Differential allelic representation (DAR) identifies candidate eQTLs and improves transcriptome analysis.

In comparisons between mutant and wild-type genotypes, transcriptome analysis can reveal the direct impacts of a mutation, together with the homeostatic responses of the biological system. Recent studies have highlighted that, when the effects of homozygosity for recessive mutations are studied in non-isogenic backgrounds, genes located proximal to the mutation on the same chromosome often appear over-represented among those genes identified as differentially expressed (DE). One hypothesis suggests that DE genes chromosomally linked to a mutation may not reflect functional responses to the mutation but, instead, result from an unequal distribution of expression quantitative trait loci (eQTLs) between sample groups of mutant or wild-type genotypes. This is problematic because eQTL expression differences are difficult to distinguish from genes that are DE due to functional responses to a mutation. Here we show that chromosomally co-located differentially expressed genes (CC-DEGs) are also observed in analyses of dominant mutations in heterozygotes. We define a method and a metric to quantify, in RNA-sequencing data, localised differential allelic representation (DAR) between those sample groups subjected to differential expression analysis. We show how the DAR metric can predict regions prone to eQTL-driven differential expression, and how it can improve functional enrichment analyses through gene exclusion or weighting-based approaches. Advantageously, this improved ability to identify probable eQTLs also reveals examples of CC-DEGs that are likely to be functionally related to a mutant phenotype. This supports a long-standing prediction that selection for advantageous linkage disequilibrium influences chromosome evolution. By comparing the genomes of zebrafish (Danio rerio) and medaka (Oryzias latipes), a teleost with a conserved ancestral karyotype, we find possible examples of chromosomal aggregation of CC-DEGs during evolution of the zebrafish lineage. Our method for DAR analysis requires only RNA-sequencing data, facilitating its application across new and existing datasets.

The Use of Zebrafish in Transcriptome Analysis of the Early Effects of Mutations Causing Early Onset Familial Alzheimer's Disease and Other Inherited Neurodegenerative Conditions.

The degree to which non-human animals can be used to model Alzheimer's disease is a contentious issue, particularly as there is still widespread disagreement regarding the pathogenesis of this neurodegenerative dementia. The currently popular transgenic models are based on artificial expression of genes mutated in early onset forms of familial Alzheimer's disease (EOfAD). Uncertainty regarding the veracity of these models led us to focus on heterozygous, single mutations of endogenous genes (knock-in models) as these most closely resemble the genetic state of humans with EOfAD, and so incorporate the fewest assumptions regarding pathological mechanism. We have generated a number of lines of zebrafish bearing EOfAD-like and non-EOfAD-like mutations in genes equivalent to human PSEN1, PSEN2, and SORL1. To analyze the young adult brain transcriptomes of these mutants, we exploited the ability of zebrafish to produce very large families of simultaneous siblings composed of a variety of genotypes and raised in a uniform environment. This "intra-family" analysis strategy greatly reduced genetic and environmental "noise" thereby allowing detection of subtle changes in gene sets after bulk RNA sequencing of entire brains. Changes to oxidative phosphorylation were predicted for all EOfAD-like mutations in the three genes studied. Here we describe some of the analytical lessons learned in our program combining zebrafish genome editing with transcriptomics to understand the molecular pathologies of neurodegenerative disease.

pHAPE: a plasmid for production of DNA size marker ladders for gel electrophoresis.

DNA size markers (also known as 'molecular weight markers' or 'DNA ladders') are an essential tool when using gel electrophoresis to identify and purify nucleic acids. However, the cost of these DNA ladders is not insignificant and, over time, impinges on the funds available for research and training in molecular biology. Here, we describe a method for the generation of 'pHAPE', a plasmid from which a variety of DNA ladders can be generated via simple restriction enzyme digestions. The pHAPE plasmid can be generated by mutagenesis of the commonly used pBluescript II SK+ phagemid followed by insertion of a 7141 bp sequence (comprised of three smaller, synthetic fragments). Our use of pHAPE allows us some small relief from the ever-rising costs of performing molecular biology experiments ('Don't worry, pHAPE').

