Candidate Genes Directly Related to SLITRK4, SLITRK5, & SLITRK6Yusra Sannah, Khadijah Jeddy, & Caitlin Benitez, Forest Hills Central High School, Grand Rapids, MI, 49546 US

BioScience Project, Wakefield, MA, 01880 US

Background

Results

Conclusions

● The SLITRK gene family is expressed via six structurally related transmembrane proteins (SLITRK1–6)

● SLITRK gene family is involved in neurodevelopmental disorders such as Tourettes Syndrome, Schizophrenia, Autism

Spectrum Disorders, and other social behavior disorders. They are also uniquely expressed in brain tumors, including

astrocytoma, oligodendroglioma, glioblastoma, medulloblastoma, and supratentorial primitive neuroectodermal tumor (PNET).

● Human SLITRK genes have been found to be potential molecular indicators of brain tumor properties.

● SLITRK4 is involved in synaptogenesis and promotes synapse differentiation (PubMed:27812321).

● SLITRK4, SLITRK5, and SLITRK6 regulate and suppress neurite outgrowth, the latter specifically required for normal vision

and hearing.

● As shown by the SLITRK4 STRING network to the right (Section 5, Figure B), the SLITRK genes do not have any known

protein interaction networks, and the goal of this research is to identify interacting genes common with the genes SLITRK4,

SLITRK5, and SLITRK6.

5. Protein Interaction Network Analysis3. KEGG Pathways

Methods● Allen Brain Atlas Database (http://www.brain-map.org/) was used to collect gene expression data for the SLITRK4, 5, & 6 genes

in the developing brain. The data came from 29 donors.

● Venn Diagrams (http://bioinfogp.cnb.csic.es/tools/venny/index.html) were used to sort through the genes found in the three

most expressive brain regions per SLITRK to find all the genes that the regions had in common, and those that were unique to

each SLITRK and region.

● These gene lists of similar genes were then analyzed in DAVID (https://david.ncifcrf.gov/tools.jsp), which is a functional

annotation tool used to cluster different genes based on their function. It also provided information on gene ontology and

pathways that are involved with the genes related to the SLITRK genes.

● Genes that formed functional groups related to neurodevelopment and other known functions of the SLITRK genes were then

filtered to form a list of genes of interest that were common between brain regions and between the SLITRK4, 5, & 6 genes,

which were then further researched with STRING (http://string-db.org/) to identify proteins networks that a common gene

interacted with.

● Cytoscape (http://www.cytoscape.org/) is a more indepth database that used to identify what proteins a gene interacts with.

● DAVID (https://david.ncifcrf.gov/tools.jsp) was used again to further strengthen the relationship of the common gene to the

SLITRK4, SLITRK5, and SLITRK6 genes.

● Information on some of the proteins were found here (https://www.genecards.org/cgi-bin/carddisp.pl?gene=RAC1)

SLITRK6 is expressed highly in the

striatum (STR) during 8pcw-5mos,

mediodorsal nucleus of the thalamus

(MD) from 16pcw-18mos, & cerebellar

cortex (CBC) from 19pcw-5yrs.

These gene pathways indicate the genes (starred in red) that were highly correlated to the SLITRK4 and

SLITRK6 in the brain regions during the corresponding periods that these genes were most expressive

from the gene expression graphs (1). Other pathways found through DAVID include neurotrophin signalling

pathway, serotonergic synapse, amphetamine addiction (both acute and chronic) and neuroactive ligand

intercepter interaction. The Cancer Pathway is shown above, and the dopaminergic synapse is below. One

point of interest was in the presence of only post-synaptic starred genes in the dopaminergic synapse

pathway.

4. Genes of Interest

Figure A: The protein interaction network of the EPHA3 gene. This string has a total of 54 nodes and 944 edges, and this is remarkable in that the

expected number of nodes is 92, which is less than 1% of what is known. There are also 12 genes of interest (boxed in yellow) on the STRING network.

The connecting nodes EFNB1, EFNB2, EFNB3, EFNA3, EFNA5, NGEF, EPHA4, EPHA5, EPHA6, CRK, RASA1, EPHB2 are all seemingly related to both

EPHA3D and SLITRK4, SLITRK5, and SLITRK6. Additionally, proteins boxed in green (FYN and RAC1) were found to be bottlenecks connecting the

networks of EPHA3 AND SEMA3D (see Figure D).

Figure B: The protein interaction network of the SLITRK4 gene. The absence of connecting proteins to the SLITRK4 node suggests the limited amount of

information known about the SLITRK gene family and its interacting proteins. The genes SLITRK5 and SLITRK6 show similar results when mapped.

Figure C: The protein interaction network of the SEMA3D gene. The connecting proteins NRP1, PLXNA1, PLXNA2, and PLXNA3 also appeared on DAVID

as genes of interest in axon guidance.

Figure D: The combined protein interaction network of SEMA3D and EPHA3 found via Cytoscape. The connecting genes are FYN and RAC1, also known

as bottlenecks, for bridging the functions and roles of the SEMA3D and EPHA3 clusters. The combined interaction network displayed 1074 connections,

indicating a high degree of connectivity given the few number of genes.

1. Gene Expression

SLITRK4 SLITRK5 SLITRK6

SLITRK4 is highly expressed in the

inferolateral temporal cortex (ITC) is

during 8-16 pcw, the striatum (STR) is

from 16-37 pcw, and in the cerebellar

cortex (CBC) from 10 mos-4 yrs of

age.Submitting the list of the

common genes from all

donors into DAVID

showed their functions,

which helps to serve as

evidence of their

connection to the

SLITRK4/5/6. Searching

the list with keywords

such as “synapse,”

“axon,” and “neur-”, two

gene families were

frequently found: EPHA

and SEMA. The specific

genes that were most

common within these

families between

SLITRK4, SLITRK5, and

SLITRK6 include EPHA3

and SEMA3D.

