Leber’s hereditary optic neuropathy (LHON) is a genetic disease that causes the patients to become blind, first in one eye and then the other, around the ages of 10-75 years. The disease is caused by mutations in the mitochondrial DNA, which disturbs the respiratory chain leading to the deterioration of the retinal ganglion cells. This study’s aim is to optimize a multiplex amplification-refractory mutation system PCR for detection of three primary mutations causing LHON. This was done through a series of PCRs, including PCR aimed at the ß-globin gene, conventional simplex PCR and a simplex ARMS-PCR aimed at the three primary mutations causing LHON. This study was, however, terminated prematurely due the Covid-19 outbreak and the optimization of the ARMS-PCR could therefore not be done. This study’s aim was adapted to the new circumstances to instead provide guidance on how to perform the optimization using the results from the PCRs that were done before the termination. The results found that for the ARMS-PCR 2 mM of magnesium would suffice as a start point overall and the need to solve the problems with the two 14484 plasmids was evident. The ARMS-PCR is one of many methods that can be used to the detect single nucleotide polymorphism, but its availability and robustness makes this a method worth optimizing. To continue with the optimization of the ARMS-PCR several factors would have to be tested, including annealing temperature, primer concentrations and magnesium concentration.

55 Abstract Frequency of selected genetic polymorphisms of cytochrome P450 in the Czech population and the influence of CYP2C9 genotype on the hypolipidemic effect of fluvastatin Introduction: One of the main factors of genetically determined variability in response of humans to administered drugs are differences in catalytic activity of metabolizing enzymes, which are caused mainly by genetic polymorphisms in cytochrom P450 family enzymes. This thesis consists of two parts and it is presented as a commentary to the original papers. The first aim was to investigate the frequency of functionally important variant alleles of three main isoenzymes of cytochrome P450 gene: CYP2D6, CYP2C9, CYP2C19, throughout the Czech population, predict the prevalence of poor metabolizer phenotypes, and then to compare the results to the data from other populations. Secondly, we analysed the correlation between the CYP2C9 genotype and cholesterol-lowering effect of fluvastatin in human hypercholesterolemic patients. Methods: Genotypes were determined by PCR-RFLP. The presence of alleles CYP2D6*1, *6, *5, *4, *3, and gene duplication was analysed in 233 healthy volunteers, CYP2C9*1, *2 and*3 in 254 subjects and CYP2C19*1, *2 and *2 in 218 subjects. Eighty seven patients on fluvastatin therapy, and 48 patients on monotherapy...

Bakgrund: Den pågående pandemin COVID-19 orsakas av viruset SARS-CoV-2. Sedan december 2020 har nya varianter av viruset med betydande genetiska förändringar upptäckts, gemensamt benämnt varianter av särskild betydelse eller variants of concern (VOC). Just nu är det tre VOC som bevakas särskilt; B.1.1.7 (först upptäckt i Storbritannien), B.1.351 (först upptäckt i Sydafrika) respektive P.1 (först upptäckt i Brasilien). Den tidigaste statistiken från Folkhälsomyndigheten om förekomsten av VOC i Region Dalarna är från februari 2021. Förekomsten av VOC innan dess är fortfarande okänd. I regionen delas analysering av prover vid misstanke om COVID-19 in i de olika kategorierna patienter, vårdpersonal, smittspårning och allmänhet. Befintlig statistik om förekomsten av VOC grundar sig nästan enbart på förekomsten bland allmänhetens prover. Syfte: Syftet med denna studie var att undersöka förekomsten av SARS-CoV-2 varianter av särskild betydelse i prover tagna från patienter, vårdpersonal och smittspårningar under december 2020-januari 2021 i Region Dalarna. Studien syftade också till att undersöka när spridningen av respektive VOC kan ha startat i regionen. Metod: Provmaterialet bestod av SARS-CoV-2 positiva prov tagna inom analyskategorierna under tidsperioden. Prover analyserades med RT-PCR och smältkurvsanalys för detektion av VOC-karaktäristiska mutationer. Resultat: Ett fåtal fall av B.1.1.7 detekterades redan i december och en stigande andel av B.1.1.7 påvisades inom analyskategorierna under januari, som tecken på att en regional spridning kan ha startat vid tidpunkten. Endast ett fåtal fall av B.1.351 och/eller P.1 detekterades inom analyskategorierna under tidsperioden, vilket tyder på att en regional spridning av dessa ännu inte hade startat i januari. / Background: The ongoing pandemic COVID-19 is caused by the virus SARS-CoV-2. Since December 2020 new variants of the virus with significant mutations have been discovered, referred to as variants of concern (VOC). At the point, the occurrence of three VOC is especially monitored; B.1.1.7 (discovered in UK), B.1.351 (discovered in South Africa) and P.1 (discovered in Brazil). The earliest statistics about the occurrence of VOC in Region Dalarna, Sweden, is from February 2021 and the occurrence before that is still unknown. In the region analysis of specimen in case of suspected COVID-19 is divided into the different categories patients, healthcare-staff, infection tracing and public. Existing statistics is based almost exclusively on the occurrence of VOC in specimen from the public. Aim: The aim of this study was to examine the occurrence of SARS-CoV-2 VOC among specimens collected from patients, healthcare staff and infection tracing in Region Dalarna during December 2020-January 2021. The study also aimed to examine when the spread of each VOC started in the region. Method: SARS-CoV-2 positive specimen collected within the categories during the time was analyzed with RT-PCR and melting curve analysis for detection of VOC-characteristic mutations. Results: A few cases of B.1.1.7 was detected already in December and an increased percentage of B.1.1.7 was detected within the categories during January, suggesting that a regional spread started at the time. Only a few cases of B.1.351 and/or P.1 was detected within the categories, suggesting that a regional spread of these had not yet started in January.

