Relating Global and Local Connectome Changes to Dementia and Targeted Gene Expression in Alzheimer’s Disease Samar S. M. Elsheikh 1 , Emile R. Chimusa 2 , Alzheimer’s Disease Neuroimaging Initiative * , Nicola J. Mulder 1,† , and Alessandro Crimi 3,4 1 Computational Biology Division, Department of Integrative Biomedical Sciences, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, 7925, South Africa 2 Division of Human Genetics, Department of Pathology, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, 7925, South Africa 3 University Hospital of Z¨ urich, Z ¨ urich, 8091, Switzerland 4 African Institute for Mathematical Sciences, Ghana † [email protected]* Data used in preparation of this article were obtained from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database (adni.loni.usc.edu). As such, the investigators within the ADNI contributed to the design and implementation of ADNI and/or provided data but did not participate in analysis or writing of this report. A complete listing of ADNI investigators can be found at: http: //adni.loni.usc.edu/wp-content/uploads/how_to_apply/ADNI_Acknowledgement_List.pdf ABSTRACT Networks are present in many aspects of our lives, and networks in neuroscience have recently gained much attention leading to novel representations of brain connectivity. The integration of neuroimaging and genetics allows a better understanding of the effects of the genetic variations on brain structural and functional connections. The current work uses whole-brain tractography in longitudinal case-control setting and measures the brain structural connectivity changes to study the neurodegeneration of Alzheimer’s. This is accomplished by examining the effect of targeted genetic risk factors on the most common local and global brain connectivity measures. Furthermore, we examined the extent to which Clinical Dementia Rating is affected by brain connections longitudinally and genetic variation. Here, we show that the expression of PLAU and HFE genes increases the change in betweenness centrality related to the fusiform gyrus and clustering coefficient of cingulum bundle over time, respectively. We also show that betweenness centrality has a high contribution to dementia in distinct brain regions. Our findings provide insights into the complex longitudinal interplay between genetics and brain characteristics and highlight the role of Alzheimer’s genetic risk factors in the estimation of regional brain connection alterations. Introduction 1 There are many factors which may affect susceptibility to 2 Alzheimer’s disease (AD) and various ways to measure the 3 disease status. However, there is no single factor which can 4 be used to predict the disease risk sufficiently 1 . Genetics is be- 5 lieved to be the most common risk factor in AD development 2 . 6 Towards studying the etiology of the disease, a number of 7 genetic variants located in about 20 genes have been reported 8 to affect the disease through many cell-type specific biologi- 9 cal functions 3 . Those efforts resulted from omic studies such 10 as Genome-Wide Associations Studies (GWAS), which high- 11 lighted dozens of multi-scale genetic variations associated 12 with AD risk 4–6 . 13 From the early stages of studying the disease, the well 14 known genetic risk factors of AD were found to lie within 15 the coding genes of proteins involved in amyloid-β (Aβ ) pro- 16 cessing. These include the well-known Apolipoprotein E 17 gene (ApoE) that increases the risk of developing AD 7 , the 18 Amyloid precursor protein (APP) 8 , presenilin-1 (PSEN1) and 19 presenilin-2 (PSEN2) 9, 10 . The advancement in technologies 20 and the integration of genetic and neuroimaging datasets have 21 taken Alzheimer’s research steps further, and produced de- 22 tailed descriptions of molecular and brain aspects 11 . Studies 23 have utilised the connectome 12 to study different brain dis- 24 eases through associating genetic variants to brain connec- 25 tivity 13 .A structural connectome is a representation of the 26 brain as a network of distinct brain regions (nodes) and their 27 structural connections (edges), calculated as the number of 28 anatomical tracts. Those anatomical tracts are generally ob- 29 tained by diffusion-weighted imaging (DWI) 14 . DWI is the 30 most commonly used method for mapping and characterizing 31 the diffusion of water molecules in three-dimensions, as a 32 function of the location in order to construct a contrast in the 33 magnetic resonance images. This representation highlighted a 34 network based organization of the brain with separated sub- 35 networks (network segregation) which are connected by few 36 nodes (network integration) 15 . Given such a “small-world” 37 representation of the brain, it is also possible to represent each 38 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/730416 doi: bioRxiv preprint
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Relating Global and Local Connectome Changes toDementia and Targeted Gene Expression inAlzheimer’s DiseaseSamar S. M. Elsheikh1, Emile R. Chimusa2, Alzheimer’s Disease NeuroimagingInitiative∗, Nicola J. Mulder1,†, and Alessandro Crimi3,4
1Computational Biology Division, Department of Integrative Biomedical Sciences, Institute of Infectious Disease andMolecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, 7925, South Africa2Division of Human Genetics, Department of Pathology, Institute of Infectious Disease and Molecular Medicine,Faculty of Health Sciences, University of Cape Town, Cape Town, 7925, South Africa3University Hospital of Zurich, Zurich, 8091, Switzerland4African Institute for Mathematical Sciences, Ghana†[email protected]∗Data used in preparation of this article were obtained from the Alzheimer’s Disease Neuroimaging Initiative (ADNI)database (adni.loni.usc.edu). As such, the investigators within the ADNI contributed to the design andimplementation of ADNI and/or provided data but did not participate in analysis or writing of this report. A completelisting of ADNI investigators can be found at: http://adni.loni.usc.edu/wp-content/uploads/how_to_apply/ADNI_Acknowledgement_List.pdf
ABSTRACT
Networks are present in many aspects of our lives, and networks in neuroscience have recently gained much attention leadingto novel representations of brain connectivity. The integration of neuroimaging and genetics allows a better understanding of theeffects of the genetic variations on brain structural and functional connections. The current work uses whole-brain tractographyin longitudinal case-control setting and measures the brain structural connectivity changes to study the neurodegenerationof Alzheimer’s. This is accomplished by examining the effect of targeted genetic risk factors on the most common local andglobal brain connectivity measures. Furthermore, we examined the extent to which Clinical Dementia Rating is affected bybrain connections longitudinally and genetic variation. Here, we show that the expression of PLAU and HFE genes increasesthe change in betweenness centrality related to the fusiform gyrus and clustering coefficient of cingulum bundle over time,respectively. We also show that betweenness centrality has a high contribution to dementia in distinct brain regions. Ourfindings provide insights into the complex longitudinal interplay between genetics and brain characteristics and highlight therole of Alzheimer’s genetic risk factors in the estimation of regional brain connection alterations.
