Tesis sobre el tema "Clinical and genetic analysis"

Malaria, an infectious disease caused by Plasmodium parasites, is one of the major world-scale health problems. Despite the efforts aimed at finding an effective way to control the disease, the success has been thwarted by the emergence of parasite drug resistance and mosquito resistance to insecticides. This thesis focuses on the genetic analysis of resistance to murine malaria induced by the lethal Plasmodium berghei ANKA using a wild-derived-inbred strain (WDIS). The aim of this thesis was to exploit the genetic diversity represented among WDIS for identifying loci contributing to resistance/susceptibility to murine malaria. The work included a genome-wide polymorphism survey using microsatellite markers performed on 10 WDIS. Comparisons of these strains to laboratory inbred strains confirmed a higher rate of polymorphism among the WDIS. We conclude that these WDIS represent repositories of unique naturally occurring genetic variability that may prove to be invaluable for the study of complex phenotypes. Next, we used the WDIS to search for novel phenotypes related to malaria pathogenesis. Whereas most laboratory strains were susceptible to experimental cerebral malaria (ECM) after infection with P. berghei ANKA, several WDIS were found to be resistant. To study the genetic inheritance of resistant/susceptibility to P. berghei ANKA infection we analysed backcross and F2 cohorts derived from crossing the WLA wild-derived strain with a laboratory mouse strain (C57BL/6). A novel phenotype represented by the cure of infection, clearance of parasitaemia and establishment of immunological memory was observed in the F2 progeny. The backcross progeny was used to genetically map one locus on chromosome 1 (Berr1) and one locus on chromosome 11 (Berr2) that mediate control of resistance to ECM induced by P. berghei ANKA. Genetic mapping using the F2 progeny showed that a locus on chromosome 1 (Berr1) and a locus on chromosome 9 (Berr3) were contributing to control survival time after infection with lethal Plasmodium. Finally, we identified, a locus on chromosome 4 (Berr4) that appears to control time of death due to hyperparasitaemia. This thesis underlines the value of using WDIS to reveal genetic factors involved in the aetiology of disease phenotypes. The characterisation of the genetic factors represented by the malaria resistance loci identified here are expected to provide a better understanding of the malaria pathology.

The study aim was to identify the population of live patients in the Lothian area of Scotland, with presenile dementia of various aetiologies, and to describe the clinical profiles of each and the patterns of decline which occur, together with any genetic characterisation possible. Cases were identified using the Lothian Psychiatric Case Register. For the demographic data, the CAMDEX (The Cambridge Examination of Mental Disorders of the Elderly) informant interview was used. The behavioural assessment comprised the CAPE-BRS (Clifton Assessment Procedure for the Elderly, Behavioural Rating Scale), the Cornell Depression Scale and the MOUSEPAD, a new behavioural and psychopathological assessment. Of 290 potential cases, 164 (57%) were excluded. Reasons: Death 50 (31%), Unsuitable 40 (24%), Refused 40 (24%), Untraced 23 (14%), Out of area 11 (7%), Of the 126 (43%) seen, 112 (89%) met DSM3R (Diagnostic and Statistical manual of Mental Disorders, Third Edition, Revised) criteria for dementia. 63 (56%) of the 112 were rated as DSM3R severe type. 80 (72%) of the 112 fulfilled the McKhann criteria for Alzheimer's Disease. Full description of the group is available. The genetic testing included Apolipoprotein E, α-1 antichymotrypsin and Very Low Density Lipoprotein Receptor (VLDL-R) allele typing. Important data concerning the services provided and used by this group of patients and their carers has been collected and can be shared with organisations working to find funding for presenile dementia in the health service. This study will provide a thoroughly documented and clinically well worked-up sample. The analysis of the work will help to identify if subgroups exist, according to the patterns of various clinical features, the rates of decline and genetic variations. This would in turn, give a greater chance to plan appropriately for all those involved in caring for and managing these illnesses.

This thesis details the genetic analysis and clinico-genetic correlates of early onset familial Alzheimer's disease (AD). Multiply affected families have been examined for linkage to markers on two candidate chromosomes, 21 and 14. The analysis demonstrates linkage between the β amyloid precursor protein (APP) gene and AD in five early onset (< 65 years) families in which three different mutations were subsequently discovered. Simulation studies were used extensively and enabled the evaluation of lod values below the accepted criterion of 3.0 in single families. Clinical details of these families are presented. In general, these families show classical AD symptoms and signs but with additional prominent features previously recognised in early onset disease. Linkage to βAPP is excluded in analyses of other early onset families which are shown to be linked to the long arm of chromosome 14. A comparison of clinical features is made between allelic variants of the βAPP locus and chromosome 14 linked families. In particular, analysis of variance of age of onset in early onset families supports the notion of clinico-genetic heterogeneity by demonstrating family specific ages of onset and correlation between genetic aetiology and age of onset; the very early onset families show collective evidence of linkage to chromosome 14 and the families with mean onset in the 50s have βAPP mutations.

Ototoxicity is the damage to the ear from exposure to medications. The inner ear is the commonest site of damage where cochlear and/or vestibular functions are affected. Ototoxic medications can cause irreversible toxicity, with aminoglycosides (AGs) and cisplatin being the most established agents. A series of studies are reported in this research under three main themes. Theme A focused on audiological assessments and assessment tools; Theme B focused on causation; and Theme C focused on the impact of ototoxicity and current service provision. The main Theme A study was a clinical observational study with a cross-sectional design assessing the auditory status of children with cystic fibrosis (CF) exposed to AGs. Theme B investigated potential risk factors and aspects in genetics that may be associated with increasing patient susceptibility to the ototoxic effect of AGs. Theme C assessed the effect of ototoxicity on the quality of life (QoL) of children surviving cancer. It also included a survey of current UK practice regarding auditory monitoring for ototoxicity. The novel outcomes of these studies included showing that the prevalence of AG ototoxicity in children with CF is higher than previously reported and evaluating the efficacy of auditory assessment tools. They stressed the importance of choosing appropriate criteria to define ototoxicity and identified potential risk factors associated with it. The genetic studies highlighted a rare case of normal hearing in a child with the m.1555 A>G mutation despite exposure to AGs. They complemented the limited research on the impact of ototoxicity in children on their QoL and on current practice. The latter identified gaps in the provision of ototoxicity monitoring services in the UK, especially due to the absence of nationally agreed guidelines. This research has generated recommendations for several future studies and has informed the clinical management of patients with CF.

Abstract Meniere’s disease (MD) is an inner ear disorder characterized by vertigo, tinnitus and sensorineural hearing impairment. An inherited form of the disease is called familial Meniere’s disease (FMD). The aim of this thesis was to describe the clinical and genetic features of Finnish FMD and to study its prevalence in Finland. In addition genetic factors previously associated with MD were studied in Finnish MD patients. A total of 38 Meniere-families were analysed in this study. In most of the families the mode of inheritance was found to be autosomal dominant. Meniere-like symptoms such as tinnitus or vertigo were common in these families even in individuals without a full triad of MD. Familial patients were affected earlier, suffered from longer spells of vertigo and had more autoimmune diseases compared to sporadic MD patients. The prevalence of FMD was studied among the patients treated in the Oulu University Hospital and Kainuu Central Hospital during the years 2005-2010. A family history of MD was probable in 23.4% of the cases, but only 9.3% could be confirmed, as it was not possible to gain information from deceased generations. Six candidate genes previously associated with MD were screened for mutations in Finnish MD patients. Two possibly adverse variations were observed in the KCNE1 gene in two patients but in none of the controls. The role of these variations in MD is still unclear and needs further study. The association of MD to the five other genes could not be confirmed, nor was Finnish FMD linked to a previously suggested locus on chromosome 12
Tiivistelmä Menieren tauti on sisäkorvan sairaus, jolle on tyypillistä huimaus, korvien soiminen ja kuulon heikkeneminen. Tauti voi esiintyä myös perinnöllisenä. Tutkimustyön tavoitteena oli selvittää perinnöllisyyden osuutta Menieren taudissa, kuvata suomalaisen perinnöllisen Menieren taudin tyypilliset piirteet ja tutkia suomalaisessa aineistossa aikaisemmin tautiin yhdistettyjä perinnöllisiä tekijöitä. Tutkimuksessa analysoitiin 38 sukua, joissa Menieren tautia esiintyi perinnöllisenä. Suurimmassa osassa tapauksista periytyminen tapahtui vallitsevasti. Suvuissa esiintyi paljon Meniere-tyypistä oirehdintaa, kuten tinnitusta ja huimausta, ilman Menieren taudin koko taudinkuvaa. Meniere-suvuissa potilaat sairastuivat keskimääräistä aikaisemmin, kärsivät pidemmistä huimauskohtauksista ja sairastivat enemmän autoimmuunitauteja. Perinnöllisen Menieren taudin yleisyyttä tutkittiin Kainuun keskussairaalassa ja Oulun yliopistollisessa sairaalassa vuosina 2005−2010 hoidettujen potilaiden keskuudessa. Potilaista 23,4 %:lla Menieren taudin sukuhistoria oli positiivinen; kuitenkin vain 9,3 % pystyttiin vahvistamaan, sillä tietojen kerääminen edesmenneiltä sukupolvilta ei ollut mahdollista. Kuuden Menieren tautiin aikaisemmin yhdistetyn geenin merkitystä tutkittiin suomalaisessa aineistossa mutaatio- ja ehdokasgeenianalyysillä. KCNE1-geenistä löydettiin kaksi mahdollisesti proteiinia vaurioittavaa sekvenssinvaihtelua, joita ei havaittu kontrollihenkilöillä. Muutosten merkitys Menieren taudin synnyssä jäi kuitenkin epävarmaksi ja vaatii jatkotutkimuksia. Muiden geenien yhteyttä sairauteen ei pystytty vahvistamaan. Suomalainen Menieren tauti ei myöskään kytkeytynyt aikaisemmin ehdotettuun lokukseen kromosomissa 12

Best macular dystrophy (BMD) is an autosomal dominant inherited eye disease with a juvenile onset. Accumulation of the pigment lipofuscin in the retinal pigment epithelium can later cause macular degeneration and loss of vision. BMD have histopathologic similarities with age-related macular degeneration, the most common cause of blindness among elderly. BMD diagnosis is made with fundus examination and electrophysiology. The VMD2 gene, causing BMD, has previously been localized to 11q13 using linkage and recombination of a 12 generation family with BMD.

