bioinformatic analysis of familial alzheimer’s

Bioinformatic analysis offamilial Alzheimer’s

disease-like zebrafishNhi Hin (@nhihin)

Zebrafish Alzheimer’s Disease Genetics Laboratory and Bioinformatics HubUniversity of Adelaide

Healthy Alzheimer’s

(Magistri et al. 2015)

https://dx.doi.org/10.3233%2FJAD-150398

Alzheimer’s-likeAlzheimer’sHealthy Before

(Rabinovici et al. 2010)

https://dx.doi.org/10.1093%2Fbrain%2Fawp326

Alzheimer’s-likeAlzheimer’sHealthy -like.

(Modelling genetic state of familial AD)

psen1Q96K97/+

psen1K97fs/+(non fAD-like)

(Newman et al. 2019, Moussavi Nik et al. 2015)

(fAD-like)

https://doi.org/10.1186/s13041-019-0467-y

https://doi.org/10.1093/hmg/ddv110

Age: 6 monthspsen1: Q96K97/+(n=4)

Age: 6 monthspsen1: +/+(n=4)

Age: 6 monthspsen1: K97fs/+(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









1. “Accelerated aging” in young adult mutants

Normoxia

Hypoxia





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

↑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

↑0.29↓0.21

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.4JH%#�)BMMNBSL�(FOF�4FUT�TIPXJOH�EJGGFSFOUJBM�FYQSFTTJPO�XJUI�G"%�MJLF�NVUBUJPO

'JHVSF��(FOF�TFUT�TIPXJOH�EJGGFSFOUJBM�FYQSFTTJPO�XJUI�BHJOH�BOE�PS�GBNJMJBM�"M[IFJNFS�T�EJTFBTF�G"%�MJLF�NVUBUJPO��7BMVFT�JO�FBDI�DFMM�BSF�UIF�FTUJNBUFE�QSPQPSUJPOT�PG�VQ��BOE�EPXO�SFHVMBUFE�HFOFT�GPS�FBDI�HFOF�TFU �GPS�BOZ�QBSUJDVMBS�QBJSXJTF�DPNQBSJTPO��$PMPVST�PG�DFMMT�BSF�FRVBM�UP�UIF�EJGGFSFODF�CFUXFFO�UIF�QSPQPSUJPO�PG�VQ��BOE�EPXO�SFHVMBUFE�HFOFT�JO�B�HFOF�TFU��%JGGFSFOUJBMMZ�FYQSFTTFE�HFOF�TFUT�IBWF�.JYFE�'%3�CFMPX��(FOF�TFUT�BSF�GSPN�UIF��)BMMNBSL��HFOF�TFU�DPMMFDUJPO�BU�UIF�.PMFDVMBS�4JHOBUVSFT�%BUBCBTF�.4JH%#��

↑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

↑0.36↓0.26

↑0.29↓0.21

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↑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

↑0.21↓0.25

↑0.28↓0.2

↑0.19↓0.24

↑0.33↓0.26

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↑0.28↓0.23

↑0.089↓0.15

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↑0.12↓0.11

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↑0.076↓0.1

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↑0.045↓0.2

↑0.21↓0.23

↑0.13↓0.12

↑0.34↓0.17

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↑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

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↑0.36↓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)













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.

https://doi.org/10.3389/fnins.2018.00533

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









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)



response)









Normoxia

Hypoxia

Negative control

Positivecontrol

p = 0.001 p = 1

p = 0.026(Using gene set enrichment analysis,p-values Bonferroni-adjusted)



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

bioinformatic analysis of familial alzheimer’s

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