# Introduction

This document explores different ways to assess the relevance of a gene for a set of human phenotypes using the PCAN package.

library(PCAN)
## Some functions can be parallelized.
## They use the bpmapply function from the BiocParallel library.
## Follow instructions provided in the BiocParallel manual to
## options(MulticoreParam=quote(MulticoreParam(workers=4)))


## Use case

This document demonstrates the capabilities of the PCAN package through the analysis of the following example.

data(traitDef, package="PCAN")
disId <- "612285"
disName <- traitDef[
which(traitDef$id==disId & traitDef$db=="OMIM"),
"name"
]


Here, we pretend that we don't know anything about the genetics of the r disName. The symptoms of this syndrome can be formally described by the following terms from the Human Phenotype Ontology [@kohler_human_2014] (according to this version of the phenotype_annotation.tab):

data(hpByTrait, hpDef, package="PCAN")
hpOfInterest <- hpByTrait[
which(hpByTrait$id==disId & hpByTrait$db=="OMIM"),
"hp"
]
knitr::kable(
hpDef[match(hpOfInterest, hpDef$id),], row.names=FALSE, col.names=c("HP", "Name") )  Let's store these phenoytpes in the hpOfInterest vector: hpOfInterest <-r paste0('c("', paste(hpOfInterest, collapse='", "'), '")') data(geneDef, package="PCAN") genId <- "57545" genSymb <- geneDef[ match(genId, geneDef$entrez),
"symbol"
]


The r genSymb is known to be associated to the r disName according to OMIM. Let's pretend in the frame of this example that we don't know this association and that this gene came out from sequencing data related to an individual suffering from the r disName.

The aim of the following analyses is to assess the relevance of this gene for the set of phenotypes under focus.

## Prior knowledge

To achieve the goal described above, we rely on prior knowledge about genetics of disorders and human phenotypes.

### Mendelian disorders and associated genes

Genes known to be associated to disorders were identified using clinVar [@landrum_clinvar:_2014]. In this package we provide part of this information taken from the ClinVarFullRelease_2015-05.xml.gz file.

The geneByTrait data frame provides the entrez gene IDs associated to disorder. Association were filtered according to the following criteria:

• they all come from OMIM;
• they all have a pathogenic clinical status;
• they all have one of the following origins: germline, de novo, inherited, maternal, paternal, biparental or uniparental.
data(geneByTrait, package="PCAN")
dim(geneByTrait)


The traitDef data frame provides the names of the different disorders:

data(traitDef, package="PCAN")


The geneDef data frame provides basic information about the genes:

data(geneDef, package="PCAN")


Since we pretend that we don't know the association between the r disName (disId <- "r disId") and r genSymb (genId <- "r genId"), let's remove it from the geneByTrait data frame:

geneByTrait <- geneByTrait[
which(geneByTrait$id!=disId | geneByTrait$entrez!=genId),
]
dim(geneByTrait)


### Human phenotype of mendelian disorders

OMIM disorders were associated to human phenotype using the phenotype_annotation.tab file. The associations are available in the hpByTrait data frame:

data(hpByTrait, package="PCAN")


Description of the different HP terms were obtained from the hp.obo file. They are available in the hpDef data frame:

data(hpDef, package="PCAN")


The same hp.obo file was used to get the descendant HP and the ancestor HP for each HP term. They are available in the hp_descendants and the hp_ancestors lists respectively:

data(hp_descendants, hp_ancestors, package="PCAN")

rootHpName <- "Phenotypic abnormality"
rootHpId <- hpDef$id[which(hpDef$name==rootHpName)]


We only kept information related to the descendants of r rootHpName (r rootHpId).

The geneByHp data frame, showing gene associated to each HP term, has been created from the geneByTrait and the hpByTraitdata frames. This data frame is available in the package: data(geneByHp, package="PCAN"). However, since we pretend in the frame of this example that we don't know the association between the r disName and r genSymb, we need to rebuild the geneByHp data frame using our filtered geneByTrait data frame:

geneByHp <- unique(merge(
geneByTrait,
hpByTrait,
by="id"
)[,c("entrez", "hp")])


### Custom prior knowledge

Several objects representing some biological knowledge are attached to this package for the convenience of the user. Nevertheless, the package functions could be used with other source of knowledge depending on the user needs and the updates of the different resources.

In the frame of this project we provide some R scripts (available in the inst/DataPackage-Generator/ folder of the package) allowing the generation of a package gathering up-to-date information from different databases:

Generating such a package is a way to get up-to-date information from these sources. However, some other means could be more convenient for some users. That's why this data resource is not tightly coupled with the PCAN package.