Differential allelic representation (DAR) identifies candidate eQTLs and improves transcriptome analysis.

In comparisons between mutant and wild-type genotypes, transcriptome analysis can reveal the direct impacts of a mutation, together with the homeostatic responses of the biological system. Recent studies have highlighted that, when homozygous mutations are studied in non-isogenic backgrounds, genes from the same chromosome as a mutation often appear over-represented among differentially expressed (DE) genes. One hypothesis suggests that DE genes chromosomally linked to a mutation may not reflect true biological responses to the mutation but, instead, result from differences in representation of expression quantitative trait loci (eQTLs) between sample groups selected on the basis of mutant or wild-type genotype. This is problematic when inclusion of spurious DE genes in a functional enrichment study results in incorrect inferences of mutation effect. Here we show that chromosomally co-located differentially expressed genes (CC-DEGs) can also be observed in analyses of dominant mutations in heterozygotes. We define a method and a metric to quantify, in RNA-sequencing data, localised differential allelic representation (DAR) between groups of samples subject to differential expression analysis. We show how the DAR metric can predict regions prone to eQTL-driven differential expression, and how it can improve functional enrichment analyses through gene exclusion or weighting of gene-level rankings. Advantageously, this improved ability to identify probable eQTLs also reveals examples of CC-DEGs that are likely to be functionally related to a mutant phenotype. This supports a long-standing prediction that selection for advantageous linkage disequilibrium influences chromosome evolution. By comparing the genomes of zebrafish (Danio rerio) and medaka (Oryzias latipes), a teleost with a conserved ancestral karyotype, we find possible examples of chromosomal aggregation of CC-DEGs during evolution of the zebrafish lineage. The DAR metric provides a solid foundation for addressing the eQTL issue in new and existing datasets because it relies solely on RNA-sequencing data.

Brain transcriptomes of zebrafish and mouse Alzheimer's disease knock-in models imply early disrupted energy metabolism.

Energy production is the most fundamentally important cellular activity supporting all other functions, particularly in highly active organs, such as brains. Here, we summarise transcriptome analyses of young adult (pre-disease) brains from a collection of 11 early-onset familial Alzheimer's disease (EOFAD)-like and non-EOFAD-like mutations in three zebrafish genes. The one cellular activity consistently predicted as affected by only the EOFAD-like mutations is oxidative phosphorylation, which produces most of the energy of the brain. All the mutations were predicted to affect protein synthesis. We extended our analysis to knock-in mouse models of APOE alleles and found the same effect for the late onset Alzheimer's disease risk allele ε4. Our results support a common molecular basis for the initiation of the pathological processes leading to both early and late onset forms of Alzheimer's disease, and illustrate the utility of zebrafish and knock-in single EOFAD mutation models for understanding the causes of this disease.

In-Frame and Frameshift Mutations in Zebrafish Presenilin 2 Affect Different Cellular Functions in Young Adult Brains.

BACKGROUND: Mutations in PRESENILIN 2 (PSEN2) cause early onset familial Alzheimer's disease (EOfAD) but their mode of action remains elusive. One consistent observation for all PRESENILIN gene mutations causing EOfAD is that a transcript is produced with a reading frame terminated by the normal stop codon-the "reading frame preservation rule". Mutations that do not obey this rule do not cause the disease. The reasons for this are debated. OBJECTIVE: To predict cellular functions affected by heterozygosity for a frameshift, or a reading frame-preserving mutation in zebrafish psen2 using bioinformatic techniques. METHODS: A frameshift mutation (psen2 N140fs ) and a reading frame-preserving (in-frame) mutation (psen2 T141 _ L142delinsMISLISV ) were previously isolated during genome editing directed at the N140 codon of zebrafish psen2 (equivalent to N141 of human PSEN2). We mated a pair of fish heterozygous for each mutation to generate a family of siblings including wild type and heterozygous mutant genotypes. Transcriptomes from young adult (6 months) brains of these genotypes were analyzed. RESULTS: The in-frame mutation uniquely caused subtle, but statistically significant, changes to expression of genes involved in oxidative phosphorylation, long-term potentiation and the cell cycle. The frameshift mutation uniquely affected genes involved in Notch and MAPK signaling, extracellular matrix receptor interactions and focal adhesion. Both mutations affected ribosomal protein gene expression but in opposite directions. CONCLUSION: A frameshift and an in-frame mutation at the same position in zebrafish psen2 cause discrete effects. Changes in oxidative phosphorylation, long-term potentiation and the cell cycle may promote EOfAD pathogenesis in humans.

