● Alzheimer's disease is a progressive neurodegenerative disease that causes brain cells

to degenerate and die. It affects 5.8 million Americans and 44 million people worldwide.

● Neurons stop functioning, lose connections with other neurons, and die. Parts of the brain

that are related to memory such as the entorhinal cortex and hippocampus are affected

first, followed by the cerebral cortex.

● Approximately ⅔ of people with Alzheimer’s are female, and females are at a higher risk

of developing Alzheimer’s. Also, women tend to decline faster after developing

Alzheimer’s than men.

● Symptoms of Alzheimer’s are memory loss and inability to carry out day to day tasks.

● In this study, the genes ADAMTS4, HESX1, ADAM10, CLNK, CNTNAP2 were analyzed

for differences in gene expression patterns in male and female Alzheimer’s patients, as

well as patients with no dementia.These 5 genes have been recently identified as risk

factors for Alzheimer’s Disease.

● This study focused on examining gene expressions patterns in the frontal white matter

and hippocampus of the brain.

Analysis of Gene Expression Patterns in Male and Female Alzheimer’s PatientsNitisha Ponnappan, Lambert High School, GA and BioScience Project, Wakefield, MA

Introduction

Methods

Discussion

Results

● A recently published paper - Genome-wide meta-analysis identifies new loci and functional

pathways influencing Alzheimer’s disease risk

(https://www.researchgate.net/publication/330203436_Genome-wide_meta-

analysis_identifies_new_loci_and_functional_pathways_influencing_Alzheimer's_disease_risk

) was used to identify risk factor genes for Alzheimer’s Disease. The five genes used in this

study: ADAMTS4, HESX1, ADAM10, CLNK, and CNTNAP2 were taken from Table 1 of the

paper.

● The Aging,Dementia and TBI Database in the Allen Brain Atlas (http://aging.brain-map.org) is

a database that explores the genomic features of disease, aging and brain injury. It was used

to collect gene expression data on the five genes (ADAMTS4, HESX1, ADAM10, CNTNAP2,

and CLNK). Gene correlates (r> l0.7l) or greater were collected for further analysis.

● DAVID is a bioinformatics clustering tool that was used to identify gene ontology classifications

fo the risk factor genes. KEGG pathways were also obtained from the functional annotation

tool in DAVID (https://david.ncifcrf.gov)

● KEGG (https://www.genome.jp/kegg/pathway.html) was used to obtain the KEGG Alzheimer’s

Pathway.

● The Gene Ontology Resource (geneontology.org), with the Panther Classification System was

used to create pie charts for the biological processes the genes were involved in.

● Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/) was used to make Venn diagrams to

show the similar and different genes between the males and females gene lists.

● Python Anywhere, a programming tool,(https://www.pythonanywhere.com) was used to search

for keywords related to Alzheimer’s Disease for the five genes of interest

● The STRING database (https://string-db.org) was used to identify protein interaction networks

for the five genes.

5. KEGG Alzheimer’s Pathway Analysis

3. Common Genes

CNTNAP2

4. Pathways Biological Processes

ADAMTS4

CLNK

1. Gene Expression Profiling

Heatmaps of the ADAMTS4 gene from Allen Brain Atlas.

Gene expression data was collected for females and males with Alzheimer’s and no dementia for two brain regions: the

frontal white matter and hippocampus. This is because the hippocampus is the one of the first areas of the brain affected

by Alzheimer’s disease, followed by the frontal white matter.

At the top of the heat map, the red bar represents the frontal white matter and blue bar represents the hippocampus. The

green bar below represents dementia/no dementia and the colors in the bar below indicate the age of the donor, which

ranged from 78 to 100+ for all the samples.

Each row represents a gene and each column represents a tissue sample. If the gene is expressed more in the sample

than the control, the box is red, if they are equal it is black, and if the sample is less than the control the box is green.

Genes with an r value, which is a measure of correlation, of 7 or greater were collected for further study.

The heat maps for males and females were similar, and only had minute differences. The only notable differences were

that the females with alzheimer's heatmap had fewer green boxes (where the sample is less than the control) than the

other heat maps and the males no dementia heat map had more red boxes (where the sample is more than the control).

2. Keyword Search of Biological Processes

Keyword searches for Amyloid in the ADAMTS4 gene lists

Several keywords were searched for in the gene lists of the five genes of interest. This was done using the functional annotation chart from

DAVID database as well as Python Anywhere. Some examples of the keywords used are Alzheimer’s, dementia, cognition, immune, female,

male, and tau. Similar searches were made for all five genes of interest. These helped identify the functions of genes from the gene lists. The

visual representations below show the frequency of the keywords that appear for the ADAMTS4 gene. Both males and females appear to be

involved in neurological processes, even more so for males. In addition, for males, there generally appears to be more involvement with lipid

metabolism processes. For females, there appears to be more involvement with glucose and hormone related functions, as well as oxidation.

