Rare Variants Association Analysis in Large-Scale Sequencing Studies at the Single Locus Level

Genetic association analyses of rare variants in next-generation sequencing (NGS) studies are fundamentally challenging due to the presence of a very large number of candidate variants at extremely low minor allele frequencies. Recent developments often focus on pooling multiple variants to provide association analysis at the gene instead of the locus level. Nonetheless, pinpointing individual variants is a critical goal for genomic researches as such information can facilitate the precise delineation of molecular mechanisms and functions of genetic factors on diseases. Due to the extreme rarity of mutations and high-dimensionality, significances of causal variants cannot easily stand out from those of noncausal ones. Consequently, standard false-positive control procedures, such as the Bonferroni and false discovery rate (FDR), are often impractical to apply, as a majority of the causal variants can only be identified along with a few but unknown number of noncausal variants. To provide informative analysis of individual variants in large-scale sequencing studies, we propose the Adaptive False-Negative Control (AFNC) procedure that can include a large proportion of causal variants with high confidence by introducing a novel statistical inquiry to determine those variants that can be confidently dispatched as noncausal. The AFNC provides a general framework that can accommodate for a variety of models and significance tests. The procedure is computationally efficient and can adapt to the underlying proportion of causal variants and quality of significance rankings. Extensive simulation studies across a plethora of scenarios demonstrate that the AFNC is advantageous for identifying individual rare variants, whereas the Bonferroni and FDR are exceedingly over-conservative for rare variants association studies. In the analyses of the CoLaus dataset, AFNC has identified individual variants most responsible for gene-level significances. Moreover, single-variant results using the AFNC have been successfully applied to infer related genes with annotation information.     

Heteroresistance to the model antimicrobial peptide polymyxin B in the emerging Neisseria meningitidis lineage 11.2 urethritis clade: mutations in the pilMNOPQ operon

Clusters of Neisseria meningitidis (Nm) urethritis among primarily heterosexual males in multiple US cities have been attributed to a unique non‐encapsulated meningococcal clade (the US Nm urethritis clade, US_NmUC) within the hypervirulent clonal complex 11. Resistance to antimicrobial peptides (AMPs) is a key feature of urogenital pathogenesis of the closely related species, Neisseria gonorrhoeae. The US_NmUC isolates were found to be highly resistant to the model AMP, polymyxin B (PmB, MICs 64–256 µg ml^–1). The isolates also demonstrated stable subpopulations of heteroresistant colonies that showed near total resistant to PmB (MICs 384–1024 µg ml^–1) and colistin (MIC 256 µg ml^–1) as well as enhanced LL‐37 resistance. This is the first observation of heteroresistance in N. meningitidis. Consistent with previous findings, overall PmB resistance in US_NmUC isolates was due to active Mtr efflux and LptA‐mediated lipid A modification. However, whole genome sequencing, variant analyses and directed mutagenesis revealed that the heteroresistance phenotypes and very high‐level AMP resistance were the result of point mutations and IS1655 element movement in the pilMNOPQ operon, encoding the type IV pilin biogenesis apparatus. Cross‐resistance to other classes of antibiotics was also observed in the heteroresistant colonies. High‐level resistance to AMPs may contribute to the pathogenesis of US_NmUC.     

Systematic identification of SH3 domain-mediated human protein-protein interactions by peptide array target screening

Systematic identification of direct protein-protein interactions is often hampered by difficulties in expressing and purifying the corresponding full-length proteins. By taking advantage of the modular nature of many regulatory proteins, we attempted to simplify protein-protein interactions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein-protein interactions, we examined a set of interactions mediated by the PLCgamma1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCgamma1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCgamma1 SH3 domain negatively regulated HPK1 kinase activity. Compared to protein interactions listed in the online predicted human interaction protein database (OPHID), the majority of interactions identified by PATS are novel, suggesting that, when extended to the large number of peptide interaction domains encoded by the human genome, PATS should aid in the mapping of the human interactome.     

Mutations in glucokinase and other genes detected in neonatal and type 1B diabetes patient using whole exome sequencing may lead to disease-causing changes in protein activity

Monogenic diabetes is caused by mutations that reduce β-cell function. While Sanger sequencing is the standard method used to detect mutated genes. Next-generation sequencing techniques, such as whole exome sequencing (WES), can be used to find multiple gene mutations in one assay. We used WES to detect genetic mutations in both permanent neonatal (PND) and type 1B diabetes (T1BD).A total of five PND and nine T1BD patients were enrolled in this study. WES variants were assessed using VarioWatch, excluding those identified previously. Sanger sequencing was used to confirm the mutations, and their pathogenicity was established via the literature or bioinformatic/functional analysis. The PND and T1BD patients were diagnosed at 0.1–0.5 and 0.8–2.7?years of age, respectively. Diabetic ketoacidosis was present at diagnosis in 60% of PND patients and 44.4% of T1BD patients. We found five novel mutations in five different genes. Notably, patient 602 had a novel homozygous missense mutation c.1295C?>?A (T432?K) in the glucokinase (GCK) gene. Compared to the wild-type recombinant protein, the mutant protein had significantly lower enzymatic activity (2.5%, p?=?0.0002) and V_max (1.23?±?0.019 vs. 0.33?±?0.016, respectively; p?=?0.005). WES is a robust technique that can be used to unravel the etiologies of genetically heterogeneous forms of diabetes. Homozygous inactivating mutations of the GCK gene may have a significant role in PND pathogenesis.     

