Research Annual Report 2015

Publication 157

1334-P
Spectral-domain optical coherence tomography reveals prelaminar membranes in
optic nerve head pallor in eyes with retinitis pigmentosa

Al Rashaed S, Khan AO, Nowilaty SR, Edward DP, Kozak I.

Graefes Arch Clin Exp Ophthalmol. 2015 Apr 22.
[Epub ahead of print]

Purpose:
To determine the relationship between prelaminar structural changes of the optic
nerve head (ONH) and optic nerve waxy pallor in retinitis pigmentosa (RP) using
spectral-domain optical coherence tomography (SD-OCT) and fundus photography.

Methods:
An observational cross-sectional case control study of patients with RP with and
without ONH waxy pallor and controls. Subjects underwent clinical examination,
fundus photography, and SD-OCT raster imaging of the ONH. Four independent
specialists reviewed the images in a masked fashion.

Results:
Fifty-five eyes of 31 subjects with RP and 28 eyes of 14 controls were included.
Optic nerve head waxy pallor was seen in 29 RP eyes (52.7 %) and none in controls.
SD-OCT showed a hyper-reflective structure suggestive of a glial membrane-like
structure on the surface of ONH in 16 of RP eyes (55.1 %). In the RP group, there was
a significant positive correlation between the Exome-based case-control association
study using extreme phenotype design reveals novel candidates with protective effect
in diabetic retinopathy

Shtir C, Aldahmesh MA, Al-Dahmash S, Abboud E, Alkuraya H, Abouammoh MA,
Nowailaty SR, Al-Thubaiti G, Naim EA, ALYounes B,Binhumaid FS, ALOtaibi
AB, Altamimi AS, Alamer FH, Hashem M, Abouelhoda M, Monies D, Alkuraya FS

Hum Genet. 2015 Dec 22.
[Epub ahead of print]

Diabetic retinopathy (DR) is a common clinical expression of diabetes mellitus-
induced vasculopathy and is a major cause of vision loss. Significant gaps remain in
our understanding of the molecular pathoetiology of DR, and it is hoped that human
genetic approaches can reveal novel targets especially since DR is a heritable trait.
Previous studies have focused on genetic risk factors of DR but their results have
been mixed. In this study, we hypothesized that the use of the extreme phenotype
design will increase the power of a genomewide search for "protective" genetic
variants. We enrolled a small yet atypical cohort of 43 diabetics who did not develop
DR a decade or more after diagnosis (cases), and 64 diabetics with DR (controls), all
of similar ethnic background (Saudi). Whole-exome sequencing of the entire cohort

Research Department Annual Report 2015

158 Publications

was followed by statistical analysis employing combined multivariate and collapsing
methods at the gene level, to identify genes that are enriched for rare variants in cases
vs.

Controls:
Three genes (NME3, LOC728699, and FASTK) reached gene-based genome-wide
significance at the 10-08 threshold (p value = 1.55 × 10-10, 6.23 × 10-10, 3.21 ×
10-08, respectively). Our results reveal novel candidate genes whose increased rare
variant burden appears to protect against DR, thus highlighting them as attractive
candidate targets, if replicated by future studies, for the treatment and prevention of
DR. Extreme phenotype design when implemented in sequencing-based genome-
wide case-control studies has the potential to reveal novel candidates with a smaller
cohort size compared to standard study designs.
ONH pallor and the presence of a prelaminar structure (p = 0.006).

Conclusions:
There is a presence of glial membrane-like structures on the optic nerve head surface
in eyes with RP compared to healthy eyes. As the presence of glial membranes
correlated with the presence of ONH waxy pallor, in such cases these membranes
might be responsible for ONH waxy pallor.

Retinal structure in young patients aged 10 years or less with Best vitelliform
macular dystrophy

Schatz P, Sharon D, Al-Hamdani S, Andréasson S, Larsen M

Graefes Arch Clin Exp Ophthalmol. 2015 May 5. pp 1-7

Purpose:
Our aim was to analyze retinal structure in young patients with Best disease with
reference to future gene therapy.

Methods:
This was a retrospective observational spectral domain optical coherence tomography
study of four patients aged 10 years or less with Best disease.

