Molecular Genetics and Genomics

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Geographic location of Dulan tombs in this study. A Map showing the location of the Dulan site. Red line represents the southern Silk Road and yellow line represents Sino-Tibetan Road. Light red shape represents Tubo territory in the ninth century and dashed ellipse represents Tuyuhun territory under the regime of the Tubo Kingdom. B Map showing the distribution of tombs and sacrifice pits. Occupants of boxed tombs were analyzed in this study. C Archaeological relics excavated from the Dulan site in 2014
Uniparental analysis for Dulan people. A NMDS plot based on slatkin FST of mitochondrial HVS-I sequences between Dulan and 12 ancient groups. B NMDS plot based on slatkin FST of whole mitogenomes between Dulan and 20 modern populations. C Projection of Dulan onto PCA plot based on Y-chromosome haplogroup frequencies for 83 worldwide modern linguistic populations. D Y-chromosome haplogroup distribution during the first millennium AD
Genetic patterns of Dulan people and present-day East Asians, data collected from Allen Ancient DNA Resource version 44.3 (AADR 2021). A Projection of Dulan onto PCA for Eurasians. PC1 and PC2 were calculated using 2217 present-day Eurasian individuals and Dulan individuals were projected onto PC1 and PC2. B Projection of Dulan onto PCA for 1212 East Asians. C PCA for Dulan and 206 ancient Asian individuals, principal components constructed from present-day East Asians. D ADMIXTURE results at best K = 5 for Dulan individuals and present-day East Asian populations sorted by linguistic families
f4-statistics between Dulan individuals and ancient Eurasian populations of the form f4(DL5/DL6/DL8, China_Upper_YR_IA; X, Mbuti). The positive values show the greater affinity of the Dulan individual and X, and the negative values show the greater affinity of China_Upper_YR_IA and X compared to the Dulan individual and X. F4-statistics with |Z score|> 4 are marked in blue
The rise of the Tubo Kingdom is considered as the key period for the formation of modern groups on the Tibetan Plateau. The ethnic origin of the residents of the Tubo Kingdom is quite complex, and their genetic structure remains unclear. The tombs of the Tubo Kingdom period in Dulan County, Qinghai Province, dating back to the seventh century, are considered to be the remains left by Tubo conquerors or the Tuyuhun people dominated by the Tubo Kingdom. The human remains of these tombs are ideal materials for studying the population dynamics in the Tubo Kingdom. In this paper, we analyzed the genome-wide data of eight remains from these tombs by shotgun sequencing and multiplex PCR panels and compared the results with data of available ancient and modern populations across East Asia. Genetic continuity between ancient Dulan people with ancient Xianbei tribes in Northeast Asia, ancient settlers on the Tibetan Plateau, and modern Tibeto-Burman populations was found. Surprisingly, one out of eight individuals showed typical genetic features of populations from Central Asia. In summary, the genetic diversity of ancient Dulan people and their affiliations with other populations provide an example of the complex origin of the residents in the Tubo Kingdom and their long-distance connection with populations in a vast geographic region across ancient Asia.
 
A DNA sequence x is shredded into fragments of the same length n as that of the probes on an nxh basis so that the total number of fragments hybridizing with each oligo can be counted for each probe to obtain a feature vector from x. The oligos for the basis are judiciously selected in such a way that no cross hybridization occurs among probes in the basis itself and, moreover, that every random fragment hybridizes to (ideally exactly) one probe. An ideal basis thus produces feature vectors that are fully reproducible and contain much of the information in the original sequence x
Performance assessment of the definition of pathogenicity of bacteria and fungi using thresholding methods, based on the decision about hybridization events between oligos in the proxies of a host and a microorganism (Top: based on Gibbs Energy and Bottom: based on h-distance.) The x-axis represents different data sets for proxies and grids (IDs are in Table 4.)
Performance assessment of the definition of pathogenicity of bacteria (top), fungi (middle) and combined (bottom) obtained using machine learning models trained on genomic signatures
The current pandemic (COVID-19) has made evident the need to approach pathogenicity from a deeper and more systematic perspective that might lead to methodologies to quickly predict new strains of microbes that could be pathogenic to humans. Here we propose as a solution a general and principled definition of pathogenicity that can be practically implemented in operational ways in a framework for characterizing and assessing the (degree of) potential pathogenicity of a microbe to a given host (e.g., a human individual) just based on DNA biomarkers, and to the point of predicting its impact on a host a priori to a meaningful degree of accuracy. The definition is based on basic biochemistry, the Gibbs free Energy of duplex formation between oligonucleotides and some deep structural properties of DNA revealed by an approximation with certain properties. We propose two operational tests based on the nearest neighbor (NN) model of the Gibbs Energy and an approximating metric (the h-distance.) Quality assessments demonstrate that these tests predict pathogenicity with an accuracy of over 80%, and sensitivity and specificity over 90%. Other tests obtained by training machine learning models on deep features extracted from DNA sequences yield scores of 90% for accuracy, 100% for sensitivity and 80% for specificity. These results hint towards the possibility of an operational, objective, and general conceptual framework for prior identification of pathogens and their impact without the cost of death or sickness in a host (e.g., humans.) Consequently, a reasonable prediction of possible pathogens might pave the way to eventually transform the way we handle and prepare for future pandemic events and mitigate the adverse impact on human health, while reducing the number of clinical trials to obtain similar results.
 
Timelines of population-level effects of viruses on humans varied from the evolutionary scale of million years to contemporary spread of viral infections. Correspondingly, these events are exemplified by: (i) emergence of human endogenous retroviruses (HERVs) from ancient germline infections leading to stable integration of viral genomes into human chromosomes; and (ii) wide-spread viral infections reaching a global pandemic state such as the COVID-19 pandemic. Despite significant efforts, understanding of HERV’s roles in governance of genomic regulatory networks, their impacts on primate evolution and development of human-specific physiological and pathological phenotypic traits remains limited. Remarkably, present analyses revealed that expression of a dominant majority of genes (1696 of 1944 genes; 87%) constituting high-confidence down-steam regulatory targets of defined HERV loci was significantly altered in cells infected with the SARS-CoV-2 coronavirus, a pathogen causing the global COVID-19 pandemic. This study focused on defined sub-sets of DNA sequences derived from HERVs that are expressed at specific stages of human preimplantation embryogenesis and exert regulatory actions essential for self-renewal and pluripotency. Evolutionary histories of LTR7/HERVH and LTR5_Hs/HERVK were charted based on evidence of the earliest presence and expansion of highly conserved (HC) LTR sequences. Sequence conservation analyses of most recent releases 17 primate species’ genomes revealed that LTR7/HERVH have entered germlines of primates in Africa after the separation of the New World Monkey lineage, while LTR5_Hs/HERVK successfully colonized primates’ germlines after the segregation of Gibbons’ species. Subsequently, both LTR7 and LTR5_Hs undergo a marked ~ fourfold–fivefold expansion in genomes of Great Apes. Timelines of quantitative expansion of both LTR7 and LTR5_Hs loci during evolution of Great Apes appear to replicate the consensus evolutionary sequence of increasing cognitive and behavioral complexities of non-human primates, which seems particularly striking for LTR7 loci and 11 distinct LTR7 subfamilies. Consistent with previous reports, identified in this study, 351 human-specific (HS) insertions of LTR7 (175 loci) and LTR5_Hs (176 loci) regulatory sequences have been linked to genes implicated in establishment and maintenance of naïve and primed pluripotent states and preimplantation embryogenesis phenotypes. Unexpectedly, HS-LTRs manifest regulatory connectivity to genes encoding markers of 12 distinct cells’ populations of fetal gonads, as well as genes implicated in physiology and pathology of human spermatogenesis, including Y-linked spermatogenic failure, oligo- and azoospermia. Granular interrogations of genes linked with 11 distinct LTR7 subfamilies revealed that mammalian offspring survival (MOS) genes seem to remain one of consistent regulatory targets throughout ~ 30 MYA of the divergent evolution of LTR7 loci. Differential GSEA of MOS versus non-MOS genes identified clearly discernable dominant enrichment patterns of phenotypic traits affected by MOS genes linked with LTR7 (562 MOS genes) and LTR5_Hs (126 MOS genes) regulatory loci across the large panel of genomics and proteomics databases reflecting a broad spectrum of human physiological and pathological traits. GSEA of LTR7-linked MOS genes identified more than 2200 significantly enriched records of human common and rare diseases and gene signatures of 466 significantly enriched records of Human Phenotype Ontology traits, including Autosomal Dominant (92 genes) and Autosomal Recessive (93 genes) Inheritance. LTR7 regulatory elements appear linked with genes implicated in functional and morphological features of central nervous system, including synaptic transmission and protein–protein interactions at synapses, as well as gene signatures differentially regulated in cells of distinct neurodevelopmental stages and morphologically diverse cell types residing and functioning in human brain. These include Neural Stem/Precursor cells, Radial Glia cells, Bergman Glia cells, Pyramidal cells, Tanycytes, Immature neurons, Interneurons, Trigeminal neurons, GABAergic neurons, and Glutamatergic neurons. GSEA of LTR7-linked genes identified significantly enriched gene sets encoding markers of more than 80 specialized types of neurons and markers of 521 human brain regions, most prominently, subiculum and dentate gyrus. Identification and characterization of 1944 genes comprising high-confidence down-steam regulatory targets of LTR7 and/or LTR5_Hs loci validated and extended these observations by documenting marked enrichments for genes implicated in neoplasm metastasis, intellectual disability, autism, multiple cancer types, Alzheimer’s, schizophrenia, and other brain disorders. Overall, genes representing down-stream regulatory targets of ancient retroviral LTRs exert the apparently cooperative and exceedingly broad phenotypic impacts on human physiology and pathology. This is exemplified by altered expression of 93% high-confidence LTR targets in cells infected by contemporary viruses, revealing a convergence of virus-inflicted aberrations on genomic regulatory circuitry governed by ancient retroviral LTR elements and interference with human cells’ differentiation programs.
 
Heat stress transcription factors (Hsfs) are known to play a vital role in protecting plants against various abiotic stresses. Among the wild wheat relatives, Aegilops tauschii offers an excellent source of abiotic stress tolerance genes for improvement of bread wheat. However, little is known about its stress tolerance mechanisms. In this study, 22 AetHsf genes were identified in the genome of Aegilops tauschii and their chromosomal location, exon–intron structures, sub-cellular localization, phylogenetic and syntenic relationship were analyzed. Based on the conserved motif analysis, these Hsfs were further divided into group A, B and C. The interaction network analysis and expression profile of AetHsfs in different tissues predicted their interaction with diverse types of proteins and suggested their involvement in different developmental processes of the plant. The promoter analysis of AetHsfs showed the presence of abiotic stress-responsive, phytohormone-responsive, plant development-related and light-related cis-elements. Thus, we investigated the expression of Hsfs in Aegilops tauchii seedlings under various abiotic stress conditions and irradiated with different monochromatic lights. Most of the AetHsfs were found to be upregulated by heat stress, while some showed expression in drought, salinity and high light stress as well. Notably, the expression pattern of various AetHsfs showed their responsiveness toward dark and various light conditions (blue red and far-red) as well. Thus, this study provides novel insights into the potential role of AetHsfs in stress and light signaling pathways, which can further facilitate understanding of the stress tolerance mechanisms in Aegilops tauschii.
 
We sought to examine epigenetic inactivation of DNA damage repair (DDR) genes as prognostic and predictive biomarkers for urothelial bladder cancer (UBC) as there are currently no reliable prognostic biomarkers that identify UBC patients who would benefit from chemotherapy. Genome-wide DNA methylome using the cancer genome atlas-bladder cancer (TCGA-BLCA) datasets (primary tumors = 374 and normal tissues = 37) was performed for 154 DDR genes. The most two significant differentially methylated genes, Retinoblastoma binding protein 8 ( RBBP8) and MutS homologue 4 (MSH4) , between primary tumors and normal tissues of TCGA–BLCA were validated by methylation-specific PCR (MSP) in UBC ( n = 70) compared to normal tissues ( n = 30). RBBP8 and MSH4 expression was measured using qRT-PCR. We developed a predictive model for therapeutic response based on the RBBP8 - and MSH4 -methylation along with patients’ clinical features . Then, we assessed the prognostic significance of RBBP8 and MSH4 . RBBP8- and MSH4 methylation and corresponding gene downregulation significantly associated with muscle-invasive phenotype, prolonged progression-free survival (PFS) and increased susceptibility to cisplatin chemotherapy in UBC. Promoter methylation of RBBP8 and MSH4 was positively correlated with each other and with their corresponding gene repression. The best machine-learning classification model predicted UBC patients’ response to cisplatin-based chemotherapy with an accuracy of 90.05 ± 4.5%. Epigenetic inactivation of RBBP8 and MSH4 in UBC could sensitize patients to DNA-damaging agents. A predictive machine-learning modeling approach based on the clinical features along with RBBP8- and MSH4 -methylation might be a promising tool for stratification of UBC responders from nonresponders to chemotherapy.
 
