Deletion Of Repeats Research Articles

Uncovering the genomic diversity of the wild forage crop Setaria sphacelata

AbstractGrasses are considered one of the most important angiosperm groups in economic terms. The Setaria sphacelata complex is an intriguing species with considerable variation in genome size (GS). It is currently being explored from a genomic perspective especially because of its great adaptability under winter periods. Repetitive DNA sequences are known to contribute significantly to GS diversity, which in turn can play a role in impacting the ecology and evolution of a species. This study is aimed to characterize the GS and repetitive elements of different germplasm accessions of S. sphacelata. We analyzed 540 plants from 70 accessions collected worldwide. Flow cytometry was used for GS estimation, together with chromosome counting, fluorescent in situ hybridization (FISH), and bioinformatic analysis. Most (i.e., 80%) accessions were tetraploids (2n = 4x = 36) with GS ranging from 3.0 to 3.7 pg/2C. Additionally, a low number of diploids (2n = 2x = 18; ∼1.6 to 2 pg/2C), pentaploids (2n = 5x = 45: ∼4 pg/2C), hexaploids (2n = 6x = 54; ∼4.4 pg/2C), and octoploids (2n = 8x = 72; ∼6 pg/2C,) were detected. The greatest variation was found in accessions from South Africa. RepeatExplorer2 showed that 39% and 43% of the genomes of two diploid accessions comprised repetitive sequences, with Ty3/gypsy retrotransposons being the most abundant repeats. However, variation in the percentage of two Ty3/gypsy sublineages, Athila and Ogre, suggests different process of amplification and deletion of repeats during the evolution of the species complex. In contrast, FISH revealed some satellite repeats with a conserved localized distribution in pericentromeric regions between accessions. Although differences in the distribution of the FISH signals over the chromosomes were observed between the different ploidy levels, the data suggest that increases in the ploidy level were associated with the occurrence of hybridization and DNA loss.

Differentiation shifts from a reversible to an irreversible heterochromatin state at the DM1 locus

Epigenetic defects caused by hereditary or de novo mutations are implicated in various human diseases. It remains uncertain whether correcting the underlying mutation can reverse these defects in patient cells. Here we show by the analysis of myotonic dystrophy type 1 (DM1)-related locus that in mutant human embryonic stem cells (hESCs), DNA methylation and H3K9me3 enrichments are completely abolished by repeat excision (CTG2000 expansion), whereas in patient myoblasts (CTG2600 expansion), repeat deletion fails to do so. This distinction between undifferentiated and differentiated cells arises during cell differentiation, and can be reversed by reprogramming of gene-edited myoblasts. We demonstrate that abnormal methylation in DM1 is distinctively maintained in the undifferentiated state by the activity of the de novo DNMTs (DNMT3b in tandem with DNMT3a). Overall, the findings highlight a crucial difference in heterochromatin maintenance between undifferentiated (sequence-dependent) and differentiated (sequence-independent) cells, thus underscoring the role of differentiation as a locking mechanism for repressive epigenetic modifications at the DM1 locus.

Two chloroplast genomes with reduced inverted repeat regions in Mammillaria series Stylothelae (Cactaceae)

Background: The chloroplast genomes of Cactaceae exhibit boundary modifications in the inverted repeat regions (IRs), gene inversions, and deletions. Among nine Mammillaria species, three distinct chloroplast structures have been identified, although not all of these correspond to the morphology-based classification of the genus.
 Question: Is there a distinct chloroplast genome structure in the species of Mammillaria series Stylothelae?
 Studied species: Mammillaria bocasana and M. erythrosperma.
 Study site and dates: Mexico from 2019 to 2023.
 Methods: Chloroplast DNA was sequenced, and chloroplast genomes were de novo assembled using the Fast-Plast program. Complete plastome sequences were annotated and verified. The sequences were aligned in MAUVE program to detect possible structural changes. A maximum likelihood phylogeny was executed to evaluate the relationships of the studied species.
 Results: The plastomes ranged from 107,368 bp in Mammillaria bocasana to 108,069 bp in M. erythrosperma. Both presented a quadripartite structure and contained 108 genes. The IRs were ~ 1,600 bp long and included the genes rpl2, rpl23 (pseudo), and trnI-CAU. MAUVE identified a ~ 21 kb inversion in the large single copy containing a block of genes related to photosynthesis. The phylogenetic analysis placed both species in a single clade separated from the other species within Mammillaria subg. Mammillaria.
 Conclusions: The studied species of Mammillaria series Stylothelae exhibited a different and synapomorphic chloroplast genome structure. Other Mammillaria chloroplast genome structures have evolved independently in different lineages.