Zebrafish Chromosome 14 Gene Differential Expression in the fmr1 h u2787 Model of Fragile X Syndrome.

Zebrafish represent a valuable model for investigating the molecular and cellular basis of Fragile X syndrome (FXS). Reduced expression of the zebrafish FMR1 orthologous gene, fmr1, causes developmental and behavioural phenotypes related to FXS. Zebrafish homozygous for the hu2787 non-sense mutation allele of fmr1 are widely used to model FXS, although FXS-relevant phenotypes seen from morpholino antisense oligonucleotide (morpholino) suppression of fmr1 transcript translation were not observed when hu2787 was first described. The subsequent discovery of transcriptional adaptation (a form of genetic compensation), whereby mutations causing non-sense-mediated decay of transcripts can drive compensatory upregulation of homologous transcripts independent of protein feedback loops, suggested an explanation for the differences reported. We examined the whole-embryo transcriptome effects of homozygosity for fmr1 h u2787 at 2 days post fertilisation. We observed statistically significant changes in expression of a number of gene transcripts, but none from genes showing sequence homology to fmr1. Enrichment testing of differentially expressed genes implied effects on lysosome function and glycosphingolipid biosynthesis. The majority of the differentially expressed genes are located, like fmr1, on Chromosome 14. Quantitative PCR tests did not support that this was artefactual due to changes in relative chromosome abundance. Enrichment testing of the "leading edge" differentially expressed genes from Chromosome 14 revealed that their co-location on this chromosome may be associated with roles in brain development and function. The differential expression of functionally related genes due to mutation of fmr1, and located on the same chromosome as fmr1, is consistent with R.A. Fisher's assertion that the selective advantage of co-segregation of particular combinations of alleles of genes will favour, during evolution, chromosomal rearrangements that place them in linkage disequilibrium on the same chromosome. However, we cannot exclude that the apparent differential expression of genes on Chromosome 14 genes was, (if only in part), caused by differences between the expression of alleles of genes unrelated to the effects of the fmr1 h u2787 mutation and made manifest due to the limited, but non-zero, allelic diversity between the genotypes compared.

PRESENILIN 1 Mutations Causing Early-Onset Familial Alzheimer's Disease or Familial Acne Inversa Differ in Their Effects on Genes Facilitating Energy Metabolism and Signal Transduction.

BACKGROUND: The most common cause of early-onset familial Alzheimer's disease (EOfAD) is mutations in PRESENILIN 1 (PSEN1) allowing production of mRNAs encoding full-length, but mutant, proteins. In contrast, a single known frameshift mutation in PSEN1 causes familial acne inversa (fAI) without EOfAD. The molecular consequences of heterozygosity for these mutation types, and how they cause completely different diseases, remains largely unexplored. OBJECTIVE: To analyze brain transcriptomes of young adult zebrafish to identify similarities and differences in the effects of heterozygosity for psen1 mutations causing EOfAD or fAI. METHODS: RNA sequencing was performed on mRNA isolated from the brains of a single family of 6-month-old zebrafish siblings either wild type or possessing a single, heterozygous EOfAD-like or fAI-like mutation in their endogenous psen1 gene. RESULTS: Both mutations downregulate genes encoding ribosomal subunits, and upregulate genes involved in inflammation. Genes involved in energy metabolism appeared significantly affected only by the EOfAD-like mutation, while genes involved in Notch, Wnt and neurotrophin signaling pathways appeared significantly affected only by the fAI-like mutation. However, investigation of direct transcriptional targets of Notch signaling revealed possible increases in γ-secretase activity due to heterozygosity for either psen1 mutation. Transcriptional adaptation due to the fAI-like frameshift mutation was evident. CONCLUSION: We observed both similar and contrasting effects on brain transcriptomes of the heterozygous EOfAD-like and fAI-like mutations. The contrasting effects may illuminate how these mutation types cause distinct diseases.

