletely understood. Here we identify sequence signatures enriched in genomic p53-binding sites modulated by the transcription cofactor iASPP. Moreover, our p53–iASPP crystal structure reveals that iASPP displaces the p53 L1 loop—which mediates sequence-specific interactions with the signature-corresponding base—without perturbing other DNA-recognizing modules of the p53 DNA-binding domain. A TF commonly uses multiple structural modules to recognize its cognate DNA, and thus this mechanism of a cofactor fine-tuning TF–DNA interactions through targeting a particular module is likely widespread. Previously, all tumor suppressors and oncoproteins that associate with the p53 DNA-binding domain—except the oncogenic E6 from human papillomaviruses (HPVs)—structurally cluster at the DNA-binding site of p53, complicating drug design. By contrast, iASPP inhibits p53 through a distinct surface overlapping the E6 footprint, opening prospects for p53-targeting precision medicine to improve cancer therapy. The recognition of specific DNA sequences by transcription factors (TFs) is instrumental for decoding genomes , 2. BeyoThe apparently 53-kDa tumor suppressor and TF p53 is the most frequently mutated protein in human cancer , 12. As 13A common landmark of virtually all p53 target genes is a stretch of specific DNA sequence, termed a response element (RE). These p53 REs share the consensus sequence motif consisting of 2 half sites, each being a 10-base pair (bp) palindrome 5′-PuPuPuC(A/T)(T/A)GPyPyPy-3′ , occasionally separated by a short spacer , 18. The19BAX and TP53I3 (PIG3) (The ASPP (apoptosis stimulating protein of p53) family of transcription cofactors represent well-recognized promoter-specific regulators of p53 , 29, and3 (PIG3) , whereas3 (PIG3) . Classic3 (PIG3) –35, and 3 (PIG3) , 37. Par3 (PIG3) , 34. ThePPP1R13L) in the HCT 116 colorectal carcinoma cell line, which expresses wild-type p53, and analyzed the effects on gene expression with RNA-seq . Gene set enrichment analysis of these DE genes showed that iASPP-regulated genes are enriched in the canonical p53 signaling pathway  and the transcripts up-regulated by iASPP RNAi are enriched in transcriptional targets of p53 (P = 8.0 × 10−15) . We observed significant albeit relatively weak up-regulation of iASPP by Nutlin  (TP53) resulted in a gene expression profile more closely resembling that of p53 RNAi than iASPP depletion   in iASPP-depleted cells compared to control cells, comprising 194 up-regulated and 281 down-regulated genes (× 10−15) C. As a pe = 1.7) A, consise = 1.7)  and contP = 1.30 × 10−5; G12: P = 2.38 × 10−6; see figure legends for detailed statistics). The concurrent presence of C9 and G12 was enriched in iASPP-regulated compared to Nutlin-induced REs (P = 1.63 × 10−5). These findings suggest that the presence of C9 and/or G12 in a typical 20-bp p53 motif likely represents the sequence basis for iASPP-regulated p53 binding, which in turn underlies gene regulation by iASPP.To assess genome-wide p53 binding regulated by iASPP, we performed ChIP-seq using an anti-p53 antibody in HCT 116 cells treated with control or iASPP RNAi. A total of 214 p53-binding sites showed elevated signals (fold change > 2) following iASPP depletion E. We useSI Appendix, Fig. S2). One of the iASPP-regulated p53 ChIP peaks is shared by the overlapping gene bodies of ACTA2 and FAS, and iASPP depletion promotes the transcription of both genes (BBC3) in addition to FAS and NOXA (PMAIP1) identified above , we integrated the RNA-seq and ChIP-seq results and found 13 iPTGs associated with 12 differential p53 ChIP-seq peaks following iASPP depletion , RAP2B , RAP2B , and TIGoperties . RPS27L  , and MDM2 had similar p53 ChIP-seq signals in the presence or absence of iASPP   and a C-terminal iASPP construct (residues 625 to 828) . These constructs enabled crystallization of the purified complex .We then set out to understand the structural basis for iASPP-mediated regulation of p53–DNA interactions. The domain organization of a p53 monomer consists of an amino (N)-terminal transactivation domain (TAD), a proline-rich domain (PRD), a central, conserved sequence-specific DBD, an oligomerization domain (OD) that mediates tetramerization, and a carboxyl (C)-terminal regulatory domain (CTD) (ig. S3A) . For cry2+ ion and iASPP residues 657 to 823; the crystallographic asymmetric unit contains 1 such complex , suggesting it is not critical for the interaction between these truncated protein constructs. The first ankyrin repeat of iASPP was not traceable in the electron density . The structures of the individual components observed in the complex are virtually identical to published structures of the p53 DBD fold (a root-mean-square deviation [RMSD] of 0.28 Å for 194 Cα pairs compared to PDB entry 2XWR chain B) and iASPP (an RMSD of 0.57 Å over 166 Cα pairs compared to PDB entry 2VGE), except for the L1 loop of p53  , as discussed below.Our final model includes p53 residues 91 to 291 with a coordinated zinc complex B. There SI Appendix, Fig. S6). Interface I has the largest  buried solvent-accessible surface area. Here, iASPP almost exclusively uses a relatively flat surface of its ankyrin stack (composed of residues exposed on the second α-helix of each ankyrin repeat) to interact with the LSH motif of p53, prizing the L1 loop away from its contacts with the S2 β-strand and the H2 α-helix . iASPP F818, situated in the C-terminal loop (tail) that is trapped between the ankyrin repeats and the SH3 domain provides an auxiliary hydrophobic shield for the interface  and is composed predominantly of hydrophilic interactions. At this interface, iASPP employs a different face of its ankyrin stack to engage p53 at a surface that overlaps with its DNA-binding area . Interface III is the smallest of the 3 (920.6 Å2) where the SH3 domain of iASPP contacts the N-terminal loop of p53 DBD, which wraps around the DBD, and the surrounding p53 residues .Interface II is the second largest  observed in the crystal lattice is required for binding, we performed an alanine scan of p53 residues whose side chains are orientated to contact iASPP exclusively at each interface and assayed the binding of corresponding full-length p53 single-point mutants to our iASPP crystallization construct. Where possible, residues with solvent-exposed side chains on the apo structure of p53 DBD that do not display apparent structural roles were chosen. Alanine substitutions of interface I p53 residues H115 and R282 diminished and enhanced p53–iASPP interactions, respectively, whereas mutations of interface II p53 residues S121, R248, R280, and N288 to alanine did not substantially affect the binding E. InterfInset. A p53 DBD monomer interacts with this DNA duplex primarily via direct, base-specific major-groove interactions involving the LSH motif. In virtually all p53–DNA structures, R280 from the H2 helix anchors p53 to the cognate DNA duplex through 2 conserved hydrogen bonds to the invariant G on the pyrimidine-rich strand  . A corresponding middle quarter site from the iASPP-regulated p53 consensus motif determined from our sequence analysis is shown in h strand A. In comh strand A.We superimposed our iASPP–p53 complex on the classical DNA complex using p53 as the reference B. OveralSI Appendix, Fig. S7A). Although