bioinformatic analysis of familial alzheimer’s
TRANSCRIPT
Bioinformatic analysis offamilial Alzheimer’s
disease-like zebrafishNhi Hin (@nhihin)
Zebrafish Alzheimer’s Disease Genetics Laboratory and Bioinformatics HubUniversity of Adelaide
Alzheimer’s-likeAlzheimer’sHealthy Before
(Rabinovici et al. 2010)
psen1Q96K97/+
psen1K97fs/+(non fAD-like)
(Newman et al. 2019, Moussavi Nik et al. 2015)
(fAD-like)
Age: 6 monthspsen1: Q96K97/+(n=4)
Age: 6 monthspsen1: +/+(n=4)
Age: 6 monthspsen1: K97fs/+(n=3)
Age: 6 monthspsen1: +/+(n=3)
Compare between groupsto look for gene expression differences
Age: 6 monthspsen1: Q96K97/+(n=4)
Age: 6 monthspsen1: +/+(n=4)
Age: 24 monthspsen1: Q96K97/+(n=4)
Age: 24 monthspsen1: +/+(n=4)
Age: 6 monthspsen1: K97fs/+(n=3)
Age: 6 monthspsen1: +/+(n=3)
Age: 24 monthspsen1: K97fs/+(n=3)
Age: 24 monthspsen1: +/+(n=3)
Compare between groupsto look for gene expression differences
Age: 6 monthspsen1: Q96K97/+(n=4)
Age: 6 monthspsen1: +/+(n=4)
Age: 24 monthspsen1: Q96K97/+(n=4)
Age: 24 monthspsen1: +/+(n=4)
Age: 6 monthspsen1: K97fs/+(n=3)
Age: 6 monthspsen1: +/+(n=3)
Age: 24 monthspsen1: K97fs/+(n=3)
Age: 24 monthspsen1: +/+(n=3)
Compare between groupsto look for gene expression differences
Age: 6 monthspsen1: Q96K97/+(n=4)
Age: 6 monthspsen1: +/+(n=4)
Age: 24 monthspsen1: Q96K97/+(n=4)
Age: 24 monthspsen1: +/+(n=4)
Age: 6 monthspsen1: K97fs/+(n=3)
Age: 6 monthspsen1: +/+(n=3)
Age: 24 monthspsen1: K97fs/+(n=3)
Age: 24 monthspsen1: +/+(n=3)
Compare between groupsto look for gene expression differences
Normoxia
Hypoxia
1. Trim adapters, quality filtering,
2. Align reads to reference zebrafish genome assembly
3. Count reads aligning to gene
Gene-by-sample matrix
Libraries of reads prepared from RNA extracted from zebrafish brains Sample 1 Sample 2 Sample 3
Gene A 6 3 2Gene B 13 4 12. . . .. . . .. . . .Gene X . . .
~20,000 genes
4. Moderated t-test approach to determine genes which are differentially expressed between conditions (FDR-adjusted p-value < 0.05).
