Repeat Motifs Research Articles

Biallelic ANKS6 null variants cause notable extrarenal phenotypes in a nephronophthisis patient and lead to hepatobiliary abnormalities by YAP1 deficiency.

The ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) gene, encoding an inversin compartment protein of the primary cilium, was recently reported as a pathogenic gene of nephronophthisis (MIM PS256100). Extrarenal manifestations are frequently observed in this disease, however, potential genotype-phenotype correlations and the underlying mechanisms remain poorly understood. Here we described an infant with kidney failure, hepatobiliary abnormalities, and heart disease, in whom whole exome sequencing identified compound heterozygous variants in ANKS6, including a novel nonsense variant p.Trp458* and a recurrent splicing variant c.2394+1G > A. mRNA expression studies showed that the splicing variant caused aberrant mRNA splicing with exon 13 skipping and the biallelic variants were predicted to cause loss of ANKS6 function. We systematically characterized the clinical and genetic spectra of the disease and revealed that biallelic null variants in ANKS6 cause more severe kidney disease and more extrarenal manifestations, thus establishing a clear genotype-phenotype correlation for the disease. Further evaluations showed that ANKS6 deficiency reduced YAP1 expression in the patient's bile duct epithelium and ANKS6 promotes YAP1 transcriptional activity in a dose-dependent manner, indicating that loss of ANKS6 function causes hepatobiliary abnormalities through YAP1 deficiency during biliary morphogenesis and development, which may offer new therapeutic targets.

STRategy: A support system for collecting and analyzing next-generation sequencing data of short tandem repeats for forensic science.

Short tandem repeats (STRs) are short repeated sequences commonly found in the human genome and valuable in forensic science, used for human identity and relatedness markers. Next-generation sequencing (NGS) technologies, e.g., ForenSeq Signature Prep, can sequence STRs, inferring length-based alleles and single nucleotide polymorphisms (SNPs) and providing valuable insights into population and sub-population structures. Despite the potential benefits of NGS for STRs, no open-source software platform integrates the collection, management, and analysis of STR data from NGS into one place. Users must use multiple programs to process their STR data and then collect the results into a separate database or a file system folder. Moreover, analyzing repeat structures (STR repeat motifs) may require learning multiple software tools, making the process inefficient and cumbersome. To address this gap, we introduce the STRategy, a standalone web-based application supporting essential STR data management and analysis capabilities. The STRategy allows users to collect their data into its database, automatically calculates forensic parameters, and visualizes the analyzed data in various forms. Users can search the database using different options, such as by profile, loci, and genotypes, with and without a specific test kit. Moreover, users can also find the nucleotide variants of a locus among the samples. We designed the STRategy for internal use in a laboratory or an organization. Hence, our system includes role-based access control that allows users to search for or access specific data based on their responsibilities. The administrator role can customize the system, for example, configure maps according to the samples' geographic data, and manage reference STR repeat motifs. A laboratory or an organization can download and install a copy of STRategy on their local system using Docker, as described in https://github.com/cucpbioinfo/STRategy. In summary, the STRategy is an end-to-end system that provides users with a database to collect the analyzed STR data from NGS, the dynamic analyses of forensic parameters, and the variants of STR patterns according to the newly added samples, which are then explorable via various search options and visualizations. The system is helpful for both forensic investigations and forensic genetics.

Normal and pathogenic variation of RFC1 repeat expansions: implications for clinical diagnosis.

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is an autosomal recessive neurodegenerative disease, usually caused by biallelic AAGGG repeat expansions in RFC1. In this study, we leveraged whole genome sequencing data from nearly 10 000 individuals recruited within the Genomics England sequencing project to investigate the normal and pathogenic variation of the RFC1 repeat. We identified three novel repeat motifs, AGGGC (n = 6 from five families), AAGGC (n = 2 from one family) and AGAGG (n = 1), associated with CANVAS in the homozygous or compound heterozygous state with the common pathogenic AAGGG expansion. While AAAAG, AAAGGG and AAGAG expansions appear to be benign, we revealed a pathogenic role for large AAAGG repeat configuration expansions (n = 5). Long-read sequencing was used to characterize the entire repeat sequence, and six patients exhibited a pure AGGGC expansion, while the other patients presented complex motifs with AAGGG or AAAGG interruptions. All pathogenic motifs appeared to have arisen from a common haplotype and were predicted to form highly stable G quadruplexes, which have previously been demonstrated to affect gene transcription in other conditions. The assessment of these novel configurations is warranted in CANVAS patients with negative or inconclusive genetic testing. Particular attention should be paid to carriers of compound AAGGG/AAAGG expansions when the AAAGG motif is very large (>500 repeats) or the AAGGG motif is interrupted. Accurate sizing and full sequencing of the satellite repeat with long-read sequencing is recommended in clinically selected cases to enable accurate molecular diagnosis and counsel patients and their families.