Loss of park7 activity has differential effects on expression of iron responsive element (IRE) gene sets in the brain transcriptome in a zebrafish model of Parkinson's disease.

Mutation of the gene PARK7 (DJ1) causes monogenic autosomal recessive Parkinson's disease (PD) in humans. Subsequent alterations of PARK7 protein function lead to mitochondrial dysfunction, a major element in PD pathology. Homozygous mutants for the PARK7-orthologous genes in zebrafish, park7, show changes to gene expression in the oxidative phosphorylation pathway, supporting that disruption of energy production is a key feature of neurodegeneration in PD. Iron is critical for normal mitochondrial function, and we have previously used bioinformatic analysis of IRE-bearing transcripts in brain transcriptomes to find evidence supporting the existence of iron dyshomeostasis in Alzheimer's disease. Here, we analysed IRE-bearing transcripts in the transcriptome data from homozygous park7-/- mutant zebrafish brains. We found that the set of genes with "high quality" IREs in their 5' untranslated regions (UTRs, the HQ5'IRE gene set) was significantly altered in these 4-month-old park7-/- brains. However, sets of genes with IREs in their 3' UTRs appeared unaffected. The effects on HQ5'IRE genes are possibly driven by iron dyshomeostasis and/or oxidative stress, but illuminate the existence of currently unknown mechanisms with differential overall effects on 5' and 3' IREs.

Transcriptome analyses of 7-day-old zebrafish larvae possessing a familial Alzheimer's disease-like mutation in psen1 indicate effects on oxidative phosphorylation, ECM and MCM functions, and iron homeostasis.

BACKGROUND: Early-onset familial Alzheimer's disease (EOfAD) is promoted by dominant mutations, enabling the study of Alzheimer's disease (AD) pathogenic mechanisms through generation of EOfAD-like mutations in animal models. In a previous study, we generated an EOfAD-like mutation, psen1Q96_K97del, in zebrafish and performed transcriptome analysis comparing entire brains from 6-month-old wild type and heterozygous mutant fish. We identified predicted effects on mitochondrial function and endolysosomal acidification. Here we aimed to determine whether similar effects occur in 7 day post fertilization (dpf) zebrafish larvae that might be exploited in screening of chemical libraries to find ameliorative drugs. RESULTS: We generated clutches of wild type and heterozygous psen1Q96_K97del 7 dpf larvae using a paired-mating strategy to reduce extraneous genetic variation before performing a comparative transcriptome analysis. We identified 228 differentially expressed genes and performed various bioinformatics analyses to predict cellular functions. CONCLUSIONS: Our analyses predicted a significant effect on oxidative phosphorylation, consistent with our earlier observations of predicted effects on ATP synthesis in adult heterozygous psen1Q96_K97del brains. The dysregulation of minichromosome maintenance protein complex (MCM) genes strongly contributed to predicted effects on DNA replication and the cell cycle and may explain earlier observations of genome instability due to PSEN1 mutation. The upregulation of crystallin gene expression may be a response to defective activity of mutant Psen1 protein in endolysosomal acidification. Genes related to extracellular matrix (ECM) were downregulated, consistent with previous studies of EOfAD mutant iPSC neurons and postmortem late onset AD brains. Also, changes in expression of genes controlling iron ion transport were observed without identifiable changes in the prevalence of transcripts containing iron responsive elements (IREs) in their 3' untranslated regions (UTRs). These changes may, therefore, predispose to the apparent iron dyshomeostasis previously observed in 6-month-old heterozygous psen1Q96_K97del EOfAD-like mutant brains.