Introduction1
There are many factors which may affect susceptibility to2
Alzheimer’s disease (AD) and various ways to measure the3
disease status. However, there is no single factor which can4
be used to predict the disease risk sufficiently1. Genetics is be-5
lieved to be the most common risk factor in AD development2.6
Towards studying the etiology of the disease, a number of7
genetic variants located in about 20 genes have been reported8
to affect the disease through many cell-type specific biologi-9
cal functions3. Those efforts resulted from omic studies such10
as Genome-Wide Associations Studies (GWAS), which high-11
lighted dozens of multi-scale genetic variations associated12
with AD risk4–6.13
From the early stages of studying the disease, the well14
known genetic risk factors of AD were found to lie within15
the coding genes of proteins involved in amyloid-β (Aβ ) pro-16
cessing. These include the well-known Apolipoprotein E17
gene (ApoE) that increases the risk of developing AD7, the18
Amyloid precursor protein (APP)8, presenilin-1 (PSEN1) and19
presenilin-2 (PSEN2)9, 10. The advancement in technologies 20
and the integration of genetic and neuroimaging datasets have 21
taken Alzheimer’s research steps further, and produced de- 22
tailed descriptions of molecular and brain aspects11. Studies 23
have utilised the connectome12 to study different brain dis- 24
eases through associating genetic variants to brain connec- 25
tivity13. A structural connectome is a representation of the 26
brain as a network of distinct brain regions (nodes) and their 27
structural connections (edges), calculated as the number of 28
anatomical tracts. Those anatomical tracts are generally ob- 29
tained by diffusion-weighted imaging (DWI)14. DWI is the 30
most commonly used method for mapping and characterizing 31
the diffusion of water molecules in three-dimensions, as a 32
function of the location in order to construct a contrast in the 33
magnetic resonance images. This representation highlighted a 34
network based organization of the brain with separated sub- 35
networks (network segregation) which are connected by few 36
nodes (network integration)15. Given such a “small-world” 37
representation of the brain, it is also possible to represent each 38
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individual brain as single scalar metrics which summarize39
peculiar properties of segregation and integration16. Alterna-40
tively, those global metrics can also be used to quantify local41
properties of specific nodes/areas.42
Early work demonstrated that ApoE-4 carriers have an ac-43
celerated age-related loss of global brain inter-connectivity44
in AD subjects17, and topological alterations of both struc-45
tural and functional brain networks are present even in healthy46
subjects carrying the ApoE gene18. A more recent study has47
shown the association between ApoE expression and brain48
segregation changes6. Going beyond the ApoE gene, Jahan-49
shad et al.19 used a data set from the Alzheimer’s Disease50
Neuroimaging Initiative (ADNI) to carry out a GWAS of brain51
connectivity measures and found an associated variant in F-52
spondin (SPON1), previously known to be associated with53
dementia severity. A meta-analysis study also showed the im-54
pact of APOE, phosphatidylinositol binding clathrin assembly55
protein (PICALM), clusterin (CLU), and bridging integrator56
1 (BIN1) gene expression on resting state functional connec-57
tivity in AD patients20. In this work we utilised the most58
commonly used metrics of network segregation and integra-59
tion. The path length, for example, was used in a plethora of60
studies as biomarker to study the complex brain connectivity61
in schizophrenia21, while the local efficiency and characteris-62
tic path lengths were used to study the structural organisation63
of the brain network in autism22. For a detailed review on64
work using such metrics we refer the reader to23. For the65
local area features, we used a measure of segregation, one of66
integration and one of centrality. These metrics are described67
in detail in the Material and Methods section. Briefly, Louvain68
modularity is a community (cluster) detection method, which69
iteratively transforms the network into a set of communities.70
Transitivity also quantifies the segregation of a network, as71
the normalized counting of the fraction of triangles around72
an individual node. Characteristic path length is the average73
shortest path, and the global efficiency is its inverse. Between-74
ness centrality is fraction of all shortest paths in the network75
that pass through a given node. Those metrics represent large-76
scale organization which in turn might be used to represent77
well functioning cognitive functions24. Measures of centrality78
(e.g. degree and betweeness centrality) can be related each79
other as they measure similar things, and indeed it has been80
shown that many of them can be simply led back to the node81
degree measurement at the point to be considered its "spran-82
gles"25. Therefore, along with using them we computed their83
inter-correlations as well as their correlation with the nodal84
degree metric which is the simplest feature we can extract85
from a graph node, to investigate this aspect.86
