Allelic expression imbalance, or AEI, is the term given to differences in the expression levels of the two alleles of a gene. AEI has been previously identified in a number of species using various techniques. Here, the genome-wide extent of allelic expression imbalance in the pathogenic yeast species, Candida albicans, was examined through use of RNA sequencing in combination with a novel computational pipeline based around the diploid reference genome. Techniques for validating these results were investigated, and the difficulties surrounding specificity and quantification are discussed. As C. albicans is a highly heterozygous species, it was hypothesised that polymorphisms within alleles lead to differences in allele expression, which are further linked to differences in allele function. The functional consequences of AEI were therefore interrogated through investigation of Gene Ontology, identification of condition specific responses in AEI, and targeted construction and phenotypic screening of heterozygous knockout strains. Together, these results strongly suggest that divergence in allele expression is not linked to differences in allele function. Investigations of the possible control mechanisms behind the differences in allele expression were considered, with a focus upon structural factors such as chromosomal location, GC content, allele length and codon usage. However, issues with establishing causality are present, and difficulties lie in distinguishing between functional differences and consequences of bias in sequencing technologies. This piece of research has advanced the understanding of gene expression mechanisms within a medically important pathogen, paving the way for further investigations into the functional consequences of allelic expression imbalance in Candida albicans.

Polymorphisms in the gene encoding SORL1, involved in cellular trafficking of APP, have been implicated in late-onset Alzheimer's disease, by a mechanism thought to affect mRNA expression. To search for regulatory polymorphisms, we have measured allele-specific mRNA expression of SORL1 in human autopsy tissues from the prefrontal cortex of 26 Alzheimer's patients, and 51 controls, using two synonymous marker SNPs (rs3824968 in exon 34 (11 heterozygous AD subjects and 16 controls), and rs12364988 in exon 6 (8 heterozygous AD subjects)). Significant allelic expression imbalance (AEI), indicative of the presence of cis-acting regulatory factors, was detected in a single control subject, while allelic ratios were near unity for all other subjects. We genotyped 7 SNPs in two haplotype blocks that had previously been implicated in Alzheimer's disease. Since each of these SNPs was heterozygous in several subjects lacking AEI, this study fails to support a regulatory role for SORL1 polymorphisms in mRNA expression.

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Recent Genome Wide Association Studies (GWAS) have identified a series of single nucleotide polymorphism (SNP)s that are associated with Alzheimer’s disease (AD). One of the SNPs, rs3851179 (G/A), is near the gene phosphatidylinositol-binding clathrin assembly protein (PICALM). To evaluate whether this SNP is associated with PICALM expression, we quantified PICALM mRNA in 56 brain cDNA samples. Using linear regression analysis, we analyzed PICALM expression relative to rs3851179, AD status, and cell type specific markers. An association was detected between rs3851179 and PICALM, microvessel mRNA, glial fibrillary acidic protein (GFAP) mRNA, and synaptophysin (SYN) mRNA. To gain clarity into other possible SNP mechanisms, we searched brain cDNA for PICALM splice variants. We identified several PICALM splice variants involving exons 13-19. To identify and gain an estimation of relative abundance of splice variants, we PCR-amplified across exons 13-20 in cDNA from six individuals, three rs3851179 GG individuals and three rs3851179 AA individuals. Sequencing the cloned isoforms we found that PICALM lacking exon 13 (delta 13) is the most abundant isoform. Other isoforms detected included deletion of exon 18-19. We targeted the latter part of the gene, exon 17-20, to investigate unequal allelic expression using next generation sequencing. Individuals heterozygous for rs76719109 (n= 35), located in exon 17, were used to study the abundance of G/T allele in cDNA and genomic DNA. When we analyzed the T:G allelic ratio, the variant lacking exons 18 and 19 showed unequal allelic expression (p-value < 0.001) in a subset of individuals. One individual was an outlier, showing overall unequal allelic expression, which maybe be harboring a rare mutation capable of modifying PICALM expression. The PICALM intronic SNP rs588076 was associated with delta 18-19 isoform splicing (p-value < 0.001). In conclusion, this study gained a greater insight into the role of AD genetics in PICALM expression and splicing.

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