Molecular Genetics and Genomics

Comparative genomic analysis unravels the transmission pattern and intra-species divergence of acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus strains Abstract Acute hepatopancreatic necrosis disease (AHPND) is a recently discovered shrimp disease that has become a severe threat to global shrimp-farming industry. The causing agents of AHPND were identified as Vibrio parahaemolyticusand other vibrios harboring a plasmid encoding binary toxins PirAvp/PirBvp. However, the epidemiological involvement of environmental vibrios in AHPND is poorly understood. In this study, with an aim to reveal the possible transmission route of AHPND-causing V. parahaemolyticus, we sequenced and analyzed the genomes of four pairs of V. parahaemolyticus strains from four representative regions of shrimp farming in China, each including one strain isolated from diseased shrimp during an AHPND outbreak and one strain isolated from sediment before AHPND outbreaks. Our results showed that all the four shrimp-isolated and three of the sediment-isolated strains encode and secret PirAvp/PirBvp toxins and, therefore, are AHPND-causing strains. In silico multilocus sequence typing and high-resolution phylogenomic analysis based on single-nucleotide polymorphisms, as well as comparison of genomic loci in association with prophages and capsular polysaccharides (CPSs) consistently pointed to a close genetic relationship between the shrimp- and sediment-isolated strains obtained from the same region. In addition, our analyses revealed that the sequences associated with prophages, CPSs, and type VI secretion system-1 are highly divergent among strains from different regions, implying that these genes may play vital roles in environmental adaptation for AHPND-causing V. parahaemolyticus and thereby be potential targets for AHPND control. Summing up, this study provides the first direct evidence regarding the transmission route of AHPND-causing V. parahaemolyticus and underscores that V. parahaemolyticus in shrimp are most likely originated from local environment. The importance of environmental disinfection measures in shrimp farming was highlighted.

Heterogeneous impact of type 2 diabetes mellitus-related genetic variants on gestational glycemic traits: review and future research needs Abstract Gestational glucose homeostasis influences mother’s metabolic health, pregnancy outcomes, fetal development and offspring growth. To understand the genetic roles in pregnant glucose metabolism and genetic predisposition for gestational diabetes (GDM), we reviewed the recent literature up to Jan, 2018 and evaluated the influence of T2DM-related genetic variants on gestational glycemic traits and glucose tolerance. A total of 140 variants of 89 genes were integrated. Their associations with glycemic traits in and outside pregnancy were compared. The genetic circumstances underlying glucose metabolism exhibit a similarity between pregnant and non-pregnant populations. While, not all of the T2DM-associated genetic variants are related to pregnant glucose tolerance, such as genes involved in fasting insulin/C-peptide regulation. Some genetic variants may have distinct effects on gestational glucose homeostasis. And certain genes may be particularly involved in this process via specific mechanisms, such as HKDC1, MTNR1B, BACE2, genes encoding cell cycle regulators, adipocyte regulators, inflammatory factors and hepatic factors related to gestational glucose sensing and insulin signaling. However, it is currently difficult to evaluate these associations with quantitative synthesis due to inadequate data, different analytical methods, varied measurements for glycemic traits, controversies in diagnosis of GDM, and unknown ethnicity- and/or sex-related influences on pregnant maternal metabolism. In conclusion, different genetic associations with glycemic traits may exist between pregnant and non-pregnant conditions. Comprehensive research on specific genetic regulation in gestation is necessary.

Disease association of human tumor suppressor genes Abstract The multifactorial disease, cancer, frequently emerges due to perturbations in tumor suppressor genes (TSGs). However, a growing number of noncanonical target genes of TSGs and the highly interconnected nature of the human interactome reveal that the functions of TSGs are not limited to cancer-specific events. The various functions of TSGs lead to the assumption that cancer is linked with other human disorders. Therefore, a disease–gene association network of TSGs (TSDN) was constructed by integrating protein–protein interaction networks of TSGs (TSN) with Morbid Map in Online Mendelian Inheritance in Man. The TSDN revealed links between TSGs and 22 different human disorders including cancer and indicated disease–disease associations. In addition, high-density functional protein clusters in the TSN showed cohesive and overlapping disease–TSG associations, which proved the prevalent role of TSGs in various human diseases beyond cancer. The presence of overlapping disease–gene modules and disease–disease associations via the TSN demonstrated that other diseases can serve as possible roots of the life-threatening disease cancer. Therefore, a disease association map of TSGs could be a promising tool for exploring intricate relationships between cancer and other diseases for the early prediction of cancer and the understanding of disease etiology.