RFC1 in an Australasian neurological disease cohort: extending the genetic heterogeneity and implications for diagnostics.

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome is a progressive, generally late-onset, neurological disorder associated with biallelic pentanucleotide expansions in Intron 2 of the RFC1 gene. The locus exhibits substantial genetic variability, with multiple pathogenic and benign pentanucleotide repeat alleles previously identified. To determine the contribution of pathogenic RFC1 expansions to neurological disease within an Australasian cohort and further investigate the heterogeneity exhibited at the locus, a combination of flanking and repeat-primed PCR was used to screen a cohort of 242 Australasian patients with neurological disease. Patients whose data indicated large gaps within expanded alleles following repeat-primed PCR, underwent targeted long-read sequencing to identify novel repeat motifs at the locus. To increase diagnostic yield, additional probes at the RFC1 repeat region were incorporated into the PathWest diagnostic laboratory targeted neurological disease gene panel to enable first-pass screening of the locus for all samples tested on the panel. Within the Australasian cohort, we detected known pathogenic biallelic expansions in 15.3% (n = 37) of patients. Thirty indicated biallelic AAGGG expansions, two had biallelic 'Māori alleles' [(AAAGG)exp(AAGGG)exp], two samples were compound heterozygous for the Māori allele and an AAGGG expansion, two samples had biallelic ACAGG expansions and one sample was compound heterozygous for the ACAGG and AAGGG expansions. Forty-five samples tested indicated the presence of biallelic expansions not known to be pathogenic. A large proportion (84%) showed complex interrupted patterns following repeat-primed PCR, suggesting that these expansions are likely to be comprised of more than one repeat motif, including previously unknown repeats. Using targeted long-read sequencing, we identified three novel repeat motifs in expanded alleles. Here, we also show that short-read sequencing can be used to reliably screen for the presence or absence of biallelic RFC1 expansions in all samples tested using the PathWest targeted neurological disease gene panel. Our results show that RFC1 pathogenic expansions make a substantial contribution to neurological disease in the Australasian population and further extend the heterogeneity of the locus. To accommodate the increased complexity, we outline a multi-step workflow utilizing both targeted short- and long-read sequencing to achieve a definitive genotype and provide accurate diagnoses for patients.

Gene co-expression network identifies critical genes, pathways and regulatory motifs mediating the progression of rift valley fever in Bostaurus

Rift Valley Fever (RVF) is a mosquito-borne viral disease caused by the Rift Valley Fever Virus. The disease is a zoonosis that largely affects domestic animals, including sheep, goats, and cattle, resulting in severe morbidity and mortality marked by massive storm abortions. To halt human and livestock deaths due to RVF, the development of efficacious vaccines and therapeutics is a compelling and urgent priority. We sought to identify potential key modules (gene clusters), hub genes, and regulatory motifs involved in the pathogenesis of RVF in Bos taurus that are amenable to inhibition. We analyzed 39 Bos taurus RNA-Seq samples using the weighted gene co-expression network analysis (WGCNA) R package and uncovered significantly enriched modules containing genes with potential pivotal roles in RVF progression. Moreover, regulatory motif analysis conducted using the Multiple Expectation Maximization for Motif Elicitation (MEME) suite identified motifs that probably modulate vital biological processes. Gene ontology terms associated with identified motifs were inferred using the GoMo human database. The gene co-expression network constructed in WGCNA using 5000 genes contained seven (7) modules, out of which four were significantly enriched for terms associated with response to viruses, response to interferon-alpha, innate immune response, and viral defense. Additionally, several biological pathways implicated in developmental processes, anatomical structure development, and multicellular organism development were identified. Regulatory motifs analysis identified short, repeated motifs whose function(s) may be amenable to disruption by novel therapeutics. Predicted functions of identified motifs include tissue development, embryonic organ development, and organ morphogenesis. We have identified several hub genes in enriched co-expressed gene modules and regulatory motifs potentially involved in the pathogenesis of RVF in B. taurus that are likely viable targets for disruption by novel therapeutics.