WebSTR: A Population-wide Database of Short Tandem Repeat Variation in Humans

Short tandem repeats (STRs) are consecutive repetitions of one to six nucleotide motifs. They are hypervariable due to the high prevalence of repeat unit insertions or deletions primarily caused by polymerase slippage during replication. Genetic variation at STRs has been shown to influence a range of traits in humans, including gene expression, cancer risk, and autism. Until recently STRs have been poorly studied since they pose significant challenges to bioinformatics analyses. Moreover, genome-wide analysis of STR variation in population-scale cohorts requires large amounts of data and computational resources. However, the recent advent of genome-wide analysis tools has resulted in multiple large genome-wide datasets of STR variation spanning nearly two million genomic loci in thousands of individuals from diverse populations.Here we present WebSTR, a database of genetic variation and other characteristics of genome-wide STRs across human populations. WebSTR is based on reference panels of more than 1.7 million human STRs created with state of the art repeat annotation methods and can easily be extended to include additional cohorts or species. It currently contains data based on STR genotypes for individuals from the 1000 Genomes Project, H3Africa, the Genotype-Tissue Expression (GTEx) Project and colorectal cancer patients from the TCGA dataset.WebSTR is implemented as a relational database with programmatic access available through an API and a web portal for browsing data. The web portal is publicly available at https://webstr.ucsd.edu.

Octapeptide repeat alteration mutations of the prion protein gene in clinically diagnosed Alzheimer's disease and frontotemporal dementia.

Studies focusing on octapeptide repeat alteration mutations in PRNP in Alzheimer's disease (AD) and frontotemporal dementia (FTD) cohorts have been rare. We aim to screen sporadic AD and FTD patients with unknown etiology for the octapeptide repeat insertions and deletions in PRNP. Two hundred and six individuals were screened for alterations to the repeat region in the PRNP gene, including 146 sporadic AD and 60 sporadic FTD patients. Our study showed a 1.5% (3/206) occurrence of the octapeptide repeat alteration mutations in PRNP in a Chinese cohort of sporadic dementia. One late-onset FTD patient and one early-onset AD patient each had a two-octapeptide repeat deletion in PRNP, while one early-onset AD patient had a five-octapeptide repeat insertion mutation. PRNP octapeptide repeat alteration mutations are present in sporadic AD and FTD patients. The genetic investigation for PRNP octapeptide repeat alteration mutations in sporadic dementia patients should be carried out in future clinical studies.

Principles of the differential diagnosis of achondroplasia and pseudoachondroplasia

BACKGROUND: Achondroplasia and pseudoachondroplasia are hereditary systemic skeletal dysplasias characterized by a certain similarity of clinical manifestations; however, they have different etiopathogenetic mechanisms and confirmation methods for molecular genetic diagnosis. Their common phenotypic features often make differential diagnosis difficult during the clinical examination of patients, planning DNA diagnostics, and appropriate time detection of neurosurgical and orthopedic complications.
 AIM: This study aimed to identify differential diagnostic criteria for achondroplasia and pseudoachondroplasia and optimize the strategy for their molecular genetic diagnosis.
 MATERIALS AND METHODS: A comprehensive examination of 76 children from 74 unrelated families aged 1 month to 18 years with phenotypic signs of achondroplasia and pseudoachondroplasia was conducted. To clarify the diagnosis through genealogical and amnestic analysis, clinical and neurological examination data according to the standard method and radiographic data were used. Molecular genetic confirmation of diseases was conducted by searching for hotspot mutations in the FGFR3 gene, assessing the number of GAC repeats located in exon 13 of the COMP gene, and new-generation sequencing of the target panel consisting of 166 genes responsible for hereditary skeletal pathology.
 RESULTS: Based on a comparative analysis of the specific phenotypic characteristics, the criteria for the differential diagnosis of achondroplasia and pseudoachondroplasia were identified. The leading signs of achondroplasia are disproportionate nanism from birth, macrocrania, and facial dysmorphism, which are not specific to pseudoachondroplasia. Certain radiological features are essential in the differential diagnosis of pseudoachondroplasia, which should be considered when referring to patients for molecular genetic analysis. A deletion of the GAC repeat c.1417_1419del in the COMP gene was identified in 27% of patients with pseudoachondroplasia. Thus, the analyses of these two mutations in FGFR3 and COMP were conducted first. In the absence of target mutations, further diagnostic search should be continued with a target panel consisting of 166 genes responsible for hereditary skeletal pathology or whole-exome sequencing.
 CONCLUSIONS: The analysis of the clinical, radiological, and molecular genetic characteristics of patients with achondroplasia and pseudoachondroplasia, together with the literature data analysis, made it possible to clarify the differential diagnostic criteria for these diseases and optimize the algorithm for their molecular genetic diagnosis.