5. Test whether biological functions / pathways are over-represented in the DE genes (FDR-adjusted p-value < 0.05).
(Law et al. 2014, Smyth 2004)
RNA-seq analysis
Age: 6 monthspsen1: Q96K97/+(n=4)
Age: 6 monthspsen1: +/+(n=4)
Age: 24 monthspsen1: Q96K97/+(n=4)
Age: 24 monthspsen1: +/+(n=4)
Age: 6 monthspsen1: K97fs/+(n=3)
Age: 6 monthspsen1: +/+(n=3)
Age: 24 monthspsen1: K97fs/+(n=3)
Age: 24 monthspsen1: +/+(n=3)
1. “Accelerated aging” in young adult mutants
Normoxia
Hypoxia
Age: 6 monthspsen1: K97fs/+(n=3)
Age: 6 monthspsen1: +/+(n=3)
Age: 24 monthspsen1: K97fs/+(n=3)
Age: 24 monthspsen1: +/+(n=3)
1. “Accelerated aging” in young adult mutants
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Differentially expressed genes between K97fs/+ and +/+ at 6 months (FDR adj. p-value < 0.05)
↑0.094↓0.058
↑0.17↓0.27
↑0.22↓0.29
↑0.099↓0.11
↑0.13↓0.32
↑0.24↓0.33
↑0.064↓0.17
↑0.12↓0.27
↑0.12↓0.099
↑0.26↓0.23
↑0.16↓0.31
↑0.26↓0.24
↑0.051↓0.19
↑0.16↓0.26
↑0.18↓0.081
↑0.28↓0.21
↑0.033↓0.17
↑0.17↓0.29
↑0.28↓0.24
↑0.029↓0.15
↑0.18↓0.24
↑0.066↓0.14
↑0.26↓0.26
↑0.1↓0.14
↑0.18↓0.3
↑0.11↓0.11
↑0.29↓0.3
↑0.15↓0.28
↑0.12↓0.094
↑0.22↓0.25
↑0.082↓0.12
↑0.16↓0.26
↑0.25↓0.29
↑0.19↓0.28
↑0.11↓0.12
↑0.24↓0.26
↑0.12↓0.14
↑0.21↓0.29
↑0.23↓0.24
↑0.088↓0.11
↑0.21↓0.21
↑0.25↓0.25
↑0.11↓0.13
↑0.17↓0.17
↑0.22↓0.22
↑0.089↓0.12
↑0.18↓0.27
↑0.16↓0.082
↑0.27↓0.26
↑0.089↓0.14
↑0.2↓0.25
↑0.26↓0.25
↑0.075↓0.14
↑0.17↓0.23
↑0.24↓0.26
↑0.1↓0.17
↑0.18↓0.18
↑0.25↓0.21
↑0.086↓0.15
↑0.21↓0.2
↑0.31↓0.29
↑0.07↓0.15
↑0.19↓0.2
↑0.077↓0.11
↑0.28↓0.22
↑0.11↓0.16
↑0.21↓0.23
↑0.28↓0.23
↑0.069↓0.097
↑0.22↓0.21
↑0.28↓0.19
↑0.082↓0.16
↑0.19↓0.19
↑0.29↓0.19
↑0.082↓0.14
↑0.21↓0.18
↑0.32↓0.23
↑0.09↓0.15
↑0.23↓0.21
↑0.29↓0.21
↑0.12↓0.13
↑0.19↓0.26
↑0.31↓0.21
↑0.066↓0.18
↑0.2↓0.25
↑0.13↓0.058
↑0.26↓0.2
↑0.048↓0.14
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↑0.28↓0.2
↑0.19↓0.24
↑0.33↓0.26
↑0.053↓0.14
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↑0.091↓0.076
↑0.28↓0.21
↑0.065↓0.14
↑0.2↓0.28
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↑0.089↓0.15
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↑0.22↓0.29
↑0.24↓0.33
↑0.26↓0.23
↑0.26↓0.24
↑0.28↓0.21
↑0.28↓0.24
↑0.26↓0.26
↑0.29↓0.3
↑0.22↓0.25
↑0.25↓0.29
↑0.24↓0.26
↑0.23↓0.24
↑0.25↓0.25
↑0.22↓0.22
↑0.27↓0.26
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↑0.28↓0.23
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↑0.32↓0.23
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↑0.26↓0.2
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↑0.28↓0.23
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↑0.36↓0.17
↑0.36↓0.22
↑0.34↓0.17
