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Migraine is a debilitating neurological disorder affecting around one in seven people worldwide, but its molecular mechanisms remain poorly understood. There is some debate about whether migraine is a disease of vascular dysfunction or a result of neuronal dysfunction with secondary vascular changes. Genome-wide association ( GWA) studies have thus far identified 13 independent loci associated with migraine. To identify new susceptibility loci, we carried out a genetic study of migraine on 59,674 affected subjects and 316,078 controls from 22 GWA studies. We identified 44 independent single-nucleotide polymorphisms ( SNPs) significantly associated with migraine risk ( P

Genotype imputation has become a standard procedure prior genome-wide association studies (GWASs). For common and low-frequency variants, genotype imputation can be performed sufficiently accurately with publicly available and ethnically heterogeneous reference datasets like 1000 Genomes Project (1000G) and Haplotype Reference Consortium panels. However, the imputation of rare variants has been shown to be significantly more accurate when ethnically matched reference panel is used. Even more, greater genetic similarity between reference panel and target samples facilitates the detection of rare (or even population-specific) causal variants. Notwithstanding, the genome-wide downstream consequences and differences of using ethnically mixed and matched reference panels have not been yet comprehensively explored.We determined and quantified these differences by performing several comparative evaluations of the discovery-driven analysis scenarios. A variant-wise GWAS was performed on seven complex diseases and body mass index by using genome-wide genotype data of ∼37,000 Estonians imputed with ethnically mixed 1000G and ethnically matched imputation reference panels. Although several previously reported common (minor allele frequency; MAF > 5%) variant associations were replicated in both resulting imputed datasets, no major differences were observed among the genome-wide significant findings or in the fine-mapping effort. In the analysis of rare (MAF < 1%) coding variants, 46 significantly associated genes were identified in the ethnically matched imputed data as compared to four genes in the 1000G panel based imputed data. All resulting genes were consequently studied in the UK Biobank data.These associations provide a solid example of how rare variants can be efficiently analysed to discover novel, potentially functional genetic variants in relevant phenotypes. Furthermore, our work serves as proof of a cost-efficient study design, demonstrating that the usage of ethnically matched imputation reference panels can enable substantially improved imputation of rare variants, facilitating novel high-confidence findings in rare variant GWAS scans.Author summaryOver the last decade, genome-wide association studies (GWASs) have been widely used for detecting genetic biomarkers in a wide range of traits. Typically, GWASs are carried out using chip-based genotyping data, which are then combined with a more densely genotyped reference panel to infer untyped genetic variants in chip-typed individuals. The latter method is called genotype imputation and its accuracy depends on multiple factors. Publicly available and ethnically heterogeneous imputation reference panels (IRPs) such as 1000 Genomes Project (1000G) are sufficiently accurate for imputation of common and low-frequency variants, but custom ethnically matched IRPs outperform these in case of rare variants. In this work, we systematically compare downstream association analysis effects on eight complex traits in ∼37,000 Estonians imputed with ethnically mixed and ethnically matched IRPs. We do not observe major differences in the single variant analysis, where both imputed datasets replicate previously reported significant loci. But in the gene-based analysis of rare protein-coding variants we show that ethnically matched panel clearly outperforms 1000G panel based imputation, providing 10-fold increase in significant gene-trait associations. Our study demonstrates empirically that imputed data based on ethnically matched panel is very promising for rare variant analysis – it captures more population-specific variants and makes it possible to efficiently identify novel findings.

Genetic variants affecting hematopoiesis can influence commonly measured blood cell traits. To identify factors that affect hematopoiesis, we performed association studies for blood cell traits in the population-based Estonian Biobank using high coverage whole genome sequencing (WGS) in 2,284 samples and SNP genotyping in an additional ~17,000 samples. Our analyses identified 17 associations across 14 blood cell traits. Integration of WGS-based fine-mapping and complementary epigenomic data sets provided evidence for causal mechanisms at several loci, including at a novel basophil count-associated locus near the master hematopoietic transcription factor CEBPA. The fine-mapped variant at this basophil count association near CEBPA overlapped an enhancer active in common myeloid progenitors and influenced its activity. In situ perturbation of this enhancer by CRISPR/Cas9 mutagenesis in hematopoietic stem and progenitor cells demonstrated that it is necessary for and specifically regulates CEBPA expression during basophil differentiation. We additionally identified basophil count-associated variation at another more pleiotropic myeloid enhancer near GATA2, highlighting regulatory mechanisms for ordered expression of master hematopoietic regulators during lineage specification. Our study illustrates how population-based genetic studies can provide key insights into poorly understood cell differentiation processes of considerable physiologic relevance.