Flap Endonuclease 1 Endonucleolytically Processes RNA to Resolve R-Loops through DNA Base Excision Repair.

Flap endonuclease 1 (FEN1) is an essential enzyme that removes RNA primers and base lesions during DNA lagging strand maturation and long-patch base excision repair (BER). It plays a crucial role in maintaining genome stability and integrity. FEN1 is also implicated in RNA processing and biogenesis. A recent study from our group has shown that FEN1 is involved in trinucleotide repeat deletion by processing the RNA strand in R-loops through BER, further suggesting that the enzyme can modulate genome stability by facilitating the resolution of R-loops. However, it remains unknown how FEN1 can process RNA to resolve an R-loop. In this study, we examined the FEN1 cleavage activity on the RNA:DNA hybrid intermediates generated during DNA lagging strand processing and BER in R-loops. We found that both human and yeast FEN1 efficiently cleaved an RNA flap in the intermediates using its endonuclease activity. We further demonstrated that FEN1 was recruited to R-loops in normal human fibroblasts and senataxin-deficient (AOA2) fibroblasts, and its R-loop recruitment was significantly increased by oxidative DNA damage. We showed that FEN1 specifically employed its endonucleolytic cleavage activity to remove the RNA strand in an R-loop during BER. We found that FEN1 coordinated its DNA and RNA endonucleolytic cleavage activity with the 3'-5' exonuclease of APE1 to resolve the R-loop. Our results further suggest that FEN1 employed its unique tracking mechanism to endonucleolytically cleave the RNA strand in an R-loop by coordinating with other BER enzymes and cofactors during BER. Our study provides the first evidence that FEN1 endonucleolytic cleavage can result in the resolution of R-loops via the BER pathway, thereby maintaining genome integrity.

The RNA export and RNA decay complexes THO and TRAMP prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions.

Expansion of structure-forming CAG/CTG repetitive sequences is the cause of several neurodegenerative disorders and deletion of repeats is a potential therapeutic strategy. Transcription-associated mechanisms are known to cause CAG repeat instability. In this study, we discovered that Thp2, an RNA export factor and member of the THO (suppressors of transcriptional defects of hpr1Δ by overexpression) complex, and Trf4, a key component of the TRAMP (Trf4/5-Air1/2-Mtr4 polyadenylation) complex involved in nuclear RNA polyadenylation and degradation, are necessary to prevent CAG fragility and repeat contractions in a Saccharomyces cerevisiae model system. Depletion of both Thp2 and Trf4 proteins causes a highly synergistic increase in CAG repeat fragility, indicating a complementary role of the THO and TRAMP complexes in preventing genome instability. Loss of either Thp2 or Trf4 causes an increase in RNA polymerase stalling at the CAG repeats and other genomic loci, as well as genome-wide transcription-replication conflicts (TRCs), implicating TRCs as a cause of CAG fragility and instability in their absence. Analysis of the effect of RNase H1 overexpression on CAG fragility, RNAPII stalling, and TRCs suggests that RNAPII stalling with associated R-loops are the main cause of CAG fragility in the thp2Δ mutants. In contrast, CAG fragility and TRCs in the trf4Δ mutant can be compensated for by RPA overexpression, suggesting that excess unprocessed RNA in TRAMP4 mutants leads to reduced RPA availability and high levels of TRCs. Our results show the importance of RNA surveillance pathways in preventing RNAPII stalling, TRCs, and DNA breaks, and show that RNA export and RNA decay factors work collaboratively to maintain genome stability.

