Research Annual Report 2015

118 Research Projects

KKESHJHU 03-11
Role of ANGPTL4 in the Promotion of Proliferative Diabetic Retinopathy

King Khaled Eye Specialist Hospital Investigators: Abdulelah Al-Abdullah, MD (PI);
Saba Al-Rashaed, MD (PI); Igor Kozak, MD; Alka Mahale, PhD

John Hopkins University Investigators: Akrit Sodhi, MD (PI); Ingrid Sodhi, MD;
Fernando Arevalo, MD; Ashley Behrens, MD; Elizabeth Wellmann; Savalan
Babapoor

Purpose :
Angiopoietin-like 4 (ANGPTL4), a unique cytokine has been recently found by our
JHU colleagues to be potently upregulated in mouse models of ischemic retinal
disease (Xin et. al. PNAS, 2013). ANGPTL4, strongly promote angiogenesis, similar
to vascular endothelial growth factor (VEGF). In this proposal, we will determine the
relative contribution of ANGPTL4 to the promotion of retinal neovascularization in
patients with proliferative diabetic retinopathy (PDR), an essential first step for the
assessment of therapies targeting ANGPTL4 for the treatment of PDR.

Objectives:
To evaluate the relative expression of ANGPTL4, VEGF, and other proteins in the
ocular fluid (aqueous and vitreous) in patients with and without diabetic retinopathy.

Methods:
This is a cross-sectional study in which will we will collect ocular samples (aqueous
and vitreous) from patients going for cataract surgery and/ or pars plana vitrectomy.
Systemically healthy patients undergoing either procedure will form the control
group, Diabetics without DR or with varying degrees of DR will form the study
groups. Informed consent will be obtained, the data collection sheets will be filled and
appropriate ocular sample will be obtained during the scheduled operations. These
samples will be analyzed to study the levels of ANGPTL4 and vascular endothelial
growth factor and to correlate them with the status of diabetic retinopathy and to
compare that with the control groups.

Outcomes:
The results of this study will help us to understand better the pathogenesis of diabetic
retinopathy, angiogenesis and diabetic maculopathy.

Progress to date:
The IRB approval has been obtained at KKESH, patient recruitment is ongoing.
Up-to-date 10 percent from the total sample was recruited. The plan to start the
analyses of the available samples as soon as the needed reagents are available.

Research Department Annual Report 2015

Research Projects 119

KKESHJHU 03-16
Genetics of Congenital Glaucoma in Saudi Arabia II: Screen for Modifiers of
CYP1B1

King Khaled Eye Specialist Hospital Investigators: Leyla Al-Jasim, MD (PI); Ohood
Owaydhah, MD; Leen Abu Safieh, PhD

John Hopkins University Investigators: Cindy Berlinicke, PhD (PI); Jiang Qian, PhD;
Randy Craven, MD; Deepak Edward, MD

Purpose:
PCG is more common in Saudi Arabia than in many other parts of the world, and
in Saudi Arabia CYP1B1 is by far the major cause of PCG.. Unlike some other
populations, in some Saudi families mutations in CYP1B1 are not completely
penetrant. One explanation for this phenomenon is that modifying loci can “protect”
against PCG-associated CYP1B1 mutations, and thereby modify the clinical
phenotype.

Objectives:
1. Identify affected and non-affected individuals with identical CYP1B1 PCG-
associated mutations.
2. Perform whole-exome sequencing on individuals identified in objective #1.
3. Conduct exome-wide association analysis of the data obtained in objective #2 to
identify candidate genetic variants associated with the PCG clinical phenotype.

Methods:
1. If the patient qualifies (PCG & JOAG) and is willing to participate, written
consent will be obtained at this time. A medical and family history questionnaire
will be given at this time.
2. Blood (2x5ml) will be obtained from the patients’ arm by standard venipuncture,
by a trained phlebotomist, and will be maintained and used for DNA analysis,
prepared according to standard protocols. The sample will be labelled with an
identifying nmber as the sole identifier. The link of this identifier to the patient
and his/her medical records will be maintained in a secure file maintained at
KKESH. Personnel at JHU will not have access to this code, or any Protected
Health Information (PHI) identifiers.
3. Charts are reviewed for characterization and phenotyping of the participant’s
PCG. Any health information seen by JHU personnel will be de-identified.