# Direct comparison of phenotypes

## Phenotypes associated to the gene candidate

Let's use our prior knowledge to find disorders, and eventually human phenotypes, associated to the gene candidate r genSymb:

genDis <- traitDef[
match(
geneByTrait[which(geneByTrait$entrez==genId), "id"], traitDef$id
),
]
genDis

genHpDef <- hpDef[
match(
geneByHp[which(geneByHp$entrez==genId), "hp"], hpDef$id
),
]
genHp <- genHpDef$id dim(genHpDef) head(genHpDef)  How many of these r length(genHp) HP terms are shared with the r length(hpOfInterest) HP related to the syndrome under focus? genHpDef[which(genHpDef$id %in% hpOfInterest),]


However some of the r sum(!genHp %in% hpOfInterest) r genSymb associated HPs which are not among the r length(hpOfInterest) HPs of interest present strong similarity with the last ones. For example the r hp1 <- "HP:0001305"; hpDef$name[which(hpDef$id==hp1)] (r hp1) phenotype, associated to r genSymb, is an indirect but closely related descendant of r hp2 <- "HP:0002119"; hpDef$name[which(hpDef$id==hp2)] (r hp2) phenotype of interest.

The following steps describe a way to measure similarity between different HP terms.

## Information content and semantic similariy

Here we measure semantic similarity between HPs using gene information content (IC). The formula below shows how information content is computed for each HP term $p$:

$$IC_{p} = -ln\left(\frac{|p|}{|root|}\right)$$

Where $|p|$ is the number of gene associated to the HP term $p$ and all its descendants. $root$, in our case, is r rootHpName (r rootHpId). By definition: $IC_{root} = -ln\left(\frac{|root|}{|root|}\right) = 0$.

Let's use the computeHpIC function to compute IC for all HP terms descendants of r rootHpName in the human phenotype ontology. This function needs to know the genes associated to each HP and the descendants of each HP term.

info <- unstack(geneByHp, entrez~hp)
ic <- computeHpIC(info, hp_descendants)


Let's have a look at the distribution of IC:

hist(
ic,
breaks=100, col="grey",
main="Distribution of Information Content",
xlab="IC base on genes associated to HP"
)


IC is an measure of the specificity of genes associated to HPs. The higher IC, the more specific.

Semantic similarity ($SS_{p_{1}p_{2}}$) between two HP terms is then defined as the IC of the most informative common ancestor (MICA) (i.e. showing the higher IC).

Let's use the clacHpSim function to compute the semantic similarity between different couples of HP terms:

hp1 <- "HP:0000518"
hp2 <- "HP:0030084"
hpDef[which(hpDef$id %in% c(hp1, hp2)), 1:2] calcHpSim(hp1, hp2, IC=ic, ancestors=hp_ancestors)  hp1 <- "HP:0002119" hp2 <- "HP:0001305" hpDef[which(hpDef$id %in% c(hp1, hp2)), 1:2]
calcHpSim(hp1, hp2, IC=ic, ancestors=hp_ancestors)


## Comparing two sets of phenotype

Now, we can compute semantic similarity between all HP of interest and r genSymb associated HPs using the compMat function:

compMat <- compareHPSets(
hpSet1=genHp, hpSet2=hpOfInterest,
IC=ic,
ancestors=hp_ancestors,
method="Resnik",
BPPARAM=SerialParam()
)
dim(compMat)


Then we compute the symmetric semantic similarity score of the matrix to get single value corresponding to similarity between the two sets of HP terms: the HP terms of interest and r genSymb associated HPs.

hpSetCompSummary(compMat, method="bma", direction="symSim")


Unfortunately it is not easy to interpret such a score and to assess it's significance. To do it we need to compare the score of the candidate gene (r genSymb) to the score of all the other genes for which we can compute it. Let's compute the score for all the genes:

## Compute semantic similarity between HP of interest and all HP terms
## This step is time consumming and can be parallelized.
## Use the BPPARAM parameter to specify your own
## back-end with appropriate number of workers.
hpGeneResnik <- compareHPSets(
hpSet1=names(ic), hpSet2=hpOfInterest,
IC=ic,
ancestors=hp_ancestors,
method="Resnik",
BPPARAM=SerialParam()
)
## Group the results by gene
hpByGene <- unstack(geneByHp, hp~entrez)
hpMatByGene <- lapply(
hpByGene,
function(x){
hpGeneResnik[x, , drop=FALSE]
}
)
## Compute the corresponding scores
resnSss <- unlist(lapply(
hpMatByGene,
hpSetCompSummary,
method="bma", direction="symSim"
))
## Get the score of the gene candidate
candScore <- resnSss[genId]
candScore