CRISPR-Cas9 mediated genome editing of Huntington's disease neurospheres.

Huntington's disease (HD) is a fatal genetic disease caused by polyglutamine aggregation encoded by an expanded CAG repeat in the huntingtin gene (HTT). In this study, we cultured neurospheres derived from R6/2 mice, a representative animal model of HD, as an in vitro model. GuideRNAs were designed to induce large deletion or frameshift indel mutation of CAG expansion. These gRNAs and Cas9 were delivered to the R6/2 neurospheres and disease-related phenotypes were observed. Deletion or indel mutation of the CAG repeat was confirmed by PCR, T7E1 assay and sequencing of the edited neurospheres. Edited neurospheres showed decreased polyglutamine aggregation compared with control HD neurospheres. In the edited neurosphere, we confirmed the upregulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and brain-derived neurotrophic factor (BDNF), whose reduced expressions are closely involved in the disease progression. In addition, flow cytometry result showed an increase in cell viability with an overall decrease in necrotic and apoptotic populations among edited R6/2 neurospheres. Additional siRNA experiments confirmed that the increased viability was decreased through inhibition of PGC-1α or BDNF. Our study confirmed that CAG repeat of R6/2 mouse-derived neurospheres can be edited through CRISPR-Cas9. Editing of CAG repeat sequence decreases polyglutamine aggregation and cellular apoptosis of HD neurospheres, which may be related to the increased expressions of PGC-1α and BDNF.Our data provide the evidence that CRISPR-Cas9 mediated genome editing has therapeutic potential on HD neuronal cells.

In Silico Genome-Scale Analysis of Molecular Mechanisms Contributing to the Development of a Persistent Infection with Methicillin-Resistant Staphylococcus aureus (MRSA) ST239.

The increasing frequency of isolation of methicillin-resistant Staphylococcus aureus (MRSA) limits the chances for the effective antibacterial therapy of staphylococcal diseases and results in the development of persistent infection such as bacteremia and osteomyelitis. The aim of this study was to identify features of the MRSAST239 0943-1505-2016 (SA943) genome that contribute to the formation of both acute and chronic musculoskeletal infections. The analysis was performed using comparative genomics data of the dominant epidemic S. aureus lineages, namely ST1, ST8, ST30, ST36, and ST239. The SA943 genome encodes proteins that provide resistance to the host's immune system, suppress immunological memory, and form biofilms. The molecular mechanisms of adaptation responsible for the development of persistent infection were as follows: amino acid substitution in PBP2 and PBP2a, providing resistance to ceftaroline; loss of a large part of prophage DNA and restoration of the nucleotide sequence of beta-hemolysin, that greatly facilitates the escape of phagocytosed bacteria from the phagosome and formation of biofilms; dysfunction of the AgrA system due to the presence of psm-mec and several amino acid substitutions in the AgrC; partial deletion of the nucleotide sequence in genomic island vSAβ resulting in the loss of two proteases of Spl-operon; and deletion of SD repeats in the SdrE amino acid sequence.

Genome-wide survey of D/E repeats in human proteins uncovers their instability and aids in identifying their role in the chromatin regulator ATAD2.

D/E repeats are stretches of aspartic and/or glutamic acid residues found in over 150 human proteins. We examined genomic stability of D/E repeats and functional characteristics of D/E repeat-containing proteins vis-à-vis the proteins with poly-Q or poly-A repeats, which are known to undergo pathologic expansions. Mining of tumor sequencing data revealed that D/E repeat-coding regions are similar to those coding poly-Qs and poly-As in increased incidence of trinucleotide insertions/deletions but differ in types and incidence of substitutions. D/E repeat-containing proteins preferentially function in chromatin metabolism and are the more likely to be nuclear and interact with core histones, the longer their repeats are. One of the longest D/E repeats of unknown function is in ATAD2, a bromodomain family ATPase frequently overexpressed in tumors. We demonstrate that D/E repeat deletion in ATAD2 suppresses its binding to nascent and mature chromatin and to the constitutive pericentromeric heterochromatin, where ATAD2 represses satellite transcription.