In this study the genetic region has been further narrowed using polymorphic markers in the BMD family. A human homolog for a C. elegans protein family, expressed in retina, was identified as the VMD2 gene. It has a 1755 bp open reading frame with 11 exons and encodes a 585 amino acid protein called bestrophin. Mutation analysis of the VMD2 gene in BMD families from Sweden, Denmark and Netherlands revealed 15 missense mutations, altering single amino acids in bestrophin, accumulating in the N-terminal half of the protein. VMD2 expression analysis with in situ hybridization revealed specific localization in the retinal pigment epithelium and Northern blot showed expression in retina and brain. Clinical and genetic analysis of a BMD family with generally late onset revealed a novel bestrophin mutation.

Analysis of mouse Vmd2 and bestrophin during development showed presence of mouse bestrophin in retinal pigment epithelium at postnatal day 10 and in photoreceptor outer segments during the entire postnatal period. Vmd2 expression levels were highest around birth.

Thesis (MSc)--Stellenbosch University, 2000.
ENGLISH ABSTRACT: Hereditary haemochromatosis (HH) is an autosomal recessive iron storage disease where the accumulation of iron in parenchymal organs may lead to diabetes, heart failure, liver cirrhosis, arthropathy, weakness and a variety of other ailments if preventive measures are not taken. HH is often not considered as a cause of these conditions, particularly not in the elderly where the background frequencies of type II diabetes, osteoarthritis and heart failure are generally high. Heterozygosity for C282Y, the HFE-mutation causing HH in approximately 80% of affected individuals worldwide, has been linked to a raised incidence of malignancies of the colon and rectum, stomach and the haematological system. One of the highest carrier-frequencies (116) in the world for this mutation has been reported in the South-African Afrikaner population, resulting in C282Y-homozygosity in approximately 1 in every 115 people in this group. A sample of 197 elderly Afrikaner volunteers was recruited for genotype/phenotype association studies. Their clinical presentation was denoted, biochemical iron-status determined and HFE genotyping performed. Either an increase or decrease in survival, or both, were proposed, depending on possible gender effects. HH has been positively associated with various cancer types, but may also protect against iron-deficiency anaemia which is by far the most frequent cause of anaemia in the older person. This study has led to the following findings: 1. The carrier frequency of mutation C282Y was found to be 1/8 in the elderly population (similar in males and females), which is slightly lower than the 1/6 reported in younger adults from the same population. Only one C282Y homozygote and two C282YIH63D compound heterozygotes were detected, all of them female. 2. The prevalence of diabetes, heart disease, arthropathy or a combination of these conditions did not differ significantly in C282Y heterozygotes and the mutationnegative group. 3. Among 24 C282Y heterozygotes only one individual with rectal carcmoma was detected compared with two cases with rectal- and seven with colonic malignancies in 153 mutation-negative individuals. The single female C282Y homozygote identified suffered from both rectal and colon carcinoma and died approximately 6 months ago as a consequence of her colon malignancy. 4. Serum ferritin appears to be a highly unreliable parameter of iron status, particularly in the elderly where a variety of factors that may influence the levels are often present in elderly individuals. This may be due to ageing alone or as a result of multiple comorbidities. 5. Serum ferritin levels were lower than expected in elderly subjects with mutation C282Y and compound heterozygotes with both C282Y and H63D, which may be related to a variable penetrance of the HFE gene mutations. It is possible that variation in other genes exist that confer protection against iron-loading by gene-gene interaction. The probability that environmental factors (e.g. a low iron diet) are more important in this respect cannot be excluded, although this is considered less likely in the light of the fact that the same trend was observed in all mutation-positive elderly individuals. It is therefore highly likely that C282Y -positive subjects with significant iron loading have died before reaching their seventies, particularly since none of the males included in this study were homozygous or compound heterozygous for the mutations analysed. In conclusion, possession of a mutant HFE gene does not appear to confer a survival advantage in old age, neither does it seem that mutation carriers with significant ironloading are overlooked by the medical fraternity. Further investigations are warranted to shed more light on the contributions of gene-gene and gene-environment interaction in the clinical manifestation of Hll, and how these processes can be manipulated to prevent the symptoms of this largely underdiagnosed disease.
AFRIKAANSE OPSOMMING: Oorerflike hemochromatose (OH) is 'n outosomaal resessiewe yster-oorladingssiekte waar akkumulasie van yster in parenkimale organe kan lei tot suikersiekte, hartversaking, lewer sirrose, artropatie, moegheid en 'n verskeidenheid van ander probleme indien voorkomende maatreëls nie getref word nie. OH word gewoonlik nie oorweeg as moontlike oorsaak vir hierdie toestande nie, veral nie in ouer mense nie waar die agtergrond-frekwensie van tipe II diabetes, osteoartritis en hartversaking in elk geval hoog is. Heterosigositeit vir die HFE mutasie C282Y, wat OH veroorsaak in ongeveer 80% van geaffekteerde gevalle wêreldwyd, is geassosieer met 'n verhoogde voorkoms van kanker van die kolon, rektum, maag en ook die hematologiese sisteem. Van die hoogste draer frekwensies ter wêreld vir hierdie mutasie (1/6) is gevind in die Afrikaner populasie van Suid-Afrika, wat daarop dui dat 1 uit elke 115 mense in die groep homosigoties vir die C282Y mutasie kan wees. Eenhonderd sewe-en-negentig bejaarde Afrikaner vrywilligers het aan die studie deelgeneem wat daarop gemik was om genotipe/fenotipe korrelasies uit te voer. Die kliniese beeld van elke individu is gedokumenteer, die yster status biochemies bepaal en HFE genotipering uitgevoer. Die a priori veronderstelling was dat oorlewing sou toeneem of afneem, of beide, afhangende van die geslag van die individu. Daar is voorheen 'n verband gevind tussen OH en die ontwikkeling van bogenoemde maligniteite, maar aan die ander kant kan dit moontlik ook beskerm teen anemie as gevolg van yster gebrek, wat juis die mees algemene oorsaak van anemie in die ouer persoon is. Hierdie studie het tot die volgende bevindings gelei: 1. Die draer frekwensie van mutasie C282Y was 1/8 in die bejaardes (dieselfde in mans en vrouens), wat effens laer is as die 1/6 wat gerappoteer is in jonger volwassenes. Slegs een C282Y homosigoot en twee C282YIH63D saamgestelde heterosigote is opgespoor, en al drie was vroulik. 2. Die voorkoms van suikersiekte, hartsiekte, gewrigspyne of 'n kombinasie van hierdie aandoenings het nie betekenisvol verskil tussen die C282Y heterosigote en die mutasienegatiewe groep nie. 3. Daar was slegs een persoon met rektum karsinoom in die groep van 24 bejaarde C282Y heterosigote, terwyl daar twee gevalle met rektum kanker en sewe gevalle met kolon kanker gevind is onder die 153 mutasie-negatiewe individue. Die enkele vroulike C282Y homosigoot wat opgespoor is het beide rektum- en kolonkanker gehad en is ongeveer 6 maande vóór voltooing van die tesis oorlede aan haar kolon karsinoom. 4. Dit wil voorkom asof serum ferritien veral in bejaardes 'n hoogs onbetroubare maatstaf is vir yster status, aangesien dit deur 'n verskeidenheid faktore beïnvloed word wat dikwels in bejaardes aanwesig is as gevolg van veroudering of veelvuldige komorbiditeite. 5. Die serum ferritien vlakke was laer as verwag in sowel die bejaarde C282Y-homosigoot as in die twee saamgestelde heterosigote met mutasies C282Y en H63D, wat moonlik die gevolg is van die wisselende graad van penetrasie van HFE mutasies. Dit is moontlik dat variasie in ander gene beskerming bied teen yster-oorlading deur middel van geen-geen interaksie. Die moontlikheid dat omgewingsfaktore (soos 'n lae-yster dieet) 'n belangrike rol speel in hierdie verband kan nie uitgesluit word nie, hoewel dit minder waarskynlik lyk te wees in die lig van die feit dat dieselfde neiging waargeneem is in alle mutasie-positiewe bejaardes. Die kans is dus redelik groot dat individue met die C282Y mutasie en betekenisvolle yster oorlading oorlede is voordat hulle die sewentiger jare kon bereik, veral omdat geeneen van die mans wat ingesluit is in die studie homosigoot of 'n saamgestelde heterosigoot was vir die mutasies wat geanaliseer is nie. Opsommend wil dit voorkom asof die teenwoordigheid van 'n mutante HFE geen nie 'n beter oorlewingskans bied op ouer leeftyd nie, en dit blyk ook dat mutasie draers met betekenisvolle ysteroorlading nie deur dokters misgekyk word nie. Verdere navorsing is nodig om meer lig te werp op die bydrae van geen-geen- en geen-omgewing interaksie in die kliniese manifestasie van OH, en ook hoe hierdie prosesse gemanipuleer kan word om die simptome van hierdie onder -gediagnoseerde siekte te voorkom.

