The Chongming white goat (CM) is an indigenous goat breed exhibits unique traits that are adapted to the local environment and artificial selection. By performing whole-genome re-sequencing, we generated 14-20× coverage sequences from 10 domestic goat breeds to explore the genomic characteristics and selection signatures of the CM breed. We identified a total of 23,508,551 single-nucleotide polymorphisms (SNPs) and 2,830,800 insertion-deletion mutations (indels) after read mapping and variant calling. We further specifically identified 1.2% SNPs (271,713) and 0.9% indels (24,843) unique to the CM breed in comparison with the other nine goat breeds. Missense (SIFT < 0.05), frameshift, splice-site, start-loss, stop-loss, and stop-gain variants were identified in 183 protein-coding genes of the CM breed. Of the 183, 36 genes, including AP4E1, FSHR, COL11A2, and DYSF, are involved in phenotype ontology terms related to the nervous system, short stature, and skeletal muscle morphology. Moreover, based on genome-wide F _ST and pooled heterozygosity (Hp) calculation, we further identified selection signature genes between the CM and the other nine goat breeds. These genes are significantly associated with the nervous system (C2CD3, DNAJB13, UCP2, ZMYND11, CEP126, SCAPER, and TSHR), growth (UCP2, UCP3, TSHR, FGFR1, ERLIN2, and ZNF703), and coat color (KITLG, ASIP, AHCY, RALY, and MC1R). Our results suggest that the CM breed may be differentiated from other goat breeds in terms of nervous system owing to natural or artificial selection. The whole-genome analysis provides an improved understanding of genetic diversity and trait exploration for this indigenous goat breed.

Keywords: Capra hircus; chongming white goat; genomic characteristics; selection signature; whole-genome re-sequencing.

"Humanized" immunodeficient mice generated via the transplantation of CD34+ human hematopoietic stem cells (hHSC) are an important preclinical model system. The triple transgenic NOD.Cg-Prkdc^scidIl2rg^tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSGS) mouse line is increasingly used as recipient for CD34+ hHSC engraftment. NSGS mice combine the features of the highly immunodeficient NSG mice with transgenic expression of the human myeloid stimulatory cytokines GM-CSF, IL-3, and Kit ligand. While generating humanized NSGS (huNSGS) mice from two independent cohorts, we encountered a fatal macrophage activation syndrome (MAS)-like phenotype resulting from the transplantation of CD34+ hHSC. huNSGS mice exhibiting this phenotype declined clinically starting at approximately 10 weeks following CD34+ hHSC engraftment, with all mice requiring euthanasia by 16 weeks. Gross changes comprised small, irregular liver, splenomegaly, cardiomegaly, and generalized pallor. Hematological abnormalities included severe thrombocytopenia and anemia. Pathologically, huNSGS spontaneously developed a disseminated histiocytosis with infiltrates of activated macrophages and hemophagocytosis predominantly affecting the liver, spleen, bone marrow, and pancreas. The infiltrates were chimeric with a mixture of human and mouse macrophages. Immunohistochemistry suggested activation of the inflammasome in both human and murine macrophages. Active Epstein-Barr virus infection was not a feature. Although the affected mice exhibited robust chimerism of the spleen and bone marrow, the phenotype often developed in the face of low chimerism of the peripheral blood. Given the high penetrance and early lethality associated with the MAS-like phenotype here described, we urge caution when considering the use of huNSGS mice for the development of long-term studies.

Keywords: Chimeric mouse; HLH; Humanized model; Inflammasome; Macrophage activation syndrome; NSG-SGM3; NSGS; PDX model.

BACKGROUND

Approximately 95% of all testicular cancers are testicular germ cell tumors (GCTTs), represented by seminoma and nonseminoma germ cell testicular cancer. There is a hypothesis that the formation of GCTTs begins in early embryogenesis being a part of testicular dysgenesis syndrome (TDS).

OBJECTIVE

To determine the role of genetic factors in the development of GCTTs.