Flavonoids and andrographolides from Andrographis paniculata

Two flavonoids, identified as 5,7,2',3'-tetramethoxyflavanone and 5-hydroxy-7,2',3'-trimethoxyflavone, as well as several other flavonoids, andrographolide diterpenoids, and polyphenols, were obtained from the phytochemical investigation of the whole plant of Andrographis paniculata, a well known medicinal plant. The structures of these compounds were established with the aid of spectroscopic methods, including analysis by 2D NMR spectroscopy.     

Synthesis and anti-inflammatory evaluation of 4-anilinofuro[2,3-b]quinoline and 4-phenoxyfuro[2,3-b]quinoline derivatives. Part 3

Mast cells, neutrophils and macrophages are important inflammatory cells that have been implicated in the pathogenesis of acute and chronic inflammatory diseases. To explore a novel anti-inflammatory agent, we have synthesized certain 4-anilinofuro[2,3-b]quinoline and 4-phenoxyfuro[2,3-b]quinoline derivatives and evaluated their anti-inflammatory activities by reaction of 3,4-dichlorofuro[2,3-b]quinoline with appropriate Ar-NH(2) or Ar-OH. Compounds 6a and 15 were proved to be more potent than the reference inhibitor, mepacrine for the inhibition of rat peritoneal mast cell degranulation with IC(50) values of 6.5 and 16.4 microM, respectively. Compounds 2b, 6a, 10, and 15 also showed potent inhibitory activity (IC(50)=7.2-29.4 microM) for the secretion of lysosomal enzyme and beta-glucuronidase from neutrophils. These results also indicated that oxime derivatives are more potent than the respective ketone precursors (6a> or =2a; 7a> or =3), and the substituent such as Me at the oxime decreased inhibitory activity (6a> or =6b; 7a> or =7b). Among these derivatives, compound 6a showed the most potent activity with IC(50) values of 6.5-11.6 microM for the inhibition of mast cell degranulation and neutrophil degranulation.     

Comparative analysis of the receptor-like kinase family in Arabidopsis and rice

Receptor-like kinases (RLKs) belong to the large RLK/Pelle gene family, and it is known that the Arabidopsis thaliana genome contains >600 such members, which play important roles in plant growth, development, and defense responses. Surprisingly, we found that rice (Oryza sativa) has nearly twice as many RLK/Pelle members as Arabidopsis does, and it is not simply a consequence of a larger predicted gene number in rice. From the inferred phylogeny of all Arabidopsis and rice RLK/Pelle members, we estimated that the common ancestor of Arabidopsis and rice had >440 RLK/Pelles and that large-scale expansions of certain RLK/Pelle members and fusions of novel domains have occurred in both the Arabidopsis and rice lineages since their divergence. In addition, the extracellular domains have higher nonsynonymous substitution rates than the intracellular domains, consistent with the role of extracellular domains in sensing diverse signals. The lineage-specific expansions in Arabidopsis can be attributed to both tandem and large-scale duplications, whereas tandem duplication seems to be the major mechanism for recent expansions in rice. Interestingly, although the RLKs that are involved in development seem to have rarely been duplicated after the Arabidopsis-rice split, those that are involved in defense/disease resistance apparently have undergone many duplication events. These findings led us to hypothesize that most of the recent expansions of the RLK/Pelle family have involved defense/resistance-related genes.     