Stratified plots to assess pleiotropic enrichment. Stratified QQ plots of nominal versus empirical –log10(p value) for A: BP as a function of significance of association with AD, and B: AD as a function of significance of association with BP. The null hypothesis of no pleiotropic enrichment is represented by the black line. Fold-enrichment plots of enrichment versus nominal –log10(p value) for C: BP as a function of significance of association with AD, and D: AD as a function of significance of association with BP. The purple line represents all SNPs and has a slope of zero
Manhattan plot of conjunction –log10ccFDR values for AD and BP. The red line marks the conjunction –log10ccFDR value of 1.3, corresponding to a ccFDR of 0.05. The figure shows the chromosomal locations of the identified shared variants, with variants above the red line being significant pleiotropic loci
Protein–protein interaction network. 34 of the 37 identified pleiotropic genes were found in the STRING database and mapped to create a protein–protein interaction network. Blue lines represent known interactions from curated databases, pink lines represent experimentally determined known interactions, red lines represent predicted interactions due to gene fusion, green lines represent interactions curated from text mining, and black lines represent co-expression
Alzheimer’s disease (AD) and high blood pressure (BP) are prevalent age-related diseases with significant unexplained heritability. A thorough analysis of genetic pleiotropy between AD and BP will lay a foundation for the study of the associated molecular mechanisms, leading to a better understanding of the development of each phenotype. We used the conditional false discovery rate (cFDR) method to identify novel genetic loci associated with both AD and BP. The cFDR approach improves the effective sample size for association testing by combining GWAS summary statistics for correlated phenotypes. We identified 50 pleiotropic SNPs for AD and BP, 7 of which are novel and have not previously been reported to be associated with either AD or BP. The novel SNPs located at STK3 are particularly noteworthy, as this gene may influence AD risk via the Hippo signaling network, which regulates cell death. Bayesian colocalization analysis demonstrated that although AD and BP are associated, they do not appear to share the same causal variants. We further performed two sample Mendelian randomization analysis, but could not detect a causal effect of BP on AD. Despite the inability to establish a causal link between AD and BP, our findings report some potential novel pleiotropic loci that may influence disease susceptibility. In summary, we identified 7 SNPs that annotate to 4 novel genes which have not previously been reported to be associated with AD nor with BP and discuss the possible role of one of these genes, STK3 in the Hippo signaling network.
 
Pairwise linkage disequilibrium plot for examined Fyn and Lyn gene polymorphisms in A thyroid cancer patients and B controls. Site 1 is for rs6916861, site 2 is for rs2182644, site 3 for rs12910, site 4 for r s2668011, site 5 for r rs45587541 and site 6 for rs45489500. The darker region shows higher r2-value
Thyroid cancer is the most common malignancy of the endocrine glands, and during last couple of decades, its incidence has risen alarmingly, across the globe. Etiology of thyroid cancer is still debatable. There are a few worth mentioning risk factors which contribute to initiation of abnormalities in thyroid gland leading to cancer. Genetic instability is major risk factors in thyroid carcinogenesis. Among the genetic factors, the Src family of genes (Src, Yes1, Fyn and Lyn) have been implicated in many cancers but there is little data regarding the association of these (Src, Yes1, Fyn and Lyn) genes with thyroid carcinogenesis. Fyn and Lyn genes of Src family found engaged in proliferation, migration, invasion, angiogenesis, and metastasis in different cancers. This study was planned to examine the effect of Fyn and Lyn SNPs on thyroid cancer risk in Pakistani population in 500 patients and 500 controls. Three polymorphisms of Fyn gene (rs6916861, rs2182644 and rs12910) and three polymorphisms of Lyn gene (rs2668011, rs45587541 and rs45489500) were analyzed using Tetra-primer ARMS-PCR followed by DNA sequencing. SNP rs6916861 of Fyn gene mutant genotype (CC) showed statistically significant threefold increased risk of thyroid cancer (P < 0.0001). In case of rs2182644 of Fyn gene, mutant genotype (AA) indicated statistically significant 17-fold increased risk of thyroid cancer (P < 0.0001). Statistically significant threefold increased risk of thyroid cancer was observed in genotype AC (P < 0.0001) of Fyn gene polymorphism rs12910. In SNP rs2668011 of Lyn gene, TT genotype showed statistically significant threefold increased risk of thyroid cancer (P < 0.0001). In case of rs45587541 of Lyn gene, GA genotypes showed statistically significant 11-fold increased risk in thyroid cancer (P < 0.0001). Haplotype analysis revealed that AAATAG*, AGACAG*, AGCCAA*, AGCCAG*, CAATAG*, CGCCAG* and CGCCGA* haplotypes of Fyn and Lyn polymorphisms are associated with increased thyroid cancer risk. These results showed that genotypes and allele distribution of Fyn and Lyn are significantly linked with increased thyroid cancer risk and could be genetic adjuster for said disease.
 
Increased fetal nuchal translucency (NT) is a common ultrasonic manifestation during pregnancy. Many studies have confirmed that NT ≥ 3 mm is a high risk factor for adverse pregnancy outcome. However, when NT is between 2.5 and 2.9 mm, will it increase the risk of fetal chromosome abnormalities and other diseases? What is the most appropriate method for prenatal chromosome evaluation? At present, it has not been widely reported in the literature, and the conclusion is also controversial. This prospective cohort study included fetal samples from women who underwent amniocentesis from 2017 to 2020. The samples of the experimental group were fetuses with NT ≥ 2.5 mm at 11 to 13 + 6 weeks of gestation, with or without ultrasonographic anomaly. The control group contained fetal NT < 2.5 mm without ultrasonographic anomalies. All amniotic fluid samples were tested by copy number variants sequencing. In 262 fetal samples with isolated NT from 2.5 to 2.9 mm, the detection rate of aneuploidy was 3.4% (9/262), and the risk of aneuploidy was significantly higher than that of the control group (1.4%, 32/2331) (relative risk 2.5, 95% CI 1.2–5.2). The detection rates of other pathogenic/likely pathogenic copy number variants in the two groups were 0.8% (2/262) and 1.3% (31/2331), respectively, which was not statistically significant (relative risk 0.6, 95% CI 0.1–2.4). Our results showed that isolated NT from 2.5 to 2.9 mm increased the risk of fetal chromosome aneuploidy. Therefore, noninvasive prenatal screening is recommended as the first choice for prenatal chromosome evaluation in this population.
 
Pictures of the male voucher specimen of Ancistrus triradiatus Eigenmann, 1918 used for the whole genome sequencing (voucher number MHNG2786065 belonging to the collection of the Museum of Natural History of Geneva, Switzerland). Lateral (top), ventral (middle) and dorsal (bottom) views. This specimen is 74.21 mm in Standard Length (SL)
Synteny analysis between the reported genome of Ancitrus triradiatus and the genome of Ictalurus punctatus (Liu et al. 2016). Only scaffolds > 250 KB are considered for the A. triraditus genome assembly (small numbered bars on the left and bottom part of the external circle) while full chromosomes were considered for the Ictalurus punctatus genome assembly (numbered grey boxes on the right side of the external circle)
Gene family expansion and contraction in seven actinopterygian species related to Ancistrus triradiatus. The number of expanded gene families is shown in green (upper part of the circle), while the number of contracted gene families is shown in purple (lower part of the circle). Gene family expansion/contraction analysis was performed with CAFE5 (Mendes et al. 2020), and using previously published genomes (see “Matherials and methods”). The phylogenetic tree was extracted from the actinopterygian phylogeny of Rabosky et al. (2018)
SCPP gene family in Ancistrus triradiatus as compared to two reference species, Danio rerio and Ictalurus punctatus
Opsin gene repertoire in Ancistrus triradiatus as compared to two reference species, Danio rerio and Silurus meridionalis
The catfish Ancistrus triradiatus belongs to the species-rich family Loricariidae. Loricariids display remarkable traits such as herbivory, a benthic lifestyle, the absence of scales but the presence of dermal bony plates. They are exported as ornamental fish worldwide, with escaped fishes becoming a threat locally. Although genetic and phylogenetic studies are continuously increasing and developmental genetic investigations are underway, no genome assembly has been formally proposed for Loricariidae yet. We report a high-quality genome assembly of Ancistrus triradiatus using long and short reads, and a newly assembled transcriptome. The genome assembly is composed of 9530 scaffolds, including 85.6% of ray-finned fish BUSCOs, and 26,885 predicted protein-coding genes. The genomic GC content is higher than in other catfishes, reflecting the higher metabolism associated with herbivory. The examination of the SCPP gene family indicates that the genes presumably triggering scale loss when absent, are present in the scaleless A. triradiatus , questioning their explanatory role. The analysis of the opsin gene repertoire revealed that gene losses associated to the nocturnal lifestyle of catfishes were not entirely found in A. triradiatus , as the UV-sensitive opsin 5 is present. Finally, most gene family expansions were related to immunity except the gamma crystallin gene family which controls pupil shape and sub-aquatic vision. Thus, the genome of A. triradiatus reveals that fish herbivory may be related to the photic zone habitat, conditions metabolism, photoreception and visual functions. This genome is the first for the catfish suborder Loricarioidei and will serve as backbone for future genetic, developmental and conservation studies.
 
Three exons are shown by boxes and α/β domains are indicated by colored boxes. Exon 1 spans codons 1 to 113, exon 2 spans 114 to 154, and exon 3 spans 155 to 213. The HIF-a binding, Site CCT complex binding site and Elongin C binding site are shown in red, orange and pink linear, respectively
Flow diagram eligibility criteria of articles on VHL Mutations in RCH
The Prevalence of RCH within Regions
Retinal capillary hemangioblastomas (RCH) is a benign tumor that represents the initial manifestation in roughly half of Von Hippel Lindau (VHL) patients. They may also occur sporadically without systemic involvement. A first meta-analysis study was investigated to estimate the prevalence of Retinal capillary hemangioblastoma (RCH) in Von Hippel Lindau (VHL) syndrome, and its relation to type and location of mutations in VHL gene. The electronic databases of PubMed, Scopus, Embase, and Google Scholar were utilized to find eligible papers published up to May 2020. Lastly, after the different prevalence of RCH in Europe compared to other continents was noted, we decided to consider European and non-European patients separately. The Random effect model was used to evaluate the relation between developing RCH and types of mutations. The overall prevalence of RCH among VHL patients is about 47%. The prevalence of RCH was significantly higher in Europe in comparison with non-Europeans (p value < 0.001). Overall, the differences between the prevalence of RCH among different mutation types were not statistically significant. However, in Europe, the prevalence of RCH was significantly higher in patients with truncation mutation (p value = 0.007). In Europe, the RCH in VHL patients who had a mutation in exon 2 was significantly lower in comparison with exon 1 (p value = 0.001); but in non-Europeans, the prevalence of RCH in VHL patients that involved exon 2 was significantly higher in comparison with VHL patients with a mutation in exon1 (p value = 0.012). The highest risk of developing RCH was reported among Europeans. Overall, this study showed that the prevalence of RCH in VHL syndrome is not related to type or location of mutations and difference of RCH prevalence is probably depends on other genetic or environmental factor that should be considered in subsequent studies.
 
Pedigrees and genetic findings in families with hereditary neurological disorders. Top bold text shows the name of each family. The double horizontal lines in the pedigrees indicate consanguinity whereas squares and circles symbolize males and females, respectively. Filled symbols indicate affected individuals; those with a diagonal line are deceased. Electropherograms of healthy (upper panel) and affected (lower panel) individuals. WT, Wild type; M, Mutant; M1–M9, mutation 1–mutation 9. Position of the variants are highlighted with red arrow
Hereditary neurological disorders (HNDs) are a clinically and genetically heterogeneous group of disorders. These disorders arise from the impaired function of the central or peripheral nervous system due to aberrant electrical impulses. More than 600 various neurological disorders, exhibiting a wide spectrum of overlapping clinical presentations depending on the organ(s) involved, have been documented. Owing to this clinical heterogeneity, diagnosing these disorders has been a challenge for both clinicians and geneticists and a large number of patients are either misdiagnosed or remain entirely undiagnosed. Contribution of genetics to neurological disorders has been recognized since long; however, the complete picture of the underlying molecular bases are under-explored. The aim of this study was to accurately diagnose 11 unrelated Pakistani families with various HNDs deploying NGS as a first step approach. Using exome sequencing and gene panel sequencing, we successfully identified disease-causing genomic variants these families. We report four novel variants, one each in, ECEL1, NALCN, TBR1 and PIGP in four of the pedigrees. In the rest of the seven families, we found five previously reported pathogenic variants in POGZ, FA2H, PLA2G6 and CYP27A1. Of these, three families segregate a homozygous 18 bp in-frame deletion of FA2H, indicating a likely founder mutation segregating in Pakistani population. Genotyping for this mutation can help low-cost population wide screening in the corresponding regions of the country. Our findings not only expand the existing repertoire of mutational spectrum underlying neurological disorders but will also help in genetic testing of individuals with HNDs in other populations.
 
Timing of stem elongation (days to elongation—DTE) among 672 individuals of red clover, cultivar “Lea”. Hundred and forty-six individuals did not start stem elongation during the 95 days of the experiment and are not shown. The shaded areas at each end of the histogram show the groups of individuals that were selected for genotyping
Principal component analysis of minor allele frequencies of 66,458 chromosomal SNPs in three early (stars) and three late elongating (triangles) pools, each consisting of equal amounts of DNA from each of 17–18 red clover individuals
Single-nucleotide polymorphism and haplotype polymorphism with significantly different allele frequencies in early versus late elongating pools of red clover according to three different methods. All non-significant markers are also plotted. X-axes, chromosomal position (Mb); y-axes, FST value of the marker, based on allele frequencies in early versus late elongating pools
Main conclusion Through selective genotyping of pooled phenotypic extremes, we identified a number of loci and candidate genes putatively controlling timing of stem elongation in red clover. Abstract We have identified candidate genes controlling the timing of stem elongation prior to flowering in red clover ( Trifolium pratense L.). This trait is of ecological and agronomic significance, as it affects fitness, competitivity, climate adaptation, forage and seed yield, and forage quality. We genotyped replicate pools of phenotypically extreme individuals (early and late-elongating) within cultivar Lea using genotyping-by-sequencing in pools (pool-GBS). After calling and filtering SNPs and GBS locus haplotype polymorphisms, we estimated allele frequencies and searched for markers with significantly different allele frequencies in the two phenotypic groups using BayeScan, an F ST -based test utilizing replicate pools, and a test based on error variance of replicate pools. Of the three methods, BayeScan was the least stringent, and the error variance-based test the most stringent. Fifteen significant markers were identified in common by all three tests. The candidate genes flanking the markers include genes with potential roles in the vernalization, autonomous, and photoperiod regulation of floral transition, hormonal regulation of stem elongation, and cell growth. These results provide a first insight into the potential genes and mechanisms controlling transition to stem elongation in a perennial legume, which lays a foundation for further functional studies of the genetic determinants regulating this important trait.
 