METHODS

We studied the frequency of alleles and genotypes KITLG (rs995030, rs1508595), SPRY4 (rs4624820, rs6897876) and BAK1 (rs210138) in 97 fertile men (control), and 73 patients with GCTTs (34 seminoma and 39 nonseminoma).

RESULTS

GCTTs were statistically significantly associated with KITLG rs1508595 gene (p=0.0003 for allele G, p=0.0014 for genotype GG), and with rs995030 gene (p=0.0031 for genotype GG). When comparing patients with seminoma and control group, statistically significant differences were found for SPRY4 rs4624820 (p=0.0226 for the A and p=0.04 for the AA), for KITLG rs995030 (p=0.0375 for the G and p=0.0282 for GG), rs1508595 (p=0.0306 for G), for BAK1 rs210138 (p=0.0329 for the G and p=0.0219 for the GG). When comparing patients with nonseminoma and fertile men, statistically significant differences were found only for KITLG rs1508595 (p=0.0005 for the G and p=0.0021 for the GG). There was no statistically significant difference between the allele and genotype frequencies of the investigated genes from seminoma and nonseminoma GCTTs patients. However, these groups differed statistically significantly when genotype combinations of the three genes were investigated (p=0,029; OR 3,709 [1.147-11.99]). The combination of genotypes of the three genes was found to increase the risk of GCTTs by 6.5 times (p=0.0005; OR 6.526 [2.078-20.5], and the risk for seminoma was over 12-fold (p<0.0001; OR 12,68 [3,731-43,11].

CONCLUSIONS

A comprehensive study of genotypes associated with GCTTs in patients with manifested TDS can be used for risk stratification to identify and follow-up high-risk patients, develop approaches to family counseling and treatment, which is the basis for predictive medicine.

Pigs are one of the earliest domesticated animals and multiple breeds have been developed to meet the various demands of consumers. EigenGWAS is a novel strategy to identify candidate genes that underlying population genetic differences and to infer candidate regions under selection as well. In this study, EigenGWAS and F_st analyses were performed using the public re-sequencing data of three typical commercial pig breeds, Duroc, Landrace and Yorkshire. The intersection of genome-wide significant SNPs detected by EigenGWAS and top-ranked 1% SNPs of F_st results were treated as signals under selection. Using the data of all three breeds, 3062 signals under selection were detected and the nearby genomic regions within 300 kb upstream and downstream covered 6.54% of whole genome. Pairs of breeds were analysed along with the pathway analysis. The gene function enrichment results indicated that many candidate genes located in the genomic regions of the signals under selection were associated with biological processes related to growth, metabolism, reproduction, sensory perception, etc. Among the candidate genes, the FSHB, AHR, PTHLH, KDR and FST genes were reported to be associated with reproductive performance; the KIT, KITLG, MITF, MC1R and EDNRB genes were previously identified to affect coat colour; the RETREG1, TXNIP, BMP5, PPARD and RBP4 genes were reported to be associated with lipid metabolism and growth traits. The identified genetic differences across the three commercial breeds will advance understanding of the artificial selection history of pigs and the signals under selection will suggest potential uses in pig genomic breeding programmes.

Objective: To investigate the expression of PRDM1 and its relationship with PI3K/AKT pathway activation in extranodal NK/T cell lymphoma-nasal type. Methods: Immunocytochemistry and Western blot were used to detect the expression of PRDM1 and p-AKT in 10 EN-NK/T-NT tissue or 3 cell lines (PRDM1-positive YT cell line, PRDM1-negative NKL and NK92 cell lines). Nanostring gene expression profiling technique was used to detect the activation of the PI3K/AKT pathway in normal nasal mucosa, PRDM1-negative and positive EN-NK/T-NT tissue. MTS was used to detect cell proliferation, and flow cytometry was used to detect cell cycle and apoptosis. Results: Nanostring gene expression profiling revealed that genes associated with PI3K/AKT signaling pathway (eg, IL-7, BRCA1, ITGA8, IL2RB, FASLG, CDK2, COL27A1, CSF3R, KITLG and IL-6) were highly expressed in EN-NK/T-NT cases (P<0.05). Also, we found that p-AKT was highly expressed in YT cell line, but lower or not expressed in NK92 and NKL cells. In addition, LY294002, a PI3K/AKT pathway inhibitor, increased PRDM1 and PTEN expression in a dose dependent manner in YT cells. More importantly, YT cell were treated with 20 μmol/L LY294002 48 h, the proliferation rate was significantly decreasing (58.18% vs 100.00%, t=12.770, P=0.006), and the proportion of cells in G(1) phase was significantly increased (30.05% vs 76.93%, t=11.570, P<0.001). However, there was no significant difference in cell proliferation and cell cycle between NKL cells and control group (P>0.05). Conclusion: The activation of PI3K/AKT pathway is positive associated with the expression of PRDM1 in EN-NK/T-NT, and inhibition of PI3K/AKT pathway may have significant therapeutic potential for PRDM1-positive EN-NK/T-NT.

Adverse childhood experiences such as maltreatment or neglect are associated with mental health problems in adulthood. Changes in the regulation of the psychological and physiological stress reaction, mediated via epigenetic modifications, are discussed as potential mechanisms. This study aimed to replicate the role of DNA methylation of the KITLG gene in mediating the association between childhood adversity and stress-induced cortisol reactivity in a sample of adults reporting childhood adversity and a matched control group (N = 60). DNA was extracted from purified CD14⁺ monocytes and genome-wide DNA methylation was assessed with the 450k BeadChip for targeted replication and exploratory analyses. As previously reported, childhood adversity was associated with significantly lower cortisol reactivity to stress. We could neither replicate the association between KITLG DNA methylation and cortisol stress reactivity nor the association with childhood adversity. Moreover, DNA methylation of the target CpG (cg27512205) was not associated with KITLG mRNA expression in monocytes. Exploratory analyses of array-wide DNA methylation patterns showed no significant results for individual sites after correction for multiple testing - neither in association with childhood trauma nor with adult cortisol stress reactivity. The analysis of differentially methylated regions (DMRs) revealed two significant regions which both mapped to non-coding genes in the association with cortisol stress reactivity. The mediating role of DNA methylation of the KITLG locus in the association between childhood adversity and cortisol stress reactivity could not be replicated in monocytes. In addition to differences in investigated tissue, reasons for non-replication might include differences between samples in age, ethnicity, trauma severity, and cortisol reactivity.

BACKGROUND

Familial progressive hyper- and hypopigmentation (FPHH) is an autosomal dominant skin condition presenting in childhood with generalized macular dyspigmentation, usually reported in patients of East Asian origin. It overlaps phenotypically with other dyschromatoses, but can now be distinguished by mutations in the KIT ligand gene (KITLG).

OBJECTIVE

We report two unrelated white families with similar phenotypic presentations of FPHH developing in early childhood in several generations.

METHODS

Sanger sequencing of the exons and flanking introns of KITLG was performed.

RESULTS

This identified a new heterozygous missense mutation in each family (p.Thr34Asn and p.Val37Gly, respectively). Of the six affected individuals examined by us, two had cancer: a 62-year-old man in family 1 had developed two primary melanomas and a pharyngeal carcinoma, and a 42-year-old woman in family 2 had developed thyroid carcinoma. All had unusually sparse lateral eyebrows, a finding not previously reported in this condition.

CONCLUSIONS

We summarize the genetic spectrum of the dyschromatoses and discuss a possible increased risk of malignancy in FPHH.

Being a hydroxylated metabolite of aflatoxin B1 (AFB1) and the most threatening aspect of AFB1 contamination, aflatoxin M1 (AFM1) can lead to hepatotoxicity and hepato-carcinogenicity, and possess intestinal cytotoxicity. However, little is known about the potential mechanisms of the extrahepatic effect. The aim of this study was to investigate intestinal dysfunction induced by AFM1 via transcriptome analysis. Gene expression profiling was analyzed to comparatively characterize the differentially expressed genes (DEGs) after differentiated Caco-2 cells were exposed to different concentrations of AFM1 for 48 h. A total of 165 DEGs were significantly clustered into two down-regulated patterns. Protein-protein interaction (PPI) network analysis based on Search Tool for Retrieval of Interacting Genes (STRING)suggested that 23 key enzymes mainly participated in the regulation of the cell cycle. Q-PCR analysis was performed to validate that key 12 genes (BUB1, BUB1B, MAD2L1, CCNA2, RB1, CDK1, ANAPC4, ATM, KITLG, PRKAA2, SIRT1, and SOS1) were involved. This study firstly revealed that the toxicity of AFM1 to intestinal functions may be partly due to the occurrence of cell cycle arrest, which is linked to changes in CDK1, SOS1/Akt, and AMPK signaling molecules.

Receptor tyrosine kinase (RTK) encoded by proto-oncogene KIT is known to be involved in different types of cancers. Reportedly, KIT expression has been associated with higher grade of gliomas. Initial RT-PCR based KIT expression observed in low grade glioma cases evoked our interest to ascertain its status in glioma patients who underwent resection during 2008-2009. Contrary to earlier reports, over-expression of the RTK was observed in 32.5% glioma cases across low/high grades (n=40). Using quantitative PCR (qPCR), an up-regulation of the receptor (KIT) and its ligand (KITLG) was detected in most of the immunopositive cases at the transcript level. Sequence analysis of KIT showed two nucleotide substitutions in exons 10 and 17, in 4 and 2 cases, respectively though their pathological significance remained unclear. qPCR detected gene amplification in 2/13 glioma and allele loss in 1/13 glioma cases. This was in accordance with FISH results of these KIT positive neoplastic tissues. The data suggest that deranged expression of KIT is independent of gene amplification (p>0.05). Aberrant KIT expression is significantly associated with transcriptional up-regulation (p<0.001), though the precise mechanism(s) for transcriptional activation remain unclear.

In a previous project, we screened the American mink Bacterial Artificial Chromosome library, CHORI-231, for genes potentially involved in various coat colour phenotypes in the American mink. Subsequently, we 454 sequenced the inserts containing these genes and developed microsatellite markers for each of these genes. Here, we describe a lack of association between three different 'roan-type' phenotypes represented by Cross, Stardust and Cinnamon in American mink and six different genes that we considered to be potentially linked to these phenotypes. Thus, c-KIT (HUGO-approved symbol KIT), ATOH-1 (HUGO-approved symbol ATOH1) and POMC were excluded as potential candidates for these three phenotypes. In addition, MITF and SLC24A5 were excluded for Cross and Cinnamon, and KITL (HUGO-approved symbol KITLG) for Cross and Stardust. Although most of these genes have been implicated as the cause of similar phenotypes in other mammals, including horses, pigs, cows, dogs, cats, mice and humans, they do not appear to be responsible for comparable phenotypes found in American mink.

Testicular cancer is the most common form of solid cancer in young men. Testicular cancer is represented by testicular germ cell tumors (TGCTs) derived from embryonic stem cells with different degrees of differentiation in about 95% of cases. The development of these tumors is related to the formation of a pool of male germ cells and gametogenesis. Clinical factors that are predisposed to the development of germ-cell tumors include cryptorchidism and testicular microlithiasis, as well as infertility associated with the gr/gr deletion within the AZFс locus. KITLG, SPRY4, and BAK1 genes affect the development of the testes and gametogenesis; mutations and polymorphisms of these genes lead to a significant increase in the risk of the TGCT development. To determine the relationship between gene polymorphisms and the development of TGCTs, we developed a system for detection and studied the allele and genotype frequencies of the KITLG (rs995030, rs1508595), SPRY4 (rs4624820, rs6897876), and BAK1 (rs210138) genes in fertile men, patients with TGCTs, and patients with infertility that have the AZFс deletion. A significant association of rs995030 of the KITLG gene with the development of TGCTs (p = 0.029 for the allele G, p = 0.0124 for the genotype GG) was revealed. Significant differences in the frequencies of the studied polymorphisms in patients with the AZFc deletion and the control group of fertile men were not found. We showed significant differences in the frequencies for the combination of all high-risk polymorphisms in the control group, patients with the AZFc deletion and patients with TGCTs (p (TGCTs-AZF-control) = 0.0207). A fivefold increase in the frequency of the combination of all genotypes in the TGCT group (p = 0.0116; OR = 5.25 [1.44-19.15]) and 3.7-fold increase was identified in patients with the AZFc deletion (p = 0.045; OR = 3.69 [1.11-12.29]) were revealed. The genotyping of patients with infertility caused by the AZFc deletion can be used to identify individuals with an increased risk of TGCTs.