Le protozoaire Leishmania est à l’origine d’une maladie tropicale négligée qui peut s’avérer mortelle si elle n’est pas traitée adéquatement. Dans les dernières années, l’efficacité des médicaments utilisés dans le combat de la leishmaniose a grandement diminué en raison de la résistance du parasite à ceux-ci. Bien que le Topotecan (TPT), présentement utilisé comme antitumoral, ait démontré une activité antileishmaniale puissante (EC50 de l’ordre des nanomolaires), son efficacité pourrait être compromise par l’émergence de la résistance du parasite d’autant plus que des résistances tumorales cliniques au TPT ont déjà été rapportées. Dans cette étude, les mécanismes de résistance au TPT par Leishmania infantum ont été caractérisés d’un point de vue moléculaire. Le génome complet des parasites sélectionnés résistants au TPT (16 x EC50) (TPT700.1, TPT700.2, TPT700.3) a été séquencé. Le rôle dans la résistance des différents éléments génétiques identifiés a été confirmé à l’aide d’une complémentation par nucléofection épisomale dans le parasite sauvage et a été étudié avec des simulations computationnelles. Aucune amplification ni délétion n’a été identifiée et seulement une variation mineure du nombre de chromosomes a été observée. Cependant, un polymorphisme d’un seul nucléotide non synonyme a été identifié dans le gène de la grande sous-unité de la topoisomérase IB (TOP IB), la cible du TPT, chez chacun des parasites mutants résistants conférant des niveaux de résistance variables (TPT700.1 F187Y > TPT700.3 W232R > TPT700.2 G191A > sauvage). De plus, des modélisations in sillico ont permis d’illustrer la proximité de ces substitutions d’acides aminés au site catalytique de TOP IB ainsi qu’au site de liaison du TPT. En conclusion, ces résultats suggèrent qu’une mutation ponctuelle dans la grande sous-unité TOP IB est suffisante pour engendrer des hauts niveaux de résistance (environ 24 x EC50) chez TPT700.1F187Y, TPT700.2G191A et TPT700.3 W232R. TPT pourrait être considéré comme un modèle pharmacologique pour l’étude de la résistance chez Leishmania. / Leishmania, a protozoan parasite, causes a neglected tropical disease that is fatal if left untreated. In recent years, the effectiveness of the drugs used to tackle leishmaniasis has decreased dramatically due to the emergence of drug resistant parasites. Even though Topetecan (TPT), currently employed as an anti-tumoral drug, has shown strong anti-leishmanial activity (its EC50 being measured in nanomoles), its efficiency may be compromised by the resistance developped by the parasites, similarly to the resistance already recorded by tumoral cells to the drug. In this study, the mecanisms of resistance to TPT by Leishmania infantum were caracterised at the molecular level. The whole genome of parasites resistant to TPT (16 x EC50) (TPT700.1, TPT700.2, TPT700.3) was sequenced. The role of various genetic elements in the resistance mecanisms was confirmed via a complementation by episomal nucleofection in the wild type and was studied with the help of computational models. Neither amplications nor deletions were identified and only a minor variation in the chromosome number was observed. However, a non-synonymous single nucleotide polymorphism was identified in the gene coding the large subunit of topoisomerase IB, TPT’s target, within each of the resistant mutant parasites confering variable levels of resistance (TPT700.1 F187Y > TPT700.3 W232R > TPT700.2 G191A > wild type). Furthermore, in sillico models highlighted the proximity of these amino acid substitutions to the catalytic site of topoisomerase IB and also to the binding pockets of TPT. In conclusion, these results suggest that a point mutation in the large subunit of TOP IB is sufficient to confer high levels of resistance (about 24 x EC50) to TPT700.1F187Y, TPT700.2G191A and TPT700.3 W232R. Therefore, TPT can be considered a pharmacological tool to study resistance in Leishmania.