And now, we can compare the score of the candidate to all the others:

candRank <- sum(resnSss >= candScore)
candRank
candRelRank <- candRank/length(resnSss)
candRelRank

hist(
resnSss,
breaks=100, col="grey",
ylim=c(0,300),
xlab=expression(Sim[sym]),
ylab="Number of genes",
main=paste(
"Distribution of symmetric semantic similarity scores\nfor all the",
length(resnSss), "genes"
)
)
polygon(
x=c(candScore, 10, 10, candScore),
y=c(-10, -10, 1000, 1000),
border="#BE0000",
col="#BE000080",
lwd=3
)
text(
x=candScore, y=mean(par()$usr[3:4]), paste0( candRank, " genes (", signif(candRank*100/length(resnSss), 2), "%)\n", "show a symmetric semantic\n", "similarity score greater than\n", "the gene candidate for\n", "for the HPs under focus." ), pos=4, cex=0.6 )  According to a direct comparison, the candidate gene r genSymb is in the top r signif(candRelRank*100,2)% genes the most relevant for the set of HPs of interest. This result can be used for candidate prioritization. # The pathway consensus approach Often, gene candidates are not known yet to be associated to any genetic disorders. In such cases the prior knowledge can not be used to associate HP terms to the gene and the direct comparison of HP sets is not possible. In such situation we can focus genes known to interact with the gene of interest or known to be involved in the same biological processes and compute a consensus score taking all of them into account. This pathway consensus approach can also be used in addition to the direct comparison to provide further confidence or insight into the relationship between the gene candidate and the syndrome under focus. ## Additional prior knowledge To be able to apply such an approach we obviously need some information about gene pathways or gene network. For the convenience of the user we provide such an information within the package. However the user can use any kind of resource depending on the needs. Gene belonging to Reactome [@croft_reactome_2014] pathways are provided in the hsEntrezByRPath object. The name of the pathway can be found in the rPath data frame. data(hsEntrezByRPath, rPath, package="PCAN") head(rPath, n=3) lapply(head(hsEntrezByRPath, 3), head)  The STRING database [@jensen_string_2009] was used to get gene interactions. This information, focused on Homo sapiens genes and on interaction with a score higher than 500, can be found in the hqStrNw data frame: data(hqStrNw, package="PCAN") head(hqStrNw, n=3)  ## Genes belonging to the pathways of the candidate Here we are going to assess the relevance of genes involved in the same pathways as r genSymb for the HP terms of interest. First, let's identify the pathways in which r genSymb is involved: candPath <- names(hsEntrezByRPath)[which(unlist(lapply( hsEntrezByRPath, function(x) genId %in% x )))] rPath[which(rPath$Pathway %in% candPath),]


Then we can retrieve the symmetric semantic similarity scores for all these genes when the information is available. Let's use the hpGeneListComp function:

rPathRes <- hpGeneListComp(
geneList=hsEntrezByRPath[[candPath]],
ssMatByGene = hpMatByGene,
geneSSScore = resnSss
)


This function returns a list with many information. Have a look at ?hpGeneListComp to get a complete description of this output. Among the scores element of this output provides the scores for the genes in the submitted list:

length(rPathRes$scores) sum(!is.na(rPathRes$scores))


Among the r length(rPathRes$scores) genes belonging to the same pathway as r genSymb, a score could be computed for only r sum(!is.na(rPathRes$scores)) of them.

The p.value element of the output provides the p-value returned by wilcox.test comparing these scores to the scores of all the genes not in the provided list.

hist(
resnSss,
breaks=100, col="grey",
ylim=c(0,5),
xlab=expression(Sim[sym]),
ylab="Density",
main=paste(
"Distribution of symmetric semantic similarity scores\nfor all the",
length(resnSss), "genes"
),
probability=TRUE
)
rPathRes$scores, breaks=100, plot=FALSE ) for(i in 1:length(toAdd$density)){
polygon(
x=toAdd$breaks[c(i, i+1, i+1, i)], y=c(0, 0, rep(toAdd$density[i], 2)),
col="#BE000040",
border="#800000FF"
)
}
legend(
"topright",
paste0(
"Genes belonging to the ", candPath," pathway:\n'",
rPath[which(rPath$Pathway %in% candPath),"Pathway_name"], "'\nand with a symmetric semantic similarity score (", sum(!is.na(rPathRes$scores)),
"/",
length(rPathRes$scores), ")\n", "p-value: ", signif(rPathRes$p.value, 2)
),
pch=15,
col="#BE000040",
bty="n",
cex=0.6
)


This result show that in general the genes belonging to the r rPath$Pathway_name[which(rPath$Pathway %in% candPath)] pathway in which r genSymb is involved are relevant for the set of phenotype of interest.