Functional and structural insights into human N‐deacetylase/N‐sulfotransferase activities

AbstractHeparan sulfate (HS) is a linear polysaccharide composed of a glucuronic acid (GlcA)‐N‐acetyl‐glucosamine (GlcNAc) disaccharide repeat motif, polymerized by the EXT1–EXT2 complex. It is extensively modified by a series of Golgi localized enzymes, that generate distinct saccharide sequences involved in the binding and the regulation of numerous protein partners. N‐deacetylase/N‐sulfotransferase (NDST), of which four isoforms have been identified in mammals, are involved in the first step of this process and catalyze both the N‐deacetylation of the GlcNAc residues into GlcNH2 and its re‐N‐sulfation into GlcNS residues. Further modifications of the HS chain depend on this first maturation event, NDST action is, therefore, key to HS biosynthesis. However, although the sulfotransferase domain of NDST1 has been characterized at the structural level some 20 years ago, information on the overall structure and activity of the enzyme are still lacking. Here, we report the characterization of the two most expressed NDSTs in humans, NDST1 and NDST2, and a model structure of NDST1 homodimer using cryoelectron microscopy combined with AlphaFold2 modeling. Structure‐driven mutagenesis along with two bioassays to follow the protein activities allowed us to characterize the kinetics of the deacetylation and sulfoaddition and to identify the residue H529 as necessary for N‐deacetylation. These results shed light on a poorly understood family of enzymes and will help deciphering the molecular basis for HS and heparin maturation.

Sequence variants affecting the genome-wide rate of germline microsatellite mutations

Microsatellites are polymorphic tracts of short tandem repeats with one to six base-pair (bp) motifs and are some of the most polymorphic variants in the genome. Using 6084 Icelandic parent-offspring trios we estimate 63.7 (95% CI: 61.9–65.4) microsatellite de novo mutations (mDNMs) per offspring per generation, excluding one bp repeats motifs (homopolymers) the estimate is 48.2 mDNMs (95% CI: 46.7–49.6). Paternal mDNMs occur at longer repeats than maternal ones, which are in turn larger with a mean size of 3.4 bp vs 3.1 bp for paternal ones. mDNMs increase by 0.97 (95% CI: 0.90–1.04) and 0.31 (95% CI: 0.25–0.37) per year of father’s and mother’s age at conception, respectively. Here, we find two independent coding variants that associate with the number of mDNMs transmitted to offspring; The minor allele of a missense variant (allele frequency (AF) = 1.9%) in MSH2, a mismatch repair gene, increases transmitted mDNMs from both parents (effect: 13.1 paternal and 7.8 maternal mDNMs). A synonymous variant (AF = 20.3%) in NEIL2, a DNA damage repair gene, increases paternally transmitted mDNMs (effect: 4.4 mDNMs). Thus, the microsatellite mutation rate in humans is in part under genetic control.

Isolation and characterization of 17 polymorphic microsatellite loci with tri- and tetra-nucleotide repeat motifs for Ayu (Plecoglossus altivelis) using next-generation sequencing.

Ayu or sweetfish, Plecoglossus altivelis, an amphidromous fish ranging in the northwestern Pacific, is economically important inland fisheries and aquaculture resources. Genetic characterization of wild Ayu and derived culture seeds with competent molecular genetic markers is still insufficient for their sustainable use. Microsatellite DNA markers with larger repeat motifs (e.g. tri- and tetra-nucleotide motifs) are convenient and accurate compared with those having mono- and di-nucleotide motifs, but the latter motifs characterized most Ayu microsatellite markers developed previously. Here, we isolated and characterized 17 polymorphic microsatellite DNA markers with tri- and tetra-nucleotide repeat motif using next-generation sequencing. Alleles per locus varied from 6 to 23. The observed and expected heterozygosities ranged from 0.542 to1.000and 0.709to0.951, respectively. Polymorphic information content (PIC) of 15 out of the 17 loci were high (≧ 0.700), suggesting them to be highly informative. Twelve of the 17 loci were used for preliminary assignment test among three collections, and successfully allocated the examined fish to the original populations. The novel polymorphic microsatellite markers developed herein will be useful to examine the genetic diversity and population structure of wild Ayu and the effect of seed transplantation on native populations, providing a tool for conservation and sustainable adaptive management of this species.

