Objective: The objective of this thesis was to estimate the impact on newborn screening (NBS) results of changing screening technology from tandem mass spectrometry (MS/MS) to an approach using targeted next-generation sequencing (T-NGS) and MS/MS in parallel. Methods: We integrated results of an analysis of MS/MS screening data for phenylketonuria (PKU) and medium-chain acyl-CoA dehydrogenase (MCAD) deficiency; and a query of genetic compendia for variants of genes associated with the two disorders. Results: The introduction of T-NGS into NBS may reduce nearly 80% of false positives that are generated using the current screening approach. Based on estimated NBS results, T-NGS may be applied using a second-tier approach, which may improve specificity while maintaining sensitivity at its current level. Discussion: T-NGS may enhance the performance of NBS for PKU by improving specificity when used as a second tier test, but may be limited by feasibility and cost under current circumstances. Future studies should consider the cost-effectiveness of T-NGS for all infants undergoing NBS.

The aim of this thesis is to identify innovation opportunities, which will serve to encourage individuals with inborn metabolic disorder phenylketunuria (also PKU) to comply with their strict dietary restrictions. The theoretical part of the thesis is devoted to the definition of basic concepts of innovation and techniques of Design thinking focusing on the Human-centered design approach. Futhermore the reader will find introduction into inborn metabolic disorder phenylketonuria. The practical part contains a plan of research, description of the current situation on the Czech market, outputs of research with experts, outputs of the workshop with individuals with PKU and the identification of innovation opportunities and recommendations.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder in which the primary defect is a mutation in the LDL receptor. Heterozygous FH is among the most common inborn errors of metabolism and remains as the best example of an inherited defect causing premature coronary artery disease (CAD). This thesis describes the physical and biochemical characteristics of heterozygous FH in a large cohort consisting of 208 women and 156 men. The influence of both genetic and environmental factors on the clinical expression of FH were investigated to better understand the phenotypic variation within FH and thus improve the prediction and treatment of CAD in affected individuals. The general incidence of CAD in this population was lower compared to previous reports but the differences between the sexes were expected. It was shown that men had a much higher frequency of CAD (31%) compared to women (13%) despite having lower concentrations of total and LDL cholesterol. In addition, the average age of onset of coronary symptoms was delayed in females, 55 years compared to 48 years for males. A greater risk of developing CAD for men was associated with low levels of HDL cholesterol and a history of smoking. In women, however, CAD was associated with elevated triglyceride levels and the presence of hypertension. In order to efficiently assess the influence of the co-inheritance of the apolipoprotein E polymorphism in this large FH population, a novel apo E phenotyping procedure was developed. Phenotypes were determined directly from plasma which was neuraminidase treated, delipidated and focused in polyacrylamide minigels. The accuracy of this method was confirmed by making a comparison to the established procedure of phenotyping by isoelectric focusing of delipidated VLDL. The low cost, speed and simplicity of the minigel methodology provided ideal conditions to phenotype a large patient population. The frequencies of the ɛ2, ɛ3 and ɛ4 alleles of apolipoprotein E in 125 unrelated FH subjects did not differ significantly from the normal population. In addition, there was no apparent relationship between apo E4 and the concentration of any of the parameters in the plasma lipid profile. However, the presence of the E2 isoform was associated with significantly elevated triglycerides in both sexes. From this study, it is evident that the mutant FH gene exerts its effect within a system of interacting environmental and polygenic factors that are known to modify atherosclerotic risk. It has been established that the dissimilarity in the frequency of CAD between men and women is related to differences between the impact of known risk factors and the incidence of CAD. Therefore, the importance of the influence of these risk factors and the differences between men and women should be emphasized when treating and predicting the development of CAD in patients with FH. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