To get further insight we can explore the score of all the genes belonging to this pathway:

pathSss <- rPathRes$scores[which(!is.na(rPathRes$scores))]
names(pathSss) <- geneDef[match(names(pathSss), geneDef$entrez), "symbol"] par(mar=c(7.1, 4.1, 4.1, 2.1)) barplot( sort(pathSss), las=2, ylab=expression(Sim[sym]), main=rPath[which(rPath$Pathway %in% candPath),"Pathway_name"]
)
p <- c(0.25, 0.5, 0.75, 0.95)
abline(
h=quantile(resnSss, probs=p),
col="#BE0000",
lty=c(2, 1, 2, 2),
lwd=c(2, 2, 2, 1)
)
text(
rep(0,4),
quantile(resnSss, probs=p),
p,
pos=3,
offset=0,
col="#BE0000",
cex=0.6
)
legend(
"topleft",
paste0(
"Quantiles of the distribution of symmetric semantic similarity\n",
"scores for all the genes."
),
lty=1,
col="#BE0000",
cex=0.6
)


Finally the hpGeneHeatmap function can be used to explore which HP term of interest are best matched to each of the genes under focus:

geneLabels <- geneDef$symbol[which(!duplicated(geneDef$entrez))]
names(geneLabels) <- geneDef$entrez[which(!duplicated(geneDef$entrez))]
hpLabels <- hpDef$name names(hpLabels) <- hpDef$id
hpGeneHeatmap(
rPathRes,
genesOfInterest=genId,
geneLabels=geneLabels,
hpLabels=hpLabels,
clustByGene=TRUE,
clustByHp=TRUE,
palFun=colorRampPalette(c("white", "red")),
goiCol="blue",
main=rPath[which(rPath$Pathway %in% candPath),"Pathway_name"] )  ## Genes interacting with the candidate The same kind of analysis can be done with genes direct neighbors of r genSymb in the STRING database network: neighbors <- unique(c( hqStrNw$gene1[which(hqStrNw$gene2==genId)], hqStrNw$gene2[which(hqStrNw$gene1==genId)], genId )) neighRes <- hpGeneListComp( geneList=neighbors, ssMatByGene = hpMatByGene, geneSSScore = resnSss )  hist( resnSss, breaks=100, col="grey", ylim=c(0,10), xlab=expression(Sim[sym]), ylab="Density", main=paste( "Distribution of symmetric semantic similarity scores\nfor all the", length(resnSss), "genes" ), probability=TRUE ) toAdd <- hist( neighRes$scores,
breaks=100,
plot=FALSE
)
for(i in 1:length(toAdd$density)){ polygon( x=toAdd$breaks[c(i, i+1, i+1, i)],
y=c(0, 0, rep(toAdd$density[i], 2)), col="#BE000040", border="#800000FF" ) } legend( "topright", paste0( "Genes interacting with ", geneDef[which(geneDef$entrez==genId),"symbol"],
" (", genId, ")",
"\nand with a symmetric semantic similarity score (",
sum(!is.na(neighRes$scores)), "/", length(neighRes$scores),
")\n",
"p-value: ", signif(neighRes$p.value, 2) ), pch=15, col="#BE000040", bty="n", cex=0.6 )  neighSss <- neighRes$scores[which(!is.na(neighRes$scores))] names(neighSss) <- geneDef[match(names(neighSss), geneDef$entrez), "symbol"]
opar <- par(mar=c(7.1, 4.1, 4.1, 2.1))
barplot(
sort(neighSss),
las=2,
ylab=expression(Sim[sym]),
main=paste0(
"Genes interacting with ",
geneDef[which(geneDef$entrez==genId),"symbol"], " (", genId, ")" ) ) p <- c(0.25, 0.5, 0.75, 0.95) abline( h=quantile(resnSss, probs=p), col="#BE0000", lty=c(2, 1, 2, 2), lwd=c(2, 2, 2, 1) ) text( rep(0,4), quantile(resnSss, probs=p), p, pos=3, offset=0, col="#BE0000", cex=0.6 ) legend( "topleft", paste0( "Quantiles of the distribution of symmetric semantic similarity\n", "scores for all the genes." ), lty=1, col="#BE0000", cex=0.6 )  hpGeneHeatmap( neighRes, genesOfInterest=genId, geneLabels=geneLabels, hpLabels=hpLabels, clustByGene=TRUE, clustByHp=TRUE, palFun=colorRampPalette(c("white", "red")), goiCol="blue", main=rPath[which(rPath$Pathway %in% candPath),"Pathway_name"]
)


# Session info

sessionInfo()


# References

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PCAN documentation built on Nov. 8, 2020, 6:47 p.m.