Amaranth Genomic Resource Database: an integrated database resource of Amaranth genes and genomics.

Amaranth (Amaranthus L.) is native to Mexico and North America, where it was cultivated thousands of years ago, but now amaranth is grown worldwide. Amaranth is one of the most promising food crops with high nutritional value and belongs to the family Amaranthaceae. The high-quality genome assembly of cultivated amaranth species (A. hypochondriacus, A. cruentus) and wild/weedy species (A. tuberculatus, A. hybridus, and A. palmeri) has already been reported; therefore, we developed an Amaranth Genomic Resource Database (AGRDB) to provide access to all the genomic information such as genes, SSRs, SNPs, TFs, miRNAs, and transporters in one place. The AGRDB database contains functionally annotated gene information with their sequence details, genic as well as genomic SSRs with their three sets of primers, transcription factors classified into different families with their sequence information and annotation details, putative miRNAs with their family, sequences, and targeted gene details, transporter genes with their superfamily, trans-membrane domain details, and details of genic as well as nongenic SNPs with 3' and 5' flanking sequence information of five amaranth species. A database search can be performed using the gene ID, sequence ID, sequence motif, motif repeat, family name, annotation keyword, scaffold or chromosome numbers, etc. This resource also includes some useful tools, including JBrowse for the visualization of genes, SSRs, SNPs, and TFs on the respective amaranth genomes and BLAST search to perform a BLAST search of the user's query sequence against the amaranth genome as well as protein sequences. The AGRDB database will serve as a potential platform for genetic improvement and characterization of this futuristic crop. The AGRDB database will be accessible via the link: http://www.nbpgr.ernet.in:8080/AmaranthGRD/.

The sequence of the repetitive motif influences the frequency of multistep mutations in Short Tandem Repeats

Microsatellites, or Short Tandem Repeats (STRs), are subject to frequent length mutations that involve the loss or gain of an integer number of repeats. This work aimed to investigate the correlation between STRs’ specific repetitive motif composition and mutational dynamics, specifically the occurrence of single- or multistep mutations. Allelic transmission data, comprising 323,818 allele transfers and 1,297 mutations, were gathered for 35 Y-chromosomal STRs with simple structure. Six structure groups were established: ATT, CTT, TCTA/GATA, GAAA/CTTT, CTTTT, and AGAGAT, according to the repetitive motif present in the DNA leading strand of the markers. Results show that the occurrence of multistep mutations varies significantly among groups of markers defined by the repetitive motif. The group of markers with the highest frequency of multistep mutations was the one with repetitive motif CTTTT (25% of the detected mutations) and the lowest frequency corresponding to the group with repetitive motifs TCTA/GATA (0.93%). Statistically significant differences (α = 0.05) were found between groups with repetitive motifs with different lengths, as is the case of TCTA/GATA and ATT (p = 0.0168), CTT (p < 0.0001) and CTTTT (p < 0.0001), as well as between GAAA/CTTT and CTTTT (p = 0.0102). The same occurred between the two tetrameric groups GAAA/CTTT and TCTA/GATA (p < 0.0001) – the first showing 5.7 times more multistep mutations than the second. When considering the number of repeats of the mutated paternal alleles, statistically significant differences were found for alleles with 10 or 12 repeats, between GATA and ATT structure groups. These results, which demonstrate the heterogeneity of mutational dynamics across repeat motifs, have implications in the fields of population genetics, epidemiology, or phylogeography, and whenever STR mutation models are used in evolutionary studies in general.

Characterization of complex structural variation in the CYP2D6-CYP2D7-CYP2D8 gene loci using single-molecule long-read sequencing.