A novel method was developed for studying the genetic relatedness of Pseudomonas aeruginosa isolates from clinical and environmental sources. This bacterium is ubiquitous in the natural environment and is an important pathogen known to infect Cystic Fibrosis (CF) patients. The transmission route of strains has not yet been defined; current theories include acquisition from an environmental source or through patient-to-patient spread. A highly discriminatory, bioinformatics based, DNA typing method was developed to investigate the relatedness of clinical and environmental isolates. This study found a similarity between the environmental and several CF clonal strains and also highlighted occurrence of environmental P. aeruginosa strains in CF infections.

Domestication has led to genetic changes that affect quantitative traits in farm animals. Both candidate gene analysis using association tests and genome scans based on linkage analysis have been performed to understand the molecular basis underlying quantitative genetic variation in horses, pigs and chickens. To test a possible association of polymorphisms in the PRKAG3 gene, previously found to be associated with excess glycogen content in pig skeletal muscle, with quantitative traits in the horse, the major coding part of the equine PRKAG3 sequence was identified. Bioinformatic characterization of the equine PRKAG3 gene was conducted. A single nucleotide polymorphism (SNP) causing a missense mutation (Pro258Leu) was found. Screening this SNP showed that the Leu258 allele was more frequent in breeds with heavy muscularity. To assess previously reported associations between polymorphisms in the MC4R gene and obesity-related traits further, we conducted linkage analysis between the MC4R locus and fatness-related traits using a Wild BoarxLarge White intercross. No significant association between segregation at the MC4R locus and fatness was detected in this pedigree. A genome scan of quantitative trait loci (QTLs) has been performed in an intercross between chicken lines divergently selected for growth. Divergent parental lines have been established by selecting for high and low 56-day body weight for over 40 generations. The selection has led to approximately a 9-fold difference in 56-day body weight between lines and resulted in correlated responses for a number of traits including appetite, immune response, body composition and metabolic traits. Phenotypic data on growth and other correlated traits were collected from more than 800 F2 individuals. Genome scans using 145 markers on 26 linkage groups have identified QTLs affecting growth and correlated responses to selection for 56-day body weight. No major QTL explaining a large portion of phenotypic variation in growth was revealed in this study.

The field of forensic genetics is growing fast and the development and optimisation of more sensitive, faster and more discriminating forensic DNA analysis methods is highly important. In this thesis, an evaluation of the use of novel DNA technologies and the development of specific applications for use in forensic casework investigations are presented.

In order to maximise the use of valuable limited DNA samples, a fast and user-friendly Real-time PCR quantification assay, of nuclear and mitochondrial DNA copies, was developed. The system is based on the 5’ exonuclease detection assay and was evaluated and successfully used for quantification of a number of different evidence material types commonly found on crime scenes. Furthermore, a system is described that allows both nuclear DNA quantification and sex determination in limited samples, based on intercalation of the SYBR Green dye to double stranded DNA.

To enable highly sensitive DNA analysis, Pyrosequencing of short stretches of mitochondrial DNA was developed. The system covers both control region and coding region variation, thus providing increased discrimination power for mitochondrial DNA analysis. Finally, due to the lack of optimal assays for quantification of mitochondrial DNA mixture, an alternative use of the Pyrosequencing system was developed. This assay allows precise ratio quantification of mitochondrial DNA in samples showing contribution from more than one individual.

In conclusion, the development of optimised forensic DNA analysis methods in this thesis provides several novel quantification assays and increased knowledge of typical DNA amounts in various forensic samples. The new, fast and sensitive mitochondrial DNA Pyrosequencing assay was developed and has the potential for increased discrimination power.

Genetics and Molecular Biology are keys for the understanding the mechanisms of many of the human diseases that have strong harmful effects. The empirical mission of Genetics is to translate these mechanisms into Clinical benefits, thus bridging in-silico findings to patient bed side: approaching this goal means achieving what is commonly referred as clinical genomics or personalized medicine. In this process, technologies are assuming an increasing role. With the introduction of new experimental platforms (microarrays, sequencing, etc), today's analyses are much more detailed and can cover a wide spectrum of applications, from gene expression to Copy Number Variants detection. The advantages of technological improvements are usually followed by data management drawbacks due to the explosion of data throughput that reflects on a real need for new systems of data rationalization and management, data access, query and extraction. Our genetic laboratories partners encountered all those issues: what they need is a tool that allows data-integration and supports biological data analysis exploiting computational infrastructures on distributed environment. From such needs, we defined two main goals: (1) Computer Science goal: to design and implement a framework that integrates and manages data and genetic analyses; (2) Genetics and Molecular Biology goal (application domains): to solve biological problems through the framework and develop new methods. Given these requirements and related specifications, we designed an extensible framework based on three inter-connected layers: (1) Experimental data layer, that provides data integration of data from high-throughput platforms (also called horizontal data integration); (2) Knowledge data layer, that provides data integration of knowledge data (also called vertical integration); (3) Computational layer, that provides access to distributed environments for data analysis, in our cases GRID and Cluster technologies. Above the three design blocks, single biological problems can be supported and custom user interfaces are implemented. From our partner laboratories, two main relevant biological problems have been addressed: (1) Linkage Analysis: given a large pedigree in which subjects were genotyped with chips of 1 million of SNPs, the linkage analysis problem presented real computational limits. We designed a heuristic method to overcome computational restrictions and implemented it within our framework, exploiting GRID and Cluster environments. Using our approach, we obtained genetic results, successfully validated by end-users. We also tested performances of the system, reporting compared results. (2) SNP selection and ranking: given the problem of ranking SNPs based on a-priori information, we developed a novel method for biological data mining on genes' annotations. The method has been implemented as a web tool, SNP Ranker, that is under deep validation by our partners laboratories. The framework here designed and implemented demonstrated that this approach is consistent and can have potential impacts on the scientific community.

The goal of this research work was to understand the clinical-pharmacology based treatment approaches for sorafenib. Treatment with sorafenib is associated with high inter-patient variability in pharmacokinetic exposures, efficacy and toxicity. We explored the demographic, laboratory, clinical and pharmacogenetic factors to elucidate the sources of variability. In addition, we examined the impact of pharmacogenetic variation in VEGFR2, an important mediator of the VEGF pathway, on risk of prostate cancer. To support these investigations, (mainly single-dose) pharmacokinetic, pharmacogenetic, efficacy and toxicity information were collected from patients with solid tumors, enrolled in five phase I / II clinical trials at National Cancer Institute. Non-compartmental analysis-general linear modeling (NCA-GLM), population pharmacokinetic analysis and several correlative studies were performed to characterize the sources of variability in pharmacokinetics and response. The role of prostate specific antigen (PSA) and ex-vivo anti-angiogenic activity as efficacy markers was evaluated, respectively, for patients with prostate cancer treated with sorafenib and patients with solid tumors treated with combination of sorafenib and bevacizumab. Sweat concentrations of sorafenib were measured to study its association with development of hand-foot skin reaction (HFSR). Only body weight was a significant covariate for volume of distribution by population pharmacokinetic analysis, while BSA, albumin and UGT1A9*3 appeared to be significant by NCA-GLM. However, the contribution of these covariates in overall exposure variability was very small; hence, these were considered clinically irrelevant. The association of sorafenib exposure with efficacy in patients with prostate cancer, colorectal cancer and combined solid tumors were not significant; exposure-efficacy relationship for lung cancer patients requires further evaluation. Sorafenib exposures appeared to be associated with incidences of rash in single agent trials and with HFSR in trials involving treatment with sorafenib and bevacizumab combination. In-vitro cell-line experiments determined that prostate specific antigen (PSA) is not a suitable marker of efficacy in patients with prostate cancer treated with sorafenib. The ex-vivo anti-angiogenic activity, measured by rat-aortic ring assay using patient serum samples, appeared to be not associated with clinical response. Sorafenib concentration in sweat, upto ≥5 ng/mL, apparently was not associated with HFSR. The VEGFR2 H472Q polymorphism was associated with progression-free survival (PFS) (with an apparent heterozygous advantage for survival) and toxicities in patients treated with drugs against the VEGF pathway. Patients who developed hypertension and HFSR on bevacizumab and sorafenib therapy, respectively, appeared to have longer PFS. Therefore, these side effects should be effectively managed to avoid/delay the treatment discontinuation. The VEGFR2 H472Q and V297I genotype were not predictive of risk of prostate cancer in Caucasian subjects.

The population of northern Sweden has previously been shown to be well suited for the mapping of monogenic diseases. In this thesis we have tested the hypothesis that this population could also be used for efficient identification of risk genes for common diseases. In Paper I we have hypothesised that despite the admixture of Swedish, Finnish and Sami, the northern Swedish population consists of sub-populations geographically restricted by the main river valleys running through the region. This geographic isolation, in combination with founder effects and genetic drift, could represent a unique resource for genetic studies. On the other hand, it also underlines the importance of accounting for this e.g. in genetic association studies. To test this hypothesis, we studied the patterns of marriage within and between river valley regions and compared allelic frequencies of genetic markers between these regions. The tendency to find a spouse and live in the river valley where one was born is strong, and allelic frequencies of genetic markers vary significantly between adjacent regions. These data support our hypothesis that the river valleys are home to distinct sub-populations and that this is likely to affect mapping of genetic diseases in these populations. In Paper II, we tested the applicability of the population in mapping HSAN V, a monogenic disease. This disease was identified in only three consanguineous individuals suffering from a severe loss of deep pain perception and an impaired perception of heat. A genome-wide scan combined with sequencing of candidate genes resulted in the identification of a causative point mutation in the nerve growth factor beta (NGFB) gene. In Paper III, a large family with multiple members affected by familial forms of type 1 diabetes mellitus (T1DM) and autoimmune thyroiditis (AITD) was studied. This syndrome was mapped to the IDDM12 region on 2q33, giving positive lodscores when conditioning on HLA haplotype. The linkage to HLA and to the IDDM12 region thus confirmed previous reports of linkage and/or association of T1DM and AITD to these loci and provided evidence that the same genetic factors may be mediating these diseases. This also supported the feasibility of mapping complex diseases in northern Sweden by the use of familial forms of these diseases. In Paper IV, we applied the same approach to study type 2 diabetes mellitus (T2DM). A non-parametric genome-wide scan was carried out on a family material from northern Sweden, and linkage was found to the calpain-10 locus, a previously described T2DM-susceptibility gene on 2q37. Together, these findings demonstrate that selecting for familial forms of even complex diseases, and choosing families from the same geographical region can efficiently reduce the genetic heterogeneity of the disease and facilitate the identification of risk genes for the disease.