Clinical features of the affected children. A Blister in the skin, B Tracheostomy, C Gastroduodenostomy, D Urostomy
Upper panel: pedigree of the affected family; Lower panel: workflow for the identification of putative disease-causing mutation
Epidermolysis-Bullosa (EB), a rare Mendelian disorder, exhibits complex phenotypic and locus-heterogeneity. We identified a nuclear family of clinically unaffected parents with two offsprings manifesting EB-Pyloric-Atresia (EB-PA), with a variable clinical severity. We generated whole exome sequence data on all four individuals to (1) identify the causal mutation behind EB-PA (2) understand the background genetic variation for phenotype variability of the siblings. We assumed an autosomal recessive mode of inheritance and used suites of bioinformatic and computational tools to collate information through global databases to identify the causal genetic variant for the disease. We also investigated variations in key genes that are likely to impact phenotype severity. We identified a novel missense mutation in the ITGB4 gene (p.Ala1227Asp), for which the parents were heterozygous and the children homozygous. The mutation in ITGB4 gene, predicted to reduce the stability of the primary alpha6beta4-plectin complex compared to all previously studied mutations on ITGB4 reported to cause EB.
 
Pedigrees and variants were identified in family 1 (a, b) and family 2 (c, d). The individuals with available DNA samples were marked with asterisks. The black-filled shapes represent patients with eye disorders. The sequences of the affected were from individuals marked by black arrows. WT wild type, VT variant type
Functional prediction by bioinformatics. a The position of variants and diagram of the GJA8 protein domains spanning the cell membrane. b Hydrophilicity analysis of the wild type and variant proteins. Protscale results showed a slight change of hydrophilicity in W45L. The x-axis represents the position of the amino acid while the y-axis represents the score of hydrophobicity. The negative value means hydrophilic while the positive is hydrophobic. c Prediction of transmembrane helices in proteins. The diagram on the left shows the probability of amino acid positions in the transmembrane region (dark red), inside (blue) or outside (light red). The text on the right shows the predicted amino acid positions in detail. In W45L, the 45th amino acid of Cx50 is converted from the transmembrane domain to the extracellular region, and values are highlighted with red boxes. Transmembrane domains (TM1–TM4), extracellular (EC1–EC2) domains, intracellular loop (IC), NH2-terminus (NT), COOH-terminus (CT), and amino acid (Aa)
Quantitative analysis by CCK-8 assay of cell proliferation. Cell growth curve of 293T (a) and HeLa (b) cells transfected with indicated plasmids for 0, 24, 48, and 72 h. Cell viability of 293T (c) and HeLa (d) cells represented by OD450nm value at 72 h. *p < 0.05, **p < 0.01, ***p < 0.001. ns not significant. The unspecified represent results were compared to the pcDNA3.1
Protein expression of Cx50-flag in 293T cells. The expression level of WT (a), W45L (b), G94E (c) at different transfection times. All the three groups basically got their highest expression level around 30–36 h and dropped to a relatively lowest level at about 48 h. d A lower expression in W45L compared with WT at 30 h after transfection. e A higher lever in G94E in contrast with WT at 48 h after transfection. WT and the variant recombinant protein were detected with the anti-flag antibody. GAPDH was served as the loading control. Cells transfected with empty vector pcDNA3.1 and cells without any treatment were served as the negative control. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. WT wild-type Cx50, W45L-Cx50 p.Try45Leu, G94E Cx50 p.Gly94Glu, NT not treated, ns not significant
Subcellular localization of Cx50-flag proteins. Cells were immunostained with anti-flag monoclonal antibody (green). DAPI shows nuclear DNA staining (blue). F-actin in Rhodamine staining was used to show the morphology of cells (red). WT (a) and G94E (c) were mainly distributed in the cytoplasm and formed abundant punctate staining. W45L (b) showed less fluorescence and few punctate staining in the cytoplasm. Cells transfected with empty vector pcDNA3.1 were served as the negative control (d). The framed areas of merged images were magnified and displayed at the bottom. Fluorescent patches around the cell membrane as indicated by the white arrows may be hemichannel signals. Bar 50 µm
In this study, we report on two different GJA8 variants related to congenital eye anomalies in two unrelated families, respectively. GJA8 (or Cx50) encoding a transmembrane protein to form lens connexons has been known as a common causative gene in congenital cataracts and its variants have recently been reported related to a wide phenotypic spectrum of eye defects. We identified two GJA8 variants, c.134G>T (p.Try45Leu, W45L) detected in a cataract family by Sanger sequencing and c.281G>A (p.Gly94Glu, G94E) found in a family with severe eye malformations including microphthalmia by whole-exome sequencing. These two variants were absent in healthy population and predicted deleterious by bioinformatic analysis. Furthermore, we compared the expression in cell lines between these mutants and the wildtype to explore their potential mechanism. Cell counting kit-8 assay showed that overexpression of either W45L or G94E decreased cell viability compared with wild-type Cx50 and the control. A lower protein level in W45L found by western blotting and fewer punctate fluorescent signals showed by fluorescence microscopy suggested that W45L may have less protein expression. A higher G94E protein level and abundant dotted distribution indicated that G94E may cause aberrant protein degradation and accumulation. Such results from in vitro assays confirmed the impact of these two variants and gave us a hint about their different pathogenic roles in different phenotypes. In conclusion, our study is the first to have the functional analysis of two GJA8 variants c.134G>T and c.281G>A in Chinese pedigrees and explore the impact of these variants, which can help in prenatal diagnosis and genetic counseling as well in basic studies on GJA8.
 
In bacteria, sigma factors are crucial in determining the plasticity of core RNA polymerase (RNAP) while promoter recognition during transcription initiation. This process is modulated through an intricate regulatory network in response to environmental cues. Previously, an extracytoplasmic function (ECF) sigma factor, AlgU, was identified to positively influence the fitness of Pseudomonas aeruginosa PGPR2 during corn root colonization. In this study, we report that the inactivation of the algU gene encoded by PGPR2_23995 hampers the root colonization ability of PGPR2. An insertion mutant in the algU gene was constructed by allele exchange mutagenesis. The mutant strains displayed threefold decreased root colonization efficiency compared with the wild-type strain when inoculated individually and in the competition assay. The mutant strain was more sensitive to osmotic and antibiotic stresses and showed higher resistance to oxidative stress. On the other hand, the mutant strain showed increased biofilm formation on the abiotic surface, and the expression of the pelB and pslA genes involved in the biofilm matrix formation were up-regulated. In contrast, the expression of algD, responsible for alginate production, was significantly down-regulated in the mutant strain, which is directly regulated by the AlgU sigma factor. The mutant strain also displayed altered motility. The expression of RNA binding protein RsmA was also impeded in the mutant strain. Further, the transcript levels of genes associated with the type III secretion system (T3SS) were analyzed, which revealed a significant down-regulation in the mutant strain. These results collectively provide evidence for the regulatory role of the AlgU sigma factor in modulating gene expression during root colonization.
 
The delayed diagnosis of pancreatic cancer has resulted in rising mortality rate and low survival rate that can be circumvented using potent theranostics biomarkers. The treatment gets complicated with delayed detection resulting in lowered 5-year relative survival rate. In our present study, we employed systems biology approach to identify central genes that play crucial roles in tumor progression. Pancreatic cancer genes collected from various databases were used to construct a statistically significant interactome with 812 genes that was further analysed thoroughly using topological parameters and functional enrichment analysis. The significant genes in the network were then identified based on the maximum degree parameter. The overall survival analysis indicated through hazard ratio [HR] and gene expression [log Fold Change] across pancreatic adenocarcinoma revealed the critical role of FN1 [HR 1.4; log2(FC) 5.748], FGA [HR 0.78; log2(FC) 1.639] FGG [HR 0.9; log2(FC) 1.597], C3 [HR 1.1; log2(FC) 2.637], and QSOX1 [HR 1.4; log2(FC) 2.371]. The functional significance of the identified hub genes signified the enrichment of integrin cell surface interactions and proteoglycan syndecan-mediated cell signaling. The differential expression, low overall survival and functional significance of FN1 gene implied its possible role in controlling metastasis in pancreatic cancer. Furthermore, alternate splice variants of FN1 gene showed 10 protein coding transcripts with conserved cell attachment site and functional domains indicating the variants’ potential role in pancreatic cancer. The strong association of the identified hub-genes can be better directed to design potential theranostics biomarkers for metastasized pancreatic tumor.
 
Sex-specific differences in human DNA methylation and its maintenance
Human evolution has shaped gender differences between males and females. Over the years, scientific studies have proposed that epigenetic modifications significantly influence sex-specific differences. The evolution of sex chromosomes with epigenetics as the driving force may have led to one sex being more adaptable than the other when exposed to various factors over time. Identifying and understanding sex-specific differences, particularly in DNA methylation, will help determine how each gender responds to factors, such as disease susceptibility, environmental exposure, brain development and neurodegeneration. From a medicine and health standpoint, sex-specific methylation studies have shed light on human disease severity, progression, and response to therapeutic intervention. Interesting findings in gender incongruent individuals highlight the role of genetic makeup in influencing DNA methylation differences. Sex-specific DNA methylation studies will empower the biotechnology and pharmaceutical industry with more knowledge to identify biomarkers, design and develop sex bias drugs leading to better treatment in men and women based on their response to different diseases.
 
The histology of tissues from patients with LSCC and control subjects. Representative photomicrographs showing the histology of tissues from patients with LSCC and control subjects (original magnification × 400). LSCC, laryngeal squamous cell carcinoma
Increased Myosin X and Cortactin expression in tissues from LSCC. A The mRNA expression levels of Myosin X and Cortactin in tissue samples in different study groups as detected by quantitative RT-PCR. Control, n = 6; poorly differentiated, n = 12; moderately differentiated, n = 9; highly differentiated, n = 9; B Myosin X mRNA expression positively correlated with Cortactin mRNA in tissues in patients with LSCC; C the protein levels of Myosin X and Cortactin in tissue samples in different study groups as detected by western blotting. Control, n = 5; poorly differentiated, n = 5; moderately differentiated, n = 5; highly differentiated, n = 5. ***P < 0.001
Increased MMP2 and MMP9 expression in tissues from LSCC. A The mRNA expression levels of MMP2 and MMP9 in tissue samples in different study groups as detected by quantitative RT-PCR. Control, n = 6; poorly differentiated, n = 12; moderately differentiated, n = 9; highly differentiated, n = 9; B the protein levels of MMP2 and MMP9 in tissue samples in different study groups as detected by ELISA. Control, n = 6; poorly differentiated, n = 12; moderately differentiated, n = 9; highly differentiated, n = 9; C Myosin X mRNA expression positively correlated with MMP2 and MMP9 mRNA in tissues in patients with LSCC. ***P < 0.001. D AMC-HN-8 cells were transfected with siRNA targeting Myosin X or control siRNA for 48 h. Cells or cell culture supernatants were subjected to RT-PCR assay (n = 5). ***P < 0.001
The expression of E-cadherin and β-catenin in tissues from LSCC. A The immunoreactivity of E-cadherin (left panel) and β-catenin (right panel) in LSCC tissues. The representative photomicrographs of a highly differentiated LSCC sample are shown (original magnification × 400); B the protein levels of E-cadherin and β-catenin in tissue samples in different study groups as detected by western blotting. ***P < 0.001
Elevated expression of Myosin X correlates with lymph node metastasis. The correlation between Myosin X expression and lymph node metastasis by a 2-year follow-up
Laryngeal Squamous Cell Carcinoma (LSCC) is one of the most common malignancy in Head and neck cancer for which the mechanism underlying its metastasis is poorly understood. Myosin X, a molecular motor in cells has been demonstrated to play an important role in cell migration. However, whether Myosin X is involved in the metastasis of LSCC remains unclear. To investigate the expression of Myosin X and its implication in the metastasis of LSCC, we recruited 30 patients with LSCC and 6 patients with vocal cord polyp range from October 2016 to October 2018. Tissue samples were obtained during surgery and the expression of Myosin X, Cortactin, MMP2, MMP9, E-cadherin, and β-catenin in tissue samples were evaluated by RT-PCR, Western blot, immunohistochemistry or ELISA. Patients with LSCC were further followed-up 2 year after surgery for metastasis analysis. We found that the level of Myosin X, Cortactin, MMP2, and MMP9 was much higher in poorly differentiated LSCC compared to that in moderately and highly LSCC, as well as the control tissues. In contrast, the expression of epithelial-mesenchymal transition related marker, E-cadherin, and β-catenin, were much lower in poorly differentiated LSCC tissues compared to that in moderately and highly differentiated LSCC tissues, as well as the control tissues. Moreover, the expression of Myosin X was positively correlated with Cortactin, MMP2, and MMP9 levels. Increased expression of Myosin X in LSCC tissues was related to higher risk of metastasis. In conclusion, our findings showed that. Myosin X augments the expression of Cortactin, MMP2 and MMP9, which could upregulate the cell migration and the matrix degradation, and consequently reduce the expression of E-cadherin and β-catenin, thereby activating epithelial-mesenchymal transformation and promoting the metastasis of LSCC. Targeting Myosin X may have potential therapeutic effect in the metastasis of LSCC.
 
