A Massively Parallel Pipeline to Clone DNA Variants and Examine Molecular Phenotypes of Human Disease Mutations

Understanding the functional relevance of DNA variants is essential for all exome and genome sequencing projects. However, current mutagenesis cloning protocols require Sanger sequencing, and thus are prohibitively costly and labor-intensive. We describe a massively-parallel site-directed mutagenesis approach, “Clone-seq”, leveraging next-generation sequencing to rapidly and cost-effectively generate a large number of mutant alleles. Using Clone-seq, we further develop a comparative interactome-scanning pipeline integrating high-throughput GFP, yeast two-hybrid (Y2H), and mass spectrometry assays to systematically evaluate the functional impact of mutations on protein stability and interactions. We use this pipeline to show that disease mutations on protein-protein interaction interfaces are significantly more likely than those away from interfaces to disrupt corresponding interactions. We also find that mutation pairs with similar molecular phenotypes in terms of both protein stability and interactions are significantly more likely to cause the same disease than those with different molecular phenotypes, validating the in vivo biological relevance of our high-throughput GFP and Y2H assays, and indicating that both assays can be used to determine candidate disease mutations in the future. The general scheme of our experimental pipeline can be readily expanded to other types of interactome-mapping methods to comprehensively evaluate the functional relevance of all DNA variants, including those in non-coding regions.     

Rapid clearance of intranasally administered DNA from rat tissues

The cold-adapted (ca) live attenuated influenza vaccine (LAIV) strains are manufactured in embryonated hens' eggs. Recently, a clonal isolate from Madin Darby Canine Kidney (MDCK) cells was derived and characterized to assess its utility as a potential cell substrate for the manufacturing of LAIV [1]. Since MDCK cells are a transformed continuous cell line [2], and low levels of residual cellular components (DNA and protein) are found in the intermediates and final filled vaccine, we sought to characterize the uptake and clearance of MDCK DNA from tissues in order to assess theoretical risks associated with manufacturing LAIV in MDCK cell culture.In order to address this concern, MDCK DNA uptake and clearance studies were performed in Sprague Dawley rats. DNA extracted from MDCK Master Cell Bank (MCB) cells was administered via an intranasal (IN) or intramuscular (IM) route. Tissue distribution and clearance of MDCK DNA were then examined in fourteen selected tissue types at selected time points post-administration using a quantitative PCR assay specific for canine (SINE) DNA.Results from these studies demonstrate that the uptake and clearance of MDCK DNA from tissues vary depending on the route of administration. When DNA was administered intranasally, as compared to intramuscularly, detectable DNA levels were lower at all time points. Thus, the intranasal route of vaccine administration appears to reduce potential risk associated with residual host cell DNA that may be present in cell culture produced final vaccine products.     

Association of Genetic Variants with Isolated Fasting Hyperglycaemia and Isolated Postprandial Hyperglycaemia in a Han Chinese Population

Background

Though multiple single nucleotide polymorphisms (SNPs) associated with type 2 diabetes have been identified, the genetic bases of isolated fasting hyperglycaemia (IFH) and isolated postprandial hyperglycaemia (IPH) were still unclear. In present study, we aimed to investigate the association of genome-wide association study-validated genetic variants and IFH or IPH in Han Chinese.

Methods/Principal Findings

We genotyped 27 validated SNPs in 6,663 unrelated individuals comprising 341 IFH, 865 IPH, 1,203 combined fasting hyperglycaemia and postprandial hyperglycaemia, and 4,254 normal glycaemic subjects of Han ancestry. The distributions of genotype frequencies of FTO, CDKAL1 and GCKR were significant different between individuals with IFH and those with IPH (SNP(p_trend): rs8050136(0.0024), rs9939609(0.0049), rs7756992(0.0122), rs780094(0.0037)). Risk allele of FTO specifically increased the risk of IFH (rs8050136: OR 1.403 [95% CI 1.125–1.750], p = 0.0027; rs9939609: 1.398 [1.120–1.744], p = 0.0030). G allele of CDKAL1 specifically increased the risk of IPH (1.217 [1.092–1.355], p = 0.0004). G allele of GCKR increased the risk of IFH (1.167 [0.999–1.362], p = 0.0513), but decreased the risk of IPH (0.891 [0.801–0.991], p = 0.0331). In addition, TCF7L2 and KCNQ1 increased the risk of both IFH and IPH. When combined, each additional risk allele associated with IFH increased the risk for IFH by 1.246-fold (p<0.0001), while each additional risk allele associated with IPH increased the risk for IPH by 1.190-fold (p<0.0001).

Conclusion/Significance

Our results indicate that genotype distributions of variants from FTO, GCKR, CDKAL1 were different between IPH and IFH in Han Chinese. Variants of genes modulating insulin sensitivity (FTO, GCKR) contributed to the risk of IFH, while variants of genes related to beta cell function (CDKAL1) increase the risk of IPH.     

Microsatellite diversity and population genetic structure of redfin culter ( Culter erythropterus ) in fragmented lakes of the Yangtze River

Redfin culter (Culter erythropterus) is a small lethic freshwater fish and widely distributed in the adjacent lakes of the Yangtze River of China. Five microsatellite loci were applied to investigate the genetic variation and population structure of redfin culter from seven lakes in the middle-and-lower reaches of the Yangtze River. The gene diversity was high among the populations (H > 0.9), the average number of alleles among seven populations was low with a range from 2.00 to 3.87. The mean observed (H _O) and expected (H _E) heterozygosity ranged from 0.111 to 0.419 and from 0.162 to 0.750, respectively. Significant deviations from Hardy-Weinberg Equilibrium expectation were found in 50% of the total locus-population combination tests in which heterozygote deficits were apparent. The analysis of molecular variance (AMOVA) indicated that the percentage of variance among and within these populations were 6.18 and 93.82, respectively. The Fst values (0.062, P < 0.001) among studied populations indicated that there were significant genetic differentiations among redfin culture populations from the scattered lakes with different connections to the Yangtze River. These results are useful for the evaluation and conservation of small freshwater fishes. The factors that may be involved in low intra-population polymorphism and the pattern of the population genetic structure of redfin culter from the Yangtze River were discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: C. Sturmbauer     

