Multivariate Prediction Models for Bio-Analytical Data

Quantitative bio-analytical techniques that enable parallel measurements of large numbers of biomolecules generate vast amounts of information for studying and characterising biological systems. These analytical methods are commonly referred to as omics technologies, and can be applied for measurements of e.g. mRNA transcript, protein or metabolite abundances in a biological sample. The work presented in this thesis focuses on the application of multivariate prediction models for modelling and analysis of biological data generated by omics technologies. Omics data commonly contain up to tens of thousands of variables, which are often both noisy and multicollinear. Multivariate statistical methods have previously been shown to be valuable for visualisation and predictive modelling of biological and chemical data with similar properties to omics data. In this thesis currently available multivariate modelling methods are used in new applications, and new methods are developed to address some of the specific challenges associated with modelling of biological data. Three closely related areas of multivariate modelling of biological data are described and demonstrated in this thesis. First, a multivariate projection method is used in a novel application for predictive modelling between omics data sets, demonstrating how data from two analytical sources can be integrated and modelled to- gether by exploring covariation patterns between the data sets. This approach is exemplified by modelling of data from two studies, the first containing proteomic and metabolic profiling data and the second containing transcriptomic and metabolic profiling data. Second, a method for piecewise multivariate modelling of short timeseries data is developed and demonstrated by modelling of simulated data as well as metabolic profiling data from a toxicity study, providing a new method for characterisation of multivariate bio-analytical time-series data. Third, a kernel-based method is developed and applied for non-linear multivariate prediction modelling of omics data, addressing the specific challenge of modelling non-linear variation in biological data

Intra-tumor heterogeneity in breast cancer has limited impact on transcriptomic-based molecular profiling

Background: Transcriptomic profiling of breast tumors provides opportunity for subtyping and molecular-based patient stratification. In diagnostic applications the specimen profiled should be representative of the expression profile of the whole tumor and ideally capture properties of the most aggressive part of the tumor. However, breast cancers commonly exhibit intra-tumor heterogeneity at molecular, genomic and in phenotypic level, which can arise during tumor evolution. Currently it is not established to what extent a random sampling approach may influence molecular breast cancer diagnostics. Methods: In this study we applied RNA-sequencing to quantify gene expression in 43 pieces (2-5 pieces per tumor) from 12 breast tumors (Cohort 1). We determined molecular subtype and transcriptomic grade for all tumor pieces and analysed to what extent pieces originating from the same tumors are concordant or discordant with each other. Additionally, we validated our finding in an independent cohort consisting of 19 pieces (2-6 pieces per tumor) from 6 breast tumors (Cohort 2) profiled using microarray technique. Exome sequencing was also performed on this cohort, to investigate the extent of intra-tumor genomic heterogeneity versus the intra-tumor molecular subtype classifications. Results: Molecular subtyping was consistent in 11 out of 12 tumors and transcriptomic grade assignments were consistent in 11 out of 12 tumors as well. Molecular subtype predictions revealed consistent subtypes in four out of six patients in this cohort 2. Interestingly, we observed extensive intra-tumor genomic heterogeneity in these tumor pieces but not in their molecular subtype classifications. Conclusions: Our results suggest that macroscopic intra-tumoral transcriptomic heterogeneity is limited and unlikely to have an impact on molecular diagnostics for most patients.Peer reviewe

Extent, causes, and consequences of small RNA expression variation in human adipose tissue.