Relative HMGA1 expression level in cancer tissue and blood samples at three time points
Relative HMGA1 expression level measured 1 year after surgery concerning tobacco smoking status
Overall survival probability according to HMGA1 expression level measured in cancer tissue (A) and blood samples collected at the time of diagnosis (B), 100 days after surgery (C), and 1 year after surgery (D)
The study aimed to assess the HMGA1 gene expression level in NSCLC patients and to evaluate its association with selected clinicopathological features and overall survival of patients. The expression of the HMGA1, coding non-histone transcription regulator HMGA1, was previously proved to correlate with the ability of cancer cells to metastasize the advancement of the disease. The prognostic value of the HMGA1 expression level was demonstrated in some neoplasms, e.g., pancreatic, gastric, endometrial, hepatocellular cancer, but the knowledge about its role in non-small cell lung cancer (NSCLC) is still limited. Thus, the HMGA1 expression level was evaluated by real-time PCR method in postoperative tumor tissue and blood samples collected at the time of diagnosis, 100 days and 1 year after surgery from 47 NSCLC patients. Mean HMGA1 expression level in blood decreased systematically from the time of cancer diagnosis to 1 year after surgery. The blood HMGA1 expression level 1 year after surgery was associated with the tobacco smoking status of patients (p= 0.0230). Patients with high blood HMGA1 expression levels measured 100 days after surgery tend to have worse overall survival than those with low expression levels (p= 0.1197). Tumor HMGA1 expression level was associated with neither features nor the overall survival of NSCLC patients. Moreover, no correlation between HMGA1 expression level measured in tumor tissue and blood samples was stated. Blood HMGA1 mRNA level could be a promising factor in the prognostication of non-small cell lung cancer patients.
 
Analysis and model construction workflow. CV cross validation. (1) A step-by-step logistic regression was used to select significant SNPs (p < 0.05). At this step, obesity status was the dependent variable. (2) Final logistic regression model using the genetic risk scores. This model was utilized instead of threshold finding for the risk scores. (3) This logistic regression model was used to determine the effect size of each SNP which was used in the construction of the weighted model. At each step of the cross-validation, the effect sizes were calculated solely based on the training dataset
Forest plot of genetic variants significantly associated with the risk of developing obesity
Receiver operating characteristics (ROC) curves of the weighted GRS (A) and unweighted GRS (B) with tenfold cross validation
Obesity is a major public health issue resulting from an interaction between genetic and environmental factors. Genetic risk scores (GRSs) are useful to summarize the effects of many genetic variants on obesity risk. In this study, we aimed to assess the association of previously well-studied genetic variants with obesity and develop a genetic risk score to anticipate the risk of obesity development in the Iranian population. Among 968 participants, 599 (61.88%) were obese, and 369 (38.12%) were considered control samples. After genotyping, an initial screening of 16 variants associated with body mass index (BMI) was performed utilizing a general linear model (p < 0.25), and seven genetic variants were selected. The association of these variants with obesity was examined using a multivariate logistic regression model (p < 0.05), and finally, five variants were found to be significantly associated with obesity. Two gene score models (weighted and unweighted), including these five loci, were constructed. To compare the discriminative power of the models, the area under the curve was calculated using tenfold internal cross‐validation. Among the studied variants, ADRB3 rs4994, FTO rs9939609, ADRB2 rs1042714, IL6 rs1800795, and MTHFR rs1801133 polymorphisms were significantly associated with obesity in the Iranian population. Both of the constructed models were significantly associated with BMI (p < 0.05) and the area under the mean curve of the weighted GRS and unweighted GRS were 70.22% ± 0.05 and 70.19% ± 0.05, respectively. Both GRSs proved to predict obesity and could potentially be utilized as genetic tools to assess the obesity predisposition in the Iranian population. Also, among the studied variants, ADRB3 rs4994 and FTO rs9939609 polymorphisms have the highest impacts on the risk of obesity.
 
Morphological analysis of MucX. Ultrathin sections of MucX cultured in BTTM medium containing Glc observed under TEM (A). Optical microscope images of gram-stained cells of MucX cultured in BTTM medium containing GlcNAc (B) and Glc (F), respectively. Negative stain TEM images of MucX cultured in BTTM containing GlcNAc (C–E) and Glc (G and H), respectively. GlcNAcN-acetylglucosamine, Glc glucose
Phylogroups of MucT. A Maximum likelihood phylogenetic tree based on 16S rRNA gene sequences. The numbers at the nodes indicate bootstrap values for 100 replicates and the tree was generated considering only unambiguously aligned nucleotide positions (n = 1372). The tree was rooted using the only other named species of the genus, Akkermansia glycaniphila Pyt. B Heatmap showing the average nucleotide identity values determined for A. muciniphila strains genomes
Circular representation of genomes of MucX and another six strains. The inner circle represents the genome of MucX as a reference. Unique regions of MucX are marked in red in the outermost circle. Colored rings represent regions that are presented on both reference and compared genomes. Conversely, white gaps indicate that certain sections are not presented on this strain
Growth curves (A) and pH changes (B) of MucX and MucT in BTTM medium containing 2′-fucosyllactose (2′-FL), galacto-oligosaccharide (GOS) and lactose (Lac), respectively. Solid lines represent MucX, dotted lines represent MucT. Results are given as the mean ± SD, N = 3 in each group
TLC profiles of degradation of 2′-FL (A), GOS and Lac (B) during in vitro fermentation for 70 h, respectively. MucT was used to be compared with MucX. (C) HPLC spectra showing the degradation of 2′-FL by MucT and MucX. Fuc fucose
Akkermansia muciniphila is considered to be a next-generation probiotic, and closely related to host metabolism and immune response. Compared with other probiotics, little is known about its genomic analysis. Therefore, further researches about isolating more A. muciniphila strains and exploring functional genes are needed. In the present study, a new strain isolated from mice feces was identified as A. muciniphila (MucX). Whole-genome sequencing and annotation revealed that MucX possesses key genes necessary for human milk oligosaccharides (HMO) utilization, including α-l-fucosidases, β-galactosidases, exo-α-sialidases, and β-acetylhexosaminidases. The complete metabolic pathways for γ-aminobutyric acid and squalene and genes encoding functional proteins, such as the outer membrane protein Amuc_1100, were annotated in the MucX genome. Comparative genome analysis was used to identify functional genes unique to MucX compared to six other A. muciniphila strains. Results showed MucX genome possesses unique genes, including sugar transporters and transferases. Single-strain incubation revealed faster utilization of 2′-fucosyllactose (2′-FL), galacto-oligosaccharides, and lactose by MucX than by A. muciniphila DSM 22959. This study isolated and identified an A. muciniphila strain that can utilize 2′-FL, and expolored the genes related to HMO utilization and special metabolites, which provided a theoretical basis for the further excavation of A. muciniphila function and the compound application with fucosylated oligosaccharides.
 
Splicing disruption is one type of mutation mechanism for disease-predisposing alleles. To date, less than 30 mutations in TTC8/BBS8 have been reported; however, mutations affecting the splice site are rare. Generally missense mutations are assumed to alter protein function; however, reports have shown that mutations in protein coding exons can disrupt splicing by altering exonic splicing silencer or enhancer motifs. Hence, a missense mutation c.1347G > C (p.Q449H) involving final base of the exon 13 in the TTC8, previously identified by us to be linked with non-syndromic autosomal recessive retinitis pigmentosa (arRP), in an Indian family, that might deleteriously affect splicing has been functionally characterized. RNA was isolated, cDNA prepared and amplified using region-specific primers. PCR products were purified and sequenced bi-directionally by Sanger sequencing. Effect of mutation (c.1347G > C) on mRNA splicing has been predicted using bioinformatics tools. We reported that missense mutation (c.1347G > C) at the last base of exon 13 of TTC8 disrupted the canonical donor splice-site resulting in aberrant RNA splicing. A cryptic donor splice-site got activated 77 bases downstream of the authentic splice donor site in intron 13, resulting in the retention of 77 bases of intron 13, and a frameshift leading to pre-mature termination codon in exon 14 at codon 486. Further, duplication of exon 15 and fusion of its duplicated copy occurred with exon 13. The binding site for SC35 protein, normally involved in splicing, also got disrupted (as predicted by SpliceAid2 software), hence, leading to alternative splicing. Our findings strongly suggest that a missense mutation c.1347G > C in TTC8 disrupted the splice donor site causing retention of 77 bases of intron 13, resulting in a frameshift and subsequently introduced a pre-mature termination codon into exon 14, hence creating an altered mRNA transcript. These findings emphasize the significance of examining missense mutations especially in TTC8, to determine their pathogenic role through alternative splicing. Present findings also reiterate the notion that mutations in the TTC8/BBS8 cause phenotypic heterogeneity and does not always follow Mendelian genetics in this ciliopathy.
 
Segregation of heat tolerance in the RIL population. a The heat tolerance phenotypes and segregation of the RILs (F2:8). Note the variation in number of dropped flowers per plant and number of pods per plant. b The heat tolerance in the RIL population observed in temperature-controlled greenhouse, College Station, Texas. c The heat tolerance in the RIL population observed under the field condition, Corpus Christi, Texas. d The heat tolerance in the RIL population observed under the field condition, Weslaco, Texas. “1” for the most heat susceptible and “5” for the most heat tolerant. The heat tolerance was scored based on a combination of the number of dropped flowers per plant and number of pods per plant at the flowering and pod setting stages
An SNP linkage map of the cowpea genome and the positions of QTLs mapped for heat tolerance (qHTs). The linkage groups (LGs) of the map were assigned to the cowpea chromosomes (2n = 2x = 22), based on Lonardi et al. (2019). Each number on the right side of a Chr is an SNP code that represents a bin consisting of a group of co-segregating SNPs, while the number on the left side is the position of the SNP in the chromosome (cM). For the consensus sequences of the SNPs, see Supplementary Data S1
Heat tolerance QTLs, qHTs, mapped on the cowpea SNP linkage map. The linkage map was aligned to cowpea chromosomes (chr). The cutoff of LOD ≥ 2.50 (the dotted line) was permutated using the IciMapping software v.4.0 and that of LOD ≥ 3.0 (the red line) is widely used for claiming a QTL
Plant tolerance to heat or high temperature is crucial to crop production, especially in the situation of elevated temperature resulting from global climate change. Cowpea, Vigna unguiculata (L.) Walp., is an internationally important legume food crop and an excellent pool of genes for numerous traits resilient to environmental extremes, particularly heat and drought. Here, we report a single nucleotide polymorphism (SNP) genetic map for cowpea and identification of the loci controlling the heat tolerance in the species. The SNP map consists of 531 bins containing 4,154 SNPs grouped into 11 linkage groups, and collectively spans 1,084.7 cM, thus having a density of one SNP in 0.26 cM or 149 kb. The 11 linkage groups of the map were aligned to the 11 cowpea chromosomes. Quantitative trait locus (QTL) mapping identified nine QTLs responsible for the cowpea heat tolerance on seven of the 11 chromosomes, with each QTL explaining 6.5—21.8% of heat tolerance phenotypic variation. Moreover, we aligned these nine QTLs to the cowpea genome. Each of the QTLs was positioned in a genomic region ranging from 209,000 bp to 12,590,450 bp, and the QTL with the largest effect (21.8%) on heat tolerance, qHT4-1, was located within an interval of only 234,195 bp. These results provide SNP markers useful for marker-assisted selection for heat tolerance and lay a foundation for cloning, characterization, and applications of the genes controlling the cowpea heat tolerance for heat tolerance genetic improvement in cowpea and related crops.
 
Breast cancer is the second leading cancer among women in terms of mortality rate. In recent years, its incidence frequency has been continuously rising across the globe. In this context, the new therapeutic strategies to manage the deadly disease attracts tremendous research focus. However, finding new prognostic predictors to refine the selection of therapy for the various stages of breast cancer is an unattempted issue. Aberrant expression of genes at various stages of cancer progression can be studied to identify specific genes that play a critical role in cancer staging. Moreover, while many schemes for subtype prediction in breast cancer have been explored in the literature, stage-wise classification remains a challenge. These observations motivated the proposed two-phased method: stage-specific gene signature selection and stage classification. In the first phase, meta-analysis of gene expression data is conducted to identify stage-wise biomarkers that were then used in the second phase of cancer classification. From the analysis, 118, 12 and 4 genes respectively in stage I, stage II and stage III are determined as potential biomarkers. Pathway enrichment, gene network and literature analysis validate the significance of the identified genes in breast cancer. In this study, machine learning methods were combined with principal component and posterior probability analysis. Such a scheme offers a unique opportunity to build a meaningful model for predicting breast cancer staging. Among the machine learning models compared, Support Vector Machine (SVM) is found to perform the best for the selected datasets with an accuracy of 92.21% during test data evaluation. Perhaps, biomarker identification performed here for stage-specific cancer treatment would be a meaningful step towards predictive medicine. Significantly, the determination of correct cancer stage using the proposed 134 gene signature set can possibly act as potential target for breast cancer therapeutics.
 
Schematic representation of stylopod, zeugopod and autopod with their corresponding limb segments
Schematic representation of a limb bud showing the relative positions of the zone of polarizing activity (ZPA), the apical ectodermal ridge (AER) and the progress zone (PZ)
Disorders that result from de-arrangement of growth, development and/or differentiation of the appendages (limbs and digit) are collectively called as inherited abnormalities of human appendicular skeleton. The bones of appendicular skeleton have central role in locomotion and movement. The different types of appendicular skeletal abnormalities are well described in the report of “Nosology and Classification of Genetic skeletal disorders: 2019 Revision”. In the current article, we intend to present the embryology, developmental pathways, disorders and the molecular genetics of the appendicular skeletal malformations. We mainly focused on the polydactyly, syndactyly, brachydactyly, split-hand–foot malformation and clubfoot disorders. To our knowledge, only nine genes of polydactyly, five genes of split-hand–foot malformation, nine genes for syndactyly, eight genes for brachydactyly and only single gene for clubfoot have been identified to be involved in disease pathophysiology. The current molecular genetic data will help life sciences researchers working on the rare skeletal disorders. Moreover, the aim of present systematic review is to gather the published knowledge on molecular genetics of appendicular skeleton, which would help in genetic counseling and molecular diagnosis.
 