Mob as tumor suppressor is activated by Hippo kinase for growth inhibition in Drosophila

Tissue growth and organ size are determined by coordinated cell proliferation and apoptosis in development. Recent studies have demonstrated that Hippo (Hpo) signaling plays a crucial role in coordinating these processes by restricting cell proliferation and promoting apoptosis. Here we provide evidence that the Mob as tumor suppressor protein, Mats, functions as a key component of the Hpo signaling pathway. We found that Mats associates with Hpo in a protein complex and is a target of the Hpo serine/threonine protein kinase. Mats phosphorylation by Hpo increases its affinity with Warts (Wts)/large tumor suppressor (Lats) serine/threonine protein kinase and ability to upregulate Wts catalytic activity to target downstream molecules such as Yorkie (Yki). Consistently, our epistatic analysis suggests that mats acts downstream of hpo. Coexpression analysis indicated that Mats can indeed potentiate Hpo-mediated growth inhibition in vivo. Our results support a model in which Mats is activated by Hpo through phosphorylation for growth inhibition, and this regulatory mechanism is conserved from flies to mammals.     

E-Cadherin Can Replace N-Cadherin during Secretory-Stage Enamel Development

Background

N-cadherin is a cell-cell adhesion molecule and deletion of N-cadherin in mice is embryonic lethal. During the secretory stage of enamel development, E-cadherin is down-regulated and N-cadherin is specifically up-regulated in ameloblasts when groups of ameloblasts slide by one another to form the rodent decussating enamel rod pattern. Since N-cadherin promotes cell migration, we asked if N-cadherin is essential for ameloblast cell movement during enamel development.

Methodology/Principal Findings

The enamel organ, including its ameloblasts, is an epithelial tissue and for this study a mouse strain with N-cadherin ablated from epithelium was generated. Enamel from wild-type (WT) and N-cadherin conditional knockout (cKO) mice was analyzed. μCT and scanning electron microscopy showed that thickness, surface structure, and prism pattern of the cKO enamel looked identical to WT. No significant difference in hardness was observed between WT and cKO enamel. Interestingly, immunohistochemistry revealed the WT and N-cadherin cKO secretory stage ameloblasts expressed approximately equal amounts of total cadherins. Strikingly, E-cadherin was not normally down-regulated during the secretory stage in the cKO mice suggesting that E-cadherin can compensate for the loss of N-cadherin. Previously it was demonstrated that bone morphogenetic protein-2 (BMP2) induces E- and N-cadherin expression in human calvaria osteoblasts and we show that the N-cadherin cKO enamel organ expressed significantly more BMP2 and significantly less of the BMP antagonist Noggin than did WT enamel organ.

Conclusions/Significance

The E- to N-cadherin switch at the secretory stage is not essential for enamel development or for forming the decussating enamel rod pattern. E-cadherin can substitute for N-cadherin during these developmental processes. Bmp2 expression may compensate for the loss of N-cadherin by inducing or maintaining E-cadherin expression when E-cadherin is normally down-regulated. Notably, this is the first demonstration of a natural endogenous increase in E-cadherin expression due to N-cadherin ablation in a healthy developing tissue.     

中华鲟组织培养的初步研究

取中华鲟的尾鳍、吻端、性腺等八种组织进行体外培养,得到来源于吻端和性腺组织的两个细胞株,分别命名为CSSn和CSG.CSSn和CSG均以成纤维样细胞为主。两种细胞都含有微染色体,染色体数目众多,在分布上具有“双峰多态”的特点,表明细胞已属异倍体。细胞生长较慢,CSG接种9d后、CSSn接种14d后数目达到最高值。CSG在16—30℃生长正常,最佳生长温度在27℃,最适pH为7.2。     

两性型天然雌核发育彭泽鲫染色体组型的研究

彭泽鲫是一种两性型天然雌核发育群体。染色体组型分析结果表明：雌、雄鱼的染色体数目均为１６６。它们的核型组成是：３２条中部着丝点染色体、４０条亚中部着丝点染色体、１８条亚端部着丝点染色体和７６条端部着丝点染色体。臂数（ＮＦ）为２３８。本研究结果与其它雌核发育类型的鲫鱼有明显的差别。     

NDRG2在人胚胎呼吸系统的表达特点

目的该实验通过对16-28周人胚胎呼吸系统NDRG2表达的研究,旨在阐明NDRG2在16-28周人胚胎呼吸系统中的表达规律,为进一步明确新的发育相关基因ndrg2的功能提供依据。方法搜集16-28周胎儿呼吸系统的肺和气管组织,制成石蜡切片,用抗NDRG2单克隆抗体,行免疫组化染色(ABC法),从蛋白质水平观察NDRG2表达情况。统计阳性细胞数,利用统计学方法,判断不同胎龄的肺和气管间NDRG2表达有无差别。结果免疫组化染色表明,NDRG2在16-28周胚胎呼吸系统中有广泛的表达,阳性产物主要位于上皮细胞的胞浆中,但是不同胎龄之间NDRG2的表达未见显著差别。结论NDRG2在16-28周胚胎呼吸系统中有广泛的表达,提示NDRG2在早期胚胎的呼吸系统上皮细胞的生长与发育过程中起一定作用。而阳性产物主要表达在上皮细胞的胞浆中,说明NDRG2可能是一种胞浆蛋白。