Since apoE allele status is the predominant Alzheimers disease (AD) genetic risk factor, functional single nucleotide polymorphisms (SNPs) in brain apoE receptors represent excellent candidates for association with AD. Therefore, three low density lipoprotein receptor (LDLR) SNPs were evaluated by TaqMan allelic discrimination assays for association with AD and I found that certain haplotypes alter the odds of AD. A SNP within LDLR exon 12, rs688, was identified in silico as neutralizing a putative exon splicing enhancer (ESE). Since LDLR is a major apoE receptor in the brain, I hypothesized that rs688 modulates LDLR splicing in neural tissues and associates with AD. To evaluate this hypothesis, I analyzed splicing patterns in human hippocampus samples and established that this SNP was associated with significantly decreased LDLR exon 12 splicing efficiency when the minor allele T is present in vivo. Lastly, I evaluated whether rs688 associates with AD by genotyping DNA from the Religious Orders Study (ROS) series. The rs688T/T genotype was associated with increased AD odds in males, but not in females, in a dataset consisting of 1,457 men and 2,055 women drawn from three case-control series. The rs688T/T genotype was associated with increased AD odds in males (recessive model, odds ratio (OR) of 1.49, 95% confidence interval (CI) of 1.13- 1.97, uncorrected p=0.005), but not in females. In summary, these studies identify a functional apoE receptor SNP that is associated with AD in a sex-dependent fashion.

Background: The ovarian response to follicle stimulating hormone (FSH) stimulation in assisted conception cycles is variable. Although it would be beneficial to predict accurately the response of patients to FSH, to date no robust predictors of ovarian performance have been identified. Recently, there have been a number of studies on the effect of single nucleotide polymorphisms (SNP) in the FSH receptor gene and its predictive value in the patients undergoing ovarian stimulation. Several reports have shown that two common SNPs at positions 307 and 680 in exon 10 of the FSH receptor gene are associated with ovarian response in in-vitro fertilization (IVF). Some authors have shown predictability of ovarian response to FSH stimulation in patients with different alleles, while others have refuted this finding. Until now, there is no clear clinical benefit in screening FSHR genotypes before IVF treatment. Objective: 1) To study the association between ovarian response and FSHR gene polymorphisms2) To study the association between FSHR gene polymorphisms and markers of ovarian reserve, including Anti Mullerian Hormone, Antral Follicle Count, Follicle Stimulating Hormone.Design: Prospective observational studyMethodology: 421 patients attending a tertiary reproductive medicine unit undergoing first cycle of IVF treatment were recruited into the study. Blood tests were taken on day 2 or 3 of the cycle for assessment of hormones and for DNA extraction. The SNP genotyping was done using Taqman analysis. Non-parametric tests were done to compare the various outcome parameters in patients with different genotypes.Results: FSHR p.Asn680Ser was not predictive of ovarian response. There was no evidence of any difference in basal FSH, AMH or AFC between the patients with different FSHR genotypes, with or without an adjustment for age or BMI. On subgroup analysis, there was no evidence that FSHR p.Asn680Ser genotypes are associated with PCOS, high AMH levels or response to clomiphene citrate. FSHR gene polymorphism was also not related to oocyte maturity or fertilization rate.Conclusions: FSHR p.Asn680Ser was not shown to be predictive of ovarian response, although clinically relevant differences cannot be ruled out. There may be an effect size but smaller than that detected for the power of this study. Other genetic markers may be relevant in the prediction of response to ovarian stimulation.

Gametophytic self-incompatibility limits the ability to derive inbred lines of potato through self-pollination and is prevalent in diploid potato. Within a population of F1 hybrids between two genotypes used in potato genome sequencing, we observed fruit set on many greenhouse-grown plants. Subsequently, after controlled self-pollinations, we confirmed fruit set in 32 of 103 F1 plants. Our goal was to identify genes responsible for self-compatibility in this population and to advance selfed progeny to develop highly homozygous inbred lines. The F1 population was genotyped using a single nucleotide polymorphism (SNP) array. Polymorphic and robust SNPs were analyzed by Fisher\'s Exact Test to identify allelic states segregating with the self-compatible phenotype. Filtering 1966 SNPs to retain only those with p-values less than 0.0001 yielded 95 highly significant SNPs, with all SNPs on anchored scaffolds located on chromosome 12. Candidate genes encoding for multiple notable proteins including an S-protein homologue were identified near highly significant SNPs on the Potato Genome Browser. Seeds obtained after self-pollination of self-compatible individuals were used to advance the population for three generations. SNP chip genotyping of the S3 generation revealed entirely different SNPs segregating for self-compatibility on nine different chromosomes. Comparison of the allelic state of SNPs in the F1 and S3 generations revealed a heterozygosity reduction by 80%, with fixation of many SNPs including those surrounding the S-protein homologue. We conclude that the genes responsible for segregation of self-compatibility in the S3 generation are different from those in the F1 generation. / Master of Science