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2. Gene expression changes in aged mutant brains,mostly distinct from young mutant brains
Rotation gene set testing method (limma::mroast) (FDR adj. p-value < 0.05)
Age: 6 monthspsen1: K97fs/+(n=3)
Age: 6 monthspsen1: +/+(n=3)
Age: 24 monthspsen1: K97fs/+(n=3)
Age: 24 monthspsen1: +/+(n=3)
↑0.094↓0.058
↑0.17↓0.27
↑0.22↓0.29
↑0.099↓0.11
↑0.13↓0.32
↑0.24↓0.33
↑0.064↓0.17
↑0.12↓0.27
↑0.12↓0.099
↑0.26↓0.23
↑0.16↓0.31
↑0.26↓0.24
↑0.051↓0.19
↑0.16↓0.26
↑0.18↓0.081
↑0.28↓0.21
↑0.033↓0.17
↑0.17↓0.29
↑0.28↓0.24
↑0.029↓0.15
↑0.18↓0.24
↑0.066↓0.14
↑0.26↓0.26
↑0.1↓0.14
↑0.18↓0.3
↑0.11↓0.11
↑0.29↓0.3
↑0.15↓0.28
↑0.12↓0.094
↑0.22↓0.25
↑0.082↓0.12
↑0.16↓0.26
↑0.25↓0.29
↑0.19↓0.28
↑0.11↓0.12
↑0.24↓0.26
↑0.12↓0.14
↑0.21↓0.29
↑0.23↓0.24
↑0.088↓0.11
↑0.21↓0.21
↑0.25↓0.25
↑0.11↓0.13
↑0.17↓0.17
↑0.22↓0.22
↑0.089↓0.12
↑0.18↓0.27
↑0.16↓0.082
↑0.27↓0.26
↑0.089↓0.14
↑0.2↓0.25
↑0.26↓0.25
↑0.075↓0.14
↑0.17↓0.23
↑0.24↓0.26
↑0.1↓0.17
↑0.18↓0.18
↑0.25↓0.21
↑0.086↓0.15
↑0.21↓0.2
↑0.31↓0.29
↑0.07↓0.15
↑0.19↓0.2
↑0.077↓0.11
↑0.28↓0.22
↑0.11↓0.16
↑0.21↓0.23
↑0.28↓0.23
↑0.069↓0.097
↑0.22↓0.21
↑0.28↓0.19
↑0.082↓0.16
↑0.19↓0.19
↑0.29↓0.19
↑0.082↓0.14
↑0.21↓0.18
↑0.32↓0.23
↑0.09↓0.15
↑0.23↓0.21
↑0.29↓0.21
↑0.12↓0.13
↑0.19↓0.26
↑0.31↓0.21
↑0.066↓0.18
↑0.2↓0.25
↑0.13↓0.058
↑0.26↓0.2
↑0.048↓0.14
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↑0.19↓0.24
↑0.33↓0.26
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↑0.17↓0.26
↑0.091↓0.076
↑0.28↓0.21
↑0.065↓0.14
↑0.2↓0.28
↑0.14↓0.12
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↑0.089↓0.15
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↑0.17↓0.13
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↑0.069↓0.14
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↑0.15↓0.097
↑0.28↓0.23
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↑0.029↓0.15
↑0.18↓0.24
↑0.066↓0.14
↑0.26↓0.26
↑0.1↓0.14
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↑0.11↓0.11
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↑0.15↓0.28
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↑0.11↓0.12
↑0.24↓0.26
↑0.12↓0.14
↑0.21↓0.29
↑0.23↓0.24
↑0.088↓0.11
↑0.21↓0.21
↑0.25↓0.25
↑0.11↓0.13
↑0.17↓0.17
↑0.22↓0.22
↑0.089↓0.12
↑0.18↓0.27
↑0.16↓0.082
↑0.27↓0.26
↑0.089↓0.14
↑0.2↓0.25
↑0.26↓0.25
↑0.075↓0.14
↑0.17↓0.23
↑0.24↓0.26
↑0.1↓0.17
↑0.18↓0.18
↑0.25↓0.21
↑0.086↓0.15
↑0.21↓0.2
↑0.31↓0.29
↑0.07↓0.15
↑0.19↓0.2
↑0.077↓0.11
↑0.28↓0.22
↑0.11↓0.16
↑0.21↓0.23
↑0.28↓0.23
↑0.069↓0.097
↑0.22↓0.21
↑0.28↓0.19
↑0.082↓0.16
↑0.19↓0.19
↑0.29↓0.19
↑0.082↓0.14
↑0.21↓0.18
↑0.32↓0.23
↑0.09↓0.15
↑0.23↓0.21
↑0.29↓0.21
↑0.12↓0.13
↑0.19↓0.26
↑0.31↓0.21
↑0.066↓0.18