Results:
Findings ranged from subtle thickening at the level of the retinal pigment
epithelium-photoreceptor interdigitation line, to subretinal fluid and precipitate-like
changes at the level of the photoreceptor outer segments, and further to choroidal
neovascularization. The photoreceptor inner segment ellipsoid layer could be
visualized seemingly undisturbed above the vitelliform lesions, except in the case of
choroidal neovascularization.

Research Department Annual Report 2015

Publication 159

Conclusions:
Clinical variability is evident even among young patients aged 10 years or less with
Best disease. The earliest structural alterations seem to occur at the level of the
retinal pigment epithelium-photoreceptor interdigitation line. The photoreceptor
inner segment seems to be unaffected unless choroidal neovascularization develops,
which seems promising regarding future gene therapy.

Exome-based case-control association study using extreme phenotype design
reveals novel candidates with protective effect in diabetic retinopathy

Shtir C, Aldahmesh MA, Al-Dahmash S, Abboud E, Alkuraya H, Abouammoh MA,
Nowailaty SR, Al-Thubaiti G, Naim EA, ALYounes B,Binhumaid FS, ALOtaibi
AB, Altamimi AS, Alamer FH, Hashem M, Abouelhoda M, Monies D, Alkuraya FS

Hum Genet. 2015 Dec 22.
[Epub ahead of print]

Diabetic retinopathy (DR) is a common clinical expression of diabetes mellitus-
induced vasculopathy and is a major cause of vision loss. Significant gaps remain in
our understanding of the molecular pathoetiology of DR, and it is hoped that human
genetic approaches can reveal novel targets especially since DR is a heritable trait.
Previous studies have focused on genetic risk factors of DR but their results have
been mixed. In this study, we hypothesized that the use of the extreme phenotype
design will increase the power of a genomewide search for "protective" genetic
variants. We enrolled a small yet atypical cohort of 43 diabetics who did not develop
DR a decade or more after diagnosis (cases), and 64 diabetics with DR (controls), all
of similar ethnic background (Saudi). Whole-exome sequencing of the entire cohort
was followed by statistical analysis employing combined multivariate and collapsing
methods at the gene level, to identify genes that are enriched for rare variants in cases
vs.

Controls:
Three genes (NME3, LOC728699, and FASTK) reached gene-based genome-wide
significance at the 10-08 threshold (p value = 1.55 × 10-10, 6.23 × 10-10, 3.21 ×
10-08, respectively). Our results reveal novel candidate genes whose increased rare
variant burden appears to protect against DR, thus highlighting them as attractive
candidate targets, if replicated by future studies, for the treatment and prevention of
DR. Extreme phenotype design when implemented in sequencing-based genome-
wide case-control studies has the potential to reveal novel candidates with a smaller
cohort size compared to standard study designs.

Research Department Annual Report 2015

160 Publications

1558-Rv
Update on wide- and ultra-wide filed retinal imaging

Shoughy SS, Arevalo JF, Kozak I.

Indian J Ophthalmol. 2015 Jul;63(7):575-81. doi: 10.4103/0301-4738.167122.

The peripheral retina is the site of pathology in many ocular diseases and ultra-
widefield (UWF) imaging is one of the new technologies available to ophthalmologists
to manage some of these diseases. Currently, there are several imaging systems used
in practice for the purpose of diagnostic, monitoring disease progression or response
to therapy, and telemedicine. These include modalities for both adults and pediatric
patients. The current systems are capable of producing wide- and UWF color fundus
photographs, fluorescein and indocyanine green angiograms, and autofluorescence
images. Using this technology, important clinical observations have been made
in diseases such as diabetic retinopathy, uveitides, retinal vascular occlusions and
tumors, intraocular tumors, retinopathy of prematurity, and age-related macular
degeneration. Widefield imaging offers excellent postoperative documentation of
retinal detachment surgery. New applications will soon be available to integrate this
technology into large volume routine clinical practice.

Causes of elevated intraocular pressure following implantation of phakic
intraocular lenses for myopia

Almalki S, Abubaker A, Alsabaani NA, Edward DP.