La sélection génomique substitue à la connaissance de la généalogie celle des séquences d’ADN et connait un succès spectaculaire dans la sélection des bovins laitiers. En équin, le gain de précision pour les valeurs génétiques en CSO a été estimé faible entre la généalogie et la génomique, éventuellement à cause des particularités des populations d’apprentissage et de validation. L’objectif est de définir pour les races équines les conditions d’efficacité et de fonctionnement de la sélection génomique. La partie théorique de la thèse a consisté en une méta-analyse afin de comprendre le lien entre précision théorique et observée en fonction des paramètres des populations. L’étude a montré l’importance du nombre efficace de marqueurs Me. Ce paramètre spécifique de la population, de la structure génomique et de la parenté doit être évalué, au même titre que l’héritabilité en génétique classique. D’un point de vue pratique, la 1ère voie d’amélioration était de rechercher des gènes à effet majeur sur l’aptitude au concours de saut d’obstacles (CSO) ou au concours complet. Aucun gène majeur n’a été localisé malgré des détections significatives. Le 2nd levier pour améliorer l’estimation des valeurs génétiques en CSO était d’utiliser le Single-Step, méthode qui combine l’information génomique des étalons génotypés et la généalogie de l’ensemble des chevaux non génotypés utilisés pour l’indexation. L’évaluation pour le CSO a donc été revisitée. Malgré le re-calcul de l’héritabilité et l’application des points sur toute la période, le gain en précision reste faible. La sélection génomique a également été testée sur des chevaux d’endurance, mais comme pour le CSO les précisions obtenues pour le moment ne sont pas assez élevées pour justifier une utilisation de la sélection génomique. Récemment, un gène majeur agissant sur l’aptitude à trotter (DMRT3) a été identifié. Malgré l’effet très négatif d’un allèle sur la qualification et les performances précoces, le Trotteur français (TF) est polymorphe pour le gène à cause d’un effet positif de ce même allèle sur les performances tardives. La sélection classique et la sélection génomique ont été comparées en incluant ou non dans le modèle un marqueur lié à DMRT3, nous permettant d’identifier la meilleure combinaison de modèle et de méthode à utiliser pour estimer les valeurs génétiques du TF. Enfin, le paramètre Me a été estimé dans les populations de chevaux utilisées au cours de la thèse, et les résultats des évaluations génomiques ont été comparés en fonction de Me et des autres paramètres influant sur la précision de la sélection génomique. Deux nouveaux projets prévoyant de génotyper des chevaux de CSO d’une part et des TF d’autre part devraient permettre respectivement d’améliorer la précision de l’évaluation génomique en CSO et de confirmer l’intérêt de la prise en compte de DMRT3 dans l’évaluation génomique des TF. / Genomic selection uses genotypes information instead of pedigree information for the estimation of breeding values. In dairy cattle, the selection schemes were greatly improved with this method. In horses, a first attempt of genomic selection showed that the evaluation accuracy was not much improved when using genotypes information compared to classic evaluation, possibly because of the structure of the reference and validation populations. The objective of the thesis was to define the theoretical and practical conditions for the use of genomic selection in horses. The theoretical work of the thesis consisted in a meta-analysis to understand the relation between observed and theoretical accuracy depending on the parameters of the population. We proved the importance of the effective number of independent segments in the genome Me. This parameter is specific of the population and of the genomic structure and relationship structure. We recommend to estimate this parameter before genomic evaluation, just like heritability that is estimated before genetic evaluation. Regarding practical tasks of the thesis, the first solution to improve the breeding values estimation for jumping performances was to look for genes having a major effect on performances in jumping competitions and three-day’s events, but no major gene was evidence in spite of significant detections. The 2nd solution was to perform a single-step evaluation. This method combines information from genotyped stallions and from the pedigree of the whole population. Even if the heritability was re-estimated and points distributed to all horses to have a homogeneous criteria, the accuracy of genomic evaluation was not much improved. Genomic selection was also tested on horses running endurance races, but as for jumping the accuracy was not high enough. Recently, a major gene having a huge effect on the ability of horses to trot was evidenced (DMRT3). Even if one allele has a negative effect on qualification and early earnings, French Trotter (FT) is still heterozygote because of a positive effect of this allele on late performances. Genetic and genomic evaluations were compared with or without using in the model a SNP linked to DMRT3 as a fixed effect. This study allowed identifying the best combination of model and method to use for estimation of FT breeding values. Finally, the parameter Me was estimated in the populations of horses used in the thesis. The results of genomic evaluations were compared according to Me and the other parameters having an influence on the accuracy of genomic evaluations. Two new projects will genotype more jumping horses and FT, they should allow to improve the accuracy of genomic evaluation for jumping horses and to acknowledge the interest of using DMRT3 in the genomic evaluation of FT.