Focusing on AD, we cannot fail to mention that it is a com-87
mon dementia-related illness; in the elderly, AD represents88
the most progressive and common form of dementia. Accord-89
ingly, incorporating and assessing dementia severity when90
studying AD provides more insights into the disease progres-91
sion from a clinical point of view. A reliable global rating of92
dementia severity is the Clinical Dementia Rating (CDR)26,93
which represents a series of evaluations specific for memory, 94
orientation, judgment and problem solving, community af- 95
fairs, home and hobbies (intellectual interests maintained at 96
home), and personal care assessed at the time of the visit. 97
Summarizing this paper focuses on the following questions: 98
• Are there brain connectivity metrics that discriminate 99
changes longitudinally in AD patients compared to 100
healthy control subjects? 101
• Is there a most representative metric, or a redundancy in 102
the chosen metrics for the case in examination? 103
• Is there a correlation between the metrics used and 104
known genes expression? 105
• Is there a correlation between connectivity metrics and 106
clinical ratings? 107
To address the above questions, we initially study the longi- 108
tudinal connectivity changes and CDR score, then we regress 109
the changes overtime in the connectivity metrics on gene ex- 110
pression, lastly we perform a ridge regression between the 111
difference in CDR scores and brain connectivity. The re- 112
lationships are investigated both for global and local brain 113
connectivity metrics, as a different impact could be possible 114
at global level overall or specific to regions. 115
This paper uses a dataset from ADNI (http://adni. 116
loni.usc.edu/) and presents an integrated association 117
study of specific AD risk genes, dementia scores and struc- 118
tural connectome characteristics. We adapted a longitudinal 119
case-control study design to mainly examine the association 120
of known AD risk gene expression with local and global con- 121
nectivity metrics. We also aim at testing the longitudinal effect 122
of brain connectivity on different CDR scores, and carry out a 123
multivariate analysis to study the longitudinal effect of gene 124
expression and connectome changes on CDR. Our underlying 125
hypothesis can be summarized in the simplistic representation 126
in Figure 1, where specific genes affect decreases in connec- 127
tivity comparing baseline and follow-up and this ultimately 128
affects intellectual abilities and CDR scores. 129
Results 130
Longitudinal Connectivity Changes and CDR 131
Initially, we used descriptive statistics plots to visualize the 132
data for the two populations; the AD and matched control sub- 133
jects. To facilitate the integrated analysis, we looked into the 134
different sets of data individually to have a better understand- 135
ing of the underlying statistical distribution of each, and chose 136
the best analysis methods accordingly. Firstly, we plotted 137
the global and local connectivity metrics in a way that illus- 138
trates the longitudinal change. Those longitudinal changes are 139
measured after 1 year followup from baseline screening. The 140
global connectivity metric box plots show the baseline and 141
follow-up distributions for both AD and controls for transitiv- 142
ity, Louvain modularity, characteristic path length and global 143
2
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Figure 2. Box plots of the distribution of brain segregation and integration global connectivity metrics comparing the twotime points. The plots compare the baseline and follow-up distributions for AD and controls for Louvain modularity (a),transitivity (b), characteristic path length (c) and global efficiency (d). The asterisk denotes that there is a significant changefrom baseline to the follow-up visit.
Association Analysis204
We studied the undirected associations of the 17 gene expres-205
sion values with the longitudinal change in global and local206
brain connectivity, as well as the associations with longitu-207
dinal CDR and connectivity changes. The total sample size208
after integrating all the datasets was 47 participants. Firstly,209
we performed an association analysis of gene expression with210
the connectivity changes locally, at each AAL brain region.211
In Table 2 we show the top results reported along with the212
Spearman correlation co-efficient. The APP gene (ρ =-0.58,213
p-value=1.9e-05) and BLMH (ρ =0.57, p-value=2.8e-05) are214
the top and only significant genes in the list, and associate215
with the change in local efficiency at the right middle tem-216
poral gyrus (Temporal_Mid_R AAL region) and clustering217
coefficient at the left Heschl gyrus (Heschl_L), respectively.218
Supplementary Figure S6 shows the scatter plots related to219
these significant associations.220
In Table 2, there is a similar pattern observed in association221
results between the clustering coefficient and local efficiency,222
e.g. both metrics are associated with BLMH at the left Heschl223
gyrus (Heschl_L), APP at the right middle temporal gyrus224
(Temporal_Mid_R) and PLAU at the right angular gyrus (An-225
Table 2. Top results of Spearman associations between ADgene expression and local connectivity metrics.