↑0.2↓0.25
↑0.13↓0.058
↑0.26↓0.2
↑0.048↓0.14
↑0.21↓0.25
↑0.28↓0.2
↑0.19↓0.24
↑0.33↓0.26
↑0.053↓0.14
↑0.17↓0.26
↑0.091↓0.076
↑0.28↓0.21
↑0.065↓0.14
↑0.2↓0.28
↑0.14↓0.12
↑0.28↓0.23
↑0.089↓0.15
↑0.19↓0.28
↑0.17↓0.13
↑0.25↓0.22
↑0.069↓0.14
↑0.2↓0.26
↑0.15↓0.097
↑0.28↓0.23
↑0.18↓0.25
↑0.28↓0.25
↑0.056↓0.15
↑0.27↓0.27
↑0.16↓0.12
↑0.39↓0.17
↑0.12↓0.17
↑0.24↓0.19
↑0.12↓0.11
↑0.36↓0.17
↑0.076↓0.1
↑0.26↓0.21
↑0.36↓0.22
↑0.045↓0.2
↑0.21↓0.23
↑0.13↓0.12
↑0.34↓0.17
↑0.05↓0.2
↑0.18↓0.27
↑0.33↓0.17
↑0.074↓0.2
↑0.21↓0.25
↑0.15↓0.074
↑0.36↓0.22
↑0.053↓0.17
↑0.21↓0.26
↑0.19↓0.099
↑0.34↓0.22
↑0.044↓0.15
↑0.21↓0.27
↑0.35↓0.24
↑0.063↓0.15
↑0.14↓0.2
↑0.12↓0.07
↑0.31↓0.19
↑0.085↓0.18
↑0.21↓0.25
↑0.35↓0.21
↑0.081↓0.17
↑0.19↓0.23
↑0.11↓0.12
↑0.32↓0.22
↑0.042↓0.15
↑0.19↓0.22
↑0.076↓0.083
↑0.29↓0.19
↑0.22↓0.26
↑0.32↓0.21
↑0.077↓0.18
↑0.25↓0.23
↑0.13↓0.092
↑0.36↓0.23
↑0.056↓0.18
↑0.22↓0.23
↑0.36↓0.26
↑0.066↓0.18
↑0.25↓0.23
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↑0.22↓0.29
↑0.24↓0.33
↑0.26↓0.23
↑0.26↓0.24
↑0.28↓0.21
↑0.28↓0.24
↑0.26↓0.26
↑0.29↓0.3
↑0.22↓0.25
↑0.25↓0.29
↑0.24↓0.26
↑0.23↓0.24
↑0.25↓0.25
↑0.22↓0.22
↑0.27↓0.26
↑0.26↓0.25
↑0.24↓0.26
↑0.25↓0.21
↑0.31↓0.29
↑0.28↓0.22
↑0.28↓0.23
↑0.28↓0.19
↑0.29↓0.19
↑0.32↓0.23
↑0.29↓0.21
↑0.31↓0.21
↑0.26↓0.2
↑0.28↓0.2
↑0.33↓0.26
↑0.28↓0.21
↑0.28↓0.23
↑0.25↓0.22
↑0.28↓0.23
↑0.28↓0.25
↑0.39↓0.17
↑0.36↓0.17
↑0.36↓0.22
↑0.34↓0.17
↑0.33↓0.17
↑0.36↓0.22
↑0.34↓0.22
↑0.35↓0.24
↑0.31↓0.19
↑0.35↓0.21
↑0.32↓0.22
↑0.29↓0.19
↑0.32↓0.21
↑0.36↓0.23
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2. Gene expression changes in aged mutant brains,mostly distinct from young mutant brains
Rotation gene set testing method (limma::mroast) (FDR adj. p-value < 0.05)
Age: 6 monthspsen1: K97fs/+(n=3)
Age: 6 monthspsen1: +/+(n=3)
Age: 24 monthspsen1: K97fs/+(n=3)
Age: 24 monthspsen1: +/+(n=3)
Age: 6 monthspsen1: Q96K97/+(n=4)
Age: 6 monthspsen1: +/+(n=4)
Age: 24 monthspsen1: Q96K97/+(n=4)
Age: 24 monthspsen1: +/+(n=4)
Age: 6 monthspsen1: K97fs/+(n=3)
Age: 6 monthspsen1: +/+(n=3)
Age: 24 monthspsen1: K97fs/+(n=3)
Age: 24 monthspsen1: +/+(n=3)
3. Q96K97 and K97fs mutations have different effectson gene expression at 6 months of age
Normoxia
Hypoxia
251 differentially expressed genes
Functionally enriched in: • Energy metabolism• Transmembrane transport
processes
105 differentially expressed genes
Functionally enriched in:• Oxidative stress• Glucocorticoid stress
responses
(FDR adjusted p-value < 0.05 for differential gene expression and GO enrichment analysis)
Is iron homeostasisdisrupted in Q96K97/+ (fAD-like) zebrafish?Can this be seen in the brain RNA-seq data?