Root-knot disease caused by Meloidogyne spp. is the most important nematode disease of peanut. Even though many management strategies have been applied to control this disease on peanut, resistance is the most recommendable. Marker-assisted selection has been used as a useful tool for screening of resistant individuals in segregating populations. However, it requires many laborious steps. Thus, there is a need for PCR - based markers, which are more practical, rapid, and efficient. In this study, we tried to develop a SCAR marker linked to root-knot nematode resistance locus in peanut based on the RFLP marker R2430E. The entire sequence of R2430E was 2217 bp and contained one putative open reading frame (ORF) of 713 nucleotides. Thirteen primers including 5 forward and 8 reverse primers were synthesized to sequence the entireR2430E. Based on the results of BLAST searches, R2430E appeared to encode an AAA ATPase containing von Willebrand factor type A (VWA) domain from Magnetococcus sp. MC-1 (106 bits). To determine if there is a portion of the R2430E that hybridizes only to a band co-segregating with the resistance locus, we generated 4 probes spanning different parts of the gene. Southern analysis using these probes revealed identical banding patterns for each probe. Therefore, we concluded that there is very limited if any sequence polymorphism between different alleles detected by the R2430E probe. Additionally, this conclusion is supported by the experiment in which we tested 25 primer pairs derived from the R2430E using genomic DNA from both resistance and susceptible genotypes. In this experiment, all primer pairs amplified identical PCR fragments, suggesting again that there is little or no sequence divergence between putative alleles as differentiated by southern blotting. To identify possible single nucleotide polymorphisms (SNPs) between polymorphic R2430E RFLP bands, we cloned several fragments that span the entire R2430E transcribed sequence. Surprisingly, no SNPs were identified in the transcribed region of this gene. We propose that polymorphism detected by this RFLP marker is outside of the R2430E.

<p>There is a continuous development of different crop varieties in the crop trade. The cultivated crops tend to be more and more alike which require an effective method for crop identification. Crop type and crop type purity has become a quality measure in crop trade both nationally and internationally. A number of well known quality attributes of interest in the crop trade can be correlated to the specific crop type and therefore it is of great importance to reliably be able to identify different crop varieties. It is well known from the literature that there exist genomic variations at the nucleotide level between different crop varieties and these variations might potentially be useful for automated variety identification.</p><p>This project deals with the crop variety identification area where the possibilities of distinguishing between different wheat varieties are investigated. Experience from performing wheat variety identification at protein level has shown unsatisfactory results and therefore DNA-based techniques are proposed instead. DNA-based techniques are dependent upon the availability of sequence data from the wheat genome and some work has concerned examining the availability of sequence data from wheat. But the focus of the work has been on defining a method for computational detection of single nucleotide variations in ESTs from wheat and to experimentally test that method. Results from these experiments show that the method defined in this project detects polymorphic variations that can be correlated to variety variations</p>