Gene Sorted by P-value. Dashed line: threshold= 0.0517×90 = 3.27e−05
gular_R). We interpret this by the strong correlation that exists 226
between the local efficiency and clustering coefficient at the 227
baseline, follow-up and also, the absolute change (see Figure 228
4). On the other hand, Table 3 reports the top results of the 229
4
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Figure 3. The figure shows three box plots respectively (a) for the AD and (b) control subjects. Each boxplot represents thecorrelation values between the degree value of a node and the related local efficiency (LE), betweenness centrality (BC) andclustering coefficient (CC). Values close to 0 represent low correlation while those close to 1 show a high correlation.
association between gene expression and the change in brain230
global connectivity. In this case, all observed associations231
were not statistically significant after correcting for multiple232
hypotheses (threshold is stated in Table 3).233
Table 3. Top 20 Spearman association results of the changein global network metrics with targeted AD gene expressionswith threshold = 0.5
Regressing Change in Local and Global Brain Con-234
nectivity on Gene Expression235
We analyzed the directed association through regressing the236
change in local connectivity (as a dependant variable), at each237
AAL region, on gene expression using (as an independent238
variable or predictor) a quantile regression model. Table 4239
reports the top results, along with the regression coefficient, p-240
values and t-test statistic. PLAU was the most significant gene241
affecting the absolute change in betweenness centrality at left 242
Fusiform gyrus (Fusiform_L) with an increase of 487.13 at 243
each unit increase in PLAU expression (p-value= 3e− 06). 244
This was followed by the expression of HFE with an effect 245
size of 0.1277 on the change in local efficiency at the right 246
anterior cingulate and paracingulate gyri (Cingulum_Ant_R). 247
Those observed associations are illustrated in Figure 5. More- 248
over, we report the protein-protein interaction27 of the afore- 249
mentioned genes in Figure 6. Supplementary Figures S7, S8 250
and S9 show the Manhattan plots for the -log10 of the p-values 251
corresponding to the quantile regression models of the change 252
in local efficiency, clustering coefficient and betweenness cen- 253
trality, respectively. 254
Similarly, we regressed the absolute change of global con- 255
nectivity measures on gene expression values and the top 256
results are shown in Table 5. All the results have p-values less 257
than the threshold we set ( 0.0517 = 0.0029). 258
Additive Genetic Effect on Brain Regions 259
To visualize the overall contribution of AD gene risk factors 260
used in this work on distinct brain areas, we added up the 261
-log10 p-values for the gene expression coefficients at each 262
of the 90 AAL regions. The p-values were obtained from 263
the quantile regression analysis between the gene expression 264
values and each of the three connectivity metrics - those are 265
the absolute difference between baseline and follow-up of lo- 266
cal efficiency, clustering coefficient and betweenness. Figure 267
7 summarizes this by 1) representing the brain connectome 268
without edges for each of the connectivity metrics, 2) each 269
node represents a distinct AAL region and is annotated with 270
the name of the region, 3) the size of each node is the sum 271
-log10 of the regression coefficient associated p-vales for all 272
the genes. It is clear from Figure 7 that local efficiency and 273
clustering coefficient show more similar patterns of associ- 274
ation with genes compared to betweenness centrality. This 275
5
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Figure 4. Spearman correlations between the three localconnectivity metrics; local efficiency, clustering coefficientand betweenness centrality, at baseline (suffix: _baseline),follow-up (suffix:_followup) and the absolute differencebetween them (no suffix). The calculation of Spearman’scoefficient combines all 90 brain regions, and both AD andcontrols. The plot illustrates the very strong relationshipbetween the clustering coefficient and local efficiency atbaseline, follow-up and the absolute difference between thetwo visits.
means that the gene expression has stronger association with276
local structure of the brain when using clustering measures277
(e.g. clustering coefficients), while the pattern of associa-278
tion tends to be weaker when using measures expressing the279
state of a region being between two others (e.g. betweenness280
centrality).281
The colors are assigned automatically by the BrainNet282
Viewer. Overall, although the gene contributions to the ab-283
solute change in local efficiency have a similar pattern to284
that of clustering coefficient, the contribution to betweenness285
centrality change is relatively small.286
Regressing the difference in CDR on the difference287
in Global and Local Connectivity288
To asses the directed and undirected association of the289
longitudinal measures of global connectivity and CDR290
scores, we calculated the difference between baseline and291
follow-up visits for both CDR and global connectivity met-292
rics , i.e. CDRbaseline −CDR f ollow−up and metricbaseline −293
metric f ollow−up, respectively. The Spearman and quantile294
regression results are both shown in Table 6. We observed295
a correlation between the increase of the transitivity score296
(global brain segregation) and the CDR memory score over297
Table 4. Top 50 quantile regression results of the change inlocal network metrics (y) and targeted AD gene expression (x)
Region Sorted by p-value. Dashed line: threshold= 0.0517×90 = 3.27e−05
time (β =−6.14e−06, p-value= 0.0034). On the other hand, 298
there is a positive correlation between global efficiency (global 299
brain integration) and the CDR "home and hobbies" score. 300
Similarly, in Supplementary Table S2 we looked at the 301
monotonic effect of local connectivity metrics on the seven 302
CDR scores, both represented as the subtraction of the follow- 303
up visit from the baseline visit. The increase in betweenness 304
centrality was shown to have different effects on the CDR 305
score over the one-year time period. For example, as the 306
betweenness centrality decreases over time, the judgement and 307
problem solving increases in severity by 1.06e-08 over time 308
(p-value=1.32e-17), in the frontal lobe (Frontal_Inf_Oper_L). 309
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Figure 5. Subfigure (a) higlights regions in the brain where significant associations - between gene expression andlongitudinal change in local connectivity metrics were found, using quantile regression (HFE and PLAU) and Spearmanassociations (APP and BLMH). Each gene is plotted at the AAL brain region where the association was significant; APP atTemporal_Mid_R, BLMH at Heschl_L, PLAU at Fusiform_L and HFE at Cingulum_Ant_R. (b) and (c) are scatter plots tovisualize the association between PLAU gene expression and betweenness centrality in the left fusiform gyrus (a), and betweenthe expression of HFE gene with local efficiency in right anterior cingulate gyrus (b). The red line on the plots represents themedian (quantile) regression line, while the blue line represents the ordinary least square line.