iASPP exhibits a generally negatively charged exterior, the p53–iASPP complex retains a continuous, positively charged surface for binding DNA .In contrast to the displaced L1 loop, the other p53 DNA-binding modules remain well suited to interact with DNA in the iASPP complex. For instance, the H2 helix from the LSH motif and the L3 loop retain essentially the same structures and surface availability in the iASPP complex compared to the DNA-binding structure B and C. Typically, p53 is held together as a tetramer by its oligomerization domain and recognizes its cognate DNA sequences through its DBD. The consensus motif of p53 REs has a striking symmetry that reflects the dimer-of-dimers architecture of p53 DBDs assembled on cognate DNA sequences, even in the absence of its oligomerization domain A 22)..22).SI Appendix, Fig. S8 A and B). Superposition of 4 iASPP complexes onto the DNA complexes of p53, on the other hand, would introduce minor clashes between the ankyrin-repeat domains of the adjacent iASPP structures related by the translational symmetry , although it is possible that a slight architectural adaptation could accommodate a 4:4 iASPP–p53 assembly on an unsplit p53 consensus sequence. Notably, there is no clash between rotational symmetry-related iASPP structures .When a single p53–iASPP complex is docked onto tetrameric p53–DNA complexes, iASPP does not appear to overlap with any of the symmetry-related p53 DBD molecules  in concordance with the iASPP-regulated p53 RE sequence signatures (C9 [G on the opposite strand] and G12) being symmetrically positioned in the middle p53 quarter sites.These structural observations suggest a functioning model of 2 centrally located iASPP molecules on tetrameric p53–DNA complexes , we originally anticipated p53–iASPP interactions to be similar to those described for p53–ASPP2 . However2 vs. ∼1,500 Å2, respectively), in line with the comparable p53-binding affinities reported for iASPP and ASPP2 . A close inspection of the p53-contacting residues in iASPP and ASPP2 shows clear differences at the interfaces. At the p53–ASPP2-binding site involving the p53 L2 loop and ASPP2 ankyrin repeat IV  to contact the major groove of cognate DNA . In conjTIGAR, an antiapoptosis and prosurvival gene, involved in metabolism and ROS control. The majority of the identified iPTGs participate in p53-mediated cellular life-or-death decisions, in line with an apoptosis-related function of iASPP. Of note, several iPTGs, such as ACTA2, FHL2, PARD6B, and SLC30A1, may be linked to iASPP-related phenotypes such as sudden cardiac death and dilated cardiomyopathy . Indeed, there is growing evidence that overexpression, rather than mutation, of iASPP conveys its oncogenic properties. In addition to our previous characterization in p53 wild-type breast cancer  polyethylene glycol 3350, 0.18 M trisodium citrate. Crystallographic data were collected and processed, and the structure was determined and analyzed, essentially as described previously  and used to perform the RNA-seq and the ChIP-seq  experiments. The sequencing data were analyzed with standard bioinformatics pipelines. p53crys (human p53 [UniProt accession code P04637] residues 62 to 292) and iASPPcrys (human iASPP [UniProt Q8WUF5] residues 625 to 828) were expressed in eviously . A detaiSupplementary File


Myceliophthora thermophila (synonym Sporotrichum thermophile), an Ascomycete fungus, expresses and secretes over a dozen different LPMOs. In this study, we report the overexpression and biochemical study of a previously uncharacterized LPMO (MtLPMO9J) from M. thermophila M77 in Aspergillus nidulans. MtLPMO9J is a single-domain LPMO and has 63% sequence similarity with the catalytic domain of NcLPMO9C from Neurospora crassa. Biochemical characterization of MtLPMO9J revealed that it performs C4-oxidation and is active against cellulose, soluble cello-oligosaccharides and xyloglucan. Moreover, biophysical studies showed that MtLPMO9J is structurally stable at pH above 5 and at temperatures up to 50°C. Importantly, LC-MS analysis of the peptides after tryptic digestion of the recombinantly produced protein revealed not only the correct processing of the signal peptide and methylation of the N-terminal histidine, but also partial autoxidation of the catalytic center. This shows that redox conditions need to be controlled, not only during LPMO reactions but also during protein production, to protect LPMOs from oxidative damage.Thermophilic fungi are a promising source of thermostable enzymes able to hydrolytically or oxidatively degrade plant cell wall components. Among these enzymes are lytic polysaccharide monooxygenases (LPMOs), enzymes capable of enhancing biomass hydrolysis through an oxidative mechanism.  Lytic Polysaccharide Monooxygenases (LPMOs) are oxidative enzymes able to boost the hydrolytic efficiency of glycoside hydrolases (GHs) during the depolymerization of recalcitrant polysaccharides (such as lignocellulose and chitin) , 2. LPMOLPMO action requires an external electron donor  and molehttp://www.cazy.org)  and 4% solution B . Peptides were eluted using a 40 min gradient developing from 4% to 15% (v/v) solution B in 2 min and from 15% to 55% (v/v) solution B in 25 min before a wash phase at 90% solution B, all at a flow rate of 300 nL/min. The column was connected to a Q-Exactive mass spectrometer  operated in data-dependent mode to switch automatically between orbitrap-MS and higher-energy collisional dissociation (HCD) orbitrap-MS/MS acquisition. Peptides were identified with the Mascot server (version 2.6.1) against MtLPMO9J and the Aspergillus nidulans proteome, the latter being the background from the expression host. Correct processing of the signal peptide (i.e. cleavage before His1) was evaluated using the SEMI-trypsin search on the full-length MtLPMO9J sequence, which allowed identification of fragments resulting from one non-tryptic cleavage  at the N-terminus of the peptide and one trypsin-specific cleavage at the C-terminus of the peptide (http://massqc.proteomesoftware.com/help/metrics/percent_semi_tryptic). Tryptic peptides and their possible modifications were identified using error tolerant Mascot searches on the MtLPMO9J sequence without the signal peptide.Purified n et al. . The pepEPR experiments were performed on a Varian E109 spectrometer equipped with a cryogenic system, which allowed for low-temperature data collection. The spectrometer was operated at 9.26 GHz, with a modulation amplitude of 4 G and microwave power of 10 mW, at 70 K. Samples were drawn into quartz tubes and then frozen in liquid nitrogen. The EPR parameters were optimized to avoid line saturation and distortion. The spectrum of the buffer only was used as a baseline and subtracted from all other spectra.MtLPMO9J in 20 mM Na-phosphate buffer pH 6.0. The sample was analysed at 25°C using a 0.1 cm path length quartz cell. The far-UV spectra were recorded using a wavelength range of 205–260 nm, with 50 nm/min scan speed and 1 nm band width. The final data were obtained by signal averaging six spectra before subtracting the buffer spectrum and are reported as mean residue ellipticity (MRW). The thermal denaturation experiment was performed following the ellipticity (θ) at wavelength 215 nm, corresponding to the minimum value of the spectra, at a constant heating rate of 1°C/min with 0.5 mg/mL MtLPMO9J from 20 to 90°C.Circular dichroism (CD) measurements were conducted on a JASCO J-815 spectropolarimeter  using 0.5 mg/mL of purified MtLPMO9J in a set of 8 buffers, each at a concentration of 50 mM, with the pH ranging from 2.0 to 9.0. Reactions with 62 μM MtLPMO9J and 0.05% (v/v) SYPRO Orange dye  were prepared in each buffer on a 96-well plate [A ThermoFluor assay was applied to study the thermal stability of ll plate . The plaThe following substrates were used: phosphoric acid swollen cellulose , as described earlier ), cellotMtLPMO9J (5 μM) in 20 mM sodium acetate buffer (pH 6.0) at 50°C and shaking at 1000 rpm, in 100 μl total volume for up to 16 h. In all experiments, 1 mM ascorbic acid was used as reducing agent. For the time course experiments, 100 μL aliquots were removed from reactions with 1 mL total volume at defined time intervals. Control reactions were set up without reducing agent or enzyme; reactions containing substrate only were also set up. As a positive control, NcLPMO9C was used [The substrates  were incubated with was used . ReactioMtLPMO9J on PASC were quantified by integrating the peak areas of C4-oxidized products.LPMO reaction products were analyzed by high-performance anion exchange chromatography (HPAEC) on a Dionex ICS3000 instrument equipped with pulsed amperometric detection (PAD) and a CarboPac PA1 column (2×250 mm) with a CarboPac PA1 guard column (2×50 mm). For analysis of cellulosic substrates, a 50-min gradient  and cellLPMO reaction products were also analyzed using MALDI-TOF mass spectrometry on an Ultraflex MALDI-ToF/ToF instrument  equipped with nitrogen 337 nm laser beam. Samples (0.8 μL) were mixed with 2,5-dihydroxybenzoic acid matrix , applied to a MTP 384 ground steel target plate TF , and air-dried. Data were collected using Bruker’s flexControl software and the spectra were analyzed using Bruker’s flexAnalysis software.MtLPMO9J and 25 different LPMO9s, including several of known structure, revealed the conservation of the N-terminal His1, His83 and Tyr168, which compose the copper-binding motif (NcLPMO9C (63% sequence identity) and PaLPMO9H (58% sequence identity), both of which have an additional CBM1 carbohydrate-binding module at the C-terminus. Phylogenetic analysis places MtLPMO9J in a cluster together with C4-oxidizing LPMOs; this cluster is divided into two groups (MtLPMO9J consists of LPMOs (NcLPMO9C and PaLPMO9H) that are active on cello-oligosaccharides; the other group contains MtLPMO9C (MYCTH_100518) [NcLPMO9A (NCU02240) and NcLPMO9D (NCU01050), LPMOs that accumulate C4-oxidized cellopentaose during their action on PASC [LsLPMO9A [CvLPMO9A [The MYCTH_79765 gene encodes a single AA9 domain of 229 residues with a theoretical molecular weight of 24.3 kDa  , which i on PASC  and, theLsLPMO9A  and CvLPCvLPMO9A , form a MtLPMO9J was built and compared with the crystal structures of CvLPMO9A (PDB: 5NLT) [NcLPMO9A (PDB:5FOH), NcLPMO9C (PDB:4D7U) [NcLPMO9D (PDB:4EIR) [LsAA9A (PDB:5ACF) [MtLPMO9J contains several of the residues that are known to be involved in substrate binding, based on the structure of LsAA9A in complex with cellohexaose [NcLPMO9C in complex with cellulose and xyloglucan [CvLPMO9A that is active on cello-oligomers)  potentially involved in substrate binding  , NcLPMO9DB:4D7U) , NcLPMO9DB:4EIR)  and LsAADB:5ACF) . The MSADB:5ACF)  Fig 1B)MtLPMO9J ohexaose  and NMR loglucan  , yielding approximately 12 mg of enzyme per L of culture medium. The enzyme was purified in two chromatographic steps to ca. 95% purity . Protein denaturation curves measured using the reporter dye SYPRO Orange at different pHs showed structural stability at pHs above 5.0 with a melting temperature  of 58°C  resulted in the formation of native (i.e. non-oxidized) and oxidized cello-oligosaccharides in the presence of ascorbic acid as electron donor. HPAEC-PAD and MALDI-TOF/MS analyses of reaction mixtures with PASC showed the accumulation of cello-oligosaccharides with a degree of polymerization (DP) of 2 to 12 (MtLPMO9J on PASC was similar to that of the C4-oxidizing NcLPMO9C [m/z 1173.45 for DP7) with minor amounts occurring in the hydrated gemdiol form (m/z 1191.46), while no double-Na+ adduct (m/z 1213.46), which is diagnostic of C1-oxidation [m/z 1171.43 could, in principle, be an oligomer oxidized at both ends, but since there are no other, and more reliable, indications for C1-oxidation , the m/z 1171.43 signal likely represents a degradation product. Notably, C4-oxidized cello-oligosaccharides are unstable, which also explains why one sees a relatively high amount of native products  degradation of the oxidized products became noticeable  to produce native and C4-oxidized trimers (Glc3 and oxGlc3) or a native tetramer and a C4-oxidized dimer (Glc4 and oxGlc2)  was converted to a native cellotriose and a C4-oxidized dimer (Glc3 and oxGlc2). Activity on cellotetraose (Glc4) and cellotriose (Glc3) was not detected , while the hydrated form (m/z 1263.46) was also detected. The lack of the m/z 1285.50 signal  indicates the absence of C1-oxidation. Of note, despite major purification efforts, the purified enzyme displayed a background xylanase activity originating from the expression host, which precluded assessment of the activity of MtLPMO9J on xylan and xylo-oligosaccharides.As some of the closest homologs of  cleaved . The proNcLPMO9C . MALDI-TMyceliophthora thermophila, are a promising source of thermostable enzymes able to hydrolytically and oxidatively degrade plant cell wall components. Application of thermostable enzymes, including LPMOs, for biomass conversion could enhance saccharification rates and decrease risk of microbial contamination. Here we report the characterization of an LPMO9 from M. thermophila, MtLPMO9J (MYCTH_79765)[A. nidulans. This enzyme has been previously identified among 11 MtLPMO9s, including the five previously characterized MtLPMO9s [M. thermophila when grown on alfalfa and barley straw [Thermophilic fungi, such as TH_79765), obtaineMtLPMO9s , 19, 27,ey straw  . In one of these reports [et al. show that LPMOs indeed can use H2O2 quite efficiently, supporting the findings by Bissaro et al [2 is the natural co-substrate, in contrast with the conclusions drawn by Bissaro et al. As we did not quantify LPMO activity on the cello-oligosaccharides, a direct comparison of our results with the reaction rates for cellohexaose degradation reported by Hangasky et al. [et al. also detected a minor activity on cellotetraose.Several questions remain as to the mechanism of LPMOs, and the question whether O in 2016 , is the or H2O2  during the expression and purification protocols. From a practical point of view, our finding of enzyme oxidation during production is potentially of major importance. Oxidatively damaged LPMOs will not be recognized as such when using common quality control methods for proteins like SDS-PAGE or gel filtration. Nevertheless, such LPMOs will be impaired in copper binding and catalytic performance.Recent work on LPMOs has revealed that these enzymes are prone to oxidative self-inactivation, e.g. under conditions where they are reduced in the absence of substrate , 54. IndS1 File(PDF)Click here for additional data file.