Results of sex chromosome by prenatal BoBs assay. The Results tab shows for each sample a numeric and graphic representation of probe and group ratios against female (F) and male (M) references. Red color in a graph indicates the ratio against female (Sample/F), while blue indicates the ratio against male (Sample/M), numerical ratios exceeding user defined thresholds are highlighted with red color
Sex chromosomal abnormalities detected in case7 by single nucleotide polymorphism microarray (SNP-array) and copy number variation sequencing (CNV-Seq). A Results of SNP-array showed the copy number of Xp22.33p21.3 region is 1.87, refers to the fragment size of 25.8 Mb. The copy number of Xp21.3q28 region was 1.4, refers to the fragment size of 129.2 Mb. B Results of CNV-Seq suggested of a female fetus that was mosaicism, and the copy number of Xp21.3q28 region was 1.71, refers to the fragment size of 129.05 Mb
Sex chromosomal abnormalities detected in case13 by single nucleotide polymorphism microarray (SNP-array) and copy number variation sequencing (CNV-Seq). A Results of SNP-array showed the copy number of Y q11.221q11.222 region is 2, refers to the fragment size of 2.8 Mb. The copy number of Y q11.222q11.23 region is 0, refers to the fragment size of 7.7 Mb. The copy number of Yp11.31q11.221 region is 0.75. B Results of SNP-array showed the copy number of Yp11.31p11.2 region is 0, refers to the fragment size of 4.65 Mb. The copy number of Yp11.2 region is 0.86, refers to the fragment size of 1.4 Mb. The copy number of Yq11.21q11.221 region is 0.75, refers to the fragment size of 2.5 Mb. The copy number of Yq11.222q11.23 region is 0, refers to the fragment size of 7.4 Mb. The copy number of Yq11.221 region is 1.44, refers to the fragment size of 2.4 Mb
The prenatal BACs-on-Beads™ (BoBs) assay was introduced for rapid detection of abnormalities of chromosomes 13, 18, 21, X, and Y and specific nine significant microdeletion syndromes. The ability of prenatal BoBs to detect mosaicism ranged from 20 to 40%. However, there have been no prenatal studies of sex chromosome mosaicism in prenatal BoBs. Therefore, the present study was performed with an aim to uncover the detection level of sex chromosome mosaicism that application of prenatal BoBs assay, and then to assess the sensitivity of prenatal BoBs assay, thereby improving the prenatal diagnostic accuracy. A total of 31 samples of amniotic fluid (AF) and umbilical cord blood (UCB) for prenatal diagnosis were collected, and the results were confirmed through karyotyping, single nucleotide polymorphism microarray (SNP-array) and copy number variation sequencing (CNV-seq). 23 cases of sex chromosome mosaicism were prompted abnormal by prenatal BoBs, the minimum detection level of mosaicism was about 6% as detected by karyotype. The overall sensitivity of prenatal BoBs in the detection of sex chromosome mosaicism was 74.2% (23/31). This study evaluated the effectiveness of prenatal BoBs for detecting sex chromosome mosaicism in prenatal diagnosis, and the results will provide valuable information for genetic counseling.
 
Expression of the selected canonical sugarcane nitrate transporter genes of the NPF/NRT (ScNPF6.3; ScNPF4.6; ScNPF6.4) and NRT2 families (ScNRT2.1; ScNRT2.5) evaluated by quantitative reverse-transcript amplification (RT–qPCR) using gene-specific primers (Supplementary Table S1). The relative expression was normalized for N-sufficient conditions (+ N) treatment, and ScUBQ2 was used as the reference gene. Bars represent the means ± standard error (SE) (n = 3)
Preferential inorganic N uptake in sugarcane roots. Sugarcane plants were grown in nutrient solution for 3 months under a continuous supply of 2 mM NH4NO3 (N sufficiency) or transferred to N-deprived (−N) solution for 72 h. N-influx rates were measured for 10 min using nitrate and ammonium supplied at either 200 μM ¹⁵ N-labeled NO3 (NH4¹⁵NO3) or NH4 (¹⁵NH4NO3). Bars indicate means ± SE (n = 4–5). Significant differences at P < 0.01 are indicated by different letters. Different uppercase letters indicate significant mean differences between ¹⁵ N-nitrate and ¹⁵ N-ammonium influx between each N treatment (+ N or −N). Different lowercase letters indicate significant mean differences between ¹⁵ N-nitrate and ¹⁵ N-ammonium influx within each N treatment (+ N or −N)
¹⁵NO3 influx and transcriptional regulation of NPF/NRT1, NRT2 and NRT3 family gene members in sugarcane roots under N deprivation (a–d); ammonium or nitrate provision (e–h); and nitrate resupply (i–l). Sugarcane plants grown for 3 months under N-sufficient conditions (2 mM NH4NO3) were subjected to the various N treatments. Root ¹⁵NO3 influx was measured using nitrate supplied at 200 μM ¹⁵ N-labeled NO3 (K¹⁵NO3). Values represent the means of 6 replicates ± SD, and significant differences at P < 0.01 are indicated by different letters. Transcript levels of ScNRT2.1, ScNRT3.1 and ScNPF6.3 in sugarcane roots were quantified by RT–qPCR analysis using gene-specific primers (Supplementary Table S1). Gene expression was normalized to the values of the initial period analyzed (0 h). Bars indicate means ± SD (n = 5), and significant differences at P < 0.05 are indicated by different letters
¹⁵NO3 influx and time-dependent expression levels of ScNRT2.1, ScNRT3.1 and ScNPF6.3/ScNRT1.1 in roots upon 5 mM NH4NO3 resupply to N-deprived sugarcane in vitro plantlets. Plantlets of cultivars SP80-3280, IAC87-3396, and IACSP96-2042 were grown under N sufficiency (2 mM NH4NO3) and precultured under N-limited conditions (0.2 mM KNO3) for 7 d prior to resupplying with 5 mM NH4NO3 for 4 or 24 h. Root ¹⁵NO3 influx was measured using nitrate supplied at 200 μM ¹⁵ N-labeled NO3 (K¹⁵NO3). Transcript levels of ScNRT2.1, ScNRT3.1 and ScNPF6.3 in sugarcane roots were quantified by RT–qPCR analysis using gene-specific primers (Supplementary Table S1). Gene expression analysis of the SP80-3280 cultivar (b–d) was normalized by values of the initial period analyzed (0 h). Gene expression analysis comparing cultivars IAC87-3396 and IACSP96-2042 (f–h) was normalized by the values of IACSP96-2042 under N-limited conditions. Bars indicate means ± SD (n = 5), and significant differences at P < 0.05 are indicated by different letters
Hypothetical model of the presumed posttranscriptional regulation of nitrate uptake process in sugarcane by Serine501 (S501) phosphorylation of ScNRT2.1. At low nitrate concentration (left panel), S501 of NRT2.1 transporter is not phosphorylated, activating the transcription of ScNRT2.1/ScNRT3.1 encoding members of the high affinity nitrate uptake complex. When nitrate levels increase by resupply (right panel), an unknown kinase is recruited to phosphorylate S501 that leads to inactivate nitrate absorption through NRT2.1/NRT3.1 complex
Key message Nitrate uptake in sugarcane roots is regulated at the transcriptional and posttranscriptional levels based on the physiological status of the plant and is likely a determinant mechanism for discrimination against nitrate. Abstract Sugarcane (Saccharum spp.) is one of the most suitable energy crops for biofuel feedstock, but the reduced recovery of nitrogen (N) fertilizer by sugarcane roots increases the crop carbon footprint. The low nitrogen use efficiency (NUE) of sugarcane has been associated with the significantly low nitrate uptake, which limits the utilization of the large amount of nitrate available in agricultural soils. To understand the regulation of nitrate uptake in sugarcane roots, we identified the major canonical nitrate transporter genes (NRTs—NITRATE TRANSPORTERS) and then determined their expression profiles in roots under contrasting N conditions. Correlation of gene expression with ¹⁵N-nitrate uptake revealed that under N deprivation or inorganic N (ammonium or nitrate) supply in N-sufficient roots, the regulation of ScNRT2.1 and ScNRT3.1 expression is the predominant mechanism for the modulation of the activity of the nitrate high-affinity transport system. Conversely, in N-deficient roots, the induction of ScNRT2.1 and ScNRT3.1 transcription is not correlated with the marked repression of nitrate uptake in response to nitrate resupply or high N provision, which suggested the existence of a posttranscriptional regulatory mechanism. Our findings suggested that high-affinity nitrate uptake is regulated at the transcriptional and presumably at the posttranscriptional levels based on the physiological N status and that the regulation of NRT2.1 and NRT3.1 activity is likely a determinant mechanism for the discrimination against nitrate uptake observed in sugarcane roots, which contributes to the low NUE in this crop species.
 
The circular genome map of Ralstonia pseudosolanacearum GRsMep. a Chromosome and b Megaplasmid. The size of the chromosome is 3,716,413 bp and the megaplasmid is 2,094,192 bp. The circles from outer to inner represent genome size, forward CDS and reverse CDS (blue), rRNA and tRNA (yellow, rRNA; red, tRNA), GC ratio (brown, outward means GC ratio of the region is higher than average GC ratio; brown, inward means GC ratio of the region is lower than average GC ratio), GC skew (pink represents a region with G content greater than C, violet represents a region with C content greater than G)
Phylogenetic tree prepared using genomes of ten Ralstonia strains members of the Zingiberaceae family based on OrthoANI values. The heat map was generated with orthoANI values calculated from OAT software
The pan-genome of Ralstonia strains infecting members of the Zingiberaceae family. a Pictorial representation of core, accessory and unique gene families of ten Ralstonia strains infecting members of the Zingiberaceae family. The number of core genes shared by all the ten R. pseudosolanacearum strains is represented in the green circle. The total number of accessory genes are represented in the blue circle and the unique genes present in the genomes are shown in non-overlapping portions of the circle (pink). b COG distribution of core, accessory and unique genes in the pangenome of ten Ralstonia strains infecting members of the Zingiberaceae family. The genes belonging to COG major categories such as cellular processing and signaling (classes D, M, N, O, T, U and V), information storage and processing (classes J, K and L), metabolic pathways (classes C, G, E, F, H, I, Q and P), poorly characterized function (classes R and S) are represented in bar diagrams
Ralstonia pseudosolanacearum causes bacterial wilt in ginger, reducing ginger production worldwide. We sequenced the whole genome of a highly virulent phylotype I, race 4, biovar 3 Ralstonia pseudosolanacearum strain GRsMep isolated from a severely infected ginger field in India. R. pseudosolanacearum GRsMep genome is organised into two replicons: chromosome and megaplasmid with a total genome size of 5,810,605 bp. This strain encodes approximately 72 effectors which include a combination of core effectors as well as highly variable, diverse repertoire of type III effectors. Comparative genome analysis with GMI1000 identified conservation in the genes involved in the general virulence mechanism. Our analysis identified type III effectors, RipBJ and RipBO as present in GRsMep but absent in the reported genomes of other strains infecting Zingiberaceae family. GRsMep contains 126 unique genes when compared to the pangenome of the Ralstonia strains that infect the Zingiberaceae family. The whole-genome data of R. pseudosolanacearum strain will serve as a resource for exploring the evolutionary processes that structure and regulate the virulence determinants of the strain. Pathogenicity testing of the transposon insertional mutant library of GRsMep through virulence assay on ginger plants identified a few candidate virulence determinants specific to bacterial wilt in ginger.
 
Autosomal recessive non-syndromic hearing loss (ARNSHL) is the most common hereditary deafness. It is genetically highly heterogeneous and about 89 gene loci and 76 gene’s mutations have been implicated in the etiology of ARNSHL. Molecular basis of ARNSHL remains unresolved in 60% of cases and gene mutations are unknown for 23 of 89 reported loci. Techniques used to identify reported ARNSHL gene mutations can be divided into position-dependent and position-independent approaches. The localization of the loci has been facilitated by homozygosity mapping or linkage studies using STR or SNP genotyping in large consanguineous families. First few genes identified for hearing loss exhibited such wide diversity of function and expression patterns that candidate gene approach was not a viable option. The mapping of the disorder to a chromosomal location has been followed by Sanger sequencing of all genes in the target region or confining of the massively parallel sequencing data analyses to the linkage region. Sometimes genes located in the linkage interval were prioritized because there was a reported orthologs with mutations causing hearing loss in mouse or when mutations in the gene caused a related disorder. Position-independent approaches involving use of mouse subtractive cochlear libraries, forward genetic screening, and position-independent analyses of massively parallel sequencing data have helped identify 17 of 68 reported ARNSHL gene mutations. A thorough study of the strategies used in the identification of reported ARNSHL genes and of their relative success can help increase the success rate of future studies.
 
Roles of LncRNA in metabolic syndrome features
Some lncRNAs play a role in the development of metabolic syndrome
Long non-coding RNAs (lncRNAs) have become important regulators of gene expression because they affect a wide range of biological processes, such as cell growth, death, differentiation, and aging. More and more evidence suggests that lncRNAs play a role in maintaining metabolic homeostasis. When certain lncRNAs are out of balance, metabolic disorders like diabetes, obesity, and heart disease get worse. In this review, we talk about what we know about how lncRNAs control metabolism, with a focus on diseases caused by long-term inflammation and the characteristics of the metabolic syndrome. We looked at lncRNAs and their molecular targets in the pathogenesis of signaling pathways. We also talked about how lncRNAs are becoming more and more interesting as diagnostic and therapeutic targets for improving metabolic homeostasis.
 