This review will focus on a systems medicine approach to neonatal abstinence syndrome (NAS). Systems medicine utilizes information gained from the application of “omics” technology and bioinformatics (1). The omic approaches we will emphasize include genomics, epigenomics, proteomics, and metabolomics. The goals of systems medicine are to provide clinically relevant and objective insights into disease diagnosis, prognosis, and stratification as well as pharmacological strategies and evidence-based individualized clinical guidance. Despite the increasing incidence of NAS and its societal and economic costs, there has been only a very modest emphasis on utilizing a systems medicine approach, and this has been primarily in the areas of genomics and epigenomics. As detailed below, proteomics and metabolomics hold great promise in advancing our knowledge of NAS and its treatment. Metabolomics, in particular, can provide a quantitative assessment of the exposome, which is a comprehensive picture of both internal and external environmental factors affecting health.

Cranberry common bean (Phaseolus vulgaris L.) is planted worldwide and consumed as a critical food source of human protein, fibre, carbohydrates, and minerals. Marsh spot (MS) is a physiogenic disorder which severely impacts seed quality in common beans. Previous studies indicate that MS involves a nutritional disorder caused by Mn deficiency. However, the inheritance and genetic mechanism of MS resistance are still not fully understood. To investigate the genetics of MS resistance, a population of 138 recombinant inbred lines (RILs) was developed from a bi-parental cross between a susceptible cultivar Messina and a resistant cultivar Cran09. The population and its two parents were evaluated for MS resistance during five consecutive years from 2015 to 2019 in both sandy and heavy clay soils in Morden, Manitoba, Canada. The severities of MS were rated and subsequently converted to MS resistance index (MSRI) and MS incidence (MSI). Statistical analyses indicated that MSI and MSRI were highly correlated (r = 0.96-0.99) and had high broad-sense heritability (H²) of 86.5% and 83.2%, respectively. Joint segregation analysis (JSA) of 18 phenotypic datasets from five years and two soil types showed that MS resistance was controlled by four major genes with genetic interactions - one of which may suppress the additive effect of the other three genes. To identify the quantitative trait loci (QTL) and the candidate genes associated with the MS resistance, the 138 RILs and the two parents were sequenced using genotyping by sequencing approach. A total of 52,676 SNPs were detected. After further filtering with a threshold of minor allele frequency > 0.01 and call rate > 20%, 2,061 SNPs were retained and then imputed for genetic map construction and QTL mapping. A genetic map consisting of 2,058 SNP markers on 11 linkage groups or chromosomes was constructed, which covered 1,004 recombination blocks with a total length of 6,449 cM and an average block of 6.42 cM. Three linkage map-based QTL-mapping models ICIM-ADD, ICIM-EPI, and GCIM and one genome-wide association study (GWAS) model RTM-GWAS for 18 phenotypic datasets from different years and soil types were used for identification of QTL. A total of 36 QTL, including 21 of additive and 15 of epistatic effects, were identified. Functional gene annotation analysis revealed 151 Mn-related candidate genes across the common bean reference genome and 17 of them harbored the six QTL discovered in this study. In conclusion, MS resistance in common bean is a highly heritable trait and controlled by several major and minor genes. The results of JSA and QTL mapping advance the current understanding of the genetic mechanisms of MS resistance in cranberry common bean, and provide additional resources for application in genomics-assisted breeding and potential isolation and functional characterization of the candidate genes.

Indiana University-Purdue University Indianapolis (IUPUI) / In the past, the present and the future, Lebanon has been an important link between the East and the West. It was always known as the ‘Switzerland of the East’. Over the years, it was a hotspot for different civilizations that uniquely shaped the genomic backbone of the current Lebanese. It is also a good representation of genetically admixed individuals with diverse phenotype characteristics and unique features. Lebanon, quite like other Middle Eastern populations, lacks sufficient genetic studies that helps to better comprehend the complex genomic composition of different traits and diseases. The lack of good representation of the Middle East and North Africa (MENA) region in global studies has led to ambiguity in discovering special ancestry markers and patterns in the Lebanese genome. Yet, in this study, a thorough investigation into a Lebanese collection shows new patterns that potentially would be helpful in forensic and genealogical applications. The investigation into the autosomal and Y-STRs revealed unique alleles that would be valuable in future forensic investigation analysis. In addition, the assessment of phenotype prediction models to predict eye, hair and skin color showed promising results in terms of prediction performance. Those results encourage the future use of intelligence tools in the regions that in return would aid in serving justice and furthering science research. In fact, ancestry and genetic distance studies confirms the presence of admixture within Lebanon between Europe and North Africa. / 2029-06-01

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