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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