KRAS variation and risk of endometriosis

Endometriosis is a common gynaecological disease with symptoms of pelvic pain and infertility which affects 7-10% of women in their reproductive years. Activation of an oncogenic allele of Kirsten rat sarcoma viral oncogene homologue (KRAS) in the reproductive tract of mice resulted in the development of endometriosis. We hypothesized that variation in KRAS may influence risk of endometriosis in humans. Thirty tagSNPs spanning a region of 60.7 kb across the KRAS locus were genotyped using iPLEX chemistry on a MALDI-TOF MassARRAY platform in 959 endometriosis cases and 959 unrelated controls, and data were analysed for association with endometriosis. Genotypes were obtained for most individuals with a mean completion rate of 99.1%. We identified six haplotype blocks across the KRAS locus in our sample. There were no significant differences between cases and controls in the frequencies of individual single-nucleotide polymorphisms (SNPs) or haplotypes. We also developed a rapid method to screen for 11 common KRAS and BRAF mutations on the Sequenom MassARRAY system. The assay detected all mutations previously identified by direct sequencing in a panel of positive controls. No germline variants for KRAS or BRAF were detected. Our results demonstrate that any risk of endometriosis in women because of common variation in KRAS must be very small.

Replication of association of IL1 gene complex members with ankylosing spondylitis in Taiwanese Chinese

Objective: To test the association of interleukin 1 ( IL1) gene family members with ankylosing spondylitis ( AS), previously reported in Europid subjects, in an ethnically remote population. Methods: 200 Taiwanese Chinese AS patients and 200 ethnically matched healthy controls were genotyped for five single nucleotide polymorphisms ( SNPs) and the IL1RN. VNTR, markers previously associated with AS. Allele, genotype, and haplotype frequencies were compared between cases and controls. Results: Association of alleles and genotypes of the markers IL1F10.3, IL1RN. 4, and IL1RN. VNTR was observed with AS ( p < 0.05). Haplotypes of pairs of these markers and of the markers IL1RN.6/1 and IL1RN.6/2 were also significantly associated with AS. The strongest associations observed were with the marker IL1RN. 4, and with the two-marker haplotype IL1RN.4-IL1RN.VNTR ( both p = 0.004). Strong linkage disequilibrium was observed between all marker pairs except those involving IL1B-511 ( D' 0.4 to 0.9, p < 0.01). Conclusions: The IL1 gene cluster is associated with AS in Taiwanese Chinese. This finding provides strong statistical support that the previously observed association of this gene cluster with AS is a true positive finding.

Genetic analysis of dyslexia candidate genes in the European cross-linguistic NeuroDys cohort

Dyslexia is one of the most common childhood disorders with a prevalence of around 5-10% in school-age children. Although an important genetic component is known to have a role in the aetiology of dyslexia, we are far from understanding the molecular mechanisms leading to the disorder. Several candidate genes have been implicated in dyslexia, including DYX1C1, DCDC2, KIAA0319, and the MRPL19/C2ORF3 locus, each with reports of both positive and no replications. We generated a European cross-linguistic sample of school-age children-the NeuroDys cohort-that includes more than 900 individuals with dyslexia, sampled with homogenous inclusion criteria across eight European countries, and a comparable number of controls. Here, we describe association analysis of the dyslexia candidate genes/locus in the NeuroDys cohort. We performed both case-control and quantitative association analyses of single markers and haplotypes previously reported to be dyslexia-associated. Although we observed association signals in samples from single countries, we did not find any marker or haplotype that was significantly associated with either case-control status or quantitative measurements of word-reading or spelling in the meta-analysis of all eight countries combined. Like in other neurocognitive disorders, our findings underline the need for larger sample sizes to validate possibly weak genetic effects. © 2014 Macmillan Publishers Limited All rights reserved.