Int Ophthalmol. 2015 Aug 12
[Epub ahead of print]

The purpose of this study is to present the causes and visual acuity outcomes in
patients with elevated intraocular pressure (IOP) following implantable collamer
lens (ICL) implantation. A chart review identified patients who developed high IOP
at any postoperative examination and a minimum follow-up period of 3 months
after ICL implantation. Data are reported out to 6 months postoperatively. Outcome
measures included causes of elevated IOP, best-corrected visual acuity (BCVA) at
last visit, number of glaucoma medications, other interventions, and glaucomatous
damage. Elevated IOP occurred in 58 (10.8 %) of 534 eyes that received ICL. The
mean age was 28 ± 7.2 years. The preoperative IOP was 16.3 ± 1.2 mmHg. Elevated
IOP most commonly occurred on the first postoperative day (23/58 (39.7 %) eyes)
due to retained viscoelastic. This was followed by steroid response in 22/58 (37.9
%) eyes at 2-4 weeks postoperatively. IOP elevation in 6 (10.3 %) eyes was related
to high ICL vault and pupillary block, and in 4 (6.9 %) eyes due to synechial angle
closure. At last visit, BCVA was 20/40 or better in 56/58 (96.6 %) eyes, and 5/58
(8.6 %) eyes remained on glaucoma medications due to persistent steroid response

Research Department Annual Report 2015







































































196 Publications

1441-CR
Vision-Threatening Massive Orbital Emphysema after Infero-Medial Orbital
Decompression associated with sneezing

Antonio Augusto V. Cruz, Patricia M. S. Akaishi, Alicia Galindo-Ferreiro,
Mohammed Al-Dufaileej

Ophthal Plast Reconstr Surg, 2015 Sep-Oct;31(5):e139; DOI: 10.1097/IOP.0000000000000393

Orbital emphysema is a well-known cause of orbital compartment syndrome. A
literature review (165 articles) shows that air can enter the orbit in a large variety
of situations including fractures, surgeries (neuro, retinal, thoracic, nose, sinus,
and dental procedures), violent Valsalva maneuvers (such as during weight lifting,
nose blowing, sneezing), accidents with highly compressed air, self-inflicted injuries
(Munchausen syndrome), infections, atmospheric pressure changes, and sinus
diseases. For most of the cases, the causative mechanism is due to a bony defect on
the medial or inferomedial orbital wall. However, it is curious to notice that orbital
emphysema is a rare complication of orbital decompression despite removal of large
portions of these walls. We are aware of just 4 patients who had orbital emphysema
right after inferomedial decompression. Figure 1 shows a Saudi male patient
with Graves orbitopathy who developed massive orbital emphysema on the fifth
postoperative day after an uneventful transconjunctival inferomedial decompression.
The patient had allergic rhinitis, and the emphysema appeared after a severe episode
of sneezing. The preoperative visual acuity, which was 20/30 OU had dropped in
the affected eye to 20/800. The impressive emphysema was successfully reduced
with cantholysis, air–needle aspiration, and nasal packing with full recovery of the
visual acuity.

Clinical Characterization of LRPAP1-Related Pediatric High Myopia

Khan AO, Aldahmesh MA, Alkuraya FS

Ophthalmology. 2015 Aug 11. pii: S0161-6420(15)00665-X. doi: 10.1016/j.
ophtha.2015.06.051
[Epub ahead of print]

No abstract available.

Research Department Annual Report 2015

Publication 197

Generating Recombinant Antibodies against Putative Biomarkers of Retinal
Injury

Kierny MR, Cunningham TD, Bouhenni RA, Edward DP, Kay BK

PLoS One 2015 Apr 22;10(4):e0124492. doi: 10.1371/journal.pone.0124492. eCollection
2015

Candidate biomarkers, indicative of disease or injury, are beginning to overwhelm
the process of validation through immunological means.Recombinant antibodies
developed through phage-display offer an alternative means of generating monoclonal
antibodies faster than traditional immunization of animals. Peptide segments of
putative biomarkers of laser induced injury in the rabbit, discovered through mass
spectrometry, were used as targets for a selection against a library of phage-displayed
human single-chain variable fragment (scFv) antibodies. Highly specificantibodies
were isolated to four of these unique peptide sequences. One antibody against
the retinal protein, Guanine Nucleotide-Binding Protein Beta 5 (GBB5), had a
dissociation constant ~300 nM and recognized the full-length endogenous protein
in retinal homogenates of three different animal species by western blot. Alanine
scanning of the peptide target identified three charged and one hydrophobic amino
acid as the critical binding residues for two different scFvs. To enhance the utility of
the reagent, one scFv was dimerized through a Fragment-crystallizable hinge region
(i.e., Fc) and expressed in HEK-293 cells. This dimeric reagent yielded a 25-fold
lower detection limit in western blots.