Ha-ras overexpression mediated cell apoptosis in the presence of 5-fluorouracil

By using a mouse NIH3T3 derivate designed 7-4 harboring the inducible Ha-ras oncogene, we demonstrated the close relationship between Ha-ras expression level and sensitization of 5-flurouracil (5-FU)-treated cells. Further studies revealed that the cells susceptible to 5-FU treatment died of apoptosis, which was demonstrated by caspase-3 activation, loss of mitochondria membrane potential (MMP), and DNA fragmentation. The 7-4 cells coexpressing dominant negative Ras (Ras(Asn17)), dominant negative Raf-1 (Raf-1(CB4)), Bcl-2, or active form of phosphatidylinositol 3-kinase (PI3K) became resistant to 5-FU, and apoptosis was prevented. In contrast, the cells coexpressing dominant negative Rac 1 (Rac1(Asn17)) or dominant negative Rho A (RhoA(Asn19)) showed no change of sensitivity to 5-FU. These results indicate that Ras, Bcl-2, as well as Raf-1 and PI3K pathways play pivotal roles in 5-FU-induced apoptosis under Ha-ras-overexpressed condition. Aberrant levels of cyclin E and p21(Cip/WAF-1) expression as well as Cdc 2 phosphorylation at Tyrosine 15 suggest that perturbation of G1/S and G2/M transitions in cell cycle might be responsible for 5-FU triggered apoptosis. Sensitization of Ha-ras-related cells to 5-FU was also demonstrated in human bladder cancer cells. Through understanding the mechanism of 5-FU induced apoptosis in tumor cells, a new direction toward the treatment of Ha-ras oncogene-related cancers with 5-FU at more optimal dosages is possible and combinational therapy with other drugs that suppress PI3K and Bcl-2 activities can also be considered.     

Morphology of a spectrum of parasporal endotoxin crystals from cultures of Bacillus thuringiensis ssp. kurstaki isolate A3-4

Morphology of the parasporal -endotoxin crystals of Bacillus thuringiensis subsp. kurstaki isolate A3-4, a native (Taiwan) strain was studied by scanning (SEM) and transmission (TEM) electron microscopy. The typical bipyramidal crystals and an unusual parasporal inclusion with an embedded body were observed. The parasporal -endotoxin crystal with an embedded body, which was in different size and shape was well demonstrated in thin sections by transmission electron microscopy. The sporulated cells had multiple individually separated inclusions up to four crystals in one cell, which was unique to this isolate, and has not been reported before. The -endotoxin crystals included in the cell or released from the cell after batch fermentation or fed-batch fermentation did not show any altered morphological characteristics. However, judging from thin-sections of TEM, cells and the included parasporal crystals from fed-batch fermentation appeared larger than those from batch fermentation. It was observed that release of spore and parasporal crystals from the bacterial cell produced from batch cultures was earlier than that of fed-batch cultures. Preliminary bioassay results showed that the isolate cultures from both types of culture were equally effective against Plutella xylostella larvae. Based on the morphological observations, this strain may have a multiple insecticidal activities toward different insect species.     

Mapping the rubella virus subgenomic promoter

Rubella virus (RUB), the sole member of the Rubivirus genus in the Togaviridae family of positive-strand RNA viruses, synthesizes a single subgenomic (SG) RNA containing sequences from the 3' end of the genomic RNA including the open reading frame (ORF) that encodes the virion proteins. The synthesis of SG RNA is initiated internally on a negative-strand, genome-length template at a site known as the SG promoter (SGP). Mapping the RUB SGP was initiated by using an infectious cDNA vector, dsRobo402/GFP, in which the region containing the SGP was duplicated (K. V. Pugachev, W.-P. Tzeng, and T. K. Frey, J. Virol. 74:10811-10815, 2000). In dsRobo402/GFP, the 5'-proximal nonstructural protein ORF (NS-ORF) is followed by the first SGP (SGP-1), the green fluorescent protein (GFP) gene, the second SGP (SGP-2), and the structural protein ORF. The duplicated SGP, SGP-2, contained nucleotides (nt) -175 to +76 relative to the SG start site, including the 3' 127 nt of the NS-ORF and 47 nt between the NS-ORF and the SG start site. 5' Deletions of SGP-2 to nt -40 (9 nt beyond the 3' end of the NS-ORF) resulted in a wild-type (wt) phenotype in terms of virus replication and RNA synthesis. Deletions beyond this point impaired viability; however, the analysis was complicated by homologous recombination between SGP-1 and SGP-2 that resulted in deletion of the GFP gene and resurrection of viable virus with one SGP. Since the NS-ORF region was not necessary for SGP activity, subsequent mapping was done by using both replicon vectors, RUBrep/GFP and RUBrep/CAT, in which the SP-ORF is replaced with the reporter GFP and chloramphenical acetyltransferase genes, respectively, and the wt infectious clone, Robo402. In the replicon vectors, 5' deletions to nt -26 resulted in the synthesis of SG RNA. In the infectious clone, deletions through nt -28 gave rise to viable virus. A series of short internal deletions confirmed that the region between nt -28 and the SG start site was essential for viability and showed that the repeated UCA triplet at the 5' end of SG RNA was also required. Thus, the minimal SGP maps from nt -26 through the SG start site and appears to extend to at least nt +6, although a larger region is required for the generation of virus with a wt phenotype. Interestingly, while the positioning of the RUB SGP immediately adjacent the SG start site is thus similar to that of members of the genus Alphavirus, the other genus in the Togaviridae family, it does not include a region of nucleotide sequence homology with the alphavirus SGP that is located between nt -48 and nt -23 with respect to the SG start site in the RUB genome.