Introduction MicroRNAs are regulatory non-coding RNAs, with their outstanding regulatory mechanism, that make them potential biomarker for disease detection and therapeutics. They play an important role in pathological state, such as cancer by acting as oncogenic microRNAs and tumor suppressor microRNAs. The expression of microRNA-206, microRNA-4477a, microRNA-4795-5p, microR-4796-3p, microRNA-451b, and microRNA-4311 has proven to be deregulated in different cancer studies. However, no comprehensive study has been reported yet regarding their role in glioma patients. Aim The present study is designed to examine the expression profiling of microRNAs, such as microRNA-206, microRNA-4477a, microRNA-4795-5p, microR-4796-3p, microRNA-451b, and microRNA-4311 in glioma patients. Furthermore, the expression deregulation of selected microRNAs was correlated with oxidative stress and proliferation rate in glioma patients. Methods For this purpose, 153 glioma tissue samples and 200 brain tissues from epilepsy patients (taken as controls) were collected in the present study. Expression analysis of selected microRNAs was carried out on collected samples using real-time PCR (qPCR). Oxidative stress and proliferation rate were measured by estimation of 8OXOG level and Ki-67 using the ELISA and IHC. Results Our results showed significant deregulation of microRNA-206 (p < 0.0001), microRNA-4477a (p < 0.01), microRNA-4311 (p < 0.0001), microRNA-4795-5p (p < 0.0001), microRNA-4796-3p (p < 0.0001), and microRNA-451b (p < 0.0001) in glioma patients compared to controls. However, significant upregulation of 8OXOG level (p < 0.0001) and Ki-67 (p < 0.0001) was observed in glioma patients compared to controls. Kaplan–Meier analysis showed that deregulated expression of selected microRNAs was associated with significant decrease in survival of glioma patients. Conclusions Our results demonstrated significant deregulation of selected microRNAs in glioma patients. This deregulated expression was found associated with significant increased risk of glioma and could be further developed as effective prognostic biomarker and therapeutic tool in said disease.
 
Development of colon adenocarcinoma (COAD) metastasis involves several mediators including fluid shear stress (FSS), intracellular ROS levels, and non-coding RNAs. In our present study, we identified and investigated the role of regulatory non-coding RNA molecules specifically involved in COAD metastasis and their association with FSS and ROS. Interactions between the mRNAs associated with FSS and ROS, the corresponding microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) in COAD metastasis were used to generate the mRNA-miRNA-lncRNA-circRNA network. Experimental validation of the identified RNA hubs using quantitative real-time PCR demonstrated a direct effect of the FSS on their expression levels in cancer cells. FSS resulted in the downregulation of HMGA1 and RAN, as well as the upregulation of HSP90AA1, PMAIP1 and BIRC5. Application of shear stress also led to downregulation of hsa-miR-26b-5p and hsa-miR-34a-5p levels in HCT116 cells. Further, functional enrichment and survival analysis of the significant miRNAs, as well as the OncoPrint and the survival analyses of the selected mRNAs were performed. Subsequently, their functional role was also corroborated with existing literature. Ten significant miRNA hubs were identified, out of which hsa-miR-17-5p and hsa-miR-20a-5p were found to interact with lncRNA (CCAT2) while hsa-miR-335 was found to interact with four circRNAs. Fifteen significant miRNAs were identified in 10 different modules suggesting their importance in FSS and ROS-mediated COAD metastasis. Finally, 10 miRNAs and 3 mRNAs associated with FSS and/or ROS were identified as significant overall survival markers; 33 mRNAs were also identified as metastasis-free survival markers whereas 15 mRNAs showed > 10% gene alterations in TCGA-COAD data and may serve as promising therapeutic biomarkers in the COAD metastasis.
 
The pedigree is composed of two branches (branches 1 and 2) of a single TPT-PS family. The black circles and squares represent the affected family members, while the white symbols—healthy individuals. The horizontal lines above the symbols indicate the family members from which a DNA sample was collected and analyzed
Clinical and radiological images of triphalangeal thumb–polysyndactyly syndrome (TPT-PS) phenotypes in the presented family. The manifestation of hand malformations before surgery in patients IV-9 (A), V-17 (H), VI-1 (J), VI-2 (K), VI-3 (L,M), VII-1 (N,O) and VII-2 (P). Except for patient V-17, who manifested complete syndactyly of fingers 4–5 in the left hand and complete syndactyly of fingers 3–5 in the right hand (H), the rest of the patients presented with complete bilateral syndactyly of fingers 3–5 (A, J–P). Note the osseous fusion of the terminal phalanges in the affected fingers of all individuals with TPT-PS. Besides, some of the affected individuals had a supernumerary, usually hypoplastic triphalangeal thumb, as present in patients VI-3 (M), VII-1 (N, O) and VII-2 (P). Bilateral feet syndactyly involving toes 3–5 was shown for patients IV-9 (B), V-11 (F, G) and V-17 (I). Additionally, the clinical and radiological images of the hands after surgical treatment are shown for patients V-1 (C) and V-11 (D, E)
The schematic localization of the pZRS in reference to ZRS and SHH. Sanger sequencing results of the patients V-1 and V-11 from the TPT-PS family. In the affected individuals, including V-1 and V-11, a C to G substitution in the pZRS was detected
3D organization of the SHH-LMBR1 locus in control and patient V-11 cells. (A) Upper panel: Hi-C map of the SHH-LMBR1 locus (hg38: chr7:155,010,000–157,460,000) derived from GM12878 cells (visualized in 3D Genome Browser: http://3dgenome.fsm.northwestern.edu/view.php). Horizontal bars represent TADs identified in GM12878 cells (Rao et al. 2014). Vertical dashed lines represent the boundaries of the above-shown TADs. Below: schematic representation of genes in the examined locus. Arrows indicate transcription start sites (TSS). CTCF ChiP-seq tracks in the GM12878 cells from the ENCODE Portal (Davis et al. 2018; ENCODE Project Consortium 2012) (https://www.encodeproject.org/) with the ENCFF847KZK identifier (dataset ENCSR000DRZ). Bottom panel: 4C-seq data from control skin fibroblasts at the pZRS and SHH TSS viewpoints (indicated by triangles) show reciprocal interactions. (B) Upper panel: schematic representation of genes in the examined locus (the identified mutation in pZRS is indicated by an asterisk). Below, 4C interaction profile in patient’s skin fibroblasts from the pZRS and SHH TSS viewpoints (indicated by triangles) and the respective ratio to control results. The pZRS viewpoint: note the gain of interactions within the SHH-LMBR1 TAD indicated by a red rectangle. Horizontal bars represent windows of restriction fragments where the 4C interaction profiles statistically significantly differ between the patient and controls
Herein, we report on a large Polish family presenting with a classical triphalangeal thumb-polysyndactyly syndrome (TPT-PS). This rare congenital limb anomaly is generally caused by microduplications encompassing the Sonic Hedgehog (SHH) limb enhancer, termed the zone of polarizing activity (ZPA) regulatory sequence (ZRS). Recently, a pathogenic variant in the pre-ZRS (pZRS), a conserved sequence located near the ZRS, has been described in a TPT-PS Dutch family. We performed targeted ZRS sequencing, array comparative genomic hybridization, and whole-exome sequencing. Next, we sequenced the recently described pZRS region. Finally, we performed a circular chromatin conformation capture-sequencing (4C-seq) assay on skin fibroblasts of one affected family member and control samples to examine potential alterations in the SHH regulatory domain and functionally characterize the identified variant. We found that all affected individuals shared a recently identified pathogenic point mutation in the pZRS region: NC_000007.14:g.156792782C>G (GRCh38/hg38), which is the same as in the Dutch family. The results of 4C-seq experiments revealed increased interactions within the whole SHH regulatory domain (SHH-LMBR1 TAD) in the patient compared to controls. Our study expands the number of TPT-PS families carrying a pathogenic alteration of the pZRS and underlines the importance of routine pZRS sequencing in the genetic diagnostics of patients with TPT-PS or similar phenotypes. The pathogenic mutation causative for TPT-PS in our patient gave rise to increased interactions within the SHH regulatory domain in yet unknown mechanism.
 
Integrating functional and population genetic approaches to infer adaptation at microsatellites (STRs). a Schematic diagram of eSTR identification using RNA-seq. Previously, we identified 479 eSTRs where allele length significantly correlated with gene expression in sunflower using an RNA-seq approach (Ranathunge et al. 2020). b Population genetic analysis. We genotyped 13 eSTRs previously identified using RNA-seq along with 19 neutral STRs and conducted population genetic analyses to test whether eSTR allele length is under selection. c Schematic diagram of a hypothetical model showing eSTR allele length variation in sunflower populations across a latitudinal cline. Alle frequencies at a specific eSTR are shown here to be significantly different among sunflower populations across a well-defined cline where populations show continuous variation in flowering time. In this hypothetical example, longer eSTR allele is favored in northern populations, whereas shorter allele is favored in the south. d Clinal variation in average population eSTR allele length. Consistent with the predictions of the tuning-knob model, eSTRs show clinal patterns of variation in allele length with shorter or longer alleles being favored in climatic extremes
Geographical locations of the natural populations of common sunflower (Helianthus annuus L.) used in this study
Expected Heterozygosity (HE) a, population genetic differentiation b, allelic diversity c, and allelic richness d estimates across 17 natural populations of common sunflower (Helianthus annuus L.) at 19 anonymous and 13 transcribed microsatellite loci (eSTRs) used in the study
The relationship between standardized lnRV and standardized lnRH. Data points represent lnRV and lnRH values calculated for each sunflower (Helianthus annuus L.) population pair at each microsatellite locus. The gray boxes indicate the regions where data points are outliers for both lnRV and lnRH (p < 0.05)
Population mean microsatellite allele length plotted as a function of latitude for eSTR loci a comp41936, b comp50288, c comp25591 d comp47993, and anonymous loci, e ORS857 and f ORS815 across 17 common sunflower (Helianthus annuus L.) populations
Microsatellites, also known as short tandem repeats (STRs), have long been considered non-functional, neutrally evolving regions of the genome. Recent findings suggest that they can function as drivers of rapid adaptive evolution. Previous work on the common sunflower identified 479 transcribed microsatellites where allele length significantly correlates with gene expression (eSTRs) in a stepwise manner. Here, a population genetic approach is used to test whether eSTR allele length variation is under selection. Genotypic variation among and within populations at 13 eSTRs was compared with that at 19 anonymous microsatellites in 672 individuals from 17 natural populations of sunflower from across a cline running from Saskatchewan to Oklahoma (distance of approximately 1600 km). Expected heterozygosity, allelic richness, and allelic diversity were significantly lower at eSTRs, a pattern consistent with higher relative rates of purifying selection. Further, an analysis of variation in microsatellite allele lengths (lnRV), and heterozygosities (lnRH), indicate recent selective sweeps at the eSTRs. Mean microsatellite allele lengths at four eSTRs within populations are significantly correlated with latitude consistent with the predictions of the tuning-knob model which predicts stepwise relationships between microsatellite allele length and phenotypes. This finding suggests that shorter or longer alleles at eSTRs may be favored in climatic extremes. Collectively, our results imply that eSTRs are likely under selection and that they may be playing a role in facilitating local adaptation across a well-defined cline in the common sunflower.
 
Lung is the most important organ in the human respiratory system, whose normal functions are quite essential for human beings. Under certain pathological conditions, the normal lung functions could no longer be maintained in patients, and lung transplantation is generally applied to ease patients’ breathing and prolong their lives. However, several risk factors exist during and after lung transplantation, including bleeding, infection, and transplant rejections. In particular, transplant rejections are difficult to predict or prevent, leading to the most dangerous complications and severe status in patients undergoing lung transplantation. Given that most common monitoring and validation methods for lung transplantation rejections may take quite a long time and have low reproducibility, new technologies and methods are required to improve the efficacy and accuracy of rejection monitoring after lung transplantation. Recently, one previous study set up the gene expression profiles of patients who underwent lung transplantation. However, it did not provide a tool to predict lung transplantation responses. Here, a further deep investigation was conducted on such profiling data. A computational framework, incorporating several machine learning algorithms, such as feature selection methods and classification algorithms, was built to establish an effective prediction model distinguishing patient into different clinical subgroups, corresponding to different rejection responses after lung transplantation. Furthermore, the framework also screened essential genes with functional enrichments and create quantitative rules for the distinction of patients with different rejection responses to lung transplantation. The outcome of this contribution could provide guidelines for clinical treatment of each rejection subtype and contribute to the revealing of complicated rejection mechanisms of lung transplantation.
 
Electrophoresis patterns of PCR analysis. M1 = DNA marker 1, II = 270 bp, ID = 270 bp + 249 bp, DD = 249 bp
Sequencing and blast analysis of the Indels. II = 270 bp, DD = 249 bp
The aim of this study was to assess the potential of 21 bp mutation in the second intron of the GSN gene as a molecular marker-assisted by exploring the effect of 21 bp mutation on growth traits in four beef cattle breeds. Gelsolin (GSN), a member of the superfamily of gel proteins, is involved in the regulation of a variety of cellular activities in the organism and plays an important function in cell motility, apoptosis, signal transduction and inflammatory responses. Gelatin can not only negatively regulate the pro-apoptotic effect of P53 protein, but also promote apoptosis by blocking the interaction between actin and deoxyribonuclease, so, the GSN gene was selected as a candidate gene in this study. In this study, a 21 bp mutation on the second intron to the GSN gene was verified in 573 individuals of Yunling (YL, n = 220), Jiaxian (JX, n = 140), Xianan (XN, n = 114) and Qinchuan (QC, n = 97) cattle breeds using Once PCR and agarose gel electrophoresis. The association analysis of polymorphisms in the GSN gene with growth traits in four breeds was revealed: in YL cattle, the heart girth and forehead size of heterozygous ID genotype were significantly higher than II genotype (P < 0.05). In JX cattle, the withers height, body length and heart girth of II and ID genotype were significantly highest than DD genotype (P < 0.01); the height at hip cross and height at sacrum of II genotype was significantly highest than DD genotype (P < 0.01), but ID genotype was significantly higher than DD genotype. In XN cattle, the abdominal girth and circumference of the cannon bone of II genotype were significantly higher than ID genotype (P < 0.05). In QC cattle, the hucklebone width of ID genotype was significantly the highest than II genotype (P < 0.01). The results suggest that GSN may be an important candidate gene and that a 21 bp mutation on the second intron to the GSN gene can be used for molecular marker-assisted selection of four beef cattle breeds.
 