(Lumsden et al. 2019)
1. Define gene set representing iron homeostasis genes (genes containing Iron Regulatory Elements in their UTRs).
2. Apply gene set enrichment analysis to assess differences in iron homeostasis in the psen1Q96K97/+ fish vs. wild type siblings.
5’ UTR 3’ UTR
Start Stop
Zebrafish transcripts from Ensembl reference genomeassembly (~50,000 transcripts)
5’ UTR 3’ UTR
Identified 3’ and 5’transcripts with IREs
Summarised transcripts to genes
These gene sets represent genes involvedin iron metabolism / homeostasis
Extracted 3’ and 5’UTR sequences from all zebrafish transcripts
1,082 zebrafish genes containing
IREs in 3’ UTR(Iron deficiency
response)
307 zebrafish genes containing
IREs in 5’ UTR (Iron surplus
response)
Gene set enrichment analysisin comparisons of interest
Defining sets of genes containing Iron Regulatory Elements
Age: 6 monthspsen1: Q96K97/+(n=4)
Age: 6 monthspsen1: +/+(n=4)
Age: 24 monthspsen1: Q96K97/+(n=4)
Age: 24 monthspsen1: +/+(n=4)
Age: 6 monthspsen1: K97fs/+(n=3)
Age: 6 monthspsen1: +/+(n=3)
Age: 24 monthspsen1: K97fs/+(n=3)
Age: 24 monthspsen1: +/+(n=3)
Normoxia
Hypoxia
4. Evidence of iron deficiency response in Q96K97/+brains at 6 months
p = 0.001
(Using gene set enrichment analysis,p-values Bonferroni-adjusted)
1,082 zebrafish genes containing
IREs in 3’ UTR(Iron deficiency
response)
Age: 6 monthspsen1: Q96K97/+(n=4)
Age: 6 monthspsen1: +/+(n=4)
Age: 24 monthspsen1: Q96K97/+(n=4)
Age: 24 monthspsen1: +/+(n=4)
Age: 6 monthspsen1: K97fs/+(n=3)
Age: 6 monthspsen1: +/+(n=3)
Age: 24 monthspsen1: K97fs/+(n=3)
Age: 24 monthspsen1: +/+(n=3)
Normoxia
Hypoxia
Negative control
Positivecontrol
p = 0.001 p = 1
p = 0.026(Using gene set enrichment analysis,p-values Bonferroni-adjusted)
1,082 zebrafish genes containing
IREs in 3’ UTR(Iron deficiency
response)
4. Evidence of iron deficiency response in Q96K97/+brains at 6 months
1.Analysing RNA-seq data can be an effective approach for detecting broad patterns of gene expression changesacross the brain, and also more targeted hypotheses (with a representative gene set).
2. Gene set enrichment testing shows preliminary evidence to support iron dyshomeostasis in early stages of familial Alzheimer’s disease (need to test other zebrafish models/mutations).
Summary
Acknowledgements
Zebrafish Alzheimer’s Disease Genetics Laboratory Bioinformatics HubA/Prof. Michael Lardelli Dr. Stephen Pederson
Morgan NewmanKarissa Barthelson
Yang DongJiayu Ruan
Ewen GerkenAoya Chen
Tanya Jayne
Jimmy BreenRick TearleNathan Dan KortschakAlastair LudingtonCharlotte SaiMelanie SmithNing LiuJustin BogiasJacqui Rehn
+ Dave Adelson, Sabrina Ng, Shanice Mah, Arwa Nawaz