Small RNAs are functional molecules that modulate mRNA transcripts and have been implicated in the aetiology of several common diseases. However, little is known about the extent of their variability within the human population. Here, we characterise the extent, causes, and effects of naturally occurring variation in expression and sequence of small RNAs from adipose tissue in relation to genotype, gene expression, and metabolic traits in the MuTHER reference cohort. We profiled the expression of 15 to 30 base pair RNA molecules in subcutaneous adipose tissue from 131 individuals using high-throughput sequencing, and quantified levels of 591 microRNAs and small nucleolar RNAs. We identified three genetic variants and three RNA editing events. Highly expressed small RNAs are more conserved within mammals than average, as are those with highly variable expression. We identified 14 genetic loci significantly associated with nearby small RNA expression levels, seven of which also regulate an mRNA transcript level in the same region. In addition, these loci are enriched for variants significant in genome-wide association studies for body mass index. Contrary to expectation, we found no evidence for negative correlation between expression level of a microRNA and its target mRNAs. Trunk fat mass, body mass index, and fasting insulin were associated with more than twenty small RNA expression levels each, while fasting glucose had no significant associations. This study highlights the similar genetic complexity and shared genetic control of small RNA and mRNA transcripts, and gives a quantitative picture of small RNA expression variation in the human population

Human metabolic profiles are stably controlled by genetic and environmental variation

A comprehensive variation map of the human metabolome identifies genetic and stable-environmental sources as major drivers of metabolite concentrations. The data suggest that sample sizes of a few thousand are sufficient to detect metabolite biomarkers predictive of disease

A Genome-Wide Metabolic QTL Analysis in Europeans Implicates Two Loci Shaped by Recent Positive Selection

We have performed a metabolite quantitative trait locus (mQTL) study of the 1H nuclear magnetic resonance spectroscopy (1H NMR) metabolome in humans, building on recent targeted knowledge of genetic drivers of metabolic regulation. Urine and plasma samples were collected from two cohorts of individuals of European descent, with one cohort comprised of female twins donating samples longitudinally. Sample metabolite concentrations were quantified by 1H NMR and tested for association with genome-wide single-nucleotide polymorphisms (SNPs). Four metabolites' concentrations exhibited significant, replicable association with SNP variation (8.6×10−11<p<2.8×10−23). Three of these—trimethylamine, 3-amino-isobutyrate, and an N-acetylated compound—were measured in urine. The other—dimethylamine—was measured in plasma. Trimethylamine and dimethylamine mapped to a single genetic region (hence we report a total of three implicated genomic regions). Two of the three hit regions lie within haplotype blocks (at 2p13.1 and 10q24.2) that carry the genetic signature of strong, recent, positive selection in European populations. Genes NAT8 and PYROXD2, both with relatively uncharacterized functional roles, are good candidates for mediating the corresponding mQTL associations. The study's longitudinal twin design allowed detailed variance-components analysis of the sources of population variation in metabolite levels. The mQTLs explained 40%–64% of biological population variation in the corresponding metabolites' concentrations. These effect sizes are stronger than those reported in a recent, targeted mQTL study of metabolites in serum using the targeted-metabolomics Biocrates platform. By re-analysing our plasma samples using the Biocrates platform, we replicated the mQTL findings of the previous study and discovered a previously uncharacterized yet substantial familial component of variation in metabolite levels in addition to the heritability contribution from the corresponding mQTL effects

A Genome-Wide Metabolic QTL Analysis in Europeans Implicates Two Loci Shaped by Recent Positive Selection

We have performed a metabolite quantitative trait locus (mQTL) study of the 1H nuclear magnetic resonance spectroscopy (1H NMR) metabolome in humans, building on recent targeted knowledge of genetic drivers of metabolic regulation. Urine and plasma samples were collected from two cohorts of individuals of European descent, with one cohort comprised of female twins donating samples longitudinally. Sample metabolite concentrations were quantified by 1H NMR and tested for association with genome-wide single-nucleotide polymorphisms (SNPs). Four metabolites' concentrations exhibited significant, replicable association with SNP variation (8.6×10−11<p<2.8×10−23). Three of these—trimethylamine, 3-amino-isobutyrate, and an N-acetylated compound—were measured in urine. The other—dimethylamine—was measured in plasma. Trimethylamine and dimethylamine mapped to a single genetic region (hence we report a total of three implicated genomic regions). Two of the three hit regions lie within haplotype blocks (at 2p13.1 and 10q24.2) that carry the genetic signature of strong, recent, positive selection in European populations. Genes NAT8 and PYROXD2, both with relatively uncharacterized functional roles, are good candidates for mediating the corresponding mQTL associations. The study's longitudinal twin design allowed detailed variance-components analysis of the sources of population variation in metabolite levels. The mQTLs explained 40%–64% of biological population variation in the corresponding metabolites' concentrations. These effect sizes are stronger than those reported in a recent, targeted mQTL study of metabolites in serum using the targeted-metabolomics Biocrates platform. By re-analysing our plasma samples using the Biocrates platform, we replicated the mQTL findings of the previous study and discovered a previously uncharacterized yet substantial familial component of variation in metabolite levels in addition to the heritability contribution from the corresponding mQTL effects