A previous model of morphological pathogenesis assumed that cervical carcinoma is of monoclonal origin and progresses through multiple steps from normal epithelium via CINS into invasive carcinomas. The aim of this study was to investigate the molecular mechanism of pathogenesis of cervical neoplasia.

In the clonality study, we found that 75% (6/8) of informative cases of cervical carcinoma had identical patterns of loss of heterozygosity (LOH) in the multiple synchronous lesions, while the remaining cases had different LOU patterns. In an extensively studied "golden case", the multiple carcinoma and cervical intraepithelial neoplasia (CIN) lesions could be divided into several different clonal groups by the X-chromosome inactivation patterns, HPV 16 mutations and LOH patterns. The biggest clonal family included one CIN II, one CIN III and four carcinoma samples, while four other monoclonal families of carcinoma did not include CIN lesions. These results suggested that cervical carcinoma can be either monoclonal or polygonal and contains clones developing either directly or via multiple steps. In the study of HPV types and HPV16 variations, the results confirmed that specific HPV types are the cause of cervical carcinoma but failed to support the previous opinion that HPV16 E6 variants are more malignant than the prototype. We established a novel classification called oncogene lineage of HPV16, and found that additional variations of HPV 16 oncogenes might be a weak further risk factor for cervical carcinoma. In the study of LOH, we found that interstitial deletion of two common regions of chromosome 3p, i.e., 3p2l.1-3p2l.3, and 3p22, was an early event in the development of cervical carcinoma. The results showed that the hMLH1 gene, located in 3p22 and showing LOH in 43% of the studied cases, was not involved in the development of cervical carcinoma because neither the expression level of protein nor the gene sequence was altered in these cases.

In summary, a suggested model of molecular pathogenesis of cervical carcinoma is as follows. Specific types of HPV infect one or more committed stem cells in the basal layer of the epithelium. Fully efficient LOH events turn one (monoclonal origin) or more (polyclonal origin) HPV-infected stem cells into carcinoma cells without CIN steps. Less efficient LOH events would lead to CIN steps where some other unknown factors require to be added to facilitate the formation of carcinoma. In the absence of LOH events no carcinoma develops from the HPV-infected stem cells.

B cell lymphoma (BCL) comprises a biologically and clinically heterogeneous group of tumors deriving from different stages of B cell development. The immunoglobulin (Ig) variable heavy chain (V_H) gene rearrangement is unique for each BCL and can be used to reveal cellular origin, to study signs of antigen selection and to quantify tumor cell load.

The normal counterpart of mantle cell lymphoma (MCL) has been postulated to be a naïve B cell and in hairy cell leukemia (HCL) it is considered to be a post-germinal centre B cell. We analyzed the V_H gene rearrangements in 110 MCLs and 32 HCLs by PCR amplification and sequencing. Most MCLs (84%) displayed V_H genes lacking somatic hypermutation (SHM), thus correlating to a naïve cell origin, whereas a subgroup (16%) showed SHM, implying derivation from a more differentiated B cell. In HCL, a majority of cases (84%) displayed SHM with signs of intraclonal heterogeneity and 16% had unmutated V_H genes, thus questioning the cell of origin in HCL. Biased usage of particular V_H genes was detected in both HCL (V_H3-30) and MCL (V_H3-21 and V_H4-34), which indicates that antigen selection may be involved in lymphoma development. Furthermore, V_H3-21⁺ MCLs showed a highly restricted V_λ3-19 gene use and they also had a superior outcome compared to other MCLs.

Rearrangement analysis of 67 V_H3-21⁺ chronic lymphocytic leukemia (CLL) cases from three different countries verified, regardless of geographical origin, the short and highly homologous complementarity determining region 3s and the strikingly biased usage of the V_λ2-14 gene (75%), as previously reported in CLL. This further supports that antigen selection by a common antigenic epitope may have occurred in V_H3-21⁺ CLLs.

In an autologous transplantation study of 30 multiple myeloma patients, we quantified the tumor content in the autografts before and after stem cell selection using clone-specific PCR. We conclude that stem cell selection reduced the number of clonal cells linearly, but purging could not totally eliminate the tumor cells from the graft, thus increasing the risk of a relapse.

Altogether, our data allowed us to define new BCL subsets and to gain insights into the potential role of antigen selection in BCL development as well as the monitoring of tumor cell load using Ig gene rearrangements analysis.

Sudden cardiac death (SCD) is defined as sudden unexpected death occurring within one hour of onset of symptoms in an individual with or without pre-existing heart disease. The main hypothesis underlying this PhD thesis was that inherited heart diseases are responsible for a significant number of SCD in the young, including those in which the heart appears completely “normal” at postmortem (i.e. “sudden unexplained death or SUD”). Postmortem genetic studies, specifically an exome sequencing based molecular autopsy, will offer a comprehensive analysis of cardiac disease-related genes in SUD cases and families. Exome sequencing in a subgroup of 28 SUD cases (aged 1 to 40 years) revealed 3 rare variations in the common LQTS genes and 6 rare variations in an additional 25 common genes of cardiac arrhythmias and cardiomyopathies. Further, whole exome sequencing performed in a multigenerational family with two SUD cases, subtle post-mortem abnormalities and 12-lead ECG abnormalities identified two rare missense variants in non-cardiac genes and a novel missense variant in a cardiomyopathy-associated mitochondrial gene. These variants needs to be further evaluated by screening additional family members, tissue expression and functional studies. Probands seen at a multidisciplinary cardiac genetic clinic with a reported pathogenic mutation or variants of uncertain significance were further reviewed for any single nucleotide variant (SNV) reclassification by a blinded analysis. Five variants in six hypertrophic cardiomyopathy probands and four variants in probands with other inherited heart conditions were reclassified. In summary, exome based molecular autopsy ensures a comprehensive review of cardiac genes, has an increased likelihood of identifying pathogenic gene mutations including novel genes that may be associated with cardiac disease and sudden death. Periodic reassessment of SNV data is vital in all inherited cardiac disorders.

The presence of TP53 mutations has been associated with inferior outcome in diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL). In DLBCL, the impact of the TP53 codon 72 polymorphism and MDM2 SNP309 has not been clearly elucidated, whereas MDM2 SNP309 was suggested as a poor-prognostic marker in CLL. In addition, p16INK4a promoter hypermethylation has been implicated as a negative prognostic factor in DLBCL. The aim of this thesis was to further evaluate these molecular markers in well-characterised materials of DLBCL and CLL. In paper I, we investigated the prognostic role of TP53 mutation, codon 72 polymorphism and MDM2 SNP309 in DLBCL (n=102). The presence of TP53 mutations (12.7%) correlated with a poor lymphoma-specific and progression-free survival, and a particularly pronounced effect was observed in the germinal center subtype. Neither the MDM2 SNP309 nor the TP53 codon 72 polymorphism had an impact on age of onset or survival. In paper II, we applied pyrosequencing to measure the level of p16INK4a methylation in DLBCL (n=113). Thirty-seven percent of cases displayed p16INK4a methylation; however, no clear association could be observed between degree of methylation and clinical characteristics or lymphoma-specific survival. In papers III–IV, we investigated the prognostic role of MDM2 SNP309 (n=418) and TP53 mutation (n=268) in CLL. No correlation was observed between any particular MDM2 SNP309 genotype and time to treatment and overall survival. Furthermore, no association was found between the different MDM2 SNP309 genotypes and established CLL prognostic markers. TP53 mutations were detected in 3.7% of CLL patients; where the majority showed a concomitant 17p-deletion and only three carried TP53 mutations without 17p-deletion. We confirmed a significantly shorter overall survival and time to treatment in patients with both TP53 mutation and 17p-deletion. Altogether, our studies could confirm the negative prognostic impact of TP53 mutations in DLBCL, whereas MDM2 SNP309 and TP53 codon 72 polymorphisms appear to lack clinical relevance. We also question the role of p16INKa methylation as a poor-prognostic factor in DLBCL. Finally, the presence of TP53 mutation in CLL appears to be rare at disease onset and instead arise during disease progression.

The overall aim of this thesis was to evaluate the outcome when treating children for displaced femoral fractures with external fixation.

In a consecutive and prospective study during the period 1993-2000, 96 children aged 3-15 years with 98 displaced femoral fractures were treated with external fixation and early mobilisation. The mean age was 8.1 years, the mean hospital stay was 8.7 days and the mean treatment time was 61 days. All fractures healed. Minor complications included pin tract infections (18%), clinical insignificant malunions, heterotopic ossification and two re-reductions. Major complications (6%) included two re-fractures after significant trauma and three plastic deformations after premature fixator removal leading to an osteotomy.

Radiological evaluation was performed up to one year for the whole group and for a subgroup up to two years. The evaluation showed that malunions were few and prone to remodelling almost completely. Although the fractures were fixated without shortening, as recommended earlier, the overgrowth was far less than expected.

Isokinetic muscle strength was measured in both hamstrings and quadriceps in 31 of the patients and compared with 31 matched children without previous injury to the legs. Early mobilisation seems to prevent residual muscle weakness previously shown after treatment with traction or cast for femoral fractures in children.