Protein-protein interaction network analysis for the five genes of interest were analyzed. These show related genes and experimentally

validated interactions.

The CLNK interaction network has two genes: SOCS6 and SOCS4 which are both from the SOCS family and are both suppressors of

cytokine signaling. Also PIK3R3 and PIK3R1 are both involved in insulin simulation and with the SH2 domain.

In the CNTNAP2 interaction network, CNTN2 is a paralog, which is a result of gene duplication, of CNTN1 and both are part of the same

family of proteins.

HESX1 interacts with two genes: RBP4 and TLE1. RBP4 is involved in retinol binding and TLE1 is involved in transcription.

The ADAM10 interaction network has two genes: DLL1 and DLL4 which are both involved in NOTCH signaling pathways.The NOTCH1

gene interacts with ADAM10 as well.

The ADAMTS4 gene is involved with only DPH6 which is an amidase that catalyses the last step of diphthamide biosynthesis using

ammonium and ATP.

The pie charts show the different biological processes associated with the pathways collected from DAVID

for the five genes’ gene lists. Generally, cellular process is the most common process for most of the gene

lists, but for HESX1, stem cell processes were the most common. Most of the pie charts are nearly

identical, and only have minute differences. When comparing the processes for the males and females

gene lists, there are some processes that are evident in the gene lists for females, but not in the males,

and vice versa. For ADAM10, the females with Alzheimer’s gene list is involved in signaling, but not the

males with Alzheimer’s gene list. The males no dementia gene list is involved in biological phase and

rhythmic process but not the females no dementia gene list. For ADAMTS4, the females with Alzheimer’s

gene list is involved in rhythmic process, but not males with Alzheimer’s gene list. The males no dementia

gene list is involved in rhythmic process, but not the females no dementia gene list. For CLNK, males with

Alzheimer’s gene list is only involved in cellular process, while females with Alzheimer’s gene list is

involved in many biological processes including cellular processes. The males no dementia gene list is

involved in biological adhesion, which the females no dementia gene list is not. Also, the males no

dementia gene list was had a relatively high percentage of genes involved in immune system processes in

comparison to the females no dementia gene list, which is interesting since there has been a link between

Alzheimer’s disease and immune system processes. The females no dementia gene list is involved in

cellular component organization or biogenesis, localization, and response to stimulus, which the males no

dementia gene list is not involved in. For CNTNAP2, the females with Alzheimer’s gene list is involved in

cell proliferation, immune system processes, reproduction, and signaling, which the males with Alzheimer’s

gene list is not involved in. The females no dementia gene list is involved in biological phase and signaling,

which the males no dementia gene list is not involved in.

The venn diagrams show the common genes as well as

unique genes for male and female gene lists for the five

genes of interest. Gene lists for males and females with

Alzheimer’s were compared, as well as gene lists for

males and females with no dementia. CNTNAP2 Males

and Females no dementia had the most in common

(60.7% of genes) while CLNK Males and Females with

Alzheimer’s had the least in common (0.6% of genes).

Generally, males and females with alzheimer’s had a

fewer percentage of genes in common than males and

females with no dementia for all five genes of interest.

ADAMTS4 These genes are mainly involved in B secretase, y secretase, and the endoplasmic reticulum.

Alzheimer’s females-BACE1, EIF2AK3, PSEN1, PPP3CC: Females no dementia-BACE1, PSEN1

Alzheimer’s males-BACE1, NCSTN, PSEN1: Males no dementia-BACE1, NCSTN, PSEN1

ADAM10 These genes are also mainly involved in a secretase, B secretase, y secretase, and the endoplasmic reticulum.

Alzheimer’s, females-ADAM10, BACE1, NCSTN, PSEN1: Females no dementia-ADAM10, BACE1, PSEN1

Males with Alzheimer's-ADAM10, NCSTN: Males no dementia-ADAM10, BACE1, NCSTN, PSEN1

CNTNAP2 These genes were mainly involved in y secretase, mitochondria, endoplasmic reticulum, calcium signaling pathway, and fragmentation.