For non-syndromic cleft lip with or without cleft palate (ns-CL/P), the proportion of heritability explained by the known risk loci is estimated to be about 30% and is captured mainly by common variants identified in genome-wide association studies. To contribute to the explanation of the “missing heritability” problem for orofacial clefts, a candidate gene approach was taken to investigate the potential role of rare and private variants in the ns-CL/P risk. Using the next-generation sequencing technology, the coding sequence of a set of 423 candidate genes was analysed in 135 patients from the Polish population. After stringent multistage filtering, 37 rare coding and splicing variants of 28 genes were identified. 35% of these genetic alternations that may play a role of genetic modifiers influencing an individual's risk were detected in genes not previously associated with the ns-CL/P susceptibility, including COL11A1, COL17A1, DLX1, EFTUD2, FGF4, FGF8, FLNB, JAG1, NOTCH2, SHH, WNT5A and WNT9A. Significant enrichment of rare alleles in ns-CL/P patients compared with controls was also demonstrated for ARHGAP29, CHD7, COL17A1, FGF12, GAD1 and SATB2. In addition, analysis of panoramic radiographs of patients with identified predisposing variants may support the hypothesis of a common genetic link between orofacial clefts and dental abnormalities. In conclusion, our study has confirmed that rare coding variants might contribute to the genetic architecture of ns-CL/P. Since only single predisposing variants were identified in novel cleft susceptibility genes, future research will be required to confirm and fully understand their role in the aetiology of ns-CL/P.
 
Familial segregation of the MYO1H variant in the family. Affected family members are represented with a black circle (female) or square (male), and unaffected family members are represented with an empty circle or square symbol. The black arrow represents the proband (IC). Filled diamond Whole exome sequencing data were obtained. # Individuals were not involved in the study. X Individual is younger than the age of onset. The presence or absence of the MYO1H variant is represented as het. (heterozygote) and ref. (reference sequence), respectively. Ages of the individuals at the time of sample collection are presented at the right side of the symbols
Chromatograms of Sanger sequencing analysis of the identified MYO1H mutation in the family members. All unaffected individuals (I.1, II.1, III.1, III.5, III.8, IV.4) show a regular chromatogram pattern, while all affected (II.2, III.2, III.4, III.6, IV.1, IV.3, IV.8) and the clinically undetermined individual (IV.6) show messy sequence pattern after the deleted nucleotides because of their heterozygous state of the deletion. Individuals who carry the mutation are shown with an asterisk (*). The mutation is represented by the vertical red rectangle. Wild-type and mutant codons and corresponding amino acid sequences are given at the bottom of the chromatogram
Protein sequence homology of human Myo1c and human Myo1h. a Alignment of the amino acid sequence belonging to TH1 domain of Myo1c and Myo1h. The dark and light red highlighted residues represent the identical and chemically similar amino acids residues, respectively; b Crystal structure of Myo1c and homology model of the PH domain of Myo1h. Combined crystal structure of Myo1c tail (PDB ID: 4R8G) and motor (PDB ID: 4BYF) domains (left). Black squares show magnified images of the PH domain of Myo1c (green) and Myo1H (red). Superimposed structures of Myo1c and Myo1h are given at the bottom. The deleted amino acid in Myo1h is colored in cyan, and the corresponding amino acid in Myo1c is colored in red. 3D structure of the PH domain of Myo1h is generated using SPARKSX Fold Recognition software (Yang et al. 2011). The colored illustrations are created using PyMOL molecular visualization software
The schematic illustration of Myo1h domains and disease-related mutations. a Mandibular disorder-associated variants are represented in green boxes, and the mutation related with hypoventilation is shown in the purple box. The Glu992del mutation identified in this study is also located in the TH1 domain and represented in the magenta box. b Protein sequence homology of the mutation-affected region among diverse species and the highly conserved Glu992 is denoted with a red arrow
In this study, we aimed to determine the genetic basis of a Turkish family related to hereditary spastic paraplegia (HSP) by exome sequencing. HSP is a progressive neurodegenerative disorder and displays genetic and clinical heterogeneity. The major symptoms are muscle weakness and spasticity, especially in the lower extremities. We studied seven affected and seven unaffected family members, as well as a clinically undetermined member, to identify the disease-causing gene. Exome sequencing was performed for four affected and two unaffected individuals. The variants were firstly filtered for HSP-associated genes, and we found a common variant in the ZFYVE27 gene, which has been previously implied for association with HSP. Due to the incompletely penetrant segregation pattern of the ZFYVE27 variant, revealed by Sanger sequencing, with the disease in this family, filtering was re-performed according to the mode of inheritance and allelic frequencies. The resulting 14 rare variants were further evaluated in terms of their cellular functions, and three candidate variants in ATAD3C, VPS16, and MYO1H genes were selected as possible causative variants, which were analyzed for their familial segregation. ATAD3C and VPS16 variants were eliminated due to incomplete penetrance. Eventually, the MYO1H variant NM_001101421.3:c.2972_2974del (p.Glu992del, rs372231088) was found as the possible disease-causing deletion for HSP in this family. This is the first study reporting the possible role of a MYO1H variant in HSP pathogenesis. Further studies on the cellular roles of Myo1h protein are needed to validate the causality of MYO1H gene at the onset of HSP.
 
Supernumerary B chromosomes (Bs) are dispensable genetic elements widespread in eukaryotes and are poorly understood mainly in relation to mechanisms of maintenance and transmission. The cichlid Astatotilapia latifasciata can harbor Bs in a range of 0 (named B −) and 1–2 (named B +). The B in A. latifasciata is rich in several classes of repetitive DNA sequences, contains protein coding genes, and affects hosts in diverse ways, including sex-biased effects. To advance in the knowledge about the mechanisms of maintenance and transmission of B chromosomes in A. latifasciata, here, we studied the meiotic behavior in males and transmission rates of A. latifasciata B chromosome. We also analyzed structurally and functionally the predicted B chromosome copies of the cell cycle genes separin-like, tubb1-like and kif11-like. We identified in the meiotic structure relative to the B chromosome the presence of proteins associated with Synaptonemal Complex organization (SMC3, SYCP1 and SYCP3) and found that the B performs self-pairing. These data suggest that isochromosome formation was a step during B chromosome evolution and this element is in a stage of diversification of the two arms keeping the self-pairing behavior to protect the A chromosome complement of negative effects of recombination. Moreover, we observed no occurrence of B-drive and confirmed the presence of cell cycle genes copies in the B chromosome and their transcription in encephalon, muscle and gonads, which can indicates beneficial effects to hosts and contribute to B maintenance.
 
Congenital heart disease (CHD) surges from fetal cardiac dysmorphogenesis and chiefly contributes to perinatal morbidity and cardiovascular disease mortality. A continual rise in prevalence and prerequisite postoperative disease management creates need for better understanding and new strategies to control the disease. The interaction between genetic and non-genetic factors roots the multifactorial status of this disease, which remains incompletely explored. The small non-coding microRNAs (miRs, miRNAs) regulate several biological processes via post-transcriptional regulation of gene expression. Abnormal expression of miRs in developing and adult heart is associated with anomalous cardiac cell differentiation, cardiac dysfunction, and cardiovascular diseases. Here, we attempt to discover the changes in cardiac miRNA transcriptome in CHD patients over those without CHD (non-CHD) and find its role in CHD through functional annotation. This study explores the miRNome in three most commonly occurring CHD subtypes, namely atrial septal defect (ASD), ventricular septal defect (VSD), and tetralogy of fallot (TOF). We found 295 dysregulated miRNAs through high-throughput sequencing of the cardiac tissues. The bioinformatically predicted targets of these differentially expressed miRs were functionally annotated to know they were entailed in cell signal regulatory pathways, profoundly responsible for cell proliferation, survival, angiogenesis, migration and cell cycle regulation. Selective miRs (hsa-miR-221-3p, hsa-miR-218-5p, hsa-miR-873-5p) whose expression was validated by qRT-PCR, have been reported for cardiogenesis, cardiomyocyte proliferation, cardioprotection and cardiac dysfunction. These results indicate that the altered miRNome to be responsible for the disease status in CHD patients. Our data expand the existing knowledge on the epigenetic changes in CHD. In future, characterization of these cardiac-specific miRs will add huge potential to understand cardiac development, function, and molecular pathogenesis of heart diseases with a prospect of epigenetic manipulation for cardiac repair. Graphical abstract
 
Source of variables for analysis, and sample size at each level of analysis
Power (y axis) to detect a genetic relationship between Cross-psychiatric1 and a cellular phenotype with a common genetic component of varying size (coloured lines) at different values of genetic correlation (x-axis), for differing values of N: a 60 [not accounting for multiple measurements], b 850 [lower estimate of effective N], c 2435 [higher estimate of effective N]
Significant interactions between polygenic scores and fibronectin concentrations. Lines reflect the relationship between each polygenic score and the phenotype from the relevant model. Points reflect the fitted value of the phenotype from the relevant model, averaged for each value of the polygenic score (that is, for all cells from a given donor). a Cross-psychiatric1 and fibronectin concentration of 5 μg/mL on cell area, b BMI1 and fibronectin concentration of 25 μg/mL on cell width-to-length ratio, c height0.01 and fibronectin concentration of 25 μg/mL on cell width-to-length ratio, d height1 and fibronectin concentration of 25 μg/mL on cell width-to-length ratio. PGS polygenic score, BMI body mass index
Genome-wide association studies have identified thousands of significant associations between genetic variants and complex traits. Inferring biological insights from these associations has been challenging. One approach attempted has been to examine the effects of individual variants in cellular models. Here, I demonstrate the feasibility of examining the aggregate effect of many variants on cellular phenotypes. I examine the effects of polygenic scores for cross-psychiatric disorder risk, schizophrenia, body mass index and height on cellular morphology, using 1.5 million induced pluripotent stem cells (iPSC) from 60 European-ancestry donors from the Human iPSC Initiative dataset. I show that measuring multiple cells per donor provides sufficient power for polygenic score analyses, and that cross-psychiatric disorder risk is associated with cell area ( p = 0.004). Combined with emerging methods of high-throughput iPSC phenotyping, cellular polygenic scoring is a promising method for understanding potential biological effects of the polygenic component of complex traits.
 
Structure of primer design. Solid spots are methylated (red) or unmethylated (green) of CpG sites located in approximately the middle of the target region
Normalized melting curves and normalized melting peaks of case tissues (#6, red line) and control tissues (#6, green line), performed in duplicate. “A” is the fragment amplified by primer set A and “B” is the fragment amplified by primer set B. Melting curves (A1, B1) are generated by graphing fluorescence against temperature. Melting peaks (A2, A3 and B2, B3) are generated by taking the negative derivative of fluorescence with respect to temperature. Methylated bisulfite converted control DNA and unmethylated bisulfite converted control DNA (EpiTect Control DNA – Qiagen) shown in blue and black, respectively. In B2, the control is a mixed of methylated and un-methylated bisulfite converted control DNA to mimic the control samples. The case sample (T65) is shown in pink in B1 and B3. The experiments and analyisis has been repeated and verified by sequencing
DNA methylation is a fundamental epigenetic process and have a critical role in many biological processes. The study of DNA methylation at a large scale of genomic levels is widely conducted by several techniques that are next-generation sequencing (NGS)-based methods. Methylome data revealed by DNA methylation next-generation sequencing (mNGS), should be always verified by another technique which they usually have a high cost. In this study, we offered a low-cost approach to corroborate the mNGS data. In this regard, mNGS was performed on 6 colorectal cancer (case group) and 6 healthy individual colon tissue (control group) samples. An R-script detected differentially methylated regions (DMRs), was further validated by high resolution melting (MS-HRM) analysis. After analyzing the data, the algorithm found 194 DMRs. Two locations with the highest level of methylation difference were verified by MS-HRM, which their results were in accordance with the mNGS. Therefore, in the present study, we suggested MS-HRM as a simple, accurate and low-cost method, useful for confirming methylation sequencing results.
 
Eucalyptus urophylla is an economically important tree species that widely planted in tropical and sub-tropical areas around the world, which suffers significant losses due to Ralstonia solanacearum. However, little is known about the molecular mechanism of pathogen-response of Eucalyptus. We collected the vascular tissues of a E. urophylla clone infected by R. solanacearum in the laboratory, and combined transcriptome and metabolome analysis to investigate the defense responses of Eucalyptus. A total of 11 flavonoids that differentially accumulated at the first stage or a later stage after infection. The phenylpropanoid of p-coumaraldehyde, the two alkaloids trigonelline and dl-ephedrine, two types of traditional Chinese medicine with patchouli alcohol and 3-dihydrocadambine, and the amino acid phenylalanine were differentially accumulated after infection, which could be biomarkers indicating a response to R. solanacearum. Differentially expressed genes involved in plant hormone signal transduction, phenylpropanoids, flavonoids, mitogen-activated protein kinase (MAPK) signaling, and amino acid metabolism were activated at the first stage of infection or a later stage, indicating that they may participate in the defense against infection. This study is expected to deliver several insights into the molecular mechanism in response to pathogens in E. urophylla, and the findings have far-reaching implications in the control of E. urophylla pathogens.
 