Table 5. Top quantile regression results of the change inglobal network metrics and targeted AD gene expressions.Threshold= 0.5
We regressed the difference in CDR visits (response variable;311
Y), one score at a time, on both the difference in global brain312
connectivity (predictor; X1), one connectivity metric at a time313
and all gene expression values (predictor; X2), using the ridge314
regression model. Supplementary Table S3 reports the mean315
squared error (the score column) and shows the top hits in316
the multiple ridge regression. It shows that the α (alpha col-317
umn) could not converge, using the cross-validation, when318
the response variables were judgment or personal care. How-319
ever, the CDR score results show that genes and connectivity320
metrics have a small effect (β ) on the response variables (the321
Figure 6. Protein–protein interactions between the geneswith significant correlation to brain connectivity metrics. It isevident that the genes interact either directly with APP or viaone intermediate node/gene. In this sub-network extractedfrom STRING27, the different color lines represents differenttypes of interaction: Cyan edges are interactions from curateddatabases, purple are experimentally determined, yellow arefrom text mining, and black are from co-expression data.
change in CDR scores over time), and the larger effects were 322
observed when using the total CDR score (CDR_diff) as a 323
response variable. The connectivity metrics and expression of 324
genes have both negative and positive effects on CDR change. 325
The expression of ApoE, for example, has a negative effect 326
(β ) of −0.24 on the change in memory score, i.e. the memory 327
rating decreases by 0.24 as the ApoE expression increases. 328
While when the expression of ApoE increases one unit, the 329
home and hobbies score increases, over time, by 0.12. 330
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Figure 7. Connectome representations showing the metric additive genetic effect at each AAL node. The subfigures show theaxial (top; (a), (b) and (c)), coronal (middle; (d), (e) and (f)), and sagittal (bottom; (g), (h) and (i)) planes of the brain, the nodesize represents the local efficiency (left; (a), (d) and (g)), clustering coefficient (middle; (b), (e) and (h)) and betweennesscentrality (right; (c), (f) and (i)). Colors of the nodes are automatically assigned by the BrainNet Viewer. The acronyms of thebrain regions are explained in Supplementary Table S1.
Discussion331
In this paper we aim to analyse the longitudinal change of332
local and global brain connectivity metrics for AD and con-333
trols, using a dataset from ADNI. We evaluate the associations334
between the most commonly used connectivity metrics and 335
the expression of known AD risk genes. Finally, we study the 336
multivariate association between connectivity metrics, gene 337
expression and dementia clinical ratings. 338
8
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Our results show that Alzheimer’s risk genes can manip- 339
ulate the amount of change observed in the structural con- 340
nectome, measured as the absolute difference of longitudinal 341
connectivity metrics. Here, we show that longitudinal regional 342
connectivity metrics, global brain segregation and integration 343
have effects on the CDR scores. More specifically, we observe 344
a consistent decrease, over time, in the local efficiency (a net- 345
work connectivity metric defined as the length of the shortest 346
path between a node j and h, that contains only the neighbors 347
of a node i16) in response to the increase in APP expression, at 348
the right middle temporal gyrus (Temporal_Mid_R; see Table 349
2). The same connectivity metric increases over time as the 350
expression of HFE increases, at the right anterior cingulate 351
and paracingulate gyri (see Table 4). Furthermore, as the 352
disease progresses, we observe a correlation between brain 353
segregation and cognitive decline, the latter is measured as 354
CDR memory scores. Moreover, we noticed a correlation 355
between the increase in the global efficiency and the increase 356
in the home and hobbies CDR scores (see Table 6). 357
Prescott et al.28 have investigated the differences in the 358
structural connectome in three clinical stages of AD, using a 359
cross-sectional study design, and targeted regional brain areas 360
that are known to have increased amyloid plaque. Their work 361
suggested that connectome damage might occur at an earlier 362
preclinical stage towards developing AD. Here, we further 363
adapted a longitudinal study design and incorporated known 364
AD risk genes. We showed how the damage in the connectome 365
is affected by gene expression, and that the change in connec- 366
tome affects dementia, globally and locally at distinct brain 367
regions. Aside from our previous work6, which examined the 368
ApoE associations with longitudinal global connectivity in 369
AD using longitudinal global connectivity metrics, this study, 370
to our knowledge, is the first of its type to include gene expres- 371
sion data with global and local brain connectivity. However, 372
similar work has been done in schizophrenia structural brain 373
connectivity, where longitudinal magnetic resonance imaging 374
features, derived from the DWI, were associated with higher 375
genetic risk for schizophrenia29. 376
The results obtained here align with findings in the litera- 377
ture of genetics and neuroimaging. Specifically, Robson et 378
al.30 studied the interaction of the C282Y allele HFE - the 379
common basis of hemochromatosis - and found that carriers 380
of ApoE-4, the C2 variant in TF and C282Y are at higher 381
risk of developing AD. Moreover, the HFE gene is known for 382
regulating iron absorption, which results in recessive genetic 383