A cost analysis was performed, comparing three different treatment modalities of femoral shaft fractures: traction in hospital, traction in hospital/at home and external fixation. The analysis included both total medical costs and costs for the care provider. The most important factors were days spent at the hospital and the sick leave for the care provider. Treatment that can minimise these factors will contribute strongly to a lowering of health care costs.

Conclusion: External fixation of displaced femoral fractures in children can be used as standard treatment in children aged 3-15 years. The treatment provides satisfactory results with a low rate of major complications. Early mobilisation seems to prevent residual muscle weakness. The treatment reduce the number of days in hospital and the number of days of sick leave for the care provider and contributes strongly to lowering health care costs.

Thrombin is the key enzyme in the formation of a fibrin clot. Control of its activity and generation forms an important haemostatic function preventing occlusive blood clot formation. Deregulation of this control process results in increased thrombin generation and thus increased clot formation in thrombotic conditions. Thrombomodulin (Tm) is an important thrombin-regulatory gene expressed at the endothelial cell surface, and as such may have a role in modifying susceptibility to occlusive thrombotic disease. This thesis focuses on the role of gene variants, within Tm, in determining risk of coronary heart disease (CHD). Contribution to risk of CHD by variants in the Tm gene was assessed in a case-control study (H1FMECH), a large prospective study of heart disease (NPHSII) and a cross sectional study of type 2 diabetes (EDSC). Tm gene variant interaction with clinical and plasma markers of CHD was also studied. The consequences of Tm gene variants upon thrombin generation and inflammation were also addressed. Tm antigen levels and cofactor activity for protein C (PC) activation were assessed to determine whether the overall contribution to heart disease by dysfunctional variants could be estimated. In vitro functional studies were performed, to determine the molecular mechanisms of the effect of the variants showing strong effects on risk and to further our understanding of the role of Tm in the pathogenesis of CHD. The work included in this thesis demonstrates that genetic variation in the Tm gene may influence risk for CHD in an environment of metabolic syndrome. It adds to a growing body of evidence suggesting a contribution to CHD risk caused by variants or mutations in the Tm gene.

Mutations in the beta-globin (β-globin) gene cause beta-thalassaemia (β-thalassaemia).The screening strategy for β-thalassaemiais based on the value of mean corpuscular volume (MCV) from the complete blood count (CBC) data. Current laboratory practice considers blood samples with MCV higher than 80fL as normal. No further assessment will be done on these samples. However, there are clinically silent β-globin gene mutations with MCV higher than 80fL, for example, heterozygous haemoglobin E (HbE). The importance of finding out this kind of mutations is due to the serious outcome when they occur together with classic β thalassaemia mutations in compound heterozygous states, which may produce a condition mimicking β thalassaemia major. The method used to recognize the presence of clinically silent β-globin gene mutations should be robust and with high sensitivity. High resolution melting (HRM) is a suitable technique to screen gene mutations. It is fast and convenient. The process is completed in a closed system without any post PCR manipulation. The sensitivity is up to a single nucleotide change. Using HRM for mutations screening followed by confirmation with sequencing can reduce time and cost of testing clinically silent β-globin gene mutations on a large scale. This study first shows the ability of HRM in detecting various types of β-globin gene mutations. The technique is then applied to detect clinically silent β-globin gene mutations in a group of high school students with normal CBC data. Mutations with different clinically significance were found. The frequency of mutation found in the samples of the study suggests that screening for β-globin gene mutation may be worthwhile in subjects with MCV higher than 80fL.
published_or_final_version
Pathology
Master
Master of Medical Sciences

The unconventional myosin IC has previously been suggested to be a haploinsufficient tumour suppressor. The mechanism for this action has hitherto been unknown, however, and hence we decided to attempt to elucidate the genes involved. The first study involved knock-down of MYO1C using siRNA technology followed by whole transcriptiome microarray analysis performed on samples taken at different time points post transfection. This revealed a cornucopia of differential expressions compared to the negative control, among them we found an early up-regulation of the PI3K/AKT pathway and the pathway for prostate cancer. Among the down regulated pathways we found endometrial-, colorectal cancer and small cell lung cancer as well as the cell cycle pathway which was a little counter intuitive to the hypothesis that MYO1C suppresses cancer. For the next study six different genes (CCND1, CCND2, CDKN2B, CDKN2C, MYC, RBL1) important for the transitions into S-phase of the cell cycle were therefore chosen for validation using qPCR. These six genes and MYO1C were analysed on both the original time series and a new biological replicate as well as a well stratified set of endometrial carcinoma samples. We were able to verify the significant down-regulation of CCND2 in both time series indicating that this is caused by the depletion of MYO1C. In the tumour samples we saw a negative correlation between the expression of MYO1C and FIGO grade corroborating results previously found by our group when looking at protein expression.

With the completion of the human genome sequencing project, attention is currently shifting toward understanding how genetic variation, such as single nucleotide polymorphism (SNP), leads to disease. To identify, understand, and control biological mechanisms of living organisms, the enormous amounts of accumulated sequence information must be coupled to faster, cheaper, and more powerful technologies for DNA, RNA, and protein analysis. One approach is the miniaturization of analytical methods through the application of microfluidics, which involves the manipulation of fluids in micrometer-sized channels. Advances in microfluidic chip technology are expected to play a major role in the development of cost-effective and rapid DNA analysis methods. This thesis presents microfluidic approaches for different DNA genotyping assays. The overall goal is to combine the potential of the microfluidic lab-on-a-chip concept with biochemistry to develop and improve current methods for SNP genotyping. Three genotyping assays using miniaturized microfluidic approaches are addressed. The first two assays are based on primer extension by DNA polymerase. A microfluidic device consisting of a flow-through filter chamber for handling beads with nanoliter liquid volumes was used in these studies. The first assay involved an allelespecific extension strategy. The microfluidic approach took advantage of the different reaction kinetics of matched and mismatched configurations at the 3’-ends of a primer/template complex. The second assay consisted of adapting pyrosequencing technology, a bioluminometric DNA sequencing assay based on sequencing-bysynthesis, to a microfluidic flow-through platform. Base-by-base sequencing was performed in a microfluidic device to obtain accurate SNP scoring data on nanoliter volumes. This thesis also presents the applications of monolayer of beads immobilized by microcontact printing for chip-based DNA analysis. Single-base incorporation could be detected with pyrosequencing chemistry on these monolayers. The third assay developed is based on a hybridization technology termed Dynamic Allele-Specific Hybridization (DASH). In this approach, monolayered beads containing DNA duplexes were randomly immobilized on the surface of a microheater chip. DNA melting-curve analysis was performed by dynamically heating the chip while simultaneously monitoring the DNA denaturation profile to determine the genotype. Multiplexing based on single-bead analysis was achieved at heating rates more than 20 times faster than conventional DASH provides.
QC 20101008

B cell lymphomas and leukemias are heterogeneous tumors with different cellular origins. Analysis of immunoglobulin (Ig) genes enables insight into the B cell progenitor, as Ig somatic hypermutation correlates with antigen-related B cell transit through the germinal center (GC). Also, restricted Ig variable heavy chain (V_H) gene repertoires in B cell malignancies could imply antigen selection during tumorigenesis. The length of telomeres has been shown to differ between GC B cells and pre/post-GC B cells, possibly representing an alternative angle to investigate B cell tumor origin.

Mantle cell lymphoma (MCL), previously postulated to derive from a naïve, pre-GC B cell, was shown to have an Ig-mutated subset (18/110 MCLs, 16%), suggestive of divergent cellular origin and GC exposure. Another subset of MCL (16/110, 15%), characterized by V_H3-21/V_λ3-19 gene usage, alludes to a role for antigen(s) in pathogenesis, also possible for hairy cell leukemia (HCL) in which the V_H3-30 gene (6/32, 19%) was overused. HCL consisted mainly of Ig-mutated cases (27/32, 84%) with low level intraclonal heterogeneity, contrasting with the proposed post-GC origin, for both Ig-mutated and Ig-unmutated HCLs. For MCL and HCL, derivation from naïve or memory marginal zone B cells which may acquire mutations without GC transit are tempting speculations, but currently little is known about this alternative immunological pathway. Heavily mutated Ig genes without intraclonal heterogeneity were demonstrated in lymphoplasmacytic lymphoma/Waldenström’s macroglobulinemia (13/14, 93%), confirming that the precursor cell was transformed after GC affinity maturation. Telomere length analysis within 304 B cell tumors revealed variable lengths; shortest in the Ig-unmutated subset of chronic lymphocytic leukemia, longest in the GC-like subtype of diffuse large B cell lymphoma, and homogeneous in MCL regardless of Ig mutation status. However, telomere length is complex with regard to GC-related origin.

In summary, this thesis has provided grounds for speculation that antigens play a role in MCL and HCL pathogenesis, although the potential antigens involved are currently unknown. It has also enabled a more informed postulation about the cellular origin of B cell tumors, which will ultimately enhance understanding of the biological background of the diseases.

Neurodegenerative and neuropsychiatric disorders (NDD-NPDs) are multifactorial, polygenic and complex behavioral phenotypes caused by brain abnormalities. Most genetic studies have focused on understanding the genetic component of specific brain diseases. Several brain diseases also show similar clinical and pathological symptoms. In recen years, multiple studies have used next generation sequencing (NGS) technologies such as RNA sequencing (RNA-Seq) to investigate molecular signature of brain diseases. However, many studies have only focused on a particular disease and limited brain regions. By using the data from a broad range of cortical regions from multiple brain diseases, we will be able to dig deeper into the molecular basis of neurological diseases. The main aim of this thesis was to examine the transcriptome-wide characterization of cortical brain regions across neurological disorders. We focused our research efforts on highlighting cross-disease shared molecular signatures, and exploring co-expression networks and cell-type-specific patterns for NDD-NPDs. By processing and analyzing RNA-Seq data using a set of computational tools and statistical tests, we performed transcriptomic profiling of brain samples from eight groups of patients with Alzheimer’s disease (AD), Parkinson’s disease (PD), Progressive Supranuclear Palsy (PSP), Pathological Aging (PA), Autism Spectrum Disorder (ASD), Schizophrenia (SZ), Major Depressive Disorder (MDD), and Bipolar Disorder (BP)-in comparison with 2,078 brain samples from matched control subjects. In this thesis, we provide a transcriptomic framework to understand the molecular architecture of NPDs and NDDs through their shared- and specific gene expression in the brain.