There is a continuous development of different crop varieties in the crop trade. The cultivated crops tend to be more and more alike which require an effective method for crop identification. Crop type and crop type purity has become a quality measure in crop trade both nationally and internationally. A number of well known quality attributes of interest in the crop trade can be correlated to the specific crop type and therefore it is of great importance to reliably be able to identify different crop varieties. It is well known from the literature that there exist genomic variations at the nucleotide level between different crop varieties and these variations might potentially be useful for automated variety identification. This project deals with the crop variety identification area where the possibilities of distinguishing between different wheat varieties are investigated. Experience from performing wheat variety identification at protein level has shown unsatisfactory results and therefore DNA-based techniques are proposed instead. DNA-based techniques are dependent upon the availability of sequence data from the wheat genome and some work has concerned examining the availability of sequence data from wheat. But the focus of the work has been on defining a method for computational detection of single nucleotide variations in ESTs from wheat and to experimentally test that method. Results from these experiments show that the method defined in this project detects polymorphic variations that can be correlated to variety variations

Single nucleotide polymorphism (SNP) arrays are important tools widely used for genotyping and copy number estimation. This technology utilizes the specific affinity of fragmented DNA for binding to surface-attached oligonucleotide DNA probes. This thesis contemplates the variability of the probe signals of Affymetrix GeneChip SNP arrays as a function of the probe sequence to identify relevant sequence motifs which potentially cause systematic biases of genotyping and copy number estimates.

Indiana University-Purdue University Indianapolis (IUPUI) / Externally visible characteristics (EVCs) predicted from an unknown sample of DNA are particularly useful in forensics as they can provide information beyond that of an STR profile. Current EVCs which are highly studied and well-predicted include iris, hair, and skin color. Notably, models predicting iris color, such as IrisPlex, are the most accurate with up to ~95% accuracy; however, some inaccurate predictions occur, as is evidenced by the ~5%. Often, these are due to green or hazel eyes, which are frequently viewed as intermediate. Though, some of the inaccurate predictions are due to true-blue being predicted as brown and vice versa. Previous research has theorized the possibility of two SNPs, rs12913832 and rs1800407, acting as a functional haplotype affecting iris color. rs12913832 is recognized as the most predictive SNP for iris color and highly significant in other pigmentation phenotypes; presently, rs1800407 is the second-ranked SNP in the IrisPlex 6-SNP system. Both SNPs are highly variable in Europe, where the majority of variation in iris color originates. In the present study, we explore the SNP variation present in the genetic regions of OCA2-HERC2 as well as possible haplotypes. Our research centers around the functional haplotype and the addition of SNPs to the functional haplotype. In addition, three different ways of classifying the phenotype are assessed simultaneously. First, using a 4-point categorical phenotype—blue/blue grey, blue/green yellow, hazel/light brown, and dark brown. Second, calculating a continuous scale from a quantitative phenotype in which the percentage of each categorical color has been measured. Third, using the IrisPlex 6-SNP system to predict eye color and identify individuals which have been inaccurately predicted. Exploration of the SNP and haplotype variation resulted in two SNPs for both the categorical and quantitative phenotypes which were significantly correlated with hazel/light brown—rs1448484 and rs61335644, both as independent SNPs and when assessed in a haplotype with rs1800407-rs12913832. SNP rs1448484 has been associated with skin pigmentation previously and is located in a possible transcription factor binding site. SNP rs61335644 is not presently associated with pigmentation but is in complete LD with two SNPs in and around regulatory regions present in HERC2. Finally, the addition of rs1448484 and rs61335644 into the current IrisPlex 6-SNP system slightly improved each of the tested performance metrics for hazel/light brown and dark brown. Within the inaccurately predicted phenotypes, rs1800407 is confirmed to affect both inaccurately predicted groups and is the most significant SNP. Additionally, rs121918166, a missense variant in OCA2, is the second most significant SNP in true blue predicted as brown. Both SNPs were also the two most significant haplotypes with at least one allele being derived. Therefore, the next steps should include the addition of the functional haplotype and rs121918166 into the current IrisPlex model, and further testing of rs1448484 and rs61335644 on a molecular level. Consequently, the current IrisPlex model should also be reassessed on an independent test set using the 4-point categorical scale rather than the present 3-point scale.