Evolution and folding of repeat proteins

Repeat proteins are made with tandem copies of similar amino acid stretches that fold into elongated architectures. These proteins constitute excellent model systems to investigate how evolution relates to structure, folding, and function. Here, we propose a scheme to map evolutionary information at the sequence level to a coarse-grained model for repeat-protein folding and use it to investigate the folding of thousands of repeat proteins. We model the energetics by a combination of an inverse Potts-model scheme with an explicit mechanistic model of duplications and deletions of repeats to calculate the evolutionary parameters of the system at the single-residue level. These parameters are used to inform an Ising-like model that allows for the generation of folding curves, apparent domain emergence, and occupation of intermediate states that are highly compatible with experimental data in specific case studies. We analyzed the folding of thousands of natural Ankyrin repeat proteins and found that a multiplicity of folding mechanisms are possible. Fully cooperative all-or-none transitions are obtained for arrays with enough sequence-similar elements and strong interactions between them, while noncooperative element-by-element intermittent folding arose if the elements are dissimilar and the interactions between them are energetically weak. Additionally, we characterized nucleation-propagation and multidomain folding mechanisms. We show that the global stability and cooperativity of the repeating arrays can be predicted from simple sequence scores.

Loss of PPR protein Ppr2 induces ferroptosis-like cell death in Schizosaccharomyces pombe.

Ferroptosis is a form of iron- and lipid peroxidation-mediated programmed cell death that occurs widely in mammalian cells. However, this phenomenon is rarely reported in unicellular eukaryotes. Here, we address whether ferroptosis occurs in the model unicellular eukaryote Schizosaccharomyces pombe (S. pombe). Deletion of the pentatricopeptide repeat (PPR) gene ppr2 encoding as a general mitochondrial translation factor required for mitochondrial translation disrupts iron homeostasis and induces oxidative stress, resulting in loss of cell viability. The small-molecular ferroptosis inhibitors deferoxamine (DFO) and ferrostatin-1 (Fer-1) partially rescued the ppr2 deletion-induced cell death. The amount of malondialdehyde, a lipid peroxidation marker, in Δppr2 cells was higher than that in wild type. Using C11-BODIPY 581/591, an oxidation-sensitive fluorescent lipid peroxidation probe, we showed that Δppr2 cells have a large amount of lipid peroxidation compared to wild-type cells. Deletion of ferric reductase transmembrane component 1 (frp1) encoding S. pombe ferric reductase, which is required for ferric iron uptake, partially rescued the cell death of Δppr2 cells. Our results suggest that ppr2 deletion causes an imbalance in iron homeostasis and redox, leading to ferroptosis-like cell death in S. pombe.

Meiotic Chromosomal Abnormality Detected in a Heterozygote of Elymus nutans.

Elymus nutans is an allopolyploid with a genome constitution of StStYYHH (2n = 6x = 42). Highly frequent intergenomic translocations and chromosomal variations with repeat amplification and deletions in E. nutans have been identified in the previous studies. However, more complicated structural variations such as chromosomal inversions or intra-genomic translocations are still unknown in this species, so does the reason for the origin of the chromosomal variations. Heterozygotes with rearranged chromosomes always present irregular meiosis behaviors, which subsequently cause the secondary chromosome rearrangements. Investigation on the meiosis of heterozygotes, especially on the individual chromosome level, may provide the important clues to identify the more complicated chromosome structural variations in the populations, and clarify the origin of the chromosome variations. In this study, meiotic analysis was conducted on a heterozygote plant of Elymus nutans, which showed high intra- and inter-genome chromosomal variations, by sequential fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH), with each chromosome clearly recognized. The results showed chromosomal abnormalities at every meiotic stage and abnormalities in frequency variations between different sub-genomes and different individual chromosomes. The abnormalities were revealed as univalent, fragment, rod, or Y shape bivalent in diakinesis; univalent and rod bivalent in metaphase I; lagged and segregated chromatid, bridge, fragment of the sister chromatid, fragment, bridge accompanied with fragment, and unequal segregated chromosome in anaphase I; bridge and lagged chromatid in ana-telophase II; and micronucleus at uninucleate stage. Generally, the St and H genomes harbor more abnormalities than the Y genome. Moreover, a paracentric inversion in 2St was exclusively determined, and another paracentric inversion in 6Y was tentatively identified. In addition, novel deletions were clearly detected in 3H, 4H, 1Y, and 3Y homologous chromosomes; in particular, de novo pericentric inversion in 3H was repeatedly identified in metaphase I. The study revealed the chromosomal inversions pre-existed in parents or populations, as well as de novo inversions and deletions originated in the meiosis of the heterozygote in E. nutans. Moreover, it indicated wide range of meiosis abnormalities on different stages and different chromosomes, and suggests that secondary rearrangements contribute much to the chromosome variations in E. nutans.