Brain Transcriptome Analysis of a Protein-Truncating Mutation in Sortilin-Related Receptor 1 Associated With Early-Onset Familial Alzheimer's Disease Indicates Early Effects on Mitochondrial and Ribosome Function.

BACKGROUND: The early cellular stresses leading to Alzheimer's disease (AD) remain poorly understood because we cannot access living, asymptomatic human AD brains for detailed molecular analyses. Sortilin-related receptor 1 (SORL1) encodes a multi-domain receptor protein genetically associated with both rare, early-onset familial AD (EOfAD) and common, sporadic, late-onset AD (LOAD). SORL1 protein has been shown to act in the trafficking of the amyloid ß A4 precursor protein (AßPP) that is proteolysed to form one of the pathological hallmarks of AD, amyloid-ß (Aß) peptide. However, other functions of SORL1 in AD are less well understood. OBJECTIVE: To investigate the effects of heterozygosity for an EOfAD-like mutation in SORL1 on the brain transcriptome of young-adult mutation carriers using zebrafish as a model organism. METHODS: We performed targeted mutagenesis to generate an EOfAD-like mutation in the zebrafish orthologue of SORL1 and performed RNA-sequencing on mRNA isolated from the young adult brains of siblings in a family of fish either wild type (non-mutant) or heterozygous for the EOfAD-like mutation. RESULTS: We identified subtle differences in gene expression indicating changes in mitochondrial and ribosomal function in the mutant fish. These changes appear to be independent of changes in mitochondrial content or the expression of AßPP-related proteins in zebrafish. CONCLUSION: These findings provided evidence supporting that EOfAD mutations in SORL1 affect mitochondrial and ribosomal function and provide the basis for future investigation elucidating the nature of these effects.

No observed effect on brain vasculature of Alzheimer's disease-related mutations in the zebrafish presenilin 1 gene.

Previously, we found that brains of adult zebrafish heterozygous for Alzheimer's disease-related mutations in their presenilin 1 gene (psen1, orthologous to human PSEN1) show greater basal expression levels of hypoxia responsive genes relative to their wild type siblings under normoxia, suggesting hypoxic stress. In this study, we investigated whether this might be due to changes in brain vasculature. We generated and compared 3D reconstructions of GFP-labelled blood vessels of the zebrafish forebrain from heterozygous psen1 mutant zebrafish and their wild type siblings. We observed no statistically significant differences in vessel density, surface area, overall mean diameter, overall straightness, or total vessel length normalised to the volume of the telencephalon. Our findings do not support that changes in vascular morphology are responsible for the increased basal expression of hypoxia responsive genes in psen1 heterozygous mutant brains.

Brain transcriptome analysis reveals subtle effects on mitochondrial function and iron homeostasis of mutations in the SORL1 gene implicated in early onset familial Alzheimer's disease.