Depuis que Parsons et col. ont découvert en 2008 que le gène de l’isocitrate déhydrogénase 1 (IDH1) est fréquemment muté dans les glioblastomes (12%), de nombreuses équipes ont étudié la prévalence et les caractéristiques des mutations des gènes IDH1 et 2 dans les gliomes.Les mutations du gène IDH1 sont observées dans environ 40% des gliomes. La mutation d’IDH1 la plus fréquentes dans les gliomes (>90% des cas) est la mutation R132H. La fréquence des mutations IDH1 et 2 est inversement corrélée au grade des gliomes (grade II ~80%, III ~50%, and IV ~10%). Les mutations IDH1/2 ont une valeur diagnostique ainsi que pronostique (associées à une meilleure survie). Pendant ce travail de thèse nous avons dans une première partie analysé la distribution de ces mutations IDH1/2 dans les différents gliomes, leur association avec d’autres altérations génétiques, ainsi que leur valeur diagnostique et pronostique dans une cohorte de 1332 patients atteints de gliomes. Nous confirmons sur cette très grande cohorte les données de la littérature et affinons la valeur pronostique des mutations IDH1/2. Dans une seconde partie, nous avons mis en évidence dans les gliomes un polymorphisme (SNP) du gène IDH1 (SNP rs 11554137; C (cytosine) substituted by T (thymin)) précédemment observé dans les leucémies myéloïdes aigues. Ce SNP, codon 105, est localisé dans le même exon que le codon 132 fréquemment muté, et nous avons montré qu’il est associé à une moins bonne survie des patients atteints de gliomes. Les mutations du codon 132 causent une baisse de l’activité enzymatique normale d’IDH1/2 qui est remplacé par le gain d’une nouvelle. Les protéines IDH1/2 mutés, au lieu de produire de l’alpha-cétoglutarate de façon NADP dépendante, réduisent de façon NADPH dépendante l’alpha-cétoglutarate en 2-hydroxyglutarate (2HG). Une forte concentration de 2HG et une baisse de la quantité de NADPH peuvent sensibiliser les tumeurs au stress oxidatif et donc potentialiser l’effet de la radiothérapie, ce qui pourrait expliquer la meilleure survie de ces patients. Nous avons donc dans une troisième partie étudié in vitro l’impact de la mutation IDH1R132H sur la survie après radiothérapie de cellules tumorales exprimant de façon stable ce gène muté. Les résultats obtenus montrent que dans certaines conditions ces cellules pourraient être plus radiosensibles que les mêmes cellules exprimant le gène IDH1 non-muté.Dans ce travail de thèse, nous avons donc étudié le gène IDH1 dans les gliomes de patients et tenté par une approche fonctionnelle in vitro d’évaluer l’impact de la mutation IDH1R132H sur la radiosensibilité des cellules tumorales. / Since Parsons et al. (2008) found the frequent mutations of IDH1 (12%) in GBMs, various reports have studied the prevalence and characteristic of IDH1 and IDH2 mutations.The mutations in the isocitrate dehydrogenase 1 (IDH1) gene occur in nearly 40% of gliomas. The frequency of IDH1 mutations are inversely connected with grade II (~80%), III (~50%), and IV (~ 10%) gliomas. Importantly, the status of IDH1 mutations is associated with a better outcome and demonstrated a diagnostic value. We analyzed also these mutations in distribution, association with tumor-derived other genetic alterations and the diagnostic and prognostic value in a cohort of 1332 glioma patients.A synonymous single nucleotide polymorphism [SNP rs 11554137; C (cytosine) substituted by T (thymin)] has been studied in gliomas patients. The SNP rs 11554137 (in codon 105) are located in the same exon with the IDH1 R132 mutations (in codon 132). And gliomas patients with SNP rs 11554137: C>T had a poorer outcome than patients without SNP rs 11554137. This was observed a similarly adverse effect in survival in patients with AML. Mutations in codon 132 can cause a decrease of IDH1/2 activity and also gain a new enzyme function for the NADPH dependent reduction of alpha-ketoglutarate to 2-hydroxyglutarate. High 2HG and low NADPH levels might sensitize tumors to oxidative stress, potentiating response to radiotherapy, and may account for the prolonged survival of patients harboring the mutations. So we studied further the alterations of function in IDH1R132H mutant cells in vitro. Based on the decrease of defence and the increase of impairing factors in tumor cells, we found that the tumors harbouring IDH1 mutations may have an elevated radiosensitivity. In the present study, we described the impact of IDH1 mutations in gliomas and search for new perspectives for the treatment strategy.