Outcomes:
Identification of such a genetic modifier (or modifiers) could provide clues about the
mechanism of PCG pathogenesis and potential leads for treatment of this potentially
blinding disease.

Progress to date:
Recruitment finished, blood sample collected, sequencing finished & data collected
which will be sent to JH lab to screen for modifiers.

Research Department Annual Report 2015

120 Research Projects

KKESHJHU 03-19
Serum Biomarkers for Retinitis Pigmentosa

King Khaled Eye Specialist Hospital Investigators: Igor Kozak, MD (PI); Leen Abu
Safieh, PhD; Saba Al-Rashaed, MD

John Hopkins University Investigators: Donald Zack, PhD (PI); Fernando Arevalo,
MD; Baranda Hansen

Purpose:
Increase our understanding of and improve the treatment of Retinitis Pigmentosa,
which is a blinding and currently untreatable disease.

Objectives:
Identify sensitive and reliable blood biomarkers and molecular pathway indicators
for Retinitis Pigmentosa.

Methods:
Collect blood samples from well phenotyped Retinitis Pigmentosa patients and
perform RNA Next Generation Sequencing (RNA-seq) on these samples.

Outcomes:
Large amounts of RNA-seq data will be generated. Bioinformatic analysis will
hopefully identify changes in gene expression associated with Retinitis Pigmentosa,
potentially changes that are specific to different stages of RP and/or specific to
specific genetic forms of Retinitis Pigmentosa.

Progress to date:
After obtaining IRB approval from both KKESH and JHU, recruitment has started
at KKESH and continued with focus on autosomal recessive patients with RP and
their relatives. The recruitment consisted of signing informed consent, collection
of clinical data, ocular imaging and collection of blood samples. Blood samples
and associated medical records have been obtained from the first set of Retinitis
Pigmentosa patients, and samples have also been obtained from appropriate relatives
and control individuals. Preparation of cDNA libraries from these samples is
underway and DNA sequencing of the libraries will begin soon. Then bioinformatic
analysis of these samples, as well as new samples that we are continuing to obtain
from additional patients and control individuals, will be performed. After analysis,
we plan to present the results at ARVO and other vision science and ophthalmology
meetings.

Research Department Annual Report 2015

Research Projects 121

KKESHJHU 04-20
Identification of Key Molecules Driving Pterygium Formation and Progression

King Khaled Eye Specialist Hospital Investigators: Samar Al-Swailem, MD (PI); Omar
Kirat, MD; Alka Mahale, PhD

John Hopkins University Investigators: Elia Duh, MD (PI); Samuel Yiu, MD

Purpose :
To identify key molecules that drive and contribute to the formation and progression
of pterygium, focusing on surgical pterygium specimens. This study will investigate
the expression of multiple candidate molecules, with particular emphasis toward
molecules that are especially amenable to therapeutic modulation.

Objectives:
We will perform analyses of COX-1 and COX-2, IL-1β, TNFα, PlGF, IL-10, TSP-1,
NLRP3 inflammasome (TLR4, NLRP3, activated Caspase-1, IL-18, IL-1β), RAGE
ligands (especially HMGB1), for which drugs are currently available.

Methods:
We propose to obtain both primary and recurrent surgical pterygium specimens (20-
30), as well as control conjunctival specimens, and perform molecular analyses:

• Protein levels of each molecule of interest, using western blotting.
• In the event that a suitable antibody is not available for western blotting,

quantitative reverse-transcription/real-time PCR (qRT-PCR) will be used to
look at the RNA levels of the given molecule.
• If a given molecule is found to be increased (or decreased) by western blot/
qRT-PCR techniques, we will perform complementary immunohistochemistry
studies, to provide insights into the tissue localization of the molecules and the
cell types that express the molecules

Outcomes:
We anticipate that this research project will provide insights that will be directly
relevant to Saudi Arabia, leading to new therapeutic strategies to improve ophthalmic
care in the Saudi Arabia.

Progress to date:
We recruited specimens (pterygia & control) from 4 eyes.

Research Department Annual Report 2015