disorders, such as hereditary haemochromatosis31. According 384
to Pujol et al.32, the association between the harm avoidance 385
trait and right anterior cingulate gyrus volume was statisti- 386
cally significant. In their study, they examined the association 387
between the morphology of cingulate gyrus and personality in 388
100 healthy participants. Personality was assessed using the 389
Temperament and Character Inventory questionnaire. Higher 390
levels of harm avoidance were shown to increase the risk of 391
developing AD33. We show here that HFE expression affects 392
the local efficiency at the right anterior cingulate gyrus (see 393
9
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Table 4 and Figure 5). This might indicate a possible effect of394
HFE expression on the personality of AD patients or those at395
risk of developing the disease.396
Moreover, in this study we found that the Plasminogen acti-397
vator, urokinase (PLAU) expression affects the betweenness398
centrality (a measure of the region’s (or node) contribution to399
the flow of information in a network16) in the left fusiform400
gyrus, over time (see Table 4 and Figure 5). Although the401
functionality of this region is not fully understood, its rela-402
tionship with cognition and semantic memory was previously403
reported34. PLAU, on the other hand, was shown to be a risk404
factor in the development of late-onset AD as a result of its405
involvement in the conversion of plasminogen to plasmin - a406
contributor to the processing of APP by the urokinase-type407
plasminogen activator (uPA)35.408
When examining the linear associations between gene ex-409
pression and local connectivity (see Table 2 and Supplemen-410
tary Figure S6), we found that the right middle temporal gyrus,411
known for its involvement in cognitive processes including412
comprehension of language, negatively associates with APP413
expression. Additionally, the left Heschl gyrus positively cor-414
relates with bleomycin hydrolase (BLMH) expression. In the415
human brain, the BLMH protein is found in the neocortical416
neurons and senile plaques36, microscopic decaying nerve417
terminals around the amyloid occurring in the brain of AD418
patients. Some studies37, 38 have found that a variant in the419
BLMH gene, which leads to the Ile443→Val in the BLMH420
protein, increases the risk of AD; this was strongly marked421
in ApoE-4 carriers. The BLMH protein can process the Aβ422
protein precursor and is involved in the production of Aβ423
peptide39.424
The spatial location of expression highlighted in regions in425
Figure 5 are relatively in line with previous studies (e.g.40).426
Indeed, the regional expression of the APP gene has been427
shown to be positively correlated with the severity of regional428
amyloid deposition observed in PET studies. In40 the tem-429
poral medial region and the fusiform gyrus of the brain are430
among the most affected by the amyloid deposition and also431
have high levels of APP expression. Recent findings from432
tau-sensitive positron emission tomography data also con-433
firm the spatial correspondence between accumulation of tau434
pathology and neurodegeneration in AD patients in the same435
regions, though only correlations to the ApoE genes were436
investigated41. The BLMH protein alters the processing of437
APP and significantly increases the release of its proteolytic438
fragment. It has been previously reported to be expressed and439
have an impact on the hippocampal tissues, but not investi-440
gated in other brain regions42. To our knowledge, apart from441
the general expression in the brain parenchyma reported in442
the Allen brain atlas43, no study has shown spatial expression443
of the PLAU and HFE genes among AD patients. Neverthe-444
less, we can hypothesize that the significance of expression445
in specific nodes of the brain connectome is related to their446
interaction with the APP gene which is particularly expressed447
in the nodes highlighted in Figure 5, and this is supported by448
the protein-protein interaction shown in Figure 6. 449
Even though none of the AD risk genes showed a signifi- 450
cant effect on the longitudinal change in global connectivity 451
(see Tables 3 and 5), the genes showed significant effects on 452
local connectivity changes at regional brain areas (see Table 453
4 and Table 2). The global connectivity metrics of the brain, 454
on the other hand, have shown promising results in affect- 455
ing the change observed in CDR scores, including memory, 456
judgement and problem solving, as well as home and hobbies, 457
as shown in Table 6. Previous work studied the association 458
between genome-wide variants and global connectivity of 459
Alzheimer’s brains, represented as brain integration and segre- 460
gation, and found that some genes affect the amount of change 461
observed in global connectivity6. This suggests that a general- 462
isation of the current study at a gene-wide level might warrant 463
further analysis. 464
Considering the possible redundancy of brain connectivity 465
metrics, we looked for correlations with the nodal degrees 466
and other features and observed only a relative correlation to 467
betweenness centrality. Comparing all features to each other, 468
we found only a correlation between local efficiency and clus- 469
ter coefficient metrics. In summary, we hypothesize that the 470
degree is a simpler representation than betweennes centrality, 471
and it could be used as a substitute, but it is not showing re- 472
dundancy with the other metrics, and therefore either cluster 473
coefficient or efficiency should also be investigated in similar 474
studies. More generally, when correcting our threshold, we 475
did not consider the number of metrics, because of the high 476
co-linearity. Therefore, reporting all, or only one, did not 477
affect the results. 478