To prevent or delay the onset of Alzheimer's disease (AD), we must understand its molecular basis. The great majority of AD cases arise sporadically with a late onset after 65 years of age (LOAD). However, rare familial cases of AD can occur due to dominant mutations in a small number of genes that cause an early onset prior to 65 years of age (EOfAD). As EOfAD and LOAD share similar pathologies and disease progression, analysis of EOfAD genetic models may give insight into both subtypes of AD. Sortilin-related receptor 1 (SORL1) is genetically associated with both EOfAD and LOAD and provides a unique opportunity to investigate the relationships between both forms of AD. Currently, the role of SORL1 mutations in AD pathogenesis is unclear. To understand the molecular consequences of SORL1 mutation, we performed targeted mutagenesis of the orthologous gene in zebrafish. We generated an EOfAD-like mutation, V1482Afs, and a putatively null mutation, to investigate whether EOfAD-like mutations in sorl1 display haploinsufficiency by acting through loss-of-function mechanisms. We performed mRNA-sequencing on whole brains, comparing wild type fish with their siblings heterozygous for EOfAD-like or putatively loss-of-function mutations in sorl1, or transheterozygous for these mutations. Differential gene expression analysis identified a small number of differentially expressed genes due to the sorl1 genotypes. We also performed enrichment analysis on all detectable genes to obtain a more complete view on changes to gene expression by performing three methods of gene set enrichment analysis, then calculated an overall significance value using the harmonic mean p-value. This identified subtle effects on expression of genes involved in energy production, mRNA translation and mTORC1 signalling in both the EOfAD-like and null mutant brains, implying that these effects are due to sorl1 haploinsufficiency. Surprisingly, we also observed changes to expression of genes occurring only in the EOfAD-mutation carrier brains, suggesting gain-of-function effects. Transheterozygosity for the EOfAD-like and null mutations (i.e. lacking wild type sorl1), caused apparent effects on iron homeostasis and other transcriptome changes distinct from the single-mutation heterozygous fish. Our results provide insight into the possible early brain molecular effects of an EOfAD mutation in human SORL1. Differential effects of heterozygosity and complete loss of normal SORL1 expression are revealed.

Transcriptome analysis indicates dominant effects on ribosome and mitochondrial function of a premature termination codon mutation in the zebrafish gene psen2.

PRESENILIN 2 (PSEN2) is one of the genes mutated in early onset familial Alzheimer's disease (EOfAD). PSEN2 shares significant amino acid sequence identity with another EOfAD-related gene PRESENILIN 1 (PSEN1), and partial functional redundancy is seen between these two genes. However, the complete range of functions of PSEN1 and PSEN2 is not yet understood. In this study, we performed targeted mutagenesis of the zebrafish psen2 gene to generate a premature termination codon close downstream of the translation start with the intention of creating a null mutation. Homozygotes for this mutation, psen2S4Ter, are viable and fertile, and adults do not show any gross psen2-dependent pigmentation defects, arguing against significant loss of γ-secretase activity. Also, assessment of the numbers of Dorsal Longitudinal Ascending (DoLA) interneurons that are responsive to psen2 but not psen1 activity during embryogenesis did not reveal decreased psen2 function. Transcripts containing the S4Ter mutation show no evidence of destabilization by nonsense-mediated decay. Forced expression in zebrafish embryos of fusions of psen2S4Ter 5' mRNA sequences with sequence encoding enhanced green fluorescent protein (EGFP) indicated that the psen2S4Ter mutation permits utilization of cryptic, novel downstream translation start codons. These likely initiate translation of N-terminally truncated Psen2 proteins lacking late endosomal/lysosomal localization sequences and that obey the "reading frame preservation rule" of PRESENILIN EOfAD mutations. Transcriptome analysis of entire brains from a 6-month-old family of wild type, heterozygous and homozygous psen2S4Ter female siblings revealed profoundly dominant effects on gene expression likely indicating changes in ribosomal, mitochondrial, and anion transport functions.

Sorting Out the Role of the Sortilin-Related Receptor 1 in Alzheimer's Disease.

Sortilin-related receptor 1 (SORL1) encodes a large, multi-domain containing, membrane-bound receptor involved in endosomal sorting of proteins between the trans-Golgi network, endosomes and the plasma membrane. It is genetically associated with Alzheimer's disease (AD), the most common form of dementia. SORL1 is a unique gene in AD, as it appears to show strong associations with the common, late-onset, sporadic form of AD and the rare, early-onset familial form of AD. Here, we review the genetics of SORL1 in AD and discuss potential roles it could play in AD pathogenesis.