Can certain genotypes predispose to poor asthma control in children? A pharmacogenetic study of 9 candidate genes in children with difficult asthma

Objective - We tested the hypothesis that patients with difficult asthma have an increased frequency of certain genotypes that predispose them to asthma exacerbations and poor asthma control. Methods - A total of 180 Caucasian children with confirmed asthma diagnosis were selected from two phenotypic groups; difficult (n = 112) versus mild/moderate asthma (n = 68) groups. All patients were screened for 19 polymorphisms in 9 candidate genes to evaluate their association with difficult asthma. Key Results - The results indicated that LTA4H A-9188>G, TNFα G-308>A and IL-4Rα A1727>G polymorphisms were significantly associated with the development of difficult asthma in paediatric patients (p<0.001, p = 0.019 and p = 0.037, respectively). Haplotype analysis also revealed two haplotypes (ATA haplotype of IL-4Rα A1199>C, IL-4Rα T1570>C and IL-4Rα A1727>G and CA haplotype of TNFα C-863>A and TNFα G-308>A polymorphisms) which were significantly associated with difficult asthma in children (p = 0.04 and p = 0.018, respectively). Conclusions and Clinical Relevance - The study revealed multiple SNPs and haplotypes in LTA4H, TNFα and IL4-Rα genes which constitute risk factors for the development of difficult asthma in children. Of particular interest is the LTA4H A-9188>G polymorphism which has been reported, for the first time, to have strong association with severe asthma in children. Our results suggest that screening for patients with this genetic marker could help characterise the heterogeneity of responses to leukotriene-modifying medications and, hence, facilitate targeting these therapies to the subset of patients who are most likely to gain benefit.

Impact of ATP-binding cassette, subfamily B, member 1 pharmacogenetics on tacrolimus-associated nephrotoxicity and dosage requirements in paediatric patients with liver transplant

Importance of the field: Tacrolimus is the most commonly used immunosuppressive agent following solid-organ transplantation in children. Its clinical use, however, is complicated by side effects (mainly nephrotoxicity), narrow therapeutic index and pharmacokinetic variability which can result in an increased risk of treatment failure or toxicity. Studies examining interindividual differences in the expression of the ABCB1 (ATP-binding cassette, subfamily B, member 1) gene (which encodes the drug transporter, P-gp) and its genetic polymorphisms have attempted to elucidate variations in tacrolimus response and disposition in children. Areas covered in this review: This review explores pharmacogenetic knowledge developed over the last decade regarding the impact of ABCB1 polymorphisms on tacrolimus toxicity and dosage requirements in children. What the reader will gain: A better understanding of the role of ABCB1 genetic polymorphisms (and corresponding haplotypes) and ABCB1 expression levels in various tissues and organs on tacrolimus outcomes in children with liver transplant. Take home message: Pharmacogenetics offers significant potential for optimising tacrolimus use. ABCB1 donor genotypes and ABCB1 expression level in the intestine and leukocytes may be useful in dosage selection. Large prospective studies are, however, required to further explore the potential of genetic testing in identifying children who are at risk of toxicity and to better individualise tacrolimus therapy.

Microvariation at the human D1S80 locus

Microvariant allelic polymorphisms have been known since 1966 when Harris, Hubby and Lewontin described the huge store of genetic variation detectable at the polypeptide level. Later Jeffreys used MVR (minisatellite variant repeat) analysis to describe the variation hidden within minisatellite VNTRs and to propose a mutational mechanism.^ The questions I have asked follow these traditions: (1) How much microvariant polymorphism exists at the discrete allele minisatellite D1S80 locus? (2) Do alleles or groups of alleles associate randomly with the flanking markers to form haplotypes? (3) What mechanisms might explain mutations at this locus? What are the phylogenetic relationships among the alleles?^ The minisatellite locus D1S80 (1p35-36), GenBank sequence (Accession # D28507), is a highly polymorphic Variable Number of Tandem Repeat (VNTR) based on a 16 base core. D1S80 alleles are electrophoretically separable into discontinuous sets of equivalent length alleles. Sequence variation or minor length variation within these classes was expected: I have sought to determine the nature of this microvariant heterogeneity by sequencing nominal and variant alleles.^ Alleles were analyzed by Single-Strand Conformation Polymorphism (SSCP) analysis. Sequences were determined to ascertain whether sequence variation or size variation is the major cause of altered electrophoretic migration of microvariant D1S80 alleles. Twenty three alleles from 14 previously typed individuals were sequenced. The individuals were from African American, Caucasian, or Hispanic databases.^ A Tsp509 I restriction site, previously reported as a Hinf I flanking polymorphism, and a 3$\sp\prime$ flanking region BsoF I restriction site polymorphism were identified. There appears to be a strong association of the 5$\sp\prime$ flanking region Hinf I(+) and Tsp509 I(-) site and the 3$\sp\prime$ flanking region BsoF I(-) site with the 18 allele, while the 24 tends to be associated with the Hinf I(-), Tsp509 I(+) and BsoF I(+) sites.^ The general conclusion for this locus is clearly the closer you look, the more you find. D1S80 allelic polymorphisms are primarily due to variation in the number of repeat units and to sequence variation among repeats. The sequenced based gene tree depicts two major classes of alleles which conform to the two most common alleles, reflecting either equivalent age or population size bottlenecks. ^