The state-of-the-art approach for the genetic molecular cause research relies on massively parallel gene sequencing, which represents a challenge both in data handling and variant prioritization. The univocal assignment of disease pathogenicity to the sequence variants is often difficult, and requires the integration of different lines of evidence for a comprehensive interpretation. During my thesis, I contributed to the development of novel approaches to evaluate rare variant contribution to the clinical phenotype. These methods were presented and evaluated at the Critical Assessment of Genome Interpretation, ranking among top programs considering either performance or the number of correct assigned disease predictions. A similar strategy was employed for identification of disease genes linked to neurodevelopmental disorders (NDDs) comorbidity. In this case, computational methods were applied to select the most promising candidate genes for the design of diagnostic panel, which is currently used for patient screening at Pediatrics Clinic of the University of Padova. The variants found within the panel genes have been selected according frequency, pathogenicity prediction and variant segregation analysis within the family. Furthermore, I took advantage of different computational tools to investigate the mutated gene function, and used this information to demonstrate the impact of likely pathogenic variant on clinical phenotype. In several cases, likely pathogenic mutations mapped to intrinsically disordered regions (IDRs), which lack a fixed three-dimensional structure. Coherently, several studies demonstrate that mutations in IDRs are often associated with the pathogenesis of various human diseases. Thus, IDRs classification could represent a critical step for understanding the impact of possibly disease-causative variants mapping in these regions. Due to the influence of intrinsically disordered proteins (IDPs) in diseases, I participated to the manual curation and update of entries in the DisProt database, the primary repository of disorder-related data on sequence. Interestingly, increasing evidence from literature highlights the IDPs involvement in neuronal signal transduction. Among the proteins encoded by diagnostic panel genes, TANC2 especially emerged as intrinsically disordered protein with a possible role in synaptic signal transduction. As TANC2 and its protein family function was poorly characterized, I performed an in silico analysis to characterize the TANC protein activity, and the implicated biological processes. The functional hypothesis emerged from the bioinformatics analysis was used to drive further experimental investigations. In vitro validation of predicted TANC2-CDKL5 interaction highlighted the relevance of the IDRs in regulating degradation of CDKL5, whose mutations are associated with a heterogeneous set of NDD phenotypes. Furthermore, I demonstrated that TANC2 contributes to downregulate CDKL5 expression levels. For this reason, TANC2 protein could represent a novel therapeutic target to design new drugs for the treatment of CDKL5 over-expression associated diseases.
La strategia di elezione per l'identificazione di varianti causative di malattie genetiche consiste nell’utilizzo di piattaforme di Next Generation Sequencing. Questo tipo di approccio rappresenta una sfida, sia per quanto riguarda la gestione della mole di dati da sequenziamento, che per l’interpretazione clinica dei risultati. L’identificazione di varianti chiaramente implicate nella determinazione della patologia è un processo complesso, che richiede l'integrazione di diversi tipi di informazione. Durante il mio dottorato, ho contributo all’implementazione di metodi computazionali per predire la probabilità che un determinato genotipo sia associato al fenotipo clinico di interesse. Questi metodi sono stati presentati, e valutati, in occasione del Critical Assessment of Genome Interpretation (CAGI), dove si sono posizionati tra i migliori classificati sia per prestazioni che numero di predizioni corrette. Una strategia analoga è stata applicata all’identificazione di geni implicati nella comorbidità tra disordini del neurosviluppo. Anche in questo caso, l’utilizzo di tecniche bioinformatiche si è reso fondamentale per la selezione di geni candidati, che sono stati poi utilizzati nella progettazione di un pannello genico diagnostico attualmente in uso presso la Clinica Pediatrica dell’Università di Padova. Data la gran quantità di dati prodotti per esperimento, le varianti trovate nei geni inclusi nel pannello sono state filtrate in base alla frequenza, alla predizione di patogenicità e all'analisi di segregazione all'interno della famiglia. In alcuni casi, un ulteriore contributo a supporto dell’effettiva patogenicità della variante è stato dato dall’analisi bioinformatica della proteina mutata. Frequentemente, la variante candidata provoca alterazioni a livello di regioni intrinsecamente disordinate (IDR), caratterizzate dall’assenza di una conformazione tridimensionale stabile. Questo dato è coerente con la più recente letteratura: diversi studi, infatti, dimostrano l’implicazione di mutazioni nelle IDR in diverse patologie umane. La classificazione delle IDR, quindi, può rappresentare un primo passo per comprendere l'impatto di eventuali varianti causative all'interno di queste regioni. Data la rilevanza delle IDR a livello biologico e clinico, ho partecipato alla curazione manuale e all'aggiornamento delle voci presenti nel database DisProt, la principale banca dati relativa al disordine nelle proteine. È interessante notare che, tra i vari processi biologici in cui le IDR sono coinvolte, queste regioni svolgono un ruolo molto importante nel signaling neuronale. Tra le proteine codificate dai geni inclusi nel pannello genico, TANC2 si è distinta per essere una proteina disordinata, probabilmente implicata alla trasduzione del segnale a livello delle sinapsi neuronali. Dato che la funzione di TANC2 e della rispettiva famiglia proteica risultava ancora poco chiara, ho eseguito un’analisi in silico delle proteine TANC, grazie alla quale è stato possibile caratterizzare le funzioni e i diversi processi cellulari in cui queste sono coinvolte. Le ipotesi funzionali emerse dall'analisi bioinformatica sono state utilizzate per condurre ulteriori indagini sperimentali. In particolare, la validazione in vitro dell'interazione TANC2-CDKL5 ha evidenziato l’estrema importanza di regioni intrinsecamente disordinate nella regolazione della degradazione di CDKL5, le cui mutazioni sono associate con manifestazioni cliniche legate a disordini del neurosviluppo. Inoltre, gli esperimenti hanno dimostrato che TANC2 contribuisce alla down-regolazione dei livelli di espressione di CDKL5. Per questo motivo, TANC2 si candida a rappresentare un nuovo target terapeutico per lo sviluppo di nuovi composti per il trattamento di condizioni cliniche associate all’over-espressione di CDKL5.

DNA methylation may represent an important contributor to the missing heritability described in complex trait genetics. However, technology to measure DNA methylation has outpaced statistical methods for analysis. Novel methodologies are required to accommodate this growing volume of DNA methylation data. In this dissertation, I propose two novel methods to analyze DNA methylation data: (1) a new statistic based on spatial location information of DNA methylation sites to detect differentially methylated regions in the genome in case and control studies; and (2) a principal component approach for the detection of unknown substructure in DNA methylation data. For each method, I review existing ones and demonstrate the efficacy of my proposed method using simulation and data application. Medical research is increasingly focused on personalizing the care of patients. A better understanding of the interaction between treatment and patient specific prognostic factors will enable practitioners to expand the availability of tailored therapies improving patient outcomes. The Subpopulation Treatment Effect Pattern Plot (STEPP) approach was developed to allow researchers to investigate the heterogeneity of treatment effects on survival outcomes across increasing values of a continuously measured covariate, such as biomarker measurement. I extend the STEPP approach to continuous, binary and count outcomes which can be easily modeled with generalized linear models (GLM). The statistical significance of any observed heterogeneity of treatment effect is assessed using permutation tests. The method is implemented in the R software package (stepp) and is available in R version 3.1.1. The efficacy of my STEPP extension is demonstrated by using simulation and data application.

Male infertility is typically diagnosed upon routine semen analysis following the World Health Organisation’s (WHO) semen analysis manual. Recent editions of the manual have essentially changed the diagnosis of a semen sample, prompting debate between experts as to which edition should be followed. Deoxyribonucleic Acid (DNA) integrity analysis is proving to be a useful adjunct to semen analysis as 15% of infertile men have a normal semen analysis but they have an increased DNA fragmentation level (DFL) which has been associated with increased disease incidence in any resultant offspring. However, such tests are not endorsed by the WHO, possibly due to a lack of standardisation in the implementation, analysis and clinical interpretation of methods used to evaluate DNA integrity. Improved efficiency of testing is achieved by batch testing or sending samples to a central laboratory for analysis, requiring an effective storage system. Most current protocols for semen storage and related DNA integrity testing are complex, expensive and require specialised equipment. Nevertheless, since the Halosperm® G2 Kit, requires only standard laboratory equipment, a simple, convenient and stable storage method for the purpose of testing sperm DNA fragmentation would be advantageous. DNA has been successfully extracted from air‐dried semen and one particular study has investigated the use of air‐dried semen slides as a method of storage prior to DNA fragmentation testing, however, the effects of time and temperature on the integrity of spermatozoa DNA has not been considered. The first objective of this present study was to investigate the relationship between sperm DNA fragmentation (using the Halosperm® G2 Test Kit) and semen analysis results (measured according to the 4th and 5th Edition WHO semenanalysis manuals) to determine the clinical utility of the Halosperm assay. The second objective was to consider extrinsic effects on the DNA integrity of air‐dried semen in order to develop an alternative storage method for semen prior to DNA fragmentation testing using the Halosperm assay. A retrospective analysis was carried out on 905 consecutive semen samples with 4th and 5th Edition semen analysis and Halosperm result. Pearson correlations, analysis by ANOVA and post‐hoc testing by Tukey’s HSD were used for statistical analysis. Multiple aliquots of semen samples were prepared to achieve fresh, snap frozen and air‐dried samples. Samples were sequentially assessed for sperm DNA fragmentation using the Halosperm® G2 kit (Halotech DNA SL, Spain) and scored against 300 sperm, with fragmentation results ≥30% considered positive. Fragmentation levels were compared between the different protocols. Multiple aliquots of semen samples were then air‐dried to test the fragmentation levels between different slide types, reconstituting fluids, times and temperatures. Pearson’s correlation coefficient and paired t‐tests were used for statistical analysis. In summary, whilst significant associations exist between sperm DNA fragmentation and sexual abstinence, volume of the ejaculate, sperm concentration, normal sperm morphology and sperm motility, the Halosperm assay may provide an explanation for infertility where semen analysis cannot. Furthermore, air‐drying semen is a simple and stable storage method, for up to one month at ‐22 degrees, prior to DNA fragmentation testing with the Halosperm® G2 kit.