Complex regions in the human genome such as repeat motifs, pseudogenes and structural (SVs) and copy number variations (CNVs) present ongoing challenges to accurate genetic analysis, particularly for short-read Next-Generation-Sequencing (NGS) technologies. One such region is the highly polymorphic CYP2D loci, containing CYP2D6, a clinically relevant pharmacogene contributing to the metabolism of >20% of common drugs, and two highly similar pseudogenes, CYP2D7 and CYP2D8. Multiple complex SVs, including CYP2D6/CYP2D7-derived hybrid genes are known to occur in different configurations and frequencies across populations and are difficult to detect and characterize accurately. This can lead to incorrect enzyme activity assignment and impact drug dosing recommendations, often disproportionally affecting underrepresented populations. To improve CYP2D6 genotyping accuracy, we developed a PCR-free CRISPR-Cas9 based enrichment method for targeted long-read sequencing that fully characterizes the entire CYP2D6-CYP2D7-CYP2D8 loci. Clinically relevant sample types, including blood, saliva, and liver tissue were sequenced, generating high coverage sets of continuous single molecule reads spanning the entire targeted region of up to 52kb, regardless of SV present (n = 9). This allowed for fully phased dissection of the entire loci structure, including breakpoints, to accurately resolve complex CYP2D6 diplotypes with a single assay. Additionally, we identified three novel CYP2D6 suballeles, and fully characterized 17 CYP2D7 and 18 CYP2D8 unique haplotypes. This method for CYP2D6 genotyping has the potential to significantly improve accurate clinical phenotyping to inform drug therapy and can be adapted to overcome testing limitations of other clinically challenging genomic regions.

Plastomes of Sonchus (Asteraceae) endemic to the Atlantic Madeira archipelago: Genome structure, comparative analysis, and phylogenetic relationships.

The woody Sonchus alliance, a spectacular example of adaptive radiation with six genera and approximately 31 species, is found exclusively on three Macaronesian Islands (Madeira, Canaries, and Cape Verdes) in the Atlantic Ocean. Four of the Sonchus taxa are restricted to Madeira, including shrubs and small trees at higher elevations (S. fruticosus and S. pinnatus), and caudex perennials in the lower coastal areas (S. ustulatus subsp. maderensis and S. ustulatus subsp. ustulatus). The Madeiran Sonchus stemmed from a single colonization event that originated from the Canaries < 3 million years ago. However, the plastome evolution and species relationships remains insufficiently explored. We therefore assembled and characterized the plastomes of four Sonchus taxa from Madeira and conducted a phylogenomic analysis. We found highly conserved plastome sequences among the taxa, further supporting a single and recent origin. We also found highly conserved plastomes among the cosmopolitan weedy Sonchus, Macaronesian Sonchus in the Atlantic, and Juan Fernández Islands Dendroseris in the Pacific. Furthermore, we identified four mutation hotspot regions (trnK-rps16, petN-psbM, ndhF-Ψycf1, and ycf1) and simple sequence repeat motifs. This study strongly supports the monophyly of Madeiran Sonchus. However, its relationship with the remaining woody Sonchus alliance from the Canary Islands requires further investigation.

Circulating tumor DNA in plasma as an early predictor for pathological lymph node involvement and (neo-)adjuvant chemotherapy in patients with colon cancer.