Research Department Annual Report 2015

198 Publications

1232-R
Altered expression of fibrosis genes in capsules of failed ahmed glaucoma valve
implants

Mahale A, Othman MW, Al Shahwan S, Al Jadaan I, Owaydha O, Khan Z, Edward
DP.

PLoS One. 2015 Apr 16;10(4):e0122409. doi: 10.1371/journal.pone.0122409. eCollection
2015.

Purpose:
Ahmed glaucoma valve (AGV) implant is an aqueous shunt device used to
control intraocular pressure in glaucoma. Implant failure results from impervious
encapsulation of the shunt plate causing increased hydraulic resistance and raised
intraocular pressure. We hypothesized that deregulation of fibrosis pathway
contributes to capsular resistance. We tested this by studying fibrosis related gene
expression in failed AGV implants.

Methods:
Differential gene expression was examined in failed AGV capsules and compared
to normal control tenon. Following total RNA extraction, 84 key genes in fibrosis
pathway were examined by real-time PCR using RT2 Profiler PCR Array. Relative
gene expression was calculated using δδct method. Gene specific taqman assays
were used to validate select genes with ≥2 fold differential expression in the array
expression profile.

Results:
We observed differential expression in several genes in the fibrosis pathway. Almost
half (39/84) of examined genes showed ≥2 fold differential expression in majority
of capsules examined on the array. Taqman assays for select genes including CCN2
(CTGF), THBS1, SERPINE1, THBS2, COL3A1, MMP3, and IL1A in an increased
validation sample set showed significant changes in expression (p value from <0.001
to 0.022) at a high frequency in concurrence with our array results.

Conclusions:
Pathway-focused analyses identified candidate genes with altered expression
providing molecular evidence for deregulation of the fibrosis pathway in AGV
failure.

Research Department Annual Report 2015

Publication 199

1244-R
MicroRNA Profiling in Intraocular Medulloepitheliomas

Edward DP, Alkatan H, Rafiq Q, Eberhart C, Al Mesfer S, Ghazi N, Abu Safieh L,
Kondkar AA, Abu Amero KK.

PLoS One. 2015 Mar 25;10(3):e0121706. doi: 10.1371/journal.pone.0121706.
eCollection 2015.

Material and methods:
Total RNA was extracted from formalin fixed paraffin embedded (FFPE) intraocular
ME (n=7) and from age matched ciliary body controls (n=8). The clinical history
and phenotype was recorded. MiRNA profiles were determined using the Affymetrix
GeneChip miRNA Arrays analyzed using expression console 1.3 software. Validation
of significantly dysregulated miRNA was confimed by quantitaive real-time PCR.
The web-based DNA Intelligent Analysis (DIANA)-miRPath v2.0 was used to
perform enrichment analysis of differentially expressed (DE) miRNA gene targets in
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway.

Results:
The pathologic evaluation revealed one benign (benign non-teratoid, n=1) and six
malignant tumors (malignant teratoid, n=2; malignant non-teratoid, n = 4). A total
of 88 miRNAs were upregulated and 43 miRNAs were downregulated significantly
(P<0.05) in the tumor specimens. Many of these significantly dysregulated
miRNAs were known to play various roles in carcinogenesis and tumor behavior.
RT-PCR validated three significantly upregulated miRNAs and three significantly
downregulated miRNAs namely miR-217, miR-216a, miR-216b, miR-146a, miR-
509-3p and miR-211. Many DE miRNAs that were significant in ME tumors showed
dysregulation in retinoblastoma, glioblastoma, and precursor, normal and reactive
human cartilage. Enriched pathway analysis suggested a significant association of
upregulated miRNAs with 15 pathways involved in prion disease and several types of
cancer. The pathways involving significantly downregulated miRNAs included the
toll-like receptor (TLR) (p<4.36E-16) and Nuclear Factor kappa B (NF-κB) signaling
pathways (p<9.00E-06).

Conclusions:
We report significantly dysregulated miRNAs in intraocular ME tumors, which
exhibited abnormal profiles in other cancers as well such as retinoblastoma and
glioblastoma. Pathway analysis of all dysregulated miRNAs shared commonalities
with other cancer pathways.

Research Department Annual Report 2015