The central problem for researchers of HIV-1 evolution is explaining the apparent design of the virus for causing pandemic infection in humans: understanding how HIV-1 spreads is key to halting the pandemic. Current knowledge of how HIV-1 spreads from host to host is based upon experimental observation and indirect inferences informed by theory. The hypothesis of this thesis is that diversity of HIV-1 around the time of transmission is important for viral adaptation to a new human host, rather than intrinsic superiority of particular strains found in infectious fluids from human donor hosts, and that studying recombination is important for understanding this behaviour. To demonstrate the apparent randomness of transmission, I test the null-hypothesis that hard selection accounts for between-host viral divergence in a rare case study of contemporaneous infection. I explain how the experimental data that I have generated and the analyses I have carried out address certain basic assumptions and predictions about HIV-1 transmission and may inform current strategies for vaccine design. Specifically, my approach contributes to the current literature on HIV-1, by investigating an alternative hypothesis to the single virion theory of sexual transmission and by characterizing the role of recombination in a pseudodiploid virus following multiple-infection.

Les dystrophies rétiniennes sévères et précoces (EOSRD) et l'amaurose congénitale de Leber (ACL - MIM204000) sont les principales causes de cécité incurable chez les enfants. Ces maladies, variables sur le plan clinique, génétique et physiopathologique, peuvent être le signe de syndromes multisystémiques, tels que les ciliopathies. Elles se transmettent le plus souvent de manière autosomique récessive et l'implication de plusieurs gènes a été confirmée. Cependant, l'histoire et l'expression clinique de l'ACL sont imparfaitement comprises et de nombreuses mutations restent inconnues. Il est nécessaire de continuer à déchiffrer ces aspects pour affiner la compréhension de la physiopathologie. L'identification de nouveaux gènes responsables et les corrélations génotype-phénotype sont essentiels à la prise en charge des patients. Grâce au séquençage à haut débit des gènes connus de l'ACL/EOSRD et aux investigations dans les centres de référence cliniques, le Laboratoire de génétique ophtalmologique (LGO) a identifié les causes moléculaires de la maladie dans plus de 80 % des cas dans une cohorte de plus de 700 familles. À ce jour, 40 familles de ACL/EOSRD non résolues ont été soumises au séquençage de l'exome entier (WES), ce qui a permis d'identifier des gènes candidats, sélectionnés en vue d'une validation fonctionnelle. Des variants délétères de GPATCH11 ont été identifiés dans six familles comprenant 12 individus atteints de dystrophie rétinienne, présentant des troubles neurologiques et des anomalies squelettiques, fournissant des arguments forts que des mutations récessives dans le gène GPATCH11 sont responsables de la maladie. GPATCH11 est l'une des protéines contenant le domaine G-patch la moins étudiée, connues pour contribuer au spliceosome. Quatre mutations récessives ont été identifiées, avec la mutation du site d'épissage NM_174931.4 : c.328+1G>T commune à quatre familles sur six et affectant le site d'épissage consensus de l'intron 4, ce qui entraîne l'exclusion de l'exon 4 du transcrit sans rupture du cadre de lecture, produisant ainsi une protéine plus courte. La protéine GPATCH11, dans sa forme sauvage ou mutée, est localisée à la fois, de façon diffuse dans le nucléoplasme et dans le centrosome des cils primaires des fibroblastes, suggérant des rôles dans le métabolisme de l'ARN et des cils. Le modèle de souris (Gpatch11delta5/delta5) généré à l'Institut Imagine, portant la délétion de l'exon 5 équivalent à l'exon 4 de GPATCH11 humain, reproduit les défauts phénotypiques des patients, avec la présence d'une dystrophie rétinienne et des anomalies comportementales. Le transcriptome de la rétine a identifié des voies dérégulées dans l'expression et l'épissage des gènes, impactant des processus clés tels que les réponses à la lumière des photorécepteurs, la régulation de l'ARN et le métabolisme associé aux cils primaires. L'analyse par spectrométrie de masse a trouvé des protéines régulées à la baisse impliquées dans la perception visuelle, la fonction synaptique et les mécanismes de liaison et d'épissage de l'ARN, et des protéines régulées à la hausse impliquées principalement dans le métabolisme et l'épissage de l'ARN (Publication 1). En outre, l'implication de GPATCH11 dans le cerveau est en cours d'exploration par immunomarquage et analyse transcriptomique/protéomique, en se concentrant sur l'hippocampe, structure cérébrale responsable de la mémoire. Les souris Gpatch11delta5/delta5 sont viables et se développent normalement, toutefois les mâles sont complètement infertiles et présentent des testicules plus petits que la normale et vides, dont la cause est en cours d'étude en collaboration avec un laboratoire externe (Part 2A, B)
Early onset retinal dystrophies (EOSRD) and Leber congenital amaurosis (LCA - MIM204000) are the leading cause of incurable blindness in children. These diseases, clinically, genetically, and pathophysiologically variable, can be the sign of multisystemic syndromes, such as ciliopathies. They are mostly inherited in autosomal recessive manner, and several genes have been confirmed to be involved. However, the history and clinical expression of LCA are imperfectly understood and many mutations remain unknown. There is a need to continue deciphering these aspects to refine the understanding of pathophysiology. The identification of new responsible genes and the genotype-phenotype correlations are essential for disease management. Thanks to high-throughput gene panel-based sequencing of known LCA/EOSRD genes and investigation in clinical reference centres, the Laboratory of Genetics in Ophthalmology (LGO) has identified the molecular causes of the disease in more than 80% of cases in a cohort of over 700 families. To date, 40 unresolved LCA/EOSRD families have been submitted to whole exome sequencing (WES), leading to the identification of candidate genes, which have been selected for functional validation. Deleterious GPATCH11 variants have been identified in six families comprising 12 affected individuals with retinal dystrophy, exhibiting neurological disorders and skeletal anomalies, providing compelling evidence that recessive mutations in the GPATCH11 gene are responsible for the disease. GPATCH11 is one of the lesser-explored G-patch domain containing proteins, which are known to contribute to the spliceosome. Four recessive mutations were identified, with the splice-site NM_174931.4: c.328+1G>T being common to four out of six families and affecting the consensus splice site of intron 4, causing exon 4 to be excluded from the transcript without breaking the reading frame and producing a shorter protein. Both wild-type and mutated GPATCH11 proteins are localised in the nucleoplasm with a diffuse pattern and in the centrosome of the primary cilia of fibroblasts, suggesting roles in RNA and cilia metabolism. The mouse model (Gpatch11delta5/delta5) generated at the Institute Imagine, carrying the deletion of exon 5 equivalent to exon 4 of human GPATCH11, replicates the patients' phenotypic defects, such as retinal dystrophy and behavioural abnormalities. Retina transcriptome analysis identified deregulated pathways in gene expression and splicing, impacting key processes, such as photoreceptor light responses, RNA regulation, and primary cilia-associated metabolism. Mass-spectrometry analysis found downregulated proteins involved in vision perception, synaptic function and RNA binding and splicing pathways, and upregulated proteins mostly involved in RNA processing and splicing (Publication 1). Furthermore, the involvement of GPATCH11 in the brain is currently being explored through immunostaining and transcriptome/proteome analysis, focusing on the hippocampus, a brain structure responsible for memory. Gpatch11delta5/delta5 mice are viable and develop normally, except that males are completely infertile and exhibit smaller than normal and empty testis. The cause of this infertility is under investigation in collaboration with an external laboratory (Part 2A, B)

Osteonecrosis of the jaw (ONJ) is a potentially severe adverse effect of bisphosphonates. It can cause persistent pain and infection to the jawbones, and is currently considered incurable. ONJ occurs in a subset of individuals exposed to bisphosphonates (≤7%). Although a number of clinical risk factors, such as dentoalveolar surgery and dental infection, can increase the risk of ONJ development, there remains a number of patients who do not present with these clinical risk factors. Therefore, a genetic predisposition has been proposed. Genome-wide association studies (GWAS), widely performed in pharmacogenomics and successful in other drug side effects, have also been attempted in bisphosphonates-associated ONJ. However, possibly due to small cohort sizes (≤30 cases), these studies failed to detect any significant genetic risk factors. The aim of this thesis is to present the results of a large, multicentre GWAS, coupled with detailed analyses of clinical phenotype. 393 ONJ cases were recruited from 23 clinical centres worldwide. All cases were thoroughly phenotyped and adjudicated by specialist multidisciplinary teams. Random effects logistic regressions (Stata v12.1) were used for clinical risk factor analyses. All samples were genotyped using Illumina® Human1M Omni Express Beadchip (1,072,820 probes) and were compared with 2,554 genetically-matched population controls from publicly available sources. Genotype statistical analysis was performed in PLINK. Risk factors including advanced age, longer bisphosphonates duration, other cancers and use of steroids were found statistically significant (p < 0.05). With extreme phenotyping, i.e. non-surgery triggered ONJ cases versus the population controls, for the first time, a genome-wide significant single nucleotide polymorphism was identified: rs12440268 at TJP1 gene (p=1.21E-8). Individuals positive for this marker were nearly three times more likely to develop ONJ than those negative for it (OR=2.66). TJP1 encodes protein at the tight junctions, which maintain epithelial integrity. Its polymorphism may contribute to ONJ pathogenesis through impaired mucosal healing.