3532 Background: Postoperative chemotherapy is standard for colon cancer patients with pathological lymph node involvement (pN+). If the presence of pN+ could be accurately predicted prior to surgery, the initiation of neoadjuvant chemotherapy could potentially reduce the presence of micrometastasis and enhance survival. The objective of this study was to assess the utility of preoperative methylated circulating tumor DNA (meth-ctDNA) as a predictive marker for pN+ and its clinical value as a marker for the initiation of neoadjuvant chemotherapy. Methods: The clinical data of 203 colon cancer patients (stage I-III) was collected from the Colorectal Cancer Database established at Danish Colorectal Cancer Center South, Vejle Hospital, Denmark. Patients were randomized into a discovery and a validation cohort. Plasma collected preoperatively was analyzed for three different meth-ctDNAs (Neuropeptide Y, Galactose-3-O-Sulfotransferase 3, and KN Motif and Ankyrin Repeat Domains 1) by droplet digital PCR (ddPCR). Results: Based on analysis of the discovery cohort, samples were considered positive if two or more of the three methylation markers had at least three positive droplets in the ddPCR analysis. 40% of patients in the discovery cohort and 46% of patients in the validation cohort were considered positive. When using meth-ctDNA as a diagnostic tool to predict pN+, the sensitivity and specificity in the discovery cohort were 42% and 61% respectively. In the validation cohort the values were 54% and 59%, respectively. AUC was &lt; 60% in both cohorts. Meth-ctDNA was associated to clinical tumor (T)- and node (N)-category in both cohorts (p≤0.01). Four-year disease free survival (DFS) in patients with and without detectable meth-ctDNA was 60% and 90% in the discovery cohort (p = 0.01), and 62% and 78% in the validation cohort (p = 0.04). Meth-ctDNA remained the strongest prognostic factor of DFS in the multivariate analysis compared to clinical T- or N-category, gender and age (p = 0.065, HR 2.46, 95% CI 1.03-5.88), however not statistically significant. Four-year overall survival (OS) in patients with and without detectable meth-ctDNA was 75% and 95% in the discovery cohort (p = 0.006), and 78% and 91% in the validation cohort (p = 0.02). In the multivariate analysis, meth-ctDNA was the strongest prognostic factor of OS compared to clinical T- or N-category, gender and age (p = 0.016, HR 4.54, 95% CI 1.32-15.6). Conclusions: The preoperative measurement of meth-ctDNA was not found to be a clinically significant predictor of pN+ or a reliable indicator for the initiation of neoadjuvant chemotherapy. Nonetheless, the results showed that preoperative ctDNA is a powerful prognostic indicator, suggesting that randomized controlled trials should be conducted to determine the efficacy of neoadjuvant chemotherapy in colon cancer patients with preoperative detectable ctDNA.

Plakophilin-3 Binds the Membrane and Filamentous Actin without Bundling F-Actin.

Plakophilin-3 is a ubiquitously expressed protein found widely in epithelial cells and is a critical component of desmosomes. The plakophilin-3 carboxy-terminal domain harbors nine armadillo repeat motifs with largely unknown functions. Here, we report the 5 Å cryogenic electron microscopy (cryoEM) structure of the armadillo repeat motif domain of plakophilin-3, one of the smaller cryoEM structures reported to date. We find that this domain is a monomer or homodimer in solution. In addition, using an in vitro actin co-sedimentation assay, we show that the armadillo repeat domain of plakophilin-3 directly interacts with F-actin. This feature, through direct interactions with actin filaments, could be responsible for the observed association of extra-desmosomal plakophilin-3 with the actin cytoskeleton directly attached to the adherens junctions in A431 epithelial cells. Further, we demonstrate, through lipid binding analyses, that plakophilin-3 can effectively be recruited to the plasma membrane through phosphatidylinositol-4,5-bisphosphate-mediated interactions. Collectively, we report on novel properties of plakophilin-3, which may be conserved throughout the plakophilin protein family and may be behind the roles of these proteins in cell-cell adhesion.

Inhibiting eukaryotic initiation factor 5A (eIF5A) hypusination attenuated activation of the SIK2 (salt-inducible kinase 2)-p4E-BP1 pathway involved in ovarian cancer cell proliferation and migration.

Eukaryotic initiation factor 5A hypusine (eIF5AHyp) stimulates the translation of proline repeat motifs. Salt inducible kinase 2 (SIK2) containing a proline repeat motif is overexpressed in ovarian cancers, in which it promotes cell proliferation, migration, and invasion. Western blotting and dual luciferase analyses showed that depletion of eIF5AHyp by GC7 or eIF5A-targeting siRNA downregulated SIK2 level and decreased luciferase activity in cells transfected with a luciferase-based reporter construct containing consecutive proline residues, whereas the activity of the mutant control reporter construct (replacing P825L, P828H, and P831Q) did not change. According to the MTT assay, GC7, which has a potential antiproliferative effect, reduced the viability of several ovarian cancer cell lines by 20-35% at high concentrations (ES2 > CAOV-3 > OVCAR-3 > TOV-112D) but not at low concentrations. In a pull-down assay, we identified eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and 4E-BP1 (p4E-BP1) phosphorylated at Ser 65 as downstream binding partners of SIK2, and we validated that the level of p4E-BP1(Ser 65) was downregulated by SIK2-targeting siRNA. Conversely, in ES2 cells overexpressing SIK2, the p4E-BP1(Ser65) level was increased but decreased in the presence of GC7 or eIF5A-targeting siRNA. Finally, the migration, clonogenicity, and viability of ES2 ovarian cancer cells were reduced by GC7 treatment as well as by siRNA for eIF5A gene silencing and siRNA for SIK2 and 4E-BP1 gene silencing. Conversely, those activities were increased in cells overexpressing SIK2 or 4E-BP1 and decreased again in the presence of GC7. The depletion of eIF5AHyp by GC7 or eIF5A-targeting siRNA attenuated activation of the SIK2-p4EBP1 pathway. In that way, eIF5AHyp depletion reduces the migration, clonogenicity, and viability of ES2 ovarian cancer cells.