Previous genome mining of the strains Bacillus pumilus 7PB, Bacillus safensis 1TAz, 8Taz, and 32PB, and Priestia megaterium 16PB isolated from canola revealed differences in the profile of antimicrobial biosynthetic genes when compared to the species type strains. To evaluate not only the similarities among B. pumilus, B. safensis, and P. megaterium genomes but also the specificities found in the canola bacilli, we performed comparative genomic analyses through the pangenome evaluation of each species. Besides that, other genome features were explored, especially focusing on plant-associated and biotechnological characteristics. The combination of the genome metrics Average Nucleotide Identity and digital DNA–DNA hybridization formulas 1 and 3 adopting the universal thresholds of 95 and 70%, respectively, was suitable to verify the identification of strains from these groups. On average, core genes corresponded to 45%, 52%, and 34% of B. pumilus, B. safensis, and P. megaterium open pangenomes, respectively. Many genes related to adaptations to plant-associated lifestyles were predicted, especially in the Bacillus genomes. These included genes for acetoin production, polyamines utilization, root exudate chemoreceptors, biofilm formation, and plant cell-wall degrading enzymes. Overall, we could observe that strains of these species exhibit many features in common, whereas most of their variable genome portions have features yet to be uncovered. The observed antifungal activity of canola bacilli might be a result of the synergistic action of secondary metabolites, siderophores, and chitinases. Genome analysis confirmed that these species and strains have biotechnological potential to be used both as agricultural inoculants or hydrolases producers. Up to our knowledge, this is the first work that evaluates the pangenome features of P. megaterium.
 
Molecular diagnostic results and clinical features in patients with NL/NC. A A summary of testing results is illustrated. AD and AR are the most common genetic mode, and the most frequent variant genes are SLC4A1 and KMT2D. Among them, SLC4A1 had two genetic patterns of AD and AR. B A total of 35 variant mutations have been detected, including 23 missense mutations, 4 nonsense mutations, 1 non-frameshift mutation, three frameshift mutations, two splicing mutations, and one exon loss. C A summary of manifestations revealed that there were 13 cases had isolated NC, 19 cases had NL, among them, 10 had multiple stones, 6 had NL merges NC, and 3 had a single stone. D Gross hematuria, abdominal pain, and recurrent infection are more likely to exist if symptoms are present. However, most of the patients are asymptomatic; presentations leading to diagnosis include osteonosus, retardation of intelligence or motor and anorexia. E The two most frequent diseases were dRTA and Kabuki syndrome, followed by Dent disease and PH. F The patients with failed urinary acidification and hypercalciuria (group 1) were more likely to show NC (P = 0.0009 for chi-squared test), and the patients with hyperuricosuria, cystinuria, and hyperglycinuria (group 2) were more likely to have NL (P = 0.0289). There was no statistical difference in the proportion of NL in other genes (group 3) and gene negative group (group 4). (P = 0.6664, P = 0.4012). NL plus NC are marked in yellow; NC is marked in blue; NL is marked in yellow
The strategy of gene testing for nephrolithiasis (NL)/nephrocalcinosis (NC) patients in our center: A flow chart showing the metabolic evaluation and the standard of gene testing of NL/NC
Objective Hereditary factors are the main cause of pediatric nephrolithiasis (NL)/nephrocalcinosis (NC). We summarized the genotype–phenotype correlation of hereditary NL/NC in our center, to evaluate the role of genetic testing in early diagnosis. Methods The clinical data of 32 NL/NC cases, which were suspected to have an inherited basis, were retrospectively analyzed from May 2017 to August 2020. The trio-whole exome sequencing was used as the main approach for genetic testing, variants were confirmed by Sanger sequencing, and pathogenicity analysis according to protein function was predicted with custom-developed software. Results Causative monogenic mutations were detected in 24 of 32 NL/NC patients, and copy number variation was detected in one patient. A summary of manifestations in patients with inherited diseases revealed a significant degree of growth retardation, increased urinary excretion of the low-molecular weight protein, hypercalciuria, electrolyte imbalances, and young age of onset to be common in heredity disease. In addition, some patients had abnormal renal function (3 ppm 25). The most frequent pathology identified was distal renal tubular acidosis (with inclusion of SLC4A1 , ATP6V1B1 , and ATP6VOA4 genes), followed by Dent disease ( CLCN5 and OCRL1 genes), primary hyperoxaluria (PH) ( AGXT and HOGA1 genes) and Kabuki syndrome ( KMT2D gene), which was more likely to present as NC or recurrent stone and having a higher correlation with a specific biochemical phenotype and extrarenal phenotype. Conclusion The etiology of NL/NC is heterogeneous. This study explored in depth the relationship between phenotype and genotype in 32 patients, and confirmed that genetic testing and clinical phenotype evaluation enable the precision medicine approach to treating patients.
 
Flowchart of the applied analytical approach and primers designed for the enrichment of SMN genes. a Schematic of primers designed. b Key processes in the workflow describing the details for distinguishing SMN1 from highly homologous SMN2, identifying transcripts and detecting potentially pathogenic variants. c The amount of CSS reads judged by up- and downstream anchor sequences to separate SMN1 and SMN2. d Marking of the A/G SNP counts in each sequenced individual. When up- and downstream anchor sequences pointed to A, the reads were attributed to SMN2. When up- and downstream anchor sequences pointed to G, the reads were attributed to SMN1
Long-read sequencing SMN1 and SMN2.a Heatmap showing the hierarchical clustering of transcripts, including known and novel transcripts, from individuals with different SMN1 and SMN2. b Schematic diagram of all SMN transcript structures within the PacBio sequences. These known structures of transcripts in NCBI are identified on the bottom with their gene name, GI and RefSeq transcript number. Transcripts with new exon structures are defined as novel transcripts. Novel transcripts detected in long-read SMRT are on the upper side, which are accordingly placed exons. c Percentage of different transcripts of SMN1 in individuals with one or two SMN1 copy numbers. d Percentage of different transcripts of SMN2 in individuals with one or two SMN2 copy numbers
The survival of motor neuron (SMN) genes, SMN1 and SMN2, are two highly homologous genes related to spinal muscular atrophy (SMA). Different patterns of alternative splicing have been observed in the SMN genes. In this study, the long-read sequencing technique for distinguishing SMN1 and SMN2 without any assembly were developed and applied to reveal multiple alternative splicing patterns and to comprehensively identify transcript variants of the SMN genes. In total, 36 types of transcript variants were identified, with an equal number of variants generated from both SMN1 and SMN2. Of these, 18 were novel SMN transcripts that have never been reported. The structures of SMN transcripts were revealed to be much more complicated and diverse than previously discovered. These novel transcripts were derived from diverse splicing events, including skipping of one or more exons, intron retention, and exon shortening or addition. SMN1 mainly produces FL-SMN1, SMN1Δ7, SMN1Δ5 and SMN1Δ3. The distribution of SMN2 transcripts was significantly different from those of SMN1, with the majority transcripts to be SMN2Δ7, followed by FL-SMN2, SMN2Δ3,5 and SMN2Δ5,7. Targeted long-read sequencing approach could accurately distinguish sequences of SMN1 from those of SMN2. Our study comprehensively addressed naturally occurring SMN1 and SMN2 transcript variants and splicing patterns in peripheral blood mononuclear cells (PBMCs). The novel transcripts identified in our study expanded knowledge of the diversity of transcript variants generated from the SMN genes and showed a much more comprehensive profile of the SMN splicing spectrum. Results in our study will provide valuable information for the study of low expression level of SMN proteins and SMA pathogenesis based on transcript levels.
 
Countering prior beliefs that epistasis is rare, genomics advancements suggest the other way. Current practice often filters out genomic loci with low variant counts before detecting epistasis. We argue that this practice is far from optimal because it can throw away strong epistatic patterns. Instead, we present the compensated Sharma–Song test to infer genetic epistasis in genome-wide association studies by differential departure from independence. The test does not require a minimum number of replicates for each variant. We also introduce algorithms to simulate epistatic patterns that differentially depart from independence. Using two simulators, the test performed comparably to the original Sharma–Song test when variant frequencies at a locus are marginally uniform; encouragingly, it has a marked advantage over alternatives when variant frequencies are marginally nonuniform. The test further revealed uniquely clean epistatic variants associated with chicken abdominal fat content that are not prioritized by other methods. Genes involved in most numbers of inferred epistasis between single nucleotide polymorphisms (SNPs) belong to pathways known for obesity regulation; many top SNPs are located on chromosome 20 and in intergenic regions. Measuring differential departure from independence, the compensated Sharma–Song test offers a practical choice for studying epistasis robust to nonuniform genetic variant frequencies.
 
In contrast to the popular opinion that forgetting is only the opposite of learning and memory, active forgetting explains the intrinsic instability of a labile memory that lasts for hours and has its own signal transduction pathways. However, the detailed mechanisms underlying forgetting are still lacking, though the investigations available in this field offer the first insights into their regulation. To identify the alternative signaling pathways that control the process of forgetting, we used the short-term forgetting model of Caenorhabditis elegans and discovered the involvement of lev-10, a scaffolded transmembrane protein of L-AChR, by screening the candidate genes that potentially functioned in synaptic plasticity. The LEV-9/LEV-10/L-AChR functional complex was confirmed to participate in forgetting occurrence. Furthermore, EGL-9 functioned upstream of LEV-10 and negatively regulated the latter during forgetting. Meanwhile, EGL-9 was also the target of miR-51, and hence the mutation of miR-51 similarly affected the function of L-AChR and delayed the short-term forgetting. Our findings have identified an integrated signaling pathway responsible for active forgetting, which provides the new experimental evidence on the cholinergic forgetting signal.
 
Prenatal ultrasound of Fetus 1. a Fetal skull was deformed under pressure, indicating abnormal skull ossification. b Fetal extremities were short (according to the gestational age) and bowed. c Sanger sequencing validation result of Trio 1. A homozygous c.509G > A variant in the PPIB gene was identified in Fetus 1, which was inherited from the heterozygous c.509G > A from the parents
Prenatal ultrasound of Fetus 2. a Fetal skull ring was visible with slightly low echo. b Fetal thorax was bell-shaped. The ossification point of sacrococcygeal spine was unclear. c Fetal limb bones were short, and bilateral humeri and femurs were a bit curved. d Sanger sequencing validation result of Trio 2. A heterozygous c.437C > A variant and a heterozygous c.1044C > G variant in the CHST3 gene were identified in Fetus 2. The two variants were inherited from the parents respectively
Prenatal ultrasound of Fetus 3. a Fetal extremities were short for gestational age. The femurs, humeri, and fibulas were bent, some of which were telephone receiver-shaped. b Sanger sequencing validation result of Trio 3. A heterozygous c.2605G > T variant in the COL1A1 gene was identified in Fetus 3, and it was not found in the parents
Prenatal ultrasound of Fetus 4. a Fetal skull was liable to deform under pressure. b Fetal nasal bones were not shown. The thorax was narrow and bell-shaped. c Fetal limb bones were short for gestational age, and some were abnormally bending. d Sanger sequencing validation result of Trio 4. A heterozygous c.1804G > A variant in the COL1A1 gene was identified in Fetus 4, and it was not found in the parents
Prenatal ultrasound of Fetus 5. a Oval skull ring was visible with no craniosynostosis. b The long bones of the fetal limbs were short and curved, especially the lower limbs. c Sanger sequencing validation result of Trio 5. A heterozygous c.742C > T variant in the FGFR3 gene was identified in Fetus 5, and it was not found in the parents
Whole exome sequencing (WES) could yield diagnostic significance in the prenatal diagnosis of skeletal abnormalities. But the phenotypes of fetuses with skeletal abnormalities are heterogenous, and the clinical information we could obtain from an ongoing pregnancy is limited, making the prenatal diagnosis complicated. Therefore, the following interpretation and genetic counseling remain a challenge for clinicians. The aim of this study is to present and investigate the utility of trio-based WES in five fetuses with skeletal anomalies. Five trios with fetal ultrasonic skeletal anomalies were recruited in our study. Fetal specimens and parental peripheral blood were subjected to WES. The fetal skeletal abnormalities were presented through ultrasound scanning images. Fetal WES results showed variants in the PPIB, CHST3, COL1A1, and FGFR3 genes in the five trios. Inherited variants were found in two of the trios, while de novo variants were observed in three of them. Two novel compound heterozygous variants (c.437C > A and c.1044C > G) in CHST3 were identified. We presented five trios with fetal skeletal anomalies, found two novel variants and broadened the spectrum of variants associated with skeletal abnormalities, which would help the establishment of genotype–phenotype relationship in the prenatal setting. Trio-based WES could assist the prenatal diagnosis and genetic counseling of fetuses with skeletal abnormalities.
 
The genetically regulated pattern of heterocyst formation in multicellular cyanobacteria represents the simplest model to address how patterns emerge and are established, the signals that control them, and the regulatory pathways that act downstream. Although numerous factors involved in this process have been identified, the mechanisms of action of many of them remain largely unknown. The aim of this study was to identify specific relationships between 14 factors required for cell differentiation and pattern formation by exploring their putative physical interactions in the cyanobacterium model Nostoc sp. PCC 7120 and by probing their evolutionary conservation and distribution across the cyanobacterial phylum. A bacterial two-hybrid assay indicated that 10 of the 14 factors studied here are engaged in more than one protein–protein interaction. The transcriptional regulator PatB was central in this network as it showed the highest number of binary interactions. A phylum-wide genomic survey of the distribution of these factors in cyanobacteria showed that they are all highly conserved in the genomes of heterocyst-forming strains, with the PatN protein being almost restricted to this clade. Interestingly, eight of the factors that were shown to be capable of protein interactions were identified as key elements in the evolutionary genomics analysis. These data suggest that a network of 12 proteins may play a crucial role in heterocyst development and patterning. Unraveling the physical and functional interactions between these factors during heterocyst development will certainly shed light on the mechanisms underlying pattern establishment in cyanobacteria.
 
Top-cited authors
Kyonoshin Maruyama
  • Japan International Research Center for Agricultural Sciences
Yusuke Ito
  • Nagoya University
Kazuo Nakashima
  • Japan International Research Center for Agricultural Sciences
Daisuke Todaka
  • The University of Tokyo
Takumi Yoshida
  • Tokyo Women's Medical University