Genetic imputation is a cost-efficient way to improve the power and resolution of genome-wide association (GWA) studies. Current publicly accessible imputation reference panels accurately predict genotypes for common variants with minor allele frequency (MAF)≥5% and low-frequency variants (0.5≤MAF

Over the past two decades, cell-penetrating peptides (CPPs) have become increasingly popular both in research and in application. There have been numerous studies on the physiochemical characteristics and behavior of CPPs in various environments; likewise, the mechanisms of entry and delivery capabilities of these peptides have also been extensively researched. Besides the fundamental issues, there is an enormous interest in the delivery capabilities of the peptides as the family of CPPs is a promising and mostly non-toxic delivery vector candidate for numerous medical applications such as gene silencing, transgene delivery, and splice correction. Lately, however, there has been an emerging field of study besides the high-profile gene therapy applications-the use of peptides and CPPs to combat various infections caused by harmful bacteria, fungi, and viruses.In this chapter, we aim to provide a short overview of the history and properties of CPPs which is followed by more thorough descriptions of antimicrobial and antiviral peptides. To achieve this, we analyze the origin of such peptides, give an overview of the mechanisms of action and discuss the various practical applications which are ongoing or have been suggested based on research.

Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.

Migraine is a debilitating neurological disorder affecting around 1 in 7 people worldwide, but its molecular mechanisms remain poorly understood. Some debate exists over whether migraine is a disease of vascular dysfunction or a result of neuronal dysfunction with secondary vascular changes. Genome-wide association (GWA) studies have thus far identified 13 independent loci associated with migraine. To identify new susceptibility loci, we performed the largest genetic study of migraine to date, comprising 59,674 cases and 316,078 controls from 22 GWA studies. We identified 44 independent single nucleotide polymorphisms (SNPs) significantly associated with migraine risk (P < 5 × 10−8) that map to 38 distinct genomic loci, including 28 loci not previously reported and the first locus identified on chromosome X. In subsequent computational analyses, the identified loci showed enrichment for genes expressed in vascular and smooth muscle tissues, consistent with a predominant theory of migraine that highlights vascular etiologies.

Background The outcome of virus infection depends from the precise coordination of viral gene expression and genome replication. The ability to control and regulate these processes is therefore important for analysis of infection process. Viruses are also useful tools in bio- and gene technology; they can efficiently kill cancer cells and trigger immune responses to tumors. However, the methods for constructing tissue- or cell-type specific viruses typically suffer from low target-cell specificity and a high risk of reversion. Therefore novel and universal methods of regulation of viral infection are also important for therapeutic application of virus-based systems. Methods Aberrantly spliced introns were introduced into crucial gene-expression units of adenovirus vector and alphavirus DNA/RNA layered vectors and their effects on the viral gene expression, replication and/or the release of infectious genomes were studied in cell culture. Transfection of the cells with splice-switching oligonucleotides was used to correct the introduced functional defect(s). Results It was demonstrated that viral gene expression, replication and/or the release of infectious genomes can be blocked by the introduction of aberrantly spliced introns. The insertion of such an intron into an adenovirus vector reduced the expression of the targeted gene more than fifty-fold. A similar insertion into an alphavirus DNA/RNA layered vector had a less dramatic effect; here, only the release of the infectious transcript was suppressed but not the subsequent replication and spread of the virus. However the insertion of two aberrantly spliced introns resulted in an over one hundred-fold reduction in the infectivity of the DNA/RNA layered vector. Furthermore, in both systems the observed effects could be reverted by the delivery of splice-switching oligonucleotide(s), which corrected the splicing defects. Conclusions Splice-switch technology, originally developed for genetic disease therapy, can also be used to control gene expression of viral vectors. This approach represents a novel, universal and powerful method for controlling gene expression, replication, viral spread and, by extension, virus-induced cytotoxic effects and can be used both for basic studies of virus infection and in virus-based gene- and anti-cancer therapy.