Orientador: Anete Pereira de Souza.
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-27T05:07:09Z (GMT). No. of bitstreams: 1 Durigan_Mauricio_D.pdf: 7600085 bytes, checksum: 74ae2337a73b6edfb14a403af4ffa590 (MD5) Previous issue date: 2015
Resumo: Giardia duodenalis é um protozoário flagelado que parasita o homem e diversos animais domésticos e selvagens. Este parasito causa a doença giardiose que é uma das mais prevalentes doenças parasitárias de veiculação hídrica do mundo, responsável por aproximadamente 280 milhões de casos anualmente. Existe uma considerável variabilidade genética em G. duodenalis, de modo que seus isolados foram divididos em oito grupos genéticos (A-H), dois dos quais (A e B) são encontrados tanto em humanos quanto em animais. Os demais grupos (C-H) parasitam outros animais e apresentam maior especificidade a determinados hospedeiros não humanos. A contaminação ambiental por Giardia tem sido amplamente descrita embora esses estudos, em sua maioria, são realizados no nível de identificação de espécie. Há falta de estudos que correlacionam a contaminação ambiental e infecções clínicas na mesma região. O presente trabalho teve como objetivo principal contribuir para o conhecimento da diversidade genética da espécie Giardia duodenalis. Primeiramente, foi realizada a genotipagem multilocos dos principais grupos genéticos de G. duodenalis na região metropolitana de Campinas. Foram encontrados grupos genéticos associados principalmente a infecções humanas bem como isolados com potencial zoonótico em amostras ambientais e obtidas de outros animais. Foi encontrado um alto percentual (25%) de amostras com grupos genéticos mistos e um elevado número de haplótipos distintos, indicando grande diversidade genética do parasito nessa região. Na segunda parte deste trabalho, foi realizado um estudo populacional com amostras clínicas de Giardia provenientes de hospital, creche e centro de controle de zoonoses e amostras ambientais de esgoto hospitalar, efluente de estação de tratamento de esgoto e amostras hídricas de importantes rios e córregos urbanos. As análises populacionais, com exceção das amostras caninas, evidenciaram grande similaridade genética entre essas populações de Giardia. Na terceira parte do presente trabalho, foi realizada uma busca por repetições microssatélites (SSRs) nos genomas publicados de Giardia para desenvolvimento, caracterização e avaliação de polimorfismo de novos marcadores microssatélites. Foram encontrados 506, 438, 402 e 507 microssatélites correspondentes aos genomas AI, AII, B e E, respectivamente. Foram selecionados 80 SSRs específicos aos grupos genéticos A, B e E (40, 20 e 20, respectivamente), além de 36 SSRs compartilhados entre os três genomas. A análise de amplificação confirmou a existência de marcadores específicos aos grupos genéticos A, B e E, além de marcadores compartilhados entre os grupos. A caracterização dos SSRs permitiu a detecção de 12 locos SSRs polimórficos do grupo genético A e sete locos SSRs polimórficos do grupo genético B. Dentre os marcadores compartilhados, o loco GduABE01 apresentou polimorfismo. Os locos polimórficos podem servir para futuros estudos populacionais e os marcadores desenvolvidos podem ser utilizados para identificação dos principais grupos genéticos de G. duodenalis em amostras clínicas e ambientais. Os resultados apresentados contribuem para um melhor entendimento sobre a diversidade genética do parasito bem como sobre a presença de grupos com potencial zoonóticos inter-relacionados em diferentes regiões. Os novos marcadores moleculares disponibilizados podem contribuir para novos estudos populacionais, promovendo melhor discriminação entre os genótipos e possibilitando assim identificar a contaminação e promover o rastreamento da doença
Abstract: Giardia duodenalis is a flagellate protozoan that that parasites humans and several domestic and wild animals. This parasite causes giardiasis, one of the most common waterborne diseases in the world responsible for, approximately 280 million cases per year. There is a great genetic diversity in this species and its isolates have been grouped into eight distinct genetic assemblages (A-H). While groups A and B parasitize different hosts and have zoonotic potential, groups C, D, E, F, G and H usually found in animals and show greater specificity to the parasitized host. Environmental contamination for Giardia has been widely reported however, most of these studies have been performed only at species level. The present study aimed to contribute to the knowledge of the genetic diversity of the species Giardia duodenalis. In the first chapter of this document, multilocus sequence-based genotyping using three gene loci assigned most of the samples as belonging to human genotypes although isolates with zoonotic potential have also been identified in environmental and non-human clinical samples. A high percentage (25%) of mixed assemblages and a high number of different haplotypes were detected, which indicates high genetic diversity of this parasite in this region. In the second chapter, a population genetics study was performed with clinical samples from hospital, day-car center and a center for zoonosis control of the city and environmental samples from hospital sewage, effluent of a wastewater treatment plant and important water samples from rivers and urban streams. With the exception of the canine population, population genetic analysis showed consistent similarity between clinical and environmental populations. In the last chapter, we performed a search for microsatellites (SSRs) in the published genomes of Giardia to develop and characterize the polymorphism of new microsatellite markers. Our group identified 506, 438, 402 and 507 microsatellites of the genomes AI, AII, B and E, respectively. We have selected 80 markers specific to the genetic assemblages A, B and E (40, 20 and 20, respectively) and 36 shared SSRs between the three genomes. Analysis of amplification reactions confirmed the existence of specific loci of each genetic assemblage as well as shared loci among assemblages. Characterization of all loci allowed the detection of 12 polymorphic loci for group A and seven polymorphic loci for group B. Among the shared markers, GduABE01 presented polymorphism. The polymorphic markers can be used in future population genetic studies and the developed markers can contribute to the identification of the main genetic assemblages of G. duodenalis in clinical and environmental samples. The results presented here contribute to a better understanding of the genetic diversity of the parasite as well as the presence of zoonotic potential genotypes, related in different regions. The new molecular markers provided can contribute with population genetic studies in a high level of discrimination that allows identifying the source of contamination and molecular tracking of the disease
Doutorado
Genetica de Microorganismos
Doutor em Genetica e Biologia Molecular

Gilbert’s syndrome (GS) occurs in approximately 10% of the European population. The most common cause is homozygosity for UGT1A1*28, which is a TA repeat expansion in the promoter of UGT1A1. It is characterised by intermittent hyperbilirubinemia due to reduced hepatic activity of the  enzyme UDP-glucuronosyl-transferase 1A1(UGT1A1). GS also  alteres the pharmacokinetics of some drugs and increases the risk of drug toxicity. Irinotecan (Camptosar®, Campto®) is used in metastatic colorectal cancer and the active metabolite is inactivated by UGT1A1. Studies have shown that GS can be a risk factor for toxicity during irinotecan therapy. Three different methods for genotyping of UGT1A1*28 have been tested. PCR with electrophoresis used for size separation, melting temperature analysis and fluorescent PCR followed by fragment analysis on a capillary sequencer. The last method was found to be superior. This method was used for genotyping of patients with colorectal cancer treated with irinotecan and 5-fluorouracil in the Nordic VI study. A significant association between UGT1A1 genotype and plasma bilirubin level before the start of irinotecan treatment was seen (ANOVA p<0.0001). Patients with GS had an overall increased risk of adverse drug reactions (Fishers Exact test p=0.02). Gilbert’s syndrome can be diagnosed by genotyping UGT1A1*28 with a fragment analysis method. Genotyping of UGT1A1*28 can be used to identify patients with an increased risk of adverse reactions to irinotecan.
Gilberts syndrom (GS) drabbar upp till 10% av befolkningen i Västeuropa. GS beror på nedsatt aktivitet av enzymet UDP-glukuronosyltransferas 1A1 (UGT1A1) i levern. Den vanligaste orsaken är att individen är homozygot för en insertion av två baser i promotorn för genen UGT1A1. Denna genvariant kallas (TA)7TAA  eller UGT1A1*28. GS leder till intermittent stegring av bilirubin vid infektioner, men bilirubinstegring kan förekomma även utan utlösande agens. GS kan också leda till bilirubinstegring vid viss läkemedelsbehandling. Irinotekan (Campto®) används vid metastaserande colorektal cancer och dess aktiva metabolit inaktiveras av UGT1A1. Det finns rapporter om att GS ger ökad risk för toxiska biverkningar av irinotekan. Tre metoder för att bestämma UGT1A1 har jämförts: PCR med elfores, PCR med smältpunktsanalys och PCR med fragmentanalys på sekvensator. Den sista metoden var bäst och användes för att genotypa UGT1A1 hos patienter med colorektal cancer från Nordic VI-studien. De behandlades med irinotekan i kombination med bolusinjektion eller infusion av 5-fluorouracil. Vi fann att  patienter med GS hade signifikant högre S-bilirubin före behandling jämfört med övriga patienter. De hade även ökad frekvens biverkningar av irinotekan (Fishers exakta test p=0,02). Genotypning av UGT1A1 kan således användas för att diagnostisera Gilberts syndrom hos patienter med oförklarad bilirubinstegring. Det kan även användas för att identifiera patienter med ökad risk för biverkningar av irinotekan.