Mapping Blast Resistance Genes in Rice Varieties 'Minghui 63' and 'M-202'.

Rice blast caused by the fungus Magnaporthe oryzae (syn. Magnaporthe grisea) is one of the most lethal diseases for sustainable rice production worldwide. Blast resistance mediated by major resistance genes is often broken down after a short period of deployment, while minor blast resistance genes, each providing a small effect on disease reactions, are more durable. In the present study, we first evaluated disease reactions of two rice breeding parents 'Minghui 63' and 'M-202' with 11 blast races, IA45, IB1, IB45, IB49, IB54, IC1, IC17, ID1, IE1, IG1, and IH1, commonly present in the United States, under greenhouse conditions using a category disease rating resembling infection types under field conditions. 'Minghui 63' exhibited differential resistance responses in comparison with those of 'M-202' to the tested blast races. A recombinant inbred line (RIL) population of 275 lines from a cross between 'Minghui 63' and 'M-202' was also evaluated with the above-mentioned blast races. The population was genotyped with 156 simple sequence repeat (SSR) and insertion and deletion (Indel) markers. A linkage map with a genetic distance of 1,022.84 cM was constructed using inclusive composite interval mapping (ICIM) software. A total of 10 resistance QTLs, eight from 'Minghui 63' and two from 'M-202', were identified. One major QTL, qBLAST2 on chromosome 2, was identified by seven races/isolates. The remaining nine minor resistance QTLs were mapped on chromosomes 1, 3, 6, 9, 10, 11, and 12. These findings provide useful genetic markers and resources to tag minor blast resistance genes for marker-assisted selection in rice breeding program and for further studies of underlying genes.

An ARF1-binding factor triggering programmed cell death and periderm development in pear russet fruit skin.

Plants have a cuticular membrane (CM) and periderm membrane (PM), which act as barriers to terrestrial stresses. The CM covers primary organs with a continuous hydrophobic layer of waxes embedded in cutin, while the PM includes suberized cells stacked externally to the secondary tissues. The formation of native periderm is regulated by a postembryonic meristem phellogen that produces suberized phellem (cork) outwardly. However, the mechanism controlling phellogen differentiation to phellem remains to be clarified. Here, map-based cloning in a pear F1 population with segregation for periderm development in fruit skin facilitated the identification of an aspartic acid repeat deletion in Pyrus Periderm Programmed Cell Death 1.1 (PyPPCD1.1) that triggers phellogen activity for cork formation in russet fruit skin of pear. PyPPCD1.1 showed preferential expression in pear fruit skin, and the encoded protein shares a structural similarity to that of the viral capsid proteins. Aspartic acid deletion in PyPPCD1.1 weakened its nuclear localization but increased its accumulation in the chloroplast. The products of both PyPPCD1.1 and its recessive allele directly interact with ADP-ribosylation factor 1 (ARF1). PyPPCD1.1 triggered programmed cell death in an ARF1-dependent manner. Thus, this study identified the switch gene for programmed cell death and periderm development and provided a new molecular regulatory mechanism underlying the development of this trait.

Melatonin, circadian rhythms and glaucoma: current perspective.

Melatonin, circadian rhythms and glaucoma: current perspective.

Increased Pathogenicity of the Nematophagous Fungus Drechmeria coniospora Following Long-Term Laboratory Culture.