Figure C

● This project was conducted using data mining, statistical analysis, gene profiling, and protein interaction networks.

● An integrative genomic approach was used for profiling gene expression patterns to identify genes that may be related to SLITRK4/5/6.

● Our data shows commonality in function of the SLITRK4/5/6 genes in axonal growth regulation, which is one of the functions that binds these

genes together as indicated through the genes of interest found in DAVID and proteins that express these various genes.

● Based on the gene expression graphs, the brain region striatum (STR) contains a high expression of the SLITRK4/5/6 genes during various time

periods. Additionally, the cerebellar cortex (CBC) shows a high expression of SLITRK4 and SLITRK6.

● We conclude that the genes SLITRK4/5/6 are related to the gene SEMA3D. In DAVID, SEMA3D was found to be a frequently occurring gene

across SLITRK4/5/6, with similar functions as well.

● In addition, it was found that the gene EPHA3 is related to SLITRK4/5/6. This was found through the DAVID database, which displayed EPHA3

as a frequently occurring gene among SLITRK4/5/6. EPHA3 was a common element between the 3 prominent brain regions (STR, MD, and

CBC) of SLITRK6 expression.

● Since SLITRK4/5/6 are highly connected to SEMA3D and EPHA3, they are likely to take part in similar processes such as spine morphology,

cytoskeletal remodeling, and cell adhesion due to the commonality of these functions among the interacting proteins that are expressed..

● Because FYN and RAC1 are bottlenecks, or points of connection between the protein interaction networks of SEMA3D and EPHA3, we can

hypothesize that both FYN and RAC1 bridge the functions of SEMA3D and EPHA3, and that they are likely interconnected with SLITRK4/5/6

due to similar functions in axonal growth guidance and regulation.

● We found multiple connections between the SLITRK4/5/6 to cancer: one via the KEGG Pathway from the DAVID database when searching

genes with high correlation to SLITRK4, and another connection via the RASA1 gene from the protein interaction networks that is known to have

3 distinct mutations detected in basal cell carcinoma.

SLITRK5 SLITRK6

Common elements in the

STR, MD, and CBC include

the genes GNG2, NLN, and

EPHA3.

2. Common Genes

SLITRK4

Common elements in the ITC,

STR and CBC include the genes

NEGR1 and SPATA17.

SEMA3D-- Induces the collapse and paralysis of neuronal growth cones. Could

potentially act as repulsive cues toward specific neuronal populations. Binds to

neuropilin (By similarity)

PLXNA1/2/3-- Necessary for signaling by class 3 semaphorins and subsequent

remodeling of the cytoskeleton. Plays a role in axon guidance, invasive growth and cell

migration. Regulates the migration of sympathetic neurons, but not of neural crest

precursors. Required for normal dendrite spine morphology in pyramidal neurons.

NRP1-- The membrane-bound isoform 1 is a receptor involved in the development of

the cardiovascular system, in angiogenesis, in the formation of certain neuronal circuits

and in organogenesis outside the nervous system. It mediates the chemorepulsant

activity of semaphorins. It binds to semaphorin 3A

EPHA3-- Upon activation by EFNA5 regulates cell-cell adhesion, cytoskeletal

organization and cell migration. Involved in the retinotectal mapping of neurons. May

also control the segregation but not the guidance of motor and sensory axons during

neuromuscular circuit development.

EFN gene family-- A family of receptor tyrosine kinases which are crucial formigration, repulsion and adhesion during neuronal, vascular and epithelial development.Binds promiscuously transmembrane ephrin-B family ligands residing on adjacent cells,

leading to contact-dependent bidirectional signaling into neighboring cells.

NGEF-- Plays a role in axon guidance regulating ephrin-induced growth cone

collapse and dendritic spine morphogenesis

CRK-- The Crk-I and Crk-II forms differ in their biological activities. Crk-II has lesstransforming activity than Crk-I. Crk- II mediates attachment-induced MAPK8 activation,membrane ruffling and cell motility in a Rac-dependent manner. Involved inphagocytosis of apoptotic cells and cell motility via its interaction with DOCK1 andDOCK4

RASA1-- Mutations leading to changes in the binding sites of either protein are

associated with basal cell carcinomas. The protein encoded by this gene is located in

the cytoplasm and is part of the GAP1 family of GTPase-activating proteins

Common elements in the A1C,

CBC, and V1C include the

genes NLN, CNR1, KIDINS220,

and GRIA1.

During 12-25 pcw, SLITRK5 is highly

present in the primary auditory cortex

(A1C). The cerebellar cortex (CBC)

is dominant during 16-24 pcw and

the primary visual cortex (V1C) is

prominent during 16pcw-3yrs.

FYN-- Involved in several biological processes including regulation of cellgrowth and survival, cell adhesion, integrin-mediated signaling, cytoskeletalremodeling, cell motility, immune response and axon guidance.

RAC1-- The protein encoded by this gene is a GTPase which belongs to

the RAS superfamily of small GTP-binding proteins. Members of this

superfamily appear to regulate a diverse array of cellular events, including

the control of cell growth, cytoskeletal reorganization, and the activation of

protein kinases.

Figure A Figure B

Figure D