American University of Science and Technology
Faculty of Health Sciences
Department of Laboratory Science and Technology
Mutational Analysis of Keratoconus in the Lebanese Population
Final Report
CNRS Project Number ‫ ص‬/5692 (Aug 31, 2012)
Principal Investigator: Dr. Gretta Abou-Sleymane
Nov 30, 2013
2
Report
‫ كما ورد في نص وثيقة المشروع األساسية؛‬،‫ عنوان المشروع بالكامل باللغتين‬-1
‫تحليل التشوهات الجينية للقرنية المخروطية في الشعب اللبناني‬
Mutational Analysis of Keratoconus in the Lebanese population
‫ األهداف التي كانت مرجوة من المشروع والنواتج التي كانت مقررة ومدة البحث كمثا وردت في‬-5
‫النص األساسي للمشروع؛ وما تحقق منها في نهاية المشروع؛‬
We intended through this project to participate in elucidating the main genes involved
in the pathogenesis of keratoconus (KC) disease and to reveal the types of mutations
specific for Lebanese patients, as a first step in establishing a testing protocol and
potential therapeutic strategies.
A number of candidate genes has been related to KC (Bisceglia, L. et al., 2005; Eran,
P. et al., 2008; Mok, JW. et al., 2008; Paliwal, P. et al., 2009; Udar, N. et al., 2006)
Among them, two genes are mainly suspected and were investigated in different
populations; SOD1 (Superoxide Dismutase1) an antioxidant enzyme responsible for
destroying free superoxide radicals in the cell (Johnson, F. & Giulivi, C., 2005; You, Z.
et al., 2010) and VSX1 (Visual System Homeobox1) a transcription factor that plays a
critical role in ocular development (Barbaro, V. et al., 2006; Hayashi, T. et al., 2000).
Therefore, we aimed through a two years project to test for mutations in SOD1 and
VSX1 genes by direct sequencing in Lebanese patients. Moreover, we intended to select
a third candidate gene through genome wide sequencing to be conducted by our
collaborators in Leeds, UK on Lebanese patients.
Our study will therefore reveal whether the previously described mutational pattern(s)
or other genetic factors are involved in the development of KC in the Lebanese
population.
During one year of grant contract, three different phenol-chloroform DNA extraction
protocols were tested and optimized in order to determine the one that yields the highest
quantity of pure DNA. The first protocol is a 2 days long extraction procedure, the
second one is a shorter Federal Bureau of Investigation (FBI) protocol, and the third
1
one is an in-house optimized version of the FBI protocol. The extraction results were
assessed by spectrophotometric analyses and the third protocol was adopted as it
showed the highest quantity and purity of DNA. Representative results obtained from
the three protocols are represented in Table 1.
Table1. Comparison between different extraction protocols.
Concentration
A260/280
A260/230
Protocol 1
6 – 7 ng/µl
1.20 – 1.38
(-1.26) – (-1.13)
Protocol 2
18 – 27 ng/µl
0.70 – 0.78
0.1 – 0.2
Protocol 3
80 – 220 ng/µl
1.80 – 1.90
2.0 – 2.3
60 blood samples were collected from Lebanese families belonging to different ethnical
groups. The patients’ diagnosis was based on clinical examination that was conducted
in collaboration with specialized ophthalmologists. DNA was successfully isolated
from all collected samples (Table 2).
Table 2. Representative data for Spectrophotometric measurements of samples’ DNA.
Sample
1
2
3
4
5
6
7
8
9
10
Concentration
168.5 ng/µl
211.9 ng/µl
172.3 ng/µl
113.8 ng/µl
182.7 ng/µl
128.8 ng/µl
102.7 ng/µl
209.8 ng/µl
87.7 ng/µl
198.5 ng/µl
A260/280
1.82
1.85
1.82
1.88
1.83
1.83
1.80
1.86
1.85
1.90
A260/230
2.00
2.31
2.01
2.31
2.10
2.33
2.34
2.34
2.28
2.23
Subsequent to the optimization of the DNA extraction protocol, we proceeded with
mutational screening for SOD1 gene. Nine familial KC patients, one sporadic KC
patient, two related controls and one unrelated control were first tested. In some
samples, the sequencing results showed changes in either of the forward or the reverse
sequences, without confirmation by the opposite sequence. Representative results for
exon 3 are shown in table 3. Such type of changes is considered as sequencing artifacts.
Interestingly, a heterozygous change which was confirmed by both reverse and forward
primers, was detected in exon 5 (13915 T-A) of two patients belonging to the same
2
family (Table 4). We are currently conducting further studies to identify the impact of
this substitution.
Table 3. Comparison of patients and controls sequences to the RefSeq for exon 3.
Member
Patient 1
Patient 2
Family 1
Patient 3
Patient 4
Patient 1
Patient 2
Family 2
Related Control
Patient 1
Patient 2
Family 3
Patient 3
Related Control
Sporadic Case
Unrelated Control
3F
3R
11938.1 T
11769.1 C
11911 T-del
11770 A-del
11911 T-del
11771 A-del
11865 C-del
The table shows the detected changes in exon 3 of the 13 analyzed patients. In the table, F stands for forward
primer, R stands for reverse primer, deletion is represented by position Base-del, substitution is represented
by position Base.Base and insertion is represented by position Base.1.
Table 4. Comparison of patients and controls sequences to the RefSeq for exon 5.