Varicocele (VC) is the most common treatable cause of infertility, but it is difficult to distinguish fertile from infertile VC populations because the pathogenesis is unclear. In order to study the related mechanism of VC causing male sterility, we made VC rats model by surgery, analyzed the rat epididymal sperm, and use the transcriptome sequencing compared all the miRNA expression differences in testicular tissue between VC rats, surgical treatment rats and control rats. The differentially expressed miRNAs (DEMs) of testicular tissue were also screened by the edgeR package in R software. We found that rno-miR-210-3p, rno-miR-6316, rno-miR-190a-5p and rno-miR-135b-5p were key miRNAs for VC and they were all up-regulated in VC samples and they are enriched in regulation of immune system process (GO:0002683), innate immune system (R-RNO-168249) and apoptotic signaling pathway (GO:0097190). We hypothesize that negative regulation of immune system and apoptosis play an important role in the occurrence and development of VC, and it is induced the abnormal expression of target genes (such as Kitlg, Cxcl12) may involve in the development of VC associated infertility. Four key miRNAs, rno-miR-210-3p, rno-miR-6316, rno-miR-190a-5p and rno-miR-135b-5p, as well as their target genes are critical in VC, which could have attractive applications to provide new biomarkers for VC.

Keywords: Sequencing analysis; Testicular; Varicocele; miRNA profile.

Nasopharyngeal carcinoma (NPC) is a heterogeneous carcinoma whose underlying molecular mechanisms involved in tumor initiation, progression, and migration are largely unclear. The aim of the present study was to identify key biomarkers and small-molecule drugs for screening, diagnosing, and treating NPC via gene expression profile analysis. Raw microarray data was used to identify 430 differentially expressed genes (DEGs) in the Gene Expression Omnibus (GEO) database. The key modules associated with histological grade and tumor stage were identified using weighted gene co-expression network analysis. qRT-PCR was used to verify the differential expression of hub genes. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and the connectivity map database were used to identify potential mechanisms and screen small-molecule drugs targeting hub genes. Functional enrichment analysis showed that genes in the green module were enriched in the regulation of cell cycle, p53 signaling pathway, and cell part morphogenesis. Four DEG-related hub genes (CRIP1, KITLG, MARK1, and PGAP1) in the green module, which were considered potential diagnostic biomarkers, were taken as the final hub genes. The expression levels of these four hub genes were verified via qRT-PCR, and the results were consistent with findings from the GEO analysis. Screening was also conducted to identify small-molecule drugs with potential therapeutic effects against NPC. In conclusion, four potential prognostic biomarkers and several candidate small-molecule drugs, which may provide new insights for NPC therapy, were identified.

Keywords: Nasopharyngeal carcinoma; WGCNA; hub genes; signature; small-molecule drugs.

As an important indicator of sexual maturity of hens, age at first egg (AFE) is significantly associated with reproduction performance. In this study, 400 hens were divided into 6 groups based on AFE to analyze the difference of reproduction performance, reproduction hormone levels and the characterization of the hypothalamo-pituitary-gonadal axis in chickens. The results showed that the egg production of the hens in the late-maturing groups was significantly lower than that of the ones in other groups and the precocious hens had a lower egg production rate. The hens in late-maturing group had a lower fertility rate, LH levels and shorter duration of peak of egg production (PEP), the precocious hens had lower PRL levels. In addition, the characterization of the hypothalamo-pituitary-gonadal axis showed that the individuals with normal AFE had higher GNRH, GNRHR, ESR1, KITLG, and CYP11A1 expression levels than late-maturing and precocious individuals, which indicated that the chickens with normal AFE advantages on reproduction regulation system.