Our work provides new insights, though replication on a 479
larger sample size is required. Indeed, one limitation here 480
was the small sample size available. We needed to narrow 481
down our selection of participants to those with both baseline 482
and follow-up visits, and that have CDR scores, genetic and 483
imaging information available. Another limitation is given 484
by the use of only two time points, the baseline and the first 485
follow-up visit. This does not allow capturing the effects 486
of connectivity changes over a longer-term or studying the 487
survival probabilities in AD. Extending to more time points 488
would have been useful, but it would have further reduced 489
the dataset. We foresee future work in using a more complex 490
unified multi-scale model to facilitate studying the multivari- 491
ate effect of clinical and genetic factors on the connectome, 492
besides considering the complex interplay of genetic factors. 493
Nevertheless, previous studies with similar sample sizes have 494
been able to provide relevant insights for the gene and brain 495
interaction networks44. 496
In this work, we conducted an association analysis of tar- 497
geted gene expression with various longitudinal brain con- 498
nectivity features in AD. Aiming at estimating the neurode- 499
generation of the connectome, we obtained local and global 500
connectivity metrics at two visits, baseline and follow-up, af- 501
ter 12 months. We calculated the change between the two 502
visits and carried out an association analysis, using quantile 503
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and ridge regression models to study the relationship between504
gene expression and disease progression globally and region-505
ally at distinct areas of the brain. We tested the effect of506
the change in connectivity on the longitudinal CDR scores507
through quantile regression. Furthermore, using a ridge re-508
gression model, we controlled for the genetic effects in the509
previous settings to study the effect of connectivity changes510
on the CDR change.511
The present analysis was conducted in AD using a longi-512
tudinal study design and highlighted the role of PLAU, HFE,513
APP and BLMH in affecting how the pattern information is514
propagated in particular regions of the brain, which might515
have a direct effect on a person’s recognition and cognitive516
abilities. Furthermore, the results illustrated the effect of brain517
structural connections on memory and cognitive process of518
reaching a decision or drawing conclusions. The findings pre-519
sented here might have implications for better understanding520
and diagnosis of the cognitive deficits in AD and dementia521
in vivo estimation of a regional disease progression pattern is522
of high interest for the neuropathology community working523
on Alzheimers. Indeed the Braak staging hypothesis is still524
controversial, and other in-vivo studies have shown regional525
effects from positron emission tomography (PET) imaging526
(temporal lobe, the anterior cingulate gyrus, and the pari-527
etal operculum)40, and impact on the default mode network528
connectivity45. Therefore further investigations of regional529
patterns is relevant. Very recent results on treatments in mice530
showed that drug based modulated neuronal activity can re-531
duce amyloid plaques in specific locations and circuitry46. In532
view of future treatments based on specific spatial location and533
genetic influences, our study provides some initial insights534
into connectivity outcomes, or at least enhances our under-535
standing of the regions/circuits that show amyloid aggregation536
or neurodegeneration.537
Materials and Methods538
Data Description539
We used two sets of data from ADNI, which are available at540
adni.loni.usc.edu. The experiments have been con-541
ducted on the publicly available datasets described below, for542
which ethical approval has already been granted, and data543
acquisition has been conducted according to the Helsinki II544
regulations. To use this data for our analysis, we also received545
an ethical approval from the Faculty of Health Sciences Hu-546
man Research Ethics Committee.547
To fulfil our objectives, unless otherwise specified, we548
merged neuroimaging, genetic and CDR datasets for all the549
participants with those three types of data at two-time points550
available. We considered follow-up imaging and CDR ac-551
quisition one year later than the baseline visit. Given those552
constraints, we ended up with a total of 47 participants. We553
adopted a case-control study design; 11 of the participants are554
AD patients, while 36 are controls. The data were matched555
by age, and the distribution of age in AD ranges between556
76.5±7.4 for cases, and 77.0±5.1 years in controls.557
Imaging Data 558
For the imaging, we obtained the DWI volumes at two time 559
points, the baseline and follow-up visits, with one year in be- 560
tween. Along with the DWI, we used the T1-weighted images 561
which were acquired using a GE Signa scanner 3T (General 562
Electric, Milwaukee, WI, USA). The T1-weighted scans were 563
obtained with voxel size = 1.2×1.0×1.0mm3T R= 6.984ms; 564
TE = 2.848 ms; flip angle= 11◦ ), while DWI was obtained 565
with voxel size = 1.4×1.4×2.7mm3 , scan time = 9 min, and 566
46 volumes (5 T2-weighted images with no diffusion sensiti- 567
zation b0 and 41 diffusion-weighted images b= 1000s/mm2). 568
Pre-processing of Imaging Data 569
Each DWI and T1 volume have been pre-processed perform- 570
ing Eddy current correction and skull stripping. Given the 571
fact that DWI and T1 volumes were already co-registered, 572
the AAL atlas47, and the T1 reference volume are linearly 573
registered according to 12 degrees of freedom. 574
Moreover, we used the same T1 reference to get the infor- 575
mation needed to compute the partial volume effect from the 576