Alzheimer's females-ATP5O, ATP5A1, ATP5B, ATP5G1, ATP5G3, ATP2A2, NDUFS1, NDUFS3, NDUFS8, NDUFV1, NDUFV2, NDUFA2, NDUFA5, NDUFA8, NDUFA9, NDUFAB1, NDUFB8,

NDUFC2, NDUFS5, NDUFA4, CACNA1C, CACNA1D, CALM1, CALM3, CAPN1, CDK5R1, CDK5, COX4I1, COX5A, COX5B, COX6A1, COX6B1, COX6C, COX7B, COX8A, CYCS, CYC1,

GRIN1, GRIN2A, GRIN2B, GSK3B, ITPR1, MAPK3, PLCB1, PSEN2, PPP3CA, PPP3CB, PPP3R1, SNCA, UQCRQ, UQCRC1, UQCRH, UQCR10

Females no dementia-ATP5O, ATP5A1, ATP5B, ATP5G3, ATP5H, ATP2A2, NDUFS1, NDUFS3, NDUFV2, NDUFA5, NDUFA6, NDUFA8, NDUFA9, NDUFAB1, NDUFB5,, NDUFC2, NDUFS5,

NDUFA4, CACNA1C, CACNA1D, CALM1, CALM3, CAPN1, CDK5R1, CDK5, COX4I1, COX5A, COX6C, COX7A2L, COX7B, CYCS, GRIN1, GRIN2A, GSK3B, ITPR1, MAPT, PLCB1, PSEN2,

PPP3CA, PPP3CB, PPP3R1, SNCA, UQCRC1, UQCRC2, UQCRH, and UQCRFS1: Alzheimer's males-ATP5A1, ATP5B, ATP5G3, ATP2A2, NDUFS1, NDUFV2, NDUFA12, NDUFA5,

NDUFA5, NDUFA8, NDUFB3, NDUFA4, CALM3, COX5A, COX6C, COX7B, GRIN2A, GSK3B, ITPR1, PLCB1, PPP3CA, PPP3CB, PPP3R1, SNCA, UQCRC2, UQCRH: Males no dementia-

ATP5O, ATP5A1, ATP5B, ATP5G1, ATP5G3, ATP5H, ATP2A2, GNAQ, NDUFS3, NDUFA5, NDUFA8, NDUFA4, CACNA1C, CACNA1D, CALM1, CALM3, CDK5R1, CDK5, COX4I1, COX5A,

COX6C, COX7B, CYCS, GRIN1, GRIN2A, GRIN2B, GSK3B, ITPR1, MAPT, MAPK1, PLCB1, PPP3CA, PPP3CB, PPP3R1, SNCA, UQCRC2, UQCRH

CLNK and HESX1-no gene correlates from any of the four groups

Females with Alzheimer’s Males with Alzheimer’s

6. Interaction Networks

CLNK HESK1

ADAM10

ADAMTS4 CNTNAP2

Females with Alzheimer's Females No Dementia

Males with Alzheimer'sMales No Dementia

Females with Alzheimer's Females No Dementia

Males with Alzheimer's Males No Dementia

ADAMTS4 Males with Alzheimer's

CLNK

Females with Alzheimer’s Males with Alzheimer’s

ADAMTS4 Females with Alzheimer's

Protein Interaction Networks From STRING

This study was conducted to determine how gene expression patterns varied between female and male Alzheimer patients.A

bioinformatics approach was used for analysing and profiling gene expression patterns of male and female Alzheimer’s patients

as compared with a control group with no Dementia.

This was done by studying gene expression in the hippocampus and frontal white matter for five genes that were identified as risk

factor genes for Alzheimer’s disease. Based on the results collected, there are differences in gene expression patterns between

males and females.When analyzing the number of correlates (r >than l0.7l) for the gene sets from the Aging, Dementia and TBI

database, the females with Alzheimer’s group had more correlates than the males with Alzheimer’s while the females no dementia

had less correlates than the males with no dementia. This pattern suggests that there are differences in gene expression patterns

between Alzheimer’s males and females. The only exception is the CNTNAP2 gene, where females with no dementia had 1875

correlates and males no dementia had 1763 correlates.

When analyzing the results from a Venn diagram comparison, generally the males and females with Alzheimer’s gene lists had a

lower percentage of genes in common than the males and females no dementia gene lists. The females with Alzheimer’s and

males with Alzheimer’s had more unique genes which may suggest difference in gene expression patterns. For example, for

ADAM10, the gene correlates for males and females with Alzheimer’s had 219 genes in common, which is less than the 346 genes

in common for males and females no dementia.

The keyword search showed that for ADAMTS4 the gene correlates for females with Alzheimer’s and males with Alzheimer's had

only differences of a few genes for the different keywords. CNTNAP2 and HESX1 gene lists were also the same way. For

ADAM10 and CLNK, males with Alzheimer's had fewer genes overall than females with Alzheimer’s. The CLNK interaction

network had a higher involvement in immune system processes. This was evident in the protein-protein interaction network, which

showed the interactions between CLNK and other genes involved in cytokine signaling. This was also evident from the gene

enrichment, in which a large proportion of genes from the CLNK gene lists were involved in immune system processes. The key

word search also supported this. Based on the results obtained from this study, there is a difference between gene expression

patterns in males and females.