Multivariate prediction models for bio-analytical data

Quantitative bio-analytical techniques that enable parallel measurements of large numbers of biomolecules generate vast amounts of information for studying and characterising biological systems. These analytical methods are commonly referred to as omics technologies, and can be applied for measurements of e.g. mRNA transcript, protein or metabolite abundances in a biological sample. The work presented in this thesis focuses on the application of multivariate prediction models for modelling and analysis of biological data generated by omics technologies. Omics data commonly contain up to tens of thousands of variables, which are often both noisy and multicollinear. Multivariate statistical methods have previously been shown to be valuable for visualisation and predictive modelling of biological and chemical data with similar properties to omics data. In this thesis currently available multivariate modelling methods are used in new applications, and new methods are developed to address some of the specific challenges associated with modelling of biological data. Three closely related areas of multivariate modelling of biological data are described and demonstrated in this thesis. First, a multivariate projection method is used in a novel application for predictive modelling between omics data sets, demonstrating how data from two analytical sources can be integrated and modelled to- gether by exploring covariation patterns between the data sets. This approach is exemplified by modelling of data from two studies, the first containing proteomic and metabolic profiling data and the second containing transcriptomic and metabolic profiling data. Second, a method for piecewise multivariate modelling of short timeseries data is developed and demonstrated by modelling of simulated data as well as metabolic profiling data from a toxicity study, providing a new method for characterisation of multivariate bio-analytical time-series data. Third, a kernel-based method is developed and applied for non-linear multivariate prediction modelling of omics data, addressing the specific challenge of modelling non-linear variation in biological data.EThOS - Electronic Theses Online ServiceMETAGRADGBUnited Kingdo

Biological and therapeutic implications of a unique subtype of NPM1 mutated AML

In acute myeloid leukemia (AML), molecular heterogeneity across patients constitutes a major challenge for prognosis and therapy. AML with NPM1 mutation is a distinct genetic entity in the revised World Health Organization classification. However, differing patterns of co-mutation and response to therapy within this group necessitate further stratification. Here we report two distinct subtypes within NPM1 mutated AML patients, which we label as primitive and committed based on the respective presence or absence of a stem cell signature. Using gene expression (RNA-seq), epigenomic (ATAC-seq) and immunophenotyping (CyToF) analysis, we associate each subtype with specific molecular characteristics, disease differentiation state and patient survival. Using ex vivo drug sensitivity profiling, we show a differential drug response of the subtypes to specific kinase inhibitors, irrespective of the FLT3-ITD status. Differential drug responses of the primitive and committed subtype are validated in an independent AML cohort. Our results highlight heterogeneity among NPM1 mutated AML patient samples based on stemness and suggest that the addition of kinase inhibitors to the treatment of cases with the primitive signature, lacking FLT3-ITD, could have therapeutic benefit. Molecular heterogeneity of acute myeloid leukaemia (AML) across patients is a major challenge for prognosis and therapy. Here, the authors show that NPM1 mutated AML is a heterogeneous class, consisting of two subtypes which exhibit distinct molecular characteristics, differentiation state, patient survival and drug response