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Fatuma-Ayaan Rinderknecht
1701 Arena Drive
Davis,CA 95618
Harvard University
Validation
of
rare
Variants
in
the
Schizophrenia-­‐linked
gene
DPYSL2
Fatuma-­‐Ayaan
Rinderknecht,
Xuan
Pham
and
Dimitrios
Avramopoulos
Johns
Hopkins
University
Institute
of
Genetic
Medicine
*Note: This is a project I completed while doing a six-week internship at the
Johns Hopkins Institute of Genetic Medicine in the summer of 2012; this project was
originally presented in a poster form so I have transferred the information and results to
essay form. This internship was granted as part of acceptance into the CTY Centers
Scholars Program. The lab I worked in had a focus on neurobiological diseases such as
Schizophrenia and Alzheimer’s. I specifically chose this lab because of its focus on
neurodegenerative diseases. In college I hope to pursue a major in Neurobiology and
minor in Psychology and/or Cognitive Sciences, and go on to a joint MD/Ph.D. program.
While working in the lab this summer, I met many individuals who had their MD/Ph.D.
who were both conducting scientific research and meeting with patients. After
experiencing this, I was inspired to do the same and able to concrete my career goals. The
lab is continuing this research.
Abstract
The Dihydropyrimidinase-like protein-2 (DPYSL2) gene has become increasingly
interesting to researchers in recent years after several studies have linked it to
Schizophrenia (SZ)1
. We have collected DNA samples from a homogenous population of

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Ashkenazi Jews to search for variations in the DPYSL2 gene of case and control subjects
via Next Generation Sequencing. We chose four rare variants to validate with Sanger
Sequencing, in hopes to identify functional coding sequence variation in DPYSL2 that
might be involved in SZ for further study. One out of these four variants was positively
validated.
Introduction
Schizophrenia is a chronic psychiatric disorder that affects around 1% of the
population worldwide. The disorder is characterized by a breakdown of thought processes
and by poor emotional responsiveness. SZ is at least 70% heritable but it has been
difficult to identify susceptibility loci because of its environmental influences, phenotypic
variations, and genetic heterogeneity. A recent study suggested linkage to SZ
susceptibility region on chromosome 8p21. This 8p21 peak region was tested for linkage
by using a family-based association study using a European-Caucasian population and
also a case-control association study in Ashkenazim Jew population, which is more
homogenous. The study concluded that linkage was strongest in the region of the
chromosome containing rs1561817 to rs9797. DPYSL2 lie within this region of
chromosome 8, which is why it has become of interest to those researching linkage in
SZ2
.
DPYSL2 codes for a protein that resides predominantly within the central nervous
system. DPYSL2 has been shown to affect Ca2+
homeostasis, which when deregulated
becomes a primary disorder of SZ3
.

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Next Generation Sequencing (NGS) is a new high-throughput technology that
allows for more DNA to be sequenced quicker and at a lower cost. Next Gen Sequencing
can be used to identify rare and potentially functional variants4
. We identified variants in
exons; splice sites and highly conserved regions, as candidate Single Nucleotide Variants
or SNVs for validation. These variants were verified using Sanger Sequencing and Codon
Code Aligner.
Samples and Methods
The DNA was collected from 384 affected and 384 unaffected, unrelated
individuals of Ashkenazi Jewish descent. Fourteen exons and 27 conserved non-coding
regions in the DPYSL2 gene were amplified and combined into 24 tagged libraries
containing 32 case or 32 control samples per pool, each pool was sequenced using Next
Generation Sequencing. The reads from the sequencing were aligned to a reference
genome using aligner software Bowtie. They were then processed using SAMtools and
visualized with IGV (Integrative Genome Viewer).
Four Single Nucleotide Variants were chosen to be verified if they met certain
requirements such as location in exons, splice sites, or in highly conserved regions. To
make sure that the variants were true, Sanger sequencing was performed on each
individual from certain pools containing the SNV, using the same primers used to
amplify the sequences. Sanger Sequencing is necessary to determine which subject in the
pool carries the SNV, and find out the true frequency of the SNV in that pool. Sanger
Sequencing is also more exact than NGS, which allows researchers to be surer of their

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results with Sanger. The four SNVs that were re-sequenced were then visualized and
analyzed with Codon Code Aligner.
Results
One out of the four variants was validated using Sanger Sequencing and Codon
Code aligner. Not all the samples were sequenced successfully, which also contributed to
the lack of validation in three out of the four variants. The inability to verify the other
variants could also point to errors in Next Generation Sequencing as it might have
identified variants that were false positives. . Each variant had an expected frequency
shown in IGV, and the confirmed variant did have a frequency in Sanger sequencing
close to its expected one from IGV. (See Table 1, Figure 1) The variant that was
confirmed is rs113199330 and is expected to be involved in exon-splice enhancement, as
predicted by the bioinformatics software, ESEFinder. (see Figure 2)
Conclusion
In this project, the DNA from a controlled population was sequenced and scanned
for variants in the coding regions of DPYSL2. Four SNVs were chosen for validation,
and one out of the four was confirmed as a true SNV in our case pool. This variant is of
interest to researchers because of its location in the gene. It is at the beginning of the exon
4 and the rare allele is predicted to have a lowered SRSF1 binding score, as predicted
using the bioinformatics software ESEFinder. (see Figure 2).
Future work includes plans to find SNVs in the conserved non-coding regions of
DPYSL2, and validate those using similar criteria and methods. Future work concerning

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this specific SNV includes plans to generate constructs to determine the exact effect of
this variant on the transcript, study cell morphology in response to the SNV, and relate
genotype with phenotype. With new information about these variants, researchers can
continue to discover more about the DPYSL2 gene and its effect on Schizophrenia.
Figures:
Table.1. Results for each variant
Figure.1.Verified Heterozygous Variant in Pool 12, shown in Codon Code
Fig.2. Predicted Splicing Factor Binding Score for variant rs113199330 without SNV
(left) and with SNV (right) (ESEFinder 3.0.)
Figure.3.Key

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References:
1. 13q32 and 8p21." Nature Genetics (1998): 70-74.
2. Fallin, Daniele, Virginia Lasseter, Yaping Liu, and Dimitrios Avramopoulos.
"Linkage and Association on 8p21.2-p21.1 in Schizophrenia." American Journal
of Medical Genetics (2010): 188-95.
3. Hensley, Kenneth, Kalina Venkova, Alexandar Christov, William Gunning, and
Joshua Park. "Collapsin Response Mediator Protein-2: An Emerging Pathologic
Feature and Therapeutic Target for Neurodisease Indications." Molecular
Nurobiology (2011): 180-89.
4. Metzker, Michael L. "Sequencing Technologies — the next Generation." Nature
Reviews Genetics 11.1 (2009): 31-46.
Contact Information for Instructor:
Dimitrios Avramopoulos M.D. Ph.D
733 N.Broadway, BRB-507, Baltimore,MD 21205
tel 410 955-8323
Email: adimitr1@jhmi.edu