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This study set out to express, purify and characterize twelve ammonia lyase enzymes for potential application as a supplement to a treatment of an inborn error of metabolism disease. The DNA sequence for two wild-type ammonia lyases, three modified ammonia lyases and seven resurrected ancestral ammonia lyases had been synthesized and cloned in vectors. These were transformed into Escherichia coli, expressed, purified using immobilized metal affinity chromatography and size exclusion chromatography and characterized. Ten of the enzymes were successfully expressed and purified. All enzymes had a higher turnover number with substrate 1 than with substrate 2. The wild-types showed the highest catalytic turnover and one of them displayed substrate cooperativity. The modified enzymes were inactive. Some ancestral enzymes were active and had decreasing kcat with age. A promising ancestral enzymes was found that showed a kcat of 2,85 s-1 with substrate 1 and 1,82 s-1 with substrate 2. The ancestral enzymes had a lower Km with substrate 2 compared to substrate 1, while one of the wild-types had a higher Km with substrate 2 than with substrate 1, indicating that the substrate affinity has switched. The ancestral enzymes had increased thermostability compared to the wild-types which increased with age. Ranging from a +7C increase in melting temperature with the youngest ancestral enzyme to +10,7C with the oldest tested enzyme, comparing with one of the wild-types. The promising ancestral enzyme displayed a higher stability than the wild-types during long term incubation in 37_C and 25_C, since it did not become prone to aggregation,it did not show visible degradation on SDS-PAGE and it retained the highest activity following incubation. It was also demonstrated that neither wild-types nor the promising ancestral enzyme were stable in a simulated gut environment. The promising ancestral enzyme and one of the wild-types degraded substrate 1 and 2 in serum. Using the resurrection of ancestral sequences a promising enzyme has been produced and characterized, displaying properties that are desired in therapeutic enzymes. The enzyme did not aggregate or become prone to aggregation over time, it was thermostable, it was active in serum and had acceptable catalytic properties. For therapeutic application of the ancestral enzyme, immunogenicty should be analyzed in silico and in vitro followed by further investigation in vivo. / Målet med denna studie var att uttrycka, rena och karaktärisera tolv ammonia lyase enzymer, för potentiell användning som komplement till en behandling utav en sjukdom, som tillhör sjukdomsgruppen medfödda ämnesomsättningsrubbningar. DNA sekvensen för två vild-typammonia lyaser, tre modifierade ammonia lyaser och sju återuppväckta ammonia lyaser hade blivit syntetiserade och klonade i vektorer. E.coli celler blev transformerade med vektorerna, vilka uttryckte enzymerna, som renades med hjälp av immobilized metal affinity chromatography och gelfiltrering och karaktäriserades. Tio utav enzymerna kunde uttryckas och renas. Alla enzymer hade högre katalytisk omsättning av substrat 1 än substrat 2. Vildtyperna hade högst kcat med båda substrat och en utav dem uppvisade substratsammarbete. De modifierade enzymerna var inaktiva. Några av de återuppväckta ammonia lyaserna var aktiva och kcat minskade med ålder. Ett av de återuppväckta enzymerna var lovande och hade ett kcat värde av 2,85 s-1 med substrat 1 och 1,82 s-1 med substrat 2. De återuppväckta enzymerna hade ett lägre Km värde för substrat 2 än substrat 1, jämfört med en utav vildtyperna som hade ett högre Km värde för substrat 2 än substrat 1, vilket indikerar ett skifte i substrataffinitet. De återuppväckta enzymerna var mer termostabilia än vild-typerna och termostabiliteten ökar med ålder. Ökningen i smälttemperatur låg i spannet av +7C för de yngsta återuppväckta enzymerna till + 10,7C för det äldsta testade återuppväckta enzymet, vid jämförelse med en utav vild-typerna. Det lovande återuppväckta enzymet demonstrerade även en högre stabilitet än vild-typerna under långtidsinkubering, eftersom den inte blev benägen att aggregera, den uppvisade ingen nedbrytning på SDS-PAGE och den behöll högst aktivitet efter inkubering. Det bevisades även att varken vild-typerna eller det lovande återuppväckta enzymet var stabila i en simulerad magsäcksmiljö. Både det lovande återuppväckta enzymet och en av vild-typerna bröt ner substrat 1 och 2 i serum. Genom att återuppväcka sekvenser kunde ett lovande enzym produceras och karaktäriseras, vilket uppvisade egenskaper som är eftertraktade i terapeutiska enzymer. Enzymet aggregerade ej, det blev inte benäget att aggregera över tid, det var termostabilt, det var aktivt i serum och hade acceptabla katalytiska egenskaper. För terapeutisk applikation av det återuppväckta enzymet, borde analys av dess immunogenicitet utföras in silico och in vitro följt av vidare undersökning in vivo.

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