Forensic Implications of the Discrepancies Caused between NGS and CE Results by New Microvariant Allele at Penta E Microsatellite.

Examination of STR markers using the MPS technology is becoming more common in forensic genetics, but scientists still have insufficient experience in dealing with ambiguous results. However, it is always essential to resolve discordant data if we want to use the technology as an accredited method in routine forensic casework. During the internal laboratory validation of the Precision ID GlobalFiler NGS STR Panel v2 kit, we observed two discrepant genotypes at Penta E locus compared to the previous capillary electrophoresis results. Each NGS software that we applied (i.e., Converge, STRaitRazor and IGV) returned the same 12,14 and 12,16 genotypes in the two samples, respectively, instead of the 11.3,14 and 11.3,16 genotypes previously observed with CE (Capillary electrophoresis) typing. In the case of the length variant 11.3 alleles, traditional Sanger sequencing confirmed a complete twelve repeat unit structure in both samples. However, after sequencing was extended to the flanking regions of the variant alleles, sequence data revealed a two-bases GG deletion downstream of the last TCTTT repeat motif in the forward strand. The determined allele variant has not been previously reported in the scientific literature and highlights the need for a careful evaluation and thorough concordance studies before using NGS STR data in forensic cases.

A robust microsatellite instability detection model for unpaired colorectal cancer tissue samples.

Microsatellite instability (MSI) is a key biomarker for cancer immunotherapy and prognosis. Integration of MSI testing into a next-generation-sequencing (NGS) panel could save tissue sample, reduce turn-around time and cost, and provide MSI status and comprehensive genomic profiling in single test. We aimed to develop an MSI calling model to detect MSI status along with the NGS panel-based profiling test using tumor-only samples. From January 2019 to December 2020, a total of 174 colorectal cancer (CRC) patients were enrolled, including 31 MSI-high (MSI-H) and 143 microsatellite stability (MSS) cases. Among them, 56 paired tumor and normal samples (10 MSI-H and 46 MSS) were used for modeling, and another 118 tumor-only samples were used for validation. MSI polymerase chain reaction (MSI-PCR) was performed as the gold standard. A baseline was built for the selected microsatellite loci using the NGS data of 56 normal blood samples. An MSI detection model was constructed by analyzing the NGS data of tissue samples. The performance of the model was compared with the results of MSI-PCR. We first intersected the target genomic regions of the NGS panels used in this study to select common microsatellite loci. A total of 42 loci including 23 mononucleotide repeat sites and 19 longer repeat sites were candidates for modeling. As mononucleotide repeat sites are more sensitive and specific for detecting MSI status than sites with longer length motif and the mononucleotide repeat sites performed even better than the total sites, a model containing 23 mononucleotide repeat sites was constructed and named Colorectal Cancer Microsatellite Instability test (CRC-MSI). The model achieved 100% sensitivity and 100% specificity when compared with MSI-PCR in both training and validation sets. Furthermore, the CRC-MSI model was robust with the tumor content as low as 6%. In addition, 8 out of 10 MSI-H samples showed alternations in the four mismatch repair genes (MLH1, MSH2, MSH6, and PMS2). MSI status can be accurately determined along the targeted NGS panels using only tumor samples. The performance of mononucleotide repeat sites surpasses loci with longer repeat motif in MSI calling.