Keywords: age at first egg; hens; mRNA expression; reproductive traits; sexual maturity.

OBJECTIVE

To detect the expressions of receptor tyrosine kinases (RTKs) mRNA and protein and to explore potentially promising tumor markers and conceivable drug target in bladder cancer.

METHODS

The expressions of RTKs mRNA and protein in tissue from invasive urothelial carcinoma of the bladder were examined by real-time quantitative PCR array and cytokine antibody array, with normal bladder tissue as control. The Results were analyzed using bioinformatic approaches.

RESULTS

The expressions of TGFA, STAB1, SERPINE1, ANGPT2, SPINK5, ANGPTL1, PROK1, MDK, CXCL9, GRN, RUNX1, VEGFA, and TGFB1 were obviously upregulated in bladder cancer tissue, while those of EDIL3, PTN, CCL2, PDGFD, FGF13, KITLG, FGF2, SERPINF1, and TNF were downregulated. ALK, Btk, EphB2, ErbB4, PDGFR-α, ROS, Tie-2, Tyk2, and VEGFR3 were over-expressed in bladder cancer, while FRK, Fyn, IGF-IR, Insulin R, Itk, JAK1, JAK3, and LCK were low-expressed.

CONCLUSIONS

Vascular endothelial growth factor/platelet-derived growth factor-targeted therapies may play an active role in treating carcinoma of bladder.

The expanding targeted therapy landscape requires combinatorial biomarkers for patient stratification and treatment selection. This requires simultaneous exploration of multiple genes of relevant networks to account for the complexity of mechanisms that govern drug sensitivity and predict clinical outcomes. We present the algorithm, Digital Display Precision Predictor (DDPP), aiming to identify transcriptomic predictors of treatment outcome. For example, 17 and 13 key genes were derived from the literature by their association with MTOR and angiogenesis pathways, respectively, and their expression in tumor versus normal tissues was associated with the progression-free survival (PFS) of patients treated with everolimus or axitinib (respectively) using DDPP. A specific eight-gene set best correlated with PFS in six patients treated with everolimus: AKT2, TSC1, FKB-12, TSC2, RPTOR, RHEB, PIK3CA, and PIK3CB (r = 0.99, p = 5.67E-05). A two-gene set best correlated with PFS in five patients treated with axitinib: KIT and KITLG (r = 0.99, p = 4.68E-04). Leave-one-out experiments demonstrated significant concordance between observed and DDPP-predicted PFS (r = 0.9, p = 0.015) for patients treated with everolimus. Notwithstanding the small cohort and pending further prospective validation, the prototype of DDPP offers the potential to transform patients' treatment selection with a tumor- and treatment-agnostic predictor of outcomes (duration of PFS).