Genetic history of Bashkirian Mari and Southern Mansi ethnic groups in the Ural region Abstract According to genetic studies, the Hungarian Y-chromosomal gene pool significantly differs from other Uralic-speaking populations. Hungarians possess a significant frequency of haplogroup R1a-Z280 and a low frequency of haplogroup N-Tat, which is common among other Uralic-speaking populations. Based on this evidence, we further worked to define the links between the linguistically related Hungarian, Mansi and Bashkirian Mari populations. Samples were collected from 45 Bashkirian Mari and 36 Southern Mansi males in the Ural region. We analyzed male-specific markers including 23 STRs and 36 SNPs, which reflect past and recent paternal genetic history. We found that the haplogroup distribution of the two population samples showed high genetic similarity to each other except for the N-Tat* and R1a-Z93 haplogroups in the Bashkirian Mari males. On the MDS plots constructed from Fst- and Rst-genetic distances, the Bashkirian Mari and Southern Mansi population groups showed close genetic affinities with the Khanty, Northern Mansi, Mari, and Estonian populations. For phylogenetic studies, networks were constructed for the most frequent haplogroups in both populations together with other Eurasian populations. Both populations shared common haplotypes within haplogroups R1a-Z280 or N-L1034 with Hungarian speakers, suggesting a common paternal genetic footprint that arose in prehistoric or historic times. Overall, the Hungarian, Mansi, and Bashkirian Mari populations have a much more complex genetic history than the traditional linguistic model or history would suggest. Further studies are needed to clarify the common genetic profiles may have been acquired directly or indirectly during the more or less known their history.

Optimization of a 2A self-cleaving peptide-based multigene expression system for efficient expression of upstream and downstream genes in silkworm Abstract The multigene expression system is highly attractive to co-express multiple genes or multi-subunit complex-based genes for their functional studies, and in gene therapy and visual tracking of expressed proteins. However, the current multiple gene co-expression strategies usually suffer from severe inefficiency and unbalanced expression of multiple genes. Here, we report on an improved 2A self-cleaving peptide (2A)-based multigene expression system (2A-MGES), by introducing an optimized Kozak region (Ck) and altering the gene arrangement, both of which contributed to the efficient expression of two fluorescent protein genes in silkworm. By co-expressing DsRed and EGFP genes in insect cells and silkworms, the potent Ck was first found to improve the translation efficiency of downstream genes, and the expression of the flanking genes of 2A were improved by altering the gene arrangement in 2A-MGES. Moreover, we showed that combining Ck and an optimized gene arrangement in 2A-MGES could synergistically improve the expression of genes in the cell. Further, these two flanking genes, regulated by modified 2A-MGES, were further co-expressed in the middle silk gland and secreted into the cocoon, and both achieved efficient expression in the transgenic silkworms and their cocoons. These results suggested that the modified Ck-2A-MGES will be a potent tool for multiple gene expression, for studies of their functions, and their applications in insect species.

Selection pressure causes differentiation of the SPL gene family in the Juglandaceae Abstract The SQUAMOSA promoter-binding protein-like (SPL) is a plant-specific transcription factor that influences flowering and vegetative development. Although the SPL genes have been functionally analyzed in many species, studies on the evolutionary history of the whole gene family, and in the Juglandaceae specifically, have been limited. Here, we conducted a phylogenetic relationship analysis of the Juglandaceae SPL gene family compared with other land plant species. Our results showed that the SPL genes were divided into three major clades, all of which were further divided into ten small clades. Selection pressure analysis suggested that all SPL genes were exposed to purifying selection pressure during evolution. The purifying selection was smaller for the Juglandaceae SPL genes than for other angiosperms, indicating a greater susceptibility to functional differentiation in the Juglandaceae. The SPL proteins encoded by Clade 1 contained a branch-specific transmembrane structure and many conserved motif combinations at the C-terminal. We also detected many selection sites in these motif combinations. Expression analysis showed that Clade 1 genes had spatial and temporal differences and were highly expressed in various organs. The expression profile was closely related to the selection sites and motif combinations at the C-terminal. These observations represent essential entry points for revealing the functional differentiation of the SPL gene family. Our data presented here may provide a basis for future investigations of SPL genes in the Juglandaceae, especially for flower development and perhaps crop yield improvement.

Identification of rare variants in cardiac sodium channel β4-subunit gene SCN4B associated with ventricular tachycardia Abstract Ventricular tachycardia (VT) causes sudden cardiac death, however, the majority of risk genes for VT remain unknown. SCN4B encodes a β-subunit, Navβ4, for the voltage-gated cardiac sodium channel complex involved in generation and conduction of the cardiac action potential. We hypothesized that genomic variants in SCN4B increase the risk of VT. We used high-resolution melt analysis followed by Sanger sequencing to screen 199 VT patients to identify nonsynonymous variants in SCN4B. Two nonsynonymous heterozygous variants in SCN4B were identified in VT patients, including p.Gly8Ser in four VT patients and p.Ala145Ser in one VT patient. Case–control association studies were used to assess the association between variant p.Gly8Ser and VT in two independent populations for VT (299 VT cases vs. 981 controls in population 1 and 270 VT patients vs. 639 controls in population 2). Significant association was identified between p.Gly8Ser and VT in population 1 (P = 1.21 × 10−4, odds ratio or OR = 11.04), and the finding was confirmed in population 2 (P = 0.03, OR = 3.62). The association remained highly significant in the combined population (P = 3.09 × 10−5, OR = 6.17). Significant association was also identified between p.Gly8Ser and idiopathic VT (P = 1.89 × 10−5, OR = 7.27). Functional analysis with Western blotting showed that both p.Gly8Ser and p.Ala145Ser variants significantly reduced the expression level of Navβ4. Based on 2015 ACMG Standards and Guidelines, p.Gly8Ser and p.Ala145Ser can be classified as the pathogenic and likely pathogenic variant, respectively. Our data suggest that SCN4B is a susceptibility gene for common VT and idiopathic VT and link rare SCN4B variants with large effects (OR = 6.17–7.27) to common VT.