Identifying novel risk conferring genes involved in glycosylation processes with familial schizophrenia in an Indian cohort: Prediction of ADAMTS9 gene variant for structural stability

Schizophrenia is a complex neuropsychiatric disorder and heritability is as high as 80 % making it the most heritable mental disorder. Although GWAS has identified numerous variants, the pathophysiology is still elusive. Here, an attempt was made to identify genetic risk factors in familial cases of schizophrenia that are associated with a common causative pathway. To achieve this objective, exome sequencing was done in 4 familial cases and identified six unique coding variants in five genes. Among these genes, PIGQ gene has two pathogenic variants, one nonsense and in-frame deletion. One missense variant in GALNT16 and one in GALNT5 have variable damaging score, however, the other variants, in ADAMTS9 and in LTBP4 have the highest damaging score. Further analysis showed that the variant of LTBP4 was not present in the functional domain. The other missense variant in the ADAMTS9 gene was found to be significant and was present in the thrombospondin repeat motif, one of the important motifs. Detailed molecular dynamics simulation study on this variant showed a damaging effect on structural stability. Since, all these genes culminated into the glycosylation process, it was evident that an aberrant glycosylation process may be one of the risk factors. Although, extracellular matrix formation through glycosylation have been shown to be associated, the involvement of ADAMTS9 and PIGQ gene mediated glycosylation has not been reported. In this paper, a novel link between ADAMTS9 and PIGQ gene with schizophrenia have been reported. Therefore, this novel observation has contributed immensely to the existing knowledge on risk factor of Schizophrenia.

Analysis of genome instability and implications for the consequent phenotype in Plasmodium falciparum containing mutated MSH2-1 (P513T).

Malarial parasites exhibit extensive genomic plasticity, which induces the antigen diversification and the development of antimalarial drug resistance. Only a few studies have examined the genome maintenance mechanisms of parasites. The study aimed at elucidating the impact of a mutation in a DNA mismatch repair gene on genome stability by maintaining the mutant and wild-type parasites through serial in vitro cultures for approximately 400 days and analysing the subsequent spontaneous mutations. A P513T mutant of the DNA mismatch repair protein PfMSH2-1 from Plasmodium falciparum 3D7 was created. The mutation did not influence the base substitution rate but significantly increased the insertion/deletion (indel) mutation rate in short tandem repeats (STRs) and minisatellite loci. STR mutability was affected by allele size, genomic category and certain repeat motifs. In the mutants, significant telomere healing and homologous recombination at chromosomal ends caused extensive gene loss and generation of chimeric genes, resulting in large-scale chromosomal alteration. Additionally, the mutant showed increased tolerance to N-methyl-N'-nitro-N-nitrosoguanidine, suggesting that PfMSH2-1 was involved in recognizing DNA methylation damage. This work provides valuable insights into the role of PfMSH2-1 in genome stability and demonstrates that the genomic destabilization caused by its dysfunction may lead to antigen diversification.

Molecular and functional properties of human Plasmodium falciparum CSP C‐terminus antibodies

Human monoclonal antibodies (mAbs) against the central repeat and junction domain of Plasmodium falciparum circumsporozoite protein (PfCSP) have been studied extensively to guide malaria vaccine design compared to antibodies against the PfCSP C terminus. Here, we describe the molecular characteristics and protective potential of 73 germline and mutated human mAbs against the highly immunogenic PfCSP C‐terminal domain. Two mAbs recognized linear epitopes in the C‐terminal linker with sequence similarity to repeat and junction motifs, whereas all others targeted conformational epitopes in the α‐thrombospondin repeat (α‐TSR) domain. Specificity for the polymorphic Th2R/Th3R but not the conserved RII+/CS.T3 region in the α‐TSR was associated with IGHV3‐21/IGVL3‐21 or IGLV3‐1 gene usage. Although the C terminus specific mAbs showed signs of more efficient affinity maturation and class‐switching compared to anti‐repeat mAbs, live sporozoite binding and inhibitory activity was limited to a single C‐linker reactive mAb with cross‐reactivity to the central repeat and junction. The data provide novel insights in the human anti‐C‐linker and anti‐α‐TSR antibody response that support exclusion of the PfCSP C terminus from malaria vaccine designs.