tissue segmentation by using FSL. 577
Genetic Data Acquisition 578
We used the Affymetrix Human Genome U219 Array profiled 579
expression dataset from ADNI. The RNA was obtained from 580
blood samples and normalised before hybridization to the 581
array plates. Partek Genomic Suite 6.6 and Affymetrix Ex- 582
pression Console were used to check the quality of expression 583
and hybridization48. The expression values were normalised 584
using the Robust Multi-chip Average49, after which the probe 585
sets were mapped according to the human genome (hg19). 586
Further quality control steps were performed by checking 587
the gender using specific gene expression, and predicting the 588
single nucleotide plymorphisms from the expression data50, 51589
In this work, we targeted specific genes which have been re- 590
ported to affect the susceptibility of AD. We used the BioMart 591
software from Ensembl to choose those genes by specifying 592
the phenotype as AD52. We obtained a total of 17 unique gene 593
names and retrieved a total of 56 probe sets from the genetic 594
dataset. 595
Clinical Dementia Rating 596
The Clinical Dementia Rating, or CDR score is an ordinal 597
scale used to rate the condition of dementia symptoms. It 598
range from 0 to 3, and is defined by four values: 0, 0.5, 599
1, 2 and 3, ordered by severity, which stand for none, very 600
mild, mild and severe, respectively. The scores evaluate the 601
cognitive state and functionality of participants. Here, we used 602
the main six scores of CDR; memory, orientation, judgement 603
and problem solving, community affairs, home and hobbies, 604
and personal care. Besides the latter, we used a global score, 605
calculated as the sum of the six scores. We obtained the CDR 606
scores at two time points in accordance with the connectivity 607
metrics time points. 608
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to measure the segregation of the brain, that is, the capability638
of the network to shape sub-communities which are loosely639
connected to one another while forming a densely connected640
sub-network within communities15, 16.641
Suppose that n is the number of nodes in the network, N isthe set of all nodes, the link (i, j) connects node i with nodej and ai j define the connection status between node i and j,such that ai j = 1 if the link (i, j) exist, and ai j = 0 otherwise.We define the global connectivity metrics as;
E =1
n(n−1) ∑i∈N
∑j∈N, j 6=i
d−1i j , (1)
where, di j = ∑auv∈gi↔ j auv, is the shortest path length betweennode i and j, and gi↔ j is the geodesic between i and j.
L =1
n(n−1) ∑i∈N
∑j∈N, j 6=i
di j. (2)
Q =1l ∑
i j∈N
[ai j−
kik j
l
]δ (ci,c j), (3)
where l = ∑i, j∈N ai j, mi and m j are the modules containingnode i and j, respectively, and δ (ci,c j) = 1 if ci = c j and 0otherwise.
T =∑i∈N 2ti
∑i∈N ki(ki−1), (4)
where ti = 12 ∑ j,h∈N(ai jaiha jh) is the number of triangles
around node i.
Using the AAL atlas, we constructed the following local 642
brain network metrics at each region or node. We used the 643
local efficiency (Eloc,i; Equation 5), clustering coefficient (Ci; 644
Equation 6) and betweenness centrality (bi; Equation 7) at 645
each node to quantify the local connectivity. Both local ef- 646
ficiency and clustering coefficient measure the presence of 647
well-connected clusters around the node, and they are highly 648
correlated to each other. The betweenness centrality is the 649
number of shortest paths which pass through the node, and 650
measures the effect of the node on the overall flow of infor- 651
mation in the network16. The local connectivity metrics used 652
in this work, for a single node i, are defined as follows; 653
Eloc,i =∑ j,h∈N, j 6=i ai jaih
[d jh(Ni)
]−1
ki(ki−1), (5)
where, d jh(Ni), is the length of the shortest path between nodej and h - as defined in Equation, and contains only neighboursof h 1.
Ci =2tw
iki(ki−1)
. (6)
bi =1
(n−1)(n−2) ∑h, j∈N,h6= j,h6=i,i 6= j
ρh j(i)ρh j
, (7)
where ρh j(i) is the weights of shortest path between h and jthat passes through i.
Statistical Analysis 654
We used different statistical methods as described below; how- 655
ever, for the multiple testing we relied on the Bonferroni 656
correction57, 58. Where applicable, the thresholds were ob- 657
tained by dividing 0.05 by the number of independent tests. 658
We stated the corrected threshold and number of independent 659
tests at the caption of each table in the Results section. 660
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.CC-BY-NC-ND 4.0 International licenseis made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It. https://doi.org/10.1101/730416doi: bioRxiv preprint
38. Farrer, L. A. et al. Association between bleomycin hy- 847
drolase and Alzheimer’s disease in caucasians. Annals 848
neurology 44, 808–811 (1998). 849
39. Kajiya, A., Kaji, H., Isobe, T. & Takeda, A. Process- 850
ing of amyloid β -peptides by neutral cysteine protease 851
bleomycin hydrolase. Protein peptide letters 13, 119–123 852
(2006). 853
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Pharmaceutical Company; and Transition Therapeutics. The 949
Canadian Institutes of Health Research is providing funds to 950
support ADNI clinical sites in Canada. Private sector con- 951
tributions are facilitated by the Foundation for the National 952
Institutes of Health (www.fnih.org). The grantee organiza- 953
tion is the Northern California Institute for Research and 954
Education, and the study is coordinated by the Alzheimer’s 955
Therapeutic Research Institute at the University of Southern 956
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Competing interests: The authors declare no competing in-970
terests.971
Data Availability: The data used in this work are available at972
the ADNI repository (http://adni.loni.usc.edu/).973
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