A simultaneous search for footprints of early human migration processes using the genetic and folk music data in Eurasia Abstract In this study, we aimed to illustrate the efficiency of correlation analysis of musical and genetic data for certain common ethnic and ethno-musical roots of mankind. The comparison of the results to archaeogenetic data shows that correlations of recent musical and genetic data may reveal past cultural and migration processes resulting in recent connections. The significance tests verified our hypothesis supposing that propagation of oral musical traditions can be related to early human migration processes is well-founded, because the multidimensional point system determined by the inverse rank vectors of correlating Hg–UCT pairs has a very clear structure. We found that associations of Hgs jointly propagating with associations of UCTs (Unified Contour Type) can be identified as significant complex components in both modern and ancient populations, thus, modern populations can be considered as admixtures of these ancient Hg associations. It also seems obvious to conclude that these ancient Hg associations strewed their musical “parent languages” during their migrations, and the correlating UCTs of these musical parent languages may also be basic components of the recent folk music cultures. Thus, we can draw a hypothetical picture of the main characteristics of ancient musical cultures. Modern and prehistoric populations belonging to a common Hg–UCT association are located to very similar geographical areas, consequently, recent folk music cultures are basically determined by prehistoric migrations. Our study could be considered as an initial step in analysis of the correlations of prehistoric and recent musical and genetic characteristics of human evolution history.

Transcriptional regulation of the porcine miR-17-92 cluster Abstract The miR-17-92 cluster has been involved in the cell cycle, apoptosis, and signaling. However, its transcriptional regulation has not been fully characterized. To elucidate the transcriptional regulation, the promoter of miR-17-92 was analyzed in detail in pig here. We found that, as an intronic miRNA, porcine miR-17-92 cluster was regulated by two independent promoters, an A/T-rich region directly upstream of the miR-17-92 coding sequence, and a G/C-rich region corresponding to the host gene promoter of the human miR-17-92 cluster. Several cis-regulatory elements were identified including sites for c-Myc, NFY, E2F3, and SP1, among which NFY and c-Myc sites were present in both A/T- and G/C-rich regions, while E2F3 and SP1 sites only existed in G/C-rich region. Sites for c-Myc, E2F3, and SP1 were positive for regulating transcription. NFY sites played bipartite roles, functioning as a repressor for the A/T-rich region, and as an activator for the G/C-rich region. Additionally, we found that levels of individual miRNAs in the cluster were not promoted completely in parallel with each other or with pri-miR-17-92 by the A/T-rich region, through using a self-made vector by modifying pGL3-basic in which firefly luciferase gene was replaced with an miR-17-92 cluster and a direct upstream A/T-rich region. The expression regulation of miR-17-92 is complicated and the results will contribute to further revealing the regulatory mechanisms under the expression of the miR-17-92 cluster.

Identification and classification of differentially expressed genes in pyrethroid-resistant Culex pipiens pallens Abstract Culex pipiens pallens is an important vector that transmits Bancroftian filariasis, Japanese encephalitis and other diseases that pose a serious threat to human health. Extensive and improper use of insecticides has caused insecticide resistance in mosquitoes, which has become an important obstacle to the control of mosquito-borne diseases. It is crucial to investigate the underlying mechanism of insecticide resistance. The aims of this study were to identify genes involved in insecticide resistance based on the resistance phenotype, gene expression profile and single-nucleotide polymorphisms (SNPs) and to screen for major genes controlling insecticide resistance. Using a combination of SNP and transcriptome data, gene expression quantitative trait loci (eQTLs) were studied in deltamethrin-resistant mosquitoes. The most differentially expressed pathway in the resistant group was identified, and a regulatory network was built using these SNPs and the differentially expressed genes (DEGs) in this pathway. The major candidate genes involved in the control of insecticide resistance were analyzed by qPCR, siRNA microinjection and CDC bottle bioassays. A total of 85 DEGs that encoded putative detoxification enzymes (including 61 P450s) were identified in this pathway. The resistance regulatory network was built using SNPs, and these metabolic genes, and a major gene, CYP9AL1, were identified. The functional role of CYP9AL1 in insecticide resistance was confirmed by siRNA microinjection and CDC bottle bioassays. Using the eQTL approach, we identified important genes in pyrethroid resistance that may aid in understanding the mechanism underlying insecticide resistance and in targeting new measures for resistance monitoring and management.