Small-tailed Han sheep, with different FecB genotypes, manifest distinct ovulation rates and fecundities, which are due to differences in reproductive hormones secreted by the hypothalamic-pituitary-ovarian axis. Nevertheless, the function of the hypothalamus against a FecB mutant background on increasing ovulation rate is rarely reported. Therefore, we determined the expression profiles of hypothalamus tissue collected from six wild-type (WW) and six FecB mutant homozygous (BB) ewes at the follicular and luteal phases by whole-transcriptome sequencing. We identified 53 differentially expressed mRNAs (DEGs) and 40 differentially expressed long non-coding RNAs (DELs) between the two estrus states. Functional annotation analysis revealed that one of the DEGs, PRL, was particularly enriched in the hypothalamic function, hormone-related, and reproductive pathways. The lncRNA-target gene interaction networks and KEGG analysis in combination suggest that the lncRNAs LINC-676 and WNT3-AS cis-acting on DRD2 and WNT9B in different phases may induce gonadotropin-releasing hormone (GnRH) secretion. Furthermore, there were differences of regulatory elements and WNT gene family members involved in the follicular-luteal transition in the reproductive process between wild-type (WNT7A) and FecB mutant sheep (WNT9B). We combined the DEG and DEL data sets screened from different estrus states and genotypes. The overlap of these two sets was identified to select the mRNAs and lncRNAs that have major effects on ovulation. Among the overlapping molecules, seven DEGs and four DELs were involved in the follicular-luteal transition regulated by FecB mutation. Functional annotation analysis showed that two DEGs (FKBP5 and KITLG) were enriched in melanogenesis, oxytocin, and GnRH secretion. LINC-219386 and IGF2-AS were highly expressed in the BB ewes compared with WW ewes, modulating their target genes (DMXL2 and IGF2) to produce more GnRH during follicular development, which explains why mutated ewes produced more mature follicles. These results from expression profiling of the hypothalamus with the FecB mutation at different estrus states provide new insights into how the hypothalamus regulates ovulation under the effect of the FecB mutation.

Keywords: FecB; fecundity; follicular development; hypothalamus; lncRNA; sheep.

Body size is an important indicator of growth and health in sheep. In the present study, we performed Genome-Wide Association Studies (GWAS) to detect significant single-nucleotide polymorphisms (SNPs) associated with Hu sheep's body size. After genotyping parental (G1) and offspring (G2) generation of the nucleus herd for meat production of Hu sheep and conducting GWAS on the body height, chest circumference, body length, tail length, and tail width of the two groups, 5 SNPs associated with body height and 4 SNPs correlated with chest circumference were identified at the chromosomal significance level. No SNPs were significantly correlated to body length, tail length, and width. Four out of the 9 SNPs were found to be located within the 4 genes. KITLG and CADM2 are considered as candidate functional genes related to body height; MCTP1 and COL4A6 are candidate functional genes related to chest circumference. The 9 SNPs found in GWAS were verified using the G3 generation of the nucleus herd for meat production. Nine products were amplified around the 9 sites, and 29 SNPs were found; 3 mutation sites, G > C mutation at 134 bp downstream of s554331, T > G mutation at 19 bp upstream of s26859.1, and A > G mutation at 81 bp downstream of s26859.1, were significantly correlated to the body height. Dual-luciferase reporter gene experiments showed that the 3 SNPs could significantly impact dual-luciferase and gene transcription activity.

Keywords: Hu sheep; SNPs; body size traits; genome wide association studies; population verification; transcription activity.

During the domestication of the goose a change in its feather color took place, however, the molecular mechanisms responsible for this change are not completely understood. Here, we performed whole-genome resequencing on three pooled samples of geese (feral and domestic geese), with two distinct feather colors, to identify genes that might regulate feather color. We identified around 8 million SNPs within each of the three pools and validated allele frequencies for a subset of these SNPs using PCR and Sanger sequencing. Several genomic regions with signatures of differential selection were found when we compared the gray and white feather color populations using the F _ST and Hp approaches. When we combined previous functional studies with our genomic analyses we identified 26 genes (KITLG, MITF, TYRO3, KIT, AP3B1, SMARCA2, ROR2, CSNK1G3, CCDC112, VAMP7, SLC16A2, LOC106047519, RLIM, KIAA2022, ST8SIA4, LOC106044163, TRPM6, TICAM2, LOC106038556, LOC106038575, LOC106038574, LOC106038594, LOC106038573, LOC106038604, LOC106047489, and LOC106047492) that potentially regulate feather color in geese. These results substantially expand the catalog of potential feather color regulators in geese and provide a basis for further studies on domestication and avian feather coloration.

Keywords: SNP; feather color; genome; goose; pool-seq.

Background: Stickleback fish are widely used to study the genetic and ecological basis of phenotypic evolution. Although several major loci have now been identified that contribute to evolutionary differences between wild populations, further study of the phenotypes associated with particular genes and mutations has been limited by the difficulty of generating targeted mutations at precise locations in the stickleback genome.