Mitochondrial DNA, segments I and II, its applications in population genetics and forensic sciences

The investigations of human mitochondrial DNA (mtDNA) have considerably contributed to human evolution and migration. The Middle East is considered to be an essential geographic area for human migrations out of Africa since it is located at the crossroads of Africa, and the rest of the world. United Arab Emirates (UAE) population inhabits the eastern part of Arabian Peninsula and was investigated in this study. Published data of 18 populations were included in the statistical analysis. The diversity indices showed (1) high genetic distance among African populations and (2) high genetic distance between African populations and non-African populations. Asian populations clustered together in the NJ tree between the African and European populations. MtDNA haplotypes database of the UAE population was generated. By incorporating UAE mtDNA dataset into the existing worldwide mtDNA database, UAE Forensic Laboratories will be able to analyze future mtDNA evidence in a more significant and consistent manner. ^

Molecular markers and human history: A tale of two haplogroup systems

To chronicle demographic movement across African Asian corridors, a variety of molecular (sequence analysis, restriction mapping and denaturing high performance liquid chromatography etc.) and statistical (correspondence analysis, AMOVA, calculation of diversity indices and phylogenetic inference, etc.) techniques were employed to assess the phylogeographic patterns of mtDNA control region and Y chromosomal variation among 14 sub-Saharan, North African and Middle Eastern populations. The patterns of genetic diversity revealed evidence of multiple migrations across several African Asian passageways as well within the African continent itself. The two-part analysis uncovered several interesting results which include the following: (1) a north (Egypt and Middle East Asia) to south (sub-Saharan Africa) partitioning of both mtDNA and Y chromosomal haplogroup diversity, (2) a genetic diversity gradient in sub-Saharan Africa from east to west, (3) evidence in favor of the Levantine Corridor over the Horn of Africa as the major genetic conduit since the Last Glacial Maximum, (4) a substantially higher mtDNA versus Y chromosomal sub-Saharan component in the Middle East collections, (5) a higher representation of East versus West African mtDNA haplotypes in the Arabian Peninsula populations versus no such bias in the Levant groups and lastly, (6) genetic remnants of the Bantu demographic expansion in sub-Saharan Africa. ^

Could Relatedness Help Explain Why Individuals Lead in Bottlenose Dolphin Groups?

In many species, particular individuals consistently lead group travel. While benefits to followers often are relatively obvious, including access to resources, benefits to leaders are often less obvious. This is especially true for species that feed on patchy mobile resources where all group members may locate prey simultaneously and food intake likely decreases with increasing group size. Leaders in highly complex habitats, however, could provide access to foraging resources for less informed relatives, thereby gaining indirect benefits by helping kin. Recently, leadership has been documented in a population of bottlenose dolphins (Tursiops truncatus) where direct benefits to leaders appear unlikely. To test whether leaders could benefit indirectly we examined relatedness between leader-follower pairs and compared these levels to pairs who associated but did not have leader-follower relationship (neither ever led the other). We found the average relatedness value for leader-follower pairs was greater than expected based on chance. The same was not found when examining non leader-follower pairs. Additionally, relatedness for leader-follower pairs was positively correlated with association index values, but no correlation was found for this measure in non leader-follower pairs. Interestingly, haplotypes were not frequently shared between leader-follower pairs (25%). Together, these results suggest that bottlenose dolphin leaders have the opportunity to gain indirect benefits by leading relatives. These findings provide a potential mechanism for the maintenance of leadership in a highly dynamic fission-fusion population with few obvious direct benefits to leaders.

(Table 1) Individual codes, haplotype codes, GenBank accession numbers for DNA sequences and sample locality of the analysed Betamorpha fusiformis specimens from the ANDEEP III expedition