The association between PHOX2B single nucleotide polymorphisms (SNPs) and Wilms tumor risk has not been investigated. Therefore, we conducted a case-control study involving 145 Wilms tumor patients and 531 controls to explore the association between the PHOX2B rs28647582 T>C polymorphism and Wilms tumor susceptibility. The association between the PHOX2B rs28647582 T>C polymorphism and Wilms tumor susceptibility was assessed by odds ratios (ORs) and 95% confidence intervals (CIs). Our results indicated that PHOX2B rs28647582 T>C polymorphism did not significantly alter Wilms tumor susceptibility. However, in the stratified analysis, we found that TC/CC genotypes significantly increased Wilms tumor risk among children older than 18 months  and those with clinical stages III+IV , when compared with those with TT genotype. Our study suggested that PHOX2B rs28647582 T>C was weakly associated with Wilms tumor susceptibility. Our conclusions need further validation with a larger sample size.Wilms tumor is one of the most common pediatric solid tumors. The  Wilms tumor, also known as nephroblastoma, is one of the most common pediatric malignant tumors, accounting for 7–8% of tumors in childhood ,2. The pFWT1 [FWT2 [BARD1 [CTR9 [HACE1 [LMO1 genes [With the development of genome-wide association studies (GWASs), more and more Wilms tumor susceptibility genes have been discovered. For example, single nucleotide polymorphisms (SNPs) in the FWT1 , FWT2 [72 [BARD1 , CTR9 [99 [HACE1  and LMO1O1 genes  have beepair-like homeobox 2b (PHOX2B) gene belongs to the homeobox gene family which locates in 4p13. This gene accounts for 4.8 k bases approximately, contains three exons and two introns, encodes a peptide containing 314 amino acids [PHOX2B is expressed during neural development in central autonomic circuits and peripheral neural crest derivatives, particularly in retrotrapezoid nucleus, noradrenergic centers and hindbrain. PHOX2B is an indispensible transcription factor in the proliferation and differentiation of neural crest tissue [TH [PHOX2B itself [RET [TLX-2 [ALK [SOX10 [MSX1 [The homeobox genes are transcription factors that play an important part in embryonic development, including cellular differentiation, migration, apoptosis, signal transduction and angiogenesis . The pait tissue . It can ssue [TH , PHOX2B B itself , RET [20T [TLX-2 , ALK [22K [SOX10  and MSX110 [MSX1 .PHOX2B polymorphisms mostly focused on exons. With the continuous development of biotechnology, many introns have been found to assume the regulation roles in genetic expression. Previous investigations have shown that the PHOX2B rs28647582 T>C polymorphism significantly alters Hirschsprung disease susceptibility [PHOX2B rs28647582 T>C polymorphism and Wilms tumor susceptibility. Considering this polymorphism may affect the differentiation of posterior renal blastocyst, we conducted this experiment about PHOX2B rs28647582 T>C polymorphism and Wilms tumor susceptibility.In the past, research about tibility . In additibility  have widIt was a case-control study with 145 patients and 531 controls (Supplementary Table 1). The patients were collected from the Guangzhou Women and Children’s Medical Center during March 2001 to June 2016 . Wilms tPHOX2B rs28647582 T>C polymorphism was genotyped by TaqMan real-time PCR [DNA was extracted from 2 ml venous blood samples using TIANamp Blood DNA Kit . The time PCR . The inf2 test. A goodness-of-fit test was used to estimate Hardy–Weinberg equilibrium (HWE) in controls. After adjusting for age and gender, we performed an unconditional multiple logistic regression model to assess the association between the PHOX2B rs28647582 T>C polymorphism and Wilms tumor susceptibility by odds ratios (ORs) and 95% confidence intervals (CIs). Finally, stratified analysis was conducted by age, gender and clinical stages. All data were analyzed by SAS statistical software, using a two-sided test. The results were considered statistically significant when P<0.05.T-test was employed to check the differences in age. Frequency distribution of gender and genotype between patients and the controls were evaluated by χPHOX2B rs28647582 T>C polymorphism in Wilms tumor patients and controls is shown in PHOX2B rs28647582 T>C polymorphism obeyed the HWE genetic balance in controls (P=0.505). Unfortunately, we could not observe significant association between PHOX2B rs28647582 T>C polymorphism and Wilms tumor susceptibility .The genotype distribution of P=0.027) and clinical stages III+IV . No significant correlation was found between PHOX2B rs28647582 T>C polymorphism and Wilms tumor susceptibility in other groups.We further conducted a stratified analysis by age, gender and clinical stages . StratifPHOX2B mutations were observed in congenital central hypoventilation syndrome, neuroblastoma and Hirschsprung disease [ disease ,37. Subs disease ,39. As f disease ,41.PHOX2B rs28647582 is located in intron 2. Existing studies had no idea how rs28647582 affected PHOX2B. In the past few years, some conflicting results about PHOX2B rs28647582 polymorphism and Hirschsprung’s disease were drawn. Liu et al. [Introns are widely distributed in the genome of eukaryotic cells. They are much longer than exons and accumulate more mutations, accounting for 95–97% in human genome. Although introns do not code for proteins, an increasing number of studies found that introns not only played a regulatory role in transcription, but also had selective splicing and promoter-like functions during mRNA maturation and translation . PHOX2B u et al.  found thu et al.  and Xiaou et al.  indicateu et al. . These rPHOX2B rs28647582 T>C polymorphism and Wilms tumor. We observed that PHOX2B rs28647582 TC/CC genotypes significantly increased Wilms tumor susceptibility in subgroups older than 18 months or clinical stages III+IV. Based on previous studies [PHOX2B structure and expression by abnormally splicing and regulating, which would weaken PHOX2B ability to promote the proliferation and differentiation of renal cells. In addition, PHOX2B rs28647582 T>C polymorphism may combine with tumor-related genes to modify Wilms tumor susceptibility.This is the first investigation about  studies ,41, rs28PHOX2B rs28647582 T>C polymorphism and Wilms tumor susceptibility in stratified analysis, some limitations existed in our study. First, our research had a relatively small sample size, containing 145 patients and 531 controls. It was difficult to discover the genuine association between PHOX2B rs28647582 T>C polymorphism and Wilms tumor. Second, we selected only one polymorphism in PHOX2B, which made genotype association analysis impossible. It may omit the combined effect of PHOX2B rs28647582 T>C polymorphism and other potential polymorphisms in Wilms tumor [PHOX2B rs28647582 T>C polymorphism and Wilms tumor susceptibility. In order to achieve reliable results, we should expand the sample size, add genotype association analysis and perform a strict quality monitoring in follow-up studies.Although we found the correlation between ms tumor . Third, PHOX2B rs28647582 T>C polymorphism is weakly associated with Wilms tumor susceptibility. Our result need further verified.In conclusion, we have exposed that Click here for additional data file.


Antimalarial drug resistance is a substantial impediment to malaria control. The spread of resistance has been described using genetic markers, which are important epidemiological tools. We carried out a temporal analysis of changes in allele frequencies of 12 drug resistance markers over 2 decades of changing antimalarial drug policy in Kenya. k13) artemisinin resistance markers; nonetheless, a single k13 allele, K189T, was maintained at a stable high frequency (>10%) over time. There was a distinct shift from chloroquine-resistant transporter (crt)-76, multidrug-resistant gene 1 (mdr1)-86 and mdr1-1246 chloroquine (CQ) resistance alleles to a 99% prevalence of CQ-sensitive alleles in the population, following the withdrawal of CQ from routine use. In contrast, the dihydropteroate synthetase (dhps) double mutant (437G and 540E) associated with sulfadoxine-pyrimethamine (SP) resistance was maintained at a high frequency (>75%), after a change from SP to artemisinin combination therapies (ACTs). The novel cysteine desulfurase (nfs) K65 allele, implicated in resistance to lumefantrine in a West African study, showed a gradual significant decline in allele frequency pre- and post-ACT introduction (from 38% to 20%), suggesting evidence of directional selection in Kenya, potentially not due to lumefantrine. The high frequency of CQ-sensitive parasites circulating in the population suggests that the reintroduction of CQ in combination therapy for the treatment of malaria can be considered in the future. However, the risk of a reemergence of CQ-resistant parasites circulating below detectable levels or being reintroduced from other regions remains.Antimalarial drug resistance is a substantial impediment to malaria control. The spread of resistance has been described using genetic markers, which are important epidemiological tools. We carried out a temporal analysis of changes in allele frequencies of 12 drug resistance markers over 2 decades of changing antimalarial drug policy in Kenya. We did not detect any of the validated kelch 13 ( Plasmodium falciparum malaria in all regions of endemicity; however, drug resistance is an impediment to malaria control. Notably, resistance to former first-line antimalarials, chloroquine (CQ) and sulfadoxine-pyrimethamine (SP), was imported to Africa via strains from South East (SE) Asia  are recommended as the first-line treatment for uncomplicated SE) Asia –3, althoE) Asia – and wereE) Asia –, 6. For E) Asia ––9, as we Vietnam , 11, are Vietnam , 13 callP. falciparum genetic mutations associated with antimalarial drug resistance has provided molecular markers for surveillance of resistance, both in real time and retrospectively, to assess geographic origins and migration patterns of drug-resistant parasites (k13), including F446I, N458Y, M476I, Y493H, R539T, I543T, P553L, R561H, and C580Y, are enabling the detection and tracking of artemisinin resistance in SE Asia (arps10) codon V127M, chloroquine resistance transporter (crt) codon I356T, ferredoxin (fd) codon D193Y, and multidrug resistance (mdr) protein 2 codon T484I. While these mutations have been identified in parasites from Africa, they occur at very low frequencies  (crt)  and multidrug resistance 1 (mdr1) (N86/184F/D1246-NFD haplotype) genes (18mdr1-NFD haplotype and an increase in mdr1 copy number have been linked with reduced susceptibility to lumefantrine (LM) and mefloquine (MQ), respectively  and the allele 1528D in the ubiquitin carboxyl-terminal hydrolase 1 gene  (nfs) gene was observed 6 years after the introduction of ACTs. Moreover, the 50% inhibitory concentration (IC50) values for LM were significantly higher in nfs-K65 wild-type field isolates than in the mutant 65Q isolates  . Nonethe genes 1821. Furthectively –24. Morem Africa . A cliniisolates .dhfr 51I, 59R, and 108N triple mutant) combined with mutations in the dihydropteroate synthetase gene (dhps 437G and 540E double mutant) are associated with clinical and parasitological SP treatment failure in East Africa (–dhps mutation (A581G) has been associated with reduced IPTp efficacy when the prevalence of sextuple-mutant (dhfr 51I/59R/108N and dhps 437G/540E/581G) parasites exceeds 37%  whose impacts are not yet fully understood.These studies highlight the need for continued surveillance of known drug resistance markers and novel markers to maintain the gains made in using ACTs and IPTp in controlling malaria. l policy . Therefok13 codon K189T was the only polymorphism maintained at frequencies of >10%, while the rest of the observed alleles were rare, including codon A578S, with frequencies barely reaching 2%. Other than K189T, the only other polymorphism observed across all time points is the asparagine (Asn) repeat at codon 137. This repeat region included insertions of between one and four asparagine residues, though at low frequencies, <3%, compared to SE Asia (>60%)  , 37. Manarasites . Compariarasites  and othearasites , 39, reventified . Of the arps10, crt, and fd. However, a high-frequency (>10%) single-nucleotide polymorphism (SNP) (I492V) was identified in mdr2 in the 1995/1996 and 2015/2016 time points, and hence, the three middle time points were included. Consequently, two additional polymorphisms were identified , with the I492V polymorphism maintaining high frequencies .There were no mutations identified in ap2-mu, no significant temporal trends were observed, and only two polymorphisms achieved frequencies of >10% across time, I100I and [7N]227[6N/8N/9N/10N]. Of note, the prevalence of S160N mutation was similar from 1995/1996 to 2015/2016, except for a 2-fold increase from 9% in 1998/1999 to 24% in 2012/2013 that later decreased to 16% in 2015/2016. Over the same period, the E163E allele showed similar increases and decreases in frequency from 8.51% in 2005/2006 to 16.36% in 2012/2013 and down to 4.55% in 2015/2016) (Table S4). A total of 38 haplotypes were assembled, with the 3D7 haplotype TRSKT[N7][Kx1]AG[N5][N4]AFI dominating across time .In falcipain-2a was found to be the most polymorphic gene; however, the S69Stop polymorphism was not identified (Table S5). Of the 71 haplotypes assembled, the 3D7 haplotype was not observed (Table S10). The only significant temporal trend found was that of codon S59F , with the 59F allele dropping from 13% in 1995 and 1996 to 5% in 2015/2016. From 2005/2006 and 2012/2013 through to 2015/2016, the following alleles showed similar 2-fold increases and decreases in frequency: N4H, A8I, P9P, H10N and E11E .ubp-1, only two polymorphisms were found to exceed 10% frequency across time , the KYD repeat at codon 1520 and the KYE at codon 1526 (Table S6). The E1528D polymorphism observed in previous studies from Kenya was not identified. Additionally, from 2005/2006, 2012/2013, to 2015/2016, the following alleles showed 2-fold increases and decreases in frequency: the KNE repeat at codon 1514  and N1518N . Of the 23 haplotypes, the 3D7 haplotype dominated throughout the sampling period, and no significant temporal trends were observed (Table S11).In crt gene in 2015/2016 and 2017/2018. Mutant alleles at codons 74I, 75E, and 76T dominated during the pre-ACT period, and there was a distinct shift to the wild-type alleles  in the post-ACT period, almost reaching fixation (99%)  were identified in the mdr1 in 2015/2016 and 2017/2018, as follows: N86Y, G102G, G182G, F184Y, and D1246Y. mdr1 codons 86Y and 1246Y also showed a distinct shift from mutant alleles in the pre-ACT period (∼40 to 70%) to wild-type alleles N86 and D1246, nearly approaching fixation (99%)  post-ACT introduction. In contrast, the mutant 184F allele increased in frequency during the post-ACT period  were not genotyped in an earlier study  alleles dominated in the post-ACT period . The SP-sensitive haplotype (SAKA) decreased in frequency over time and was no longer detectable in 2015/2016 .S436A, A437G, K540E, and A581G 015/2016 , while tnfs, only codon K65Q was found to have a significant, albeit marginal, trend pre- and post-ACT introduction . It was also in high linkage disequilibrium with codons S62N and E67G. The 2005/2006 period appears to be the point at which the mutant allele 65Q and wild-type 65K diverge in frequency in opposite directions, with the wild-type 65K decreasing and the mutant allele 65Q increasing in frequency .In Serine-tRNA ligase, a marker not associated with resistance or drug selection, had only one polymorphic codon observed across all time points (L84V), with frequencies ranging between 2 and 5%, while the rest were rare (<5%) (Table S8). A total of 15 haplotypes were observed, and only the 3D7 haplotype occurred across all time points and with frequencies of >80%, with the rest of the haplotypes being rare (<5%) (Table S14).k13 A578S African mutation and K189T  has also been identified at 100% frequency (n = 38) in Suriname   at a high frequency post-ACT introduction suggests that the NYD haplotype is likely to have risen in frequency due to the absence of CQ, restoring the wild-type parasite population, while the increase in the NFD haplotype frequency may be attributable to artemether-lumefantrine (AL) pressure  parasites exceeds 37% , outside the region we genotyped, has also been associated with artemisinin resistance on the Thai-Myanmar border . Like aWe observed 2-fold increases or decreases in the frequencies of alleles and haplotypes of several genes, followed by a shift back to the original frequency in the durations spanning 2012/2013, 2015/2016, and through to 2017/2018. This could be due to the abrupt change in drug policy from SP to ACTs and hence the initial selection of long haplotypes followed by recombination breaking down the haplotype structure as the parasite adapts to changes in drug pressure. The lack of spatial variation in allele frequency is consistent with previous reports at this geographical scale ; howeverdhps A581G locus, since SP has proved useful in IPTp, significantly reducing morbidity in pregnant women (nfs), which potentially confers resistance to LM, contrary to the observations made in The Gambia, calls for additional studies to determine its role as a potential drug target. The artemisinin resistance-conferring SE Asian mutations in k13, such as C580Y, have not been identified in Kilifi, and many of the SNPs occurred in the N-terminal region of the gene with no evidence of drug selection. Consequently, due to a lack of the validated molecular markers of artemisinin and lumefantrine resistance, there appears to be no problem of resistance in the population; however, continued surveillance remains a requirement.Following the introduction of ACTs in 2004, there has been a rapid increase in the CQ-sensitive population to near fixation, and this reignites the debate on the use of CQ for malaria treatment, such as in combination therapy. On the other hand, there is still a need for careful monitoring of the nt women . The decWe used parasite DNA extracted from frozen blood obtained in 1995/1996, 1999/2000, 2006/2007, and 2012/2013 from a previous study  and fromP. falciparum genomic DNA was extracted from frozen blood using the Qiagen DNA blood minikit , as per the manufacturer’s instructions. Amplicons were generated from the following genes using primers from previous studies and primers designed in this study (Table S1): crt (PF3D7_0709000), mdr1 (PF3D7_0523000), dhps (PF3D7_0810800), nfs (PF3D7_0727200), k13 (PF3D7_1347700), ap2mu (PF3D7_1218300), falcipain-2a (PF3D7_1347700), ubp-1 (PF3D7_0104300), and serine-tRNA ligase, putative gene (PF3D7_0717700). Additionally, four artemisinin resistance-predisposing mutations in arps10 (PF3D7_1460900), crt (PF3D7_0709000), fd (PF3D7_1318100), and mdr2 (PF3D7_1447900) were genotyped. Given that these mutations in these genes have been identified at low frequencies (<1%) in Africa , and SNPs were identified and called based on the respective 3D7 reference sequences. Nucleotide positions which displayed a peak within a peak in the sequence chromatograms were noted as “mixed.” Consensus sequences were extracted from the sequence assemblies using CLC Genomics Workbench v9.5.3 and used to construct multiple-sequence alignments in Clustal Omega v1.2.1 . SNP frecrt and mdr1 from the 1995 to 2013 time points were obtained from a previously published study  by the number of synonymous substitutions per synonymous site over time.Nucleotide sequences were translated into amino acid sequences in AliView v1.26 . Haplotyed study . The dif2 v3.1.1  and ggpu2 v3.1.1  in R v3.MN373285 to MN373771 (ap2-mu), MN373772 to MN374234 (dhps), MN374235 to MN374817 , MN374818 to MN375065 (fd), MN375066 to MN375759 (k13), MN375760 to MN376049 (mdr1), MN376050 to MN376651 (mdr2), MN376652 to MN377106 (nfs), MN377107 to MN377726 (serine tRNA ligase gene), and MN377727 to MN378298 (ubp1). However, sequences for arps10 and crt could not be deposited, as GenBank does not accept sequences under 200 bp.The nucleotide sequence data reported in this paper are available in the GenBank database under the accession numbers


Bacille Calmette-Guerin, BCG) and various intramacrophagic bacteria, fungi, parasites. Mycobacterial disease generally begins in childhood, more rarely during adolescence and adulthood. The pathogenesis of MSMD is the inherited impaired production of interferon gamma (IFN-γ) or inadequate response to it. Autosomal recessive IL12RB1 deficiency is the most common genetic etiology of MSMD. Here we identified three novel compound heterozygous mutations in IL12RB1 gene  in three Chinese families with MSMD.Mendelian Susceptibility to Mycobacterial Diseases (MSMD) is a primary immunodeficiency disease (PID) characterized by variable susceptibility to weakly virulent mycobacteria ( IFNGR1 [IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1, ISG15, IRF8, RORC and TYK2 and two X-linked  genes have been identified [Mendelian susceptibility to mycobacterial disease (MSMD) is a rare inherited disease characterized by selective vulnerability to infections by weakly virulent mycobacteria  and other intramacrophagic bacteria , fungi  and parasites  . MSMD moIFNGR1 . Up to nentified , 4. IFN-entified . Most ofentified .IL12RB1 mutations do not respond to IL-12 and produce low levels of IFN-γ [IL12RB1 gene in three MSMD patients.Autosomal recessive IL-12Rβ1 deficiency is the most common genetic etiology of MSMD , 8. IL-1of IFN-γ . In thisThree unrelated Chinese MSMD patients from Wuhan jinyintan hospital were included in this study. The project was approved by the ethics committee of Huazhong University of Science and Technology. All participants in the study agreed with informed consent to participate in the investigation.Patient 1 is a girl who received BCG vaccine when she was 4 months old, then a lump of finger size began to appear under her left armpit. When they came to the Wuhan jinyintan hospital, the lump had appeared for nearly four months. Blood routine examination displayed the number of leukocytes were increased. The patient was given debridement and treatment with anti-inflammatory drug of cephalosporins as well as isoniazid (INH). Patient 2 is a girl who received BCG vaccine when she was 3 months old. After one month, her parents found a lump about 3.0× 2.0cm in her left armpit. The patient was treated with anti-infection and external application of Chinese Medicine. Patient 3 is a boy, who received BCG vaccine, a big lump in the left armpit were found by his parents when he was 8 months old. The lump’s size like a pigeon egg. Puncture showed tuberculosis in lymph nodes and acid-fast stain is positive in these three patients, the three patients also received other test and were diagnosed with Mendelian susceptibility to mycobacterial disease (MSMD) in Wuhan Jingyintan hospital. Pedigree of the three families were shown in IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1, ISG15, IRF8, RORC, NEMO, CYBB and TYK2. DNA sequencing analysis was performed using the BigDye Terminator Cycle Sequencing v3.1 kit and an ABI PRISM 3500 Genetic Analyzer .Peripheral blood was obtained from all patients and their parents after obtaining informed consent. Genomic DNA was extracted from blood samples using standard methods. All exons and intron–exon boundaries of MSMD disease genes were sequenced: IL12RB1 cDNA were synthesized in The Beijing Genomics Institute (BGI). Mut Express II Fast Mutagenesis Kit V2 of Vazyme Biotech Co.,Ltd were used to create expression vectors for pEGFP-C1-IL12RB1 wild-type (WT), A22T, R212Q, 765delG, R211P, R283X fusion proteins. Mutations were confirmed by sanger DNA sequencing.The human Hela cells were plated on chamber slide flaskettes  and were transiently transfected with expression vectors as described above. Twenty-four hours later cells were fixed in 4% formaldehyde in phosphate-buffered saline (PBS), pH 7.4. The nucleus of cells were stained with DAPI and then visualized by confocal microscopy (×100 oil immersion lens).HEK293T cells were plated onto 6-well plate and transfected with wild type or mutant IL-12Rβ1 expression vectors using Lipofectamine 2000 (Invitrogen). Cells were used for experiments 48h after transfection.Western blot analysis of whole cell lysate were performed to check the expression level of wild and mutant IL-12Rβ1 protein. Total proteins were separated by 10% SDS/PAGE gels, and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with GFP and GAPDH antibodies. The signals were detected with Bio-RAD ChemiDoc XRS+ imaging system. .IL12RB1 using Lipofectamine 2000 (Invitrogen). 48h later after transfection, HEK293T cells were lysed and membrane proteins were extracted with biotin labeling.HEK293T cells were cultured on 10cm cell culture dish one day and transfected with pEGFP-C1, pEGFP-C1-WT and pEGFP-C1-A22T, R211P, R212Q Cells of each tissue culture dishes were incubated with 1 mg/ml Sulfo-NHS-SS-Biotin  in PBS(2ml) for 30 min. Excess biotin was quenched using 200 mM glycine for 2 min. Then, cells were collected and lysed with cold lysis buffer (50 mM Tris-HCl (pH 7.5), 1% NP-40 substitute (Sigma), 150 mM NaCl, 2 mM EDTA, protease inhibitor) in ice for 40 min, vortexed every five minutes. Then, the mixture were centrifuged at 10,000g and membrane proteins stayed in the supernatant. Finally, the supernatant were incubated with Pierce NeutrAvidin Agarose (Thermo Fisher) at room temperature for one hour and membrane proteins were obtained after the Pierce NeutrAvidin Agarose were incubated with Sample Buffer (62.5 mM Tris-HCl (pH 6.8), 1% SDS, 10% glycerol, 50mM DTT) for three hours.+/K+- ATPase and GFP antibodies. The signals were detected with Bio-RAD ChemiDoc XRS+ imaging system. .Membrane proteins were separated by 10% SDS/PAGE gels, and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with NaIL12RB1 gene in Patient 1 carry two heterozygous mutations, a missense mutations  and a deletion mutation (c.765delG)  . The misIL12RB1 gene, c.632G > C (R211P) and c.847C > T (R283X)  and her mother carry the c.847C > T (R283X) mutation. The patient’s parents have no symptoms of MSMD.DNA sequence analysis identified a novel compound heterozygous mutation in patient 2, a missense mutation and a nonsense mutation in the  (R283X) . Both ofIL12RB1 gene, we identified the patient carried a compound heterozygous mutation, c.64G > A (A22T), c.1673insGAGCTTCCTGAG  to observed the localization of wild and mutant IL-12Rβ1 protein in the cell. The localization of the proteins was visualized by confocal microscopy  respectively. Missense mutations  lead to reduce the IL12RB1 expression level in different degree, while nonsense mutation (R283X) and frameshift mutation(765delG) result in degradation of IL-12Rβ1 protein  lead to reduce the expression of IL-12Rβ1 in the cell membrane .BCG vaccine is the most commonly used to newborns. It is considered to be safe, but some complications have been reported like cellulitis, abscesses at the site of inoculation, regional lymphadenitis (BCGitis) and disseminated BCG infection (BCG-osis) . FuthermIL12RB1[IFNGR1[Since first disease gene of MSMD was reported in 1996 , mutatioIL12RB1 and IFNGB1[IFNGR1. Few relIL12RB1 gene  in three unrelated patients with MSMD. One of the heterozygous mutation c.632G > C has been discovered in a Taiwanese patient (with MSMD) who carries the homozygous mutation [BCG, we speculate that the loss of function mutations which we identified will also will affected the IL12 signaling pathway and IFN-γ production. Our study not only enriches the IL12RB1 mutation database, but also provides patients with a more accurate diagnosis as well as precise treatment choices of Chinese MSMD patients.In this study, we identified three novel compound heterozygous mutations in mutation  and hetemutation . Here, w


To compare vitamin D status in women with PCOS versus fertile women and subsequently evaluate the association between vitamin D status and metabolic disturbances in PCOS women.We conducted a cross-sectional comparison study of 639 women with PCOS and 449 fertile women. Serum 25-hydroxyvitamin D (25(OH)D) was stratified into a severe deficient (< 25 nmol/l), insufficient (25–50 nmol/l), moderate (50–75 nmol/l) and adequate (> 75 nmol/l) status. The main outcome measures were the difference in vitamin D status between PCOS and fertile women, and the association between serum 25(OH)D and metabolic disturbances in PCOS women only.versus 64.5 nmol/l). An adjusted significant difference was seen between serum 25(OH)D and homeostasis model assessment (HOMA-IR) , HDL-cholesterol  and apolipoprotein A1  between the highest vitamin D group compared to the lowest vitamin D group.Serum 25(OH)D was significantly lower in PCOS women compared to fertile controls (mean 25(OH)D of 49.0 nmol/l This study demonstrates that women with PCOS have a significantly lower serum 25(OH)D compared to fertile controls. A compromised vitamin D status in PCOS women is associated with a higher HOMA-IR and an unfavourable lipid profile. Large randomized controlled trials are necessary to explore the causality of this linkage. Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women of reproductive age, with a prevalence up to 10% depending on which diagnostic criteria are used . Metabol2D), the active metabolite of vitamin D. Circulating 1,25(OH)2D binds to vitamin D receptors (VDR) to initiate its effect. Serum 25(OH)D is the major circulating form of vitamin D and is used as the main indicator of vitamin D status. Its half-life is 2–3 weeks compared to only 4–6 hours for 1,25(OH)2D ]/22.5). Metabolic syndrome was defined according the National Cholesterol Education Program (NCEP) ADP III criteria .Venous blood samples were drawn at examination and stored at −80°C after centrifugation at 3000 rpm for 10 min at 20°C. Serum 25(OH)D was measured using the liquid chromatography tandem-mass spectrometry (LC-MS/MS) in November 2013 (PCOS women) and September 2015 (control women). Liquid-liquid extraction (hexane) of the samples and analysis, carried out with a Waters ACQUITY UPLC system couple to a Waters Xevo TQ mass spectrometer, were performed as described elsewhere . Intra-a2), testosterone, dehydroepiandrosterone sulfate (DHEAS), fasting glucose and insulin. Hormone assays have been described in detail elsewhere [2 and DHEAS were determined by radioimmunometric assays (RIAs). Intra-assay and inter-assay CV were less than 3% and less than 5% for testosterone and less than 5% and less than 7% for E2 respectively.Endocrine evaluation included serum levels of gonadotropic hormones  and estradiol D as dependent variable and PCOS versus control