Domestication provides a window into adaptive change. Over the course of 2 decades of laboratory culture, a strain of the nematode-specific fungus Drechmeria coniospora became more virulent during its infection of Caenorhabditis elegans. Through a close comparative examination of the genome sequences of the original strain and its more pathogenic derivative, we identified a small number of non-synonymous mutations in protein-coding genes. In one case, the mutation was predicted to affect a gene involved in hypoxia resistance and we provide direct corroborative evidence for such an effect. The mutated genes with functional annotation were all predicted to impact the general physiology of the fungus and this was reflected in an increased in vitro growth, even in the absence of C. elegans. While most cases involved single nucleotide substitutions predicted to lead to a loss of function, we also observed a predicted restoration of gene function through deletion of an extraneous tandem repeat. This latter change affected the regulatory subunit of a cAMP-dependent protein kinase. Remarkably, we also found a mutation in a gene for a second protein of the same, protein kinase A, pathway. Together, we predict that they result in a stronger repression of the pathway for given levels of ATP and adenylate cyclase activity. Finally, we also identified mutations in a few lineage-specific genes of unknown function that are candidates for factors that influence virulence in a more direct manner.

Aluelement in the RNA binding motif protein, X-linked 2 (RBMX2) gene found to be linked to bipolar disorder.

ObjectiveWe have used long-read single molecule, real-time (SMRT) sequencing to fully characterize a ~12Mb genomic region on chromosome Xq24-q27, significantly linked to bipolar disorder (BD) in an extended family from a genetic sub-isolate. This family segregates BD in at least four generations with 24 affected individuals.MethodsWe selected 16 family members for targeted sequencing. The selected individuals either carried the disease haplotype, were non-carriers of the disease haplotype, or served as married-in controls. We designed hybrid capture probes enriching for 5-9Kb fragments spanning the entire 12Mb region that were then sequenced to screen for candidate structural variants (SVs) that could explain the increased risk for BD in this extended family.ResultsAltogether, 201 variants were detected in the critically linked region. Although most of these represented common variants, three variants emerged that showed near-perfect segregation among all BD type I affected individuals. Two of the SVs were identified in or near genes belonging to the RNA Binding Motif Protein, X-Linked (RBMX) gene family—a 330bp Alu (subfamily AluYa5) deletion in intron 3 of the RBMX2 gene and an intergenic 27bp tandem repeat deletion between the RBMX and G protein-coupled receptor 101 (GPR101) genes. The third SV was a 50bp tandem repeat insertion in intron 1 of the Coagulation Factor IX (F9) gene.ConclusionsAmong the three genetically linked SVs, additional evidence supported the Alu element deletion in RBMX2 as the leading candidate for contributing directly to the disease development of BD type I in this extended family.

EGF repeats of epidermal growth factor‑like domain7 promote endothelial cell activation and tumor escape from the immune system.

The tumor blood vessel endothelium forms a barrier that must be crossed by circulating immune cells in order for them to reach and kill cancer cells. Epidermal growth factor‑like domain7 (Egfl7) represses this immune infiltration by lowering the expression levels of leukocyte adhesion receptors on the surface of endothelial cells. However, the protein domains involved in these properties are not completely understood. Egfl7 is structurally composed of the predicted EMI‑, EGF‑ and C‑terminal domains. The present study aimed to investigate the roles of these different domains in tumor development by designing retroviruses coding for deletion mutants and then infecting 4T1 breast cancer cell populations, which consequently overexpressed the variants. By performing invitro soft‑agar assays, it was found that Egfl7 and its deletion variants did not affect cell proliferation or anchorage‑independent growth. When 4T1cells expressing either the wild‑type Egfl7 protein or Egfl7 domain variants were implanted in mice, Egfl7 expression markedly promoted tumor development and deletion of the EGF repeats decreased the tumor growth rate. By contrast, deleting any other domain displayed no significant effect on tumor development. The overexpression of Egfl7 also decreased Tcell and natural killer cell infiltration in tumors, as determined by immunofluorescence staining of tumor sections, whereas deletion of the EGF repeats inhibited this effect. Reverse transcription‑quantitative PCR analysis of the mechanisms involved revealed that deleting the EGF repeats partially restored the expression levels of vascular cell adhesion molecule1 and E‑selectin, which were suppressed by overexpression of Egfl7 in endothelial cells invitro. This resulted in a higher number of lymphocytes bound to HUVEC expressing Egfl7‑ΔEGF compared with HUVEC expressing wild‑type Egfl7, as assessed by fluorescent‑THP‑1 adhesion assays onto endothelial cells. Overall, the present study demonstrated that the EGF repeats may participate in the protumoral and anti‑inflammatory effects of Egfl7.