Approach and aims: We compared different methods of expressing single-guide RNAs (sgRNAs) and Cas9 activity in fertilized stickleback eggs. We used an easily scored pigmentation gene (SLC24A5) to screen for molecular lesions, phenotypic effects, and possible germline transmission of newly induced alleles. We then used the optimized CRISPR methods to target two major evolutionary loci in sticklebacks, KITLG and EDA. We hypothesized that coding region mutations in the KITLG gene would alter body pigmentation and possibly sex determination, and that mutations in the EDA gene would disrupt the formation of most armor plates, fin rays, spines, teeth, and gill rakers.

Results: Targeted deletions were successfully induced at each target locus by co-injecting one-cell stage stickleback embryos with either Cas9 mRNA or Cas9 protein, together with sgRNAs designed to protein-coding exons. Founder animals were typically mosaic for multiple mutations, which they transmitted through the germline at overall rates of 21 to 100%. We found that the copy of KITLG on the X chromosome (KITLGX) has diverged from the KITLG on the Y chromosome (KITLGY). Predicted loss-of-function mutations in the KITLGX gene dramatically altered pigmentation in both external skin and internal organ, but the same was not true for KITLGY mutations. Predicted loss-of-function mutations in either the KITLGX or KITLGY genes did not lead to sex reversal or prevent fertility. Homozygous loss-of-function mutations in the EDA gene led to complete loss of armor plates, severe reduction or loss of most soft rays in the dorsal, anal, and caudal fins, and severe reductions in tooth and gill raker number. In contrast, long dorsal and pelvic spines remained intact in EDA mutant animals, suggesting that common co-segregation of plate loss and spine reduction in wild populations is unlikely to be due to pleiotropic effects of EDA mutations.

Conclusion: CRISPR-Cas9 approaches can be used to induce germline mutations in key evolutionary loci in sticklebacks. Targeted coding region mutations confirm an important role for KITLG and EDA in skin pigmentation and armor plate reduction, respectively. They also provide new information about the functions of these genes in other body structures.

Keywords: CRISPR-Cas9; EDA; KITLG; SLC24A5; genome editing; stickleback.

Background: This study aimed to perform an immunoprofiling of systemic juvenile idiopathic arthritis (sJIA) in order to define biomarkers of clinical use as well as reveal new immune mechanisms.

Methods: Immunoprofiling of plasma samples from a clinically well-described cohort consisting of 21 sJIA patients as well as 60 age and sex matched healthy controls, was performed by a highly sensitive proteomic immunoassay. Based on the biomarkers being significantly up- or down-regulated in cross-sectional and paired analysis, related canonical pathways and cellular functions were explored by Ingenuity Pathway Analysis (IPA).

Results: The well-studied sJIA biomarkers, IL6, IL18 and S100A12, were confirmed to be increased during active sJIA as compared to healthy controls. IL18 was the only factor found to be increased during inactive sJIA as compared to healthy controls. Novel factors, including CASP8, CCL23, CD6, CXCL1, CXCL11, CXCL5, EIF4EBP1, KITLG, MMP1, OSM, SIRT2, SULT1A1 and TNFSF11, were found to be differentially expressed in active and/or inactive sJIA and healthy controls. No significant pathway activation could be predicted based on the limited factor input to the IPA. High Mobility Group Box 1 (HMGB1), a damage associated molecular pattern being involved in a series of inflammatory diseases, was determined to be higher in active sJIA than inactive sJIA.

Conclusions: We could identify a novel set of biomarkers distinguishing active sJIA from inactive sJIA or healthy controls. Our findings enable a better understanding of the immune mechanisms active in sJIA and aid the development of future diagnostic and therapeutic strategies.

Keywords: Cytokines and inflammatory mediators; High mobility group Box 1; Inflammation; Ingenuity pathway analysis; Proteomics; Systemic juvenile idiopathic arthritis.

The authors wish to make the following corrections to this paper [...].