Based on our current knowledge about population genetics, phylogeography and speciation, we begin to understand that the deep sea harbours more species than suggested in the past. Deep-sea soft-sediment environment in particular hosts a diverse and highly endemic invertebrate fauna. Very little is known about evolutionary processes that generate this remarkable species richness, the genetic variability and spatial distribution of deep-sea animals. In this study, phylogeographic patterns and the genetic variability among eight populations of the abundant and widespread deep-sea isopod morphospecies Betamorpha fusiformis [Barnard, K.H., 1920. Contributions to the crustacean fauna of South Africa. 6. Further additions to the list of marine isopods. Annals of the South African Museum 17, 319-438] were examined. A fragment of the mitochondrial 16S rRNA gene of 50 specimens and the complete nuclear 18S rRNA gene of 7 specimens were sequenced. The molecular data reveal high levels of genetic variability of both genes between populations, giving evidence for distinct monophyletic groups of haplotypes with average p-distances ranging from 0.0470 to 0.1440 (d-distances: 0.0592-0.2850) of the 16S rDNA, and 18S rDNA p-distances ranging between 0.0032 and 0.0174 (d-distances: 0.0033-0.0195). Intermediate values are absent. Our results show that widely distributed benthic deep-sea organisms of a homogeneous phenotype can be differentiated into genetically highly divergent populations. Sympatry of some genotypes indicates the existence of cryptic speciation. Flocks of closely related but genetically distinct species probably exist in other widespread benthic deep-sea asellotes and other Peracarida. Based on existing data we hypothesize that many widespread morphospecies are complexes of cryptic biological species (patchwork hypothesis).

Variabilité dans les promotteurs des gènes du métabolisme du glutathion

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Caractérisation génétique d'un effet fondateur du sud-ouest du Québec : une nouvelle forme de dystrophie musculaire des ceintures

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Dissecting the Functional Impacts of Non-Coding Genetic Variation

A large proportion of the variation in traits between individuals can be attributed to variation in the nucleotide sequence of the genome. The most commonly studied traits in human genetics are related to disease and disease susceptibility. Although scientists have identified genetic causes for over 4,000 monogenic diseases, the underlying mechanisms of many highly prevalent multifactorial inheritance disorders such as diabetes, obesity, and cardiovascular disease remain largely unknown. Identifying genetic mechanisms for complex traits has been challenging because most of the variants are located outside of protein-coding regions, and determining the effects of such non-coding variants remains difficult. In this dissertation, I evaluate the hypothesis that such non-coding variants contribute to human traits and diseases by altering the regulation of genes rather than the sequence of those genes. I will specifically focus on studies to determine the functional impacts of genetic variation associated with two related complex traits: gestational hyperglycemia and fetal adiposity. At the genomic locus associated with maternal hyperglycemia, we found that genetic variation in regulatory elements altered the expression of the HKDC1 gene. Furthermore, we demonstrated that HKDC1 phosphorylates glucose in vitro and in vivo, thus demonstrating that HKDC1 is a fifth human hexokinase gene. At the fetal-adiposity associated locus, we identified variants that likely alter VEPH1 expression in preadipocytes during differentiation. To make such studies of regulatory variation high-throughput and routine, we developed POP-STARR, a novel high throughput reporter assay that can empirically measure the effects of regulatory variants directly from patient DNA. By combining targeted genome capture technologies with STARR-seq, we assayed thousands of haplotypes from 760 individuals in a single experiment. We subsequently used POP-STARR to identify three key features of regulatory variants: that regulatory variants typically have weak effects on gene expression; that the effects of regulatory variants are often coordinated with respect to disease-risk, suggesting a general mechanism by which the weak effects can together have phenotypic impact; and that nucleotide transversions have larger impacts on enhancer activity than transitions. Together, the findings presented here demonstrate successful strategies for determining the regulatory mechanisms underlying genetic associations with human traits and diseases, and value of doing so for driving novel biological discovery.

Analysis of expression quantitative trait loci in D. melanogaster

Expression Quantitative Trait Loci (eQTL) analysis allows for the identification of genetic variation associated with variation in gene expression. It is often unclear however, which of the associated variants are causal, and by what mechanism. Integrating functional genomic data with eQTL data can provide insight into the impact of natural variation in the population, and the nature of the transcriptional machinery itself. In this thesis, I integrate functional genomic data with eQTL data derived from both 5’ CAGE and 3’ TagXseq expression assays, in developing embryos. I first use both datasets to analyse the transcription landscape in embryonic D., melanogaster, and then carry out an analysis of sequence motifs associated with transcription factor binding sites, promoters, and 3’ polyadenylation sites. Finally, I integrate functional genomic data, including these novel sequence motifs, to shed light on the mechanisms of gene expression variation in D.,melanogaster. I am able to demonstrate that some variants effecting gene regulation in Drosophila are found within haplotypes which buffer their effects.

Variabilité dans les promotteurs des gènes du métabolisme du glutathion

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.