as independent variable. We adjusted for BMI, age, blood pressure, season of vitamin D measurement, and ethnicity as potential confounders or effect modifiers. Serum 25(OH)D was log transformed due to a non-gaussian distribution. The exponentiated beta-coefficients are reported as results of the regression analyses.Sub-analyses were performed to assess the association between vitamin D status and metabolic outcome parameters in women with PCOS, by applying multivariable linear regression analyses. As the relationship between serum 25(OH)D and the outcome measures was not linear, serum 25(OH)D was divided in four groups by the widely used cut-off values of vitamin D: 1) serum 25(OH)D: < 25 nmol/l, 2) serum 25(OH)D: 25–50 nmol/l, 3) serum 25(OH)D: 51–75 nmol/l, and 4) serum 25(OH)D: > 75 nmol/l . The vit2) and 24.0 (22.0–27.0 kg/m2) in PCOS and control women, respectively. Overall median serum 25(OH)D was 49.0 (27.1–74.1 nmol/l) in PCOS versus 64.5 (39.2–85.7) in the control women. A severe vitamin D deficiency was present in 136 (21%) women out of 639 PCOS women, 190 (30%) women had a serum 25(OH)D between 25.1 and 50.0 nmol/l, 165 (26%) women had a serum 25(OH)D between 50.1 and 75.0 nmol/l, and 148 (23%) women had a serum 25(OH)D > 75 nmol/l. In the control group 49 (11%) women had a serum 25(OH)D ≤ 25 nmol/l, 106 (24%) women between 25.1 and 50.0 nmol/l, 131 (29%) women between 50.1 and 75.0 nmol/l, and 163 (36%) women had a serum 25(OH)D > 75 nmol/l. Linear regression analysis showed a lower serum 25(OH)D for the PCOS women compared to control women   . CorrectDemographic, anthropometric and clinical characteristics of all PCOS women, stratified by vitamin D group are presented in 2). Overweight/obese women (BMI > 25 kg/m2) had a mean serum 25(OH)D 46.3 ± 27.9 nmol/l, which was significantly lower compared to women with a BMI < 25: serum 25(OH)D 59.7 ± 30.0 nmol/l (p < 0.01). A total of 94 (15%) women fulfilled the NCEP APT III criteria for metabolic syndrome, who had a significant lower serum 25(OH)D than women without metabolic syndrome (mean serum 25(OH)D 44.3 ± 27.4 nmol/l and 54.2 ± 29.9 nmol/l (p < 0.01), respectively).Median BMI of all PCOS women included was 25.2  /HDL ratio was 3.9 ± 1.4 mmol/L in our study population. The ratio differed significantly between the vitamin D groups with a difference of -0.79 mmol/L between the lowest and highest vitamin D group (p < 0.01). After correcting for confounders, the association did not remain significant between the lowest and highest vitamin D groups . A significant difference was seen in HDL-cholesterol between the lowest and highest vitamin D group after correction for confounders . Apolipoprotein A1 was significantly higher in the highest vitamin D group compared to the lowest group after correction for confounders . A small significant difference was seen in serum total cholesterol demonstrating higher serum total cholesterol in the highest vitamin D group compared to the lowest vitamin D group . No difference between the vitamin D groups was found in serum triglycerides, LDL-cholesterol, and apolipoprotein B .The aim of this study was 1) to explore vitamin D status in women with PCOS compared to fertile controls and 2) to evaluate the associations between vitamin D status and metabolic disturbances in PCOS women. In this study the lower vitamin D status in women with PCOS compared to fertile controls was confirmed . Low serThe strength of our study is that we have studied the largest cohort of PCOS women and compared our findings with a comparison group without PCOS, which allows a well-powered comparison. Second, all samples were measured using the LC-MS/MS method, which is the golden standard for measurement of serum 25(OH)D .A disadvantage of our study is that metabolic measurements were not available in the fertile control women. Although the control women were enrolled from the general population and not selected on fertility, it is not likely that a substantial part of the control women had PCOS due to the fact that they all had a spontaneous pregnancy and regular menstrual cycles before pregnancy. The blood collection of the control women was carried out 15 to 17 months after delivery to exclude the effect of pregnancy on vitamin D metabolism. Another limitation of our study could be that the comparison group was not originally created as a control group for this study and was matched retrospectively, resulting in a small difference in age and BMI between both groups. Taken this into account we labelled the study not as a case-control study, but as a comparison study. However, this was the best control group available in the same region and age category, of which we were certain all women had a spontaneous pregnancy which makes the chance of PCOS and thus confounding in this group very low.Although serum 25(OH)D is stable over time, theoretically long-time storage of serum samples at -80 ⁰C could have influenced the vitamin D concentration . HoweverAnother important point is the possibility of residual confounding which cannot be ruled out, despite correction for several confounders. Last limitation could be that we have used a surrogate marker of insulin resistance (HOMA-IR) and not the golden standard to measure insulin resistance, i.e. insulin clamp technique.Several epidemiologic studies have explored the effect between vitamin D status and metabolic disturbances in PCOS women and controls. These studies are summarized in an earlier systematic review , in whicThe finding of a higher prevalence of vitamin D defiency in PCOS women compared to (fertile) controls is in line with earlier reports, which observed a percentage of vitamin D deficiency (defined as serum 25(OH)D < 50nmol/l) of 67–85% among women with PCOS .The mean vitamin D level in control women of our study was comparable with the overall mean vitamin D level that was reported in control women in the earlier performed systematic review . HoweverTo date, there is still a debate what the optimal serum 25(OH)D level is. The Institute of Medicine  has dete2D. Furthermore, the direct effect of vitamin D on insulin secretion is supported by the presence of the vitamin D-response element in the human insulin promoter gene [Vitamin D is thought to influence the development of PCOS through gene transcription and influences metabolism . The effter gene . Vitaminter gene . Finally2 directly leads to the production of estrogen and progesterone in cultured human ovary and placental cells [Next to the linkage of vitamin D to the pathogenesis of PCOS, vitamin D may also effect fertility outcomes in PCOS . Vitaminal cells ,43. Moreal cells ,45. In tal cells . An undeal cells .The association found between vitamin D and dyslipidemia (i.e. HDL-cholesterol and apolipoprotein A1) had previously been described . Low HDLApart from these cross-sectional findings, several intervention trials examined the effects of vitamin D supplementation on metabolic parameters and clinical parameters including fertility in PCOS women. These studies differed in study design, population, dosing regimens, as well as study outcomes ,49. A siThis could be due to the inconsistencies in study design, included PCOS populations and duration of follow up. The largest study with a RCT design among 104 obese, vitamin D deficient PCOS women did reveal a positive effect of weekly 50.000 IU vitamin D plus calcium 1000mg/day on insulin resistance [In conclusion, this is the largest epidemiologic study showing a significant association between vitamin D status and metabolic disturbances in patients with PCOS. Moreover, PCOS women had a significant lower serum 25(OH)D compared to fertile controls. For future research large randomized controlled clinical trials are necessary to explore whether this association has a causal linkage.S1 Data(SAV)Click here for additional data file.