Novel non-synonymous mutations of PAX8 in a cohort of Chinese with congenital hypothyroidism

Background:The transcription factor paired box 8 (PAX8) was associated with type 2 congenital non-goitrous hypothyroidism (CHNG2), a clinical phenotype of congenital hypothyroidism (CH). Though studied in a few regions with different ethnicities, the incidence of PAX8 mutations varied, even among Chinese cohorts in different regions. This study aimed to identify and characterize PAX8 mutations and explore the prevalence of its mutations in another cohort of CH. Methods: The 105 unrelated Chinese patients with CH were collected from four major hospitals. Exomes of the 105 samples were sequenced by Hiseq 2000 platform to identify mutations of PAX8 on genomic DNAs extracted from peripheral blood samples. Luciferase reporter assays were used to assess the effects of mutations on the transcription of thyroid peroxidase (TPO). Results: Three PAX8 mutations in four subjects were identified in 105 samples. One variant, rsl 89229644, was detected in two subjects, and categorized as uncertain significance. The other two missense mutations (275T>C/Ile92Thr and 398G>A/Argl33Gln) were not detected in three large-scale genotyping projects, namely 1000 Genome Project, Exome Aggregation Consortium and GO Exome Sequencing Project. Functional studies for the two mutations revealed that they could impair the transcription ability of PAX8 on one of its target genes, TPO. Therefore, the two mutations were causative for the pathogenesis of CHNG2. After combining the studies of PAX8 mutations, an average frequency of 1.74%(21/1209) could be obtained in Chinese patients with CH. Conclusion: The study specifically demonstrates the role of two mutations in impairing the transcription ability of PAX8, which should be considered as pathogenic variants for CH.

Studies on the temporal,structural,and interacting features of the clubroot resistance gene Rcr1 using CRISPR/Cas9-based systems

Clubroot disease is a severe threat to Brassica crops globally,particularly in western Canada.Genetic resistance,achieved through pyramiding clubroot resistance(CR)genes with different modes of action,is the most important strategy for managing the disease.However,studies on the CR gene functions are quite limited.In this study,we have conducted investigations into the temporal,structural,and interacting features of a newly cloned CR gene,Rcr1,using CRISPR/Cas9 technology.For temporal functionality,we developed a novel CRISPR/Cas9-based binary vector,pHHIGR-Hsp18.2,to deliver Rcr1 into a susceptible canola line(DH12075)and observed that early expression of Rcr1 is critical for conferring resistance.For structural functionality,several independent mutations in specific domains of Rcr1 resulted in loss-offunction,highlighting their importance for CR phenotype.In the study of the interacting features of Rcr1,a cysteine protease gene and its homologous allele in canola were successfully disrupted via CRISPR/Cas9 as an interacting component with Rcr1 protein,resulting in the conversion from clubroot resistant to susceptible in plants carrying intact Rcr1.These results indicated an indispensable role of these two cysteine proteases in Rcr1-mediated resistance response.This study,the first of its kind,provides valuable insights into the functionality of Rcr1.Further,the new vector p HHIGR-Hsp18.2 demonstrated an inducible feature on the removal of add-on traits,which should be useful for functional genomics and other similar research in brassica crops.

Mitochondrial DNA T7719G in tRNA-Lys gene affects litter size in Small-tailed Han sheep

Background： In farm animals, mitochondrial DNA（mtDNA） effect on economic performance remains hot-topic for breeding and genetic selection. Here, 53 maternal lineages of Small-tailed Han sheep were used to investigate the association of mitochondrial DNA variations and the lambing litter size.Results： Sequence sweeping of the mitochondrial coding regions discovered 31 non-synonymous mutations, and the association study revealed that T7719G in mt DNA t RNA-Lys gene was associated with litter size（P 〈 0.05）,manifesting 0.29 lambs per litter between the G and T carriers. Furthermore, using the mixed linear model, we assayed the potential association of the ovine litter size and haplogroups and multiple-level mtDNA haplotypes,including general haplotypes, assembled haplotypes of electron transport chain contained sequences（H-ETC）,mitochondrial respiratory complex contained sequences（H-MRC） and mitochondrial genes（H-gene, including polypeptide-coding genes, rRNA genes and tRNA genes）. The strategy for assembled mitochondrial haplotypes was proposed for the first time in mtDNA association analyses on economic traits, although none of the significant relations could be concluded（P 〉 0.05）. In addition, the nuclear major gene BMPR1B was significantly correlated with litter size in the flock（P 〈 0.05）, however, did not interact with mtDNA T7719G mutation（P 〉 0.05）.Conclusions： Our results highlight mutations of ovine mitochondrial coding genes, suggesting T7719G in tRNA-Lys gene be a potentially useful marker for selection of sheep litter size.

A strategy for uncovering germline variants altering anti-tumor CD8 T cell response

Among many factors known to alter the outcomes of T cell receptor(TCR)-induced proximal signaling,the role of human germline variants in dictating the individuality of the anti-tumor CD8 T cell response has remained challenging to address.Here,we describe a convenient strategy for molecular and functional characterization of phosphotyrosine-altering non-synonymous single nucleotide variations(pTyr-SNVs)that directly impact TCR-induced proximal phosphotyrosine motif-based signaling pathways.We devise an experimental co-cultivation set-up comprising a C57BL/6 mouse-derived metastatic melanoma cell line engineered to constitutively present ovalbumin(OVA)antigens and retrovirally engineered syngeneic major histocompatibility complex(MHC)Class I restricted OVA TCR-transgenic CD8 T cells(OT-I).Using the synthetic version of pTyr-SNV rs1178800678-G/T-encoding integrin alpha 4(ITGA4)p.S1027I variant as a prototype,we show that under identical TCR stimulation conditions,genetically determined membrane-proximal immunoreceptor tyrosin activation motif(ITAM)results in increased tyrosine phosphorylation of 70 kDa zeta-chain-associated protein(ZAP70)and the levels of cytotoxic effector molecule granzyme B(GZMB),which in turn result in enhanced cytotoxic activity against metastatic melanoma cell line.This strategy paves the way for rapid molecular and functional characterization of anti-tumor immune response-linked germline pTyr-SNVs so as to improve our understanding of the genetic basis of individual-to-individual differences in anti-tumor CD8 T cell response.

The Analysis of SNP within GPR26 Gene and Tumor-Associated Latent Loci

This study aims to investigate the association of SNPs with tumor-associated latent loci. By analyzing the effect of gene mutations on the structure and function of proteins with the database, to speculate the destructive mutants. It was found that individuals with the genotype of the five key SNP loci were more susceptible to tumors; these specific markers can be used as a molecular marker to determine the susceptibility of a tumor to the individual, and to diagnose the population with high risk of tumor; and rs201154887 can be used as a new disease molecular marker.