Member
5F
Patient 1
Patient 2
13824 A-del
Family 1
Family 2
Patient 3
13823 A-del
11836 A-del
Patient 4
13824 A-del
Patient 1
13915 T-A
Patient 2
13915 T-A
13822 A-del
5R
13915 T-A
14301 T-del
13915 T-A
Related Control
Patient 1
Family 3
13824 A-del
13839 A-del
14260.1 C
14296.1 C
13986.1
13824 A-del
11245.1 C
14277.1 T
13935.1 G
13967 T-G
Patient 2
Patient 3
3
13795 G-del
13809 T-del
13813 T-G
Related Control
13824 A-del
13824 A-del
Sporadic Case
Unrelated Control
The table shows the detected changes in exon 5 of the 13 analyzed patients. In the table, F stands for forward
primer, R stands for reverse primer, deletion is represented by position Base-del, substitution is represented
by position Base.Base and insertion is represented by position Base.1. The changes that were confirmed by
both forward and reverse primers are highlighted in yellow.
Currently, the optimization of the PCR and sequencing conditions for VSX1 is ongoing. In
parallel, five samples have already been analyzed by next generation sequencing and 10
additional samples are currently being tested using the same technique. The interpretation of
the obtained data is in progress.
The outputs and achievements with their corresponding planned time as submitted in the grant
research proposal are presented in table 6.
Table 6. Summary table of the planned activities with their corresponding progress.
Output
Mutational screening
for SOD1 gene
Activity Description
DNA extraction from
patients and controls
PCR for SOD1 exons
and intron-exon
junctions
Sequencing of PCR
products
Statistical analyses
Setting up PCR and
sequencing conditions
for VSX1 gene
Mutational screening
for VSX1 gene
DNA extraction from
patients and controls
PCR for VSX1 exons
and intron-exon
junctions
Sequencing of PCR
products
Statistical analyses
Setting up PCR and
sequencing conditions
for the novelgene of
4
Planned
Progress
Completed
Year 1
Completed for 13
samples.
Further analysis is
ongoing
4th term of year 1
&
st
1 term of year 2
In progress
(note that the grant
was approved for
1 year only)
Year 1
Completed
2nd term of year 2
3rd & 4th terms of
year 2
In progress
(note that the grant
was approved for
1 year only)
2nd term of year 2
interest according to
next generation
sequencing and
transcriptome
analyses
Mutational screening
for the novel gene of
interest
DNA extraction from
patients and controls
PCR for novel gene
exons and intron-exon
junctions
Sequencing of PCR
products
Statistical analyses
Year 1
Completed
3rd term of year 2
In progress
(note that the grant
was approved for
1 year only)
3rd & 4th terms of
year 2
References
Barbaro, V, Di Iorio, E, Ferrari, S, Bisceglia, L, Ruzza, A, De Luca, M, Pellegrini, G (2006).
Expression of VSX1 in human corneal keratocytes during differentiation into myofibroblasts
in response to wound healing. InvestOphthalmol Vis Sci., 47(12):5243-50.
Bisceglia, L, De Bonis, P, Pizzicoli, C, Fischetti, L, Laborante, A, Di Perna, M, Giuliani, F,
DelleNoci, N, Buzzonetti, L, Zelante, L (2009).Linkageanalysis in keratoconus: replication
of locus5q21.2 and identification of othersuggestiveLoci. Invest Ophthalmol Vis Sci.,
50(3):1081-6.
Eran, P, Abu Almogit, David, Z, ReznikWolf, H, Hana, G, Yaniv, B, Elon, P, Isaac, A
(2008). The D144E Substitution in the VSX1 Gene: A Non-pathogenic Variant or a Disease
Causing Mutation? Ophthalmic Genetics, 29:53–59.
Hayashi, T, Huang, J, Deeb, SS (2000). RINX (VSX1), a novel homeobox gene expressed
in the inner nuclear layer of the adult retina. Genomics, 67:128-39.
Johnson, F and Giulivi, C (2005). Superoxide dismutases and their impact upon human
health. Mol Aspects Med., 26(4-5):340-52.
Mok, JW, Baek, SJ, Joo, CK (2008). VSX1 gene variants are associated with keratoconus in
unrelated Korean patients. J Hum Genet, 53:842–849.
Paliwal, P, Singh, A, Tandon, R, Titiyal, JS, Sharma, A (2009). A novel VSX1 mutation
identified in an individual with keratoconus in India. Molecular Vision, 15:2475-2479.
Udar, N, Kenney, MC, Chalukya, M, Anderson, T, Morales, L, Brown, D, Nesburn, A,
Small. K (2004). Keratoconus—No Association with the Transforming Growth Factorβ–
Induced Gene in a Cohort of American Patients. Cornea, 23(1):13-7.
You, Z, Cao, X, Taylor, AB, Hart, PH, Levine, RL (2010). Characterization of a Covalent
Polysulfane Bridge in Cu, Zn Superoxide Dismutase. Biochemistry, 16; 49(6): 1191.
5
‫ مثج ا شثارة لث وث وبات‬،‫ ولكثن بولثوو‬،‫ الخطثوات التثي نجث ت عمليثاو باختوثار‬-3
،‫ ن وجدت‬،‫واجهثت الباحثين‬
Sampling: Blood samples were collected from 60 Lebanese males and females that were
selected from different ethnical groups and whose diagnosis was based on clinical
examination. The samples included 34 familial patients and 22 related controls belonging to
13 Lebanese families, one sporadic KC patient and 3 unrelated controls.
The collection of the 60 samples representative of the Lebanese population required
extensive collaboration with several ophthalmologists from different regions in Lebanon in
order to cover the major ethnical groups.
DNA Extraction: Three different phenol-chloroform based DNA extraction protocols were
tested in order to select the one that yields the highest quantity of isolated DNA with the
maximum purity. The first protocol is a 2 days long extraction protocol, the second one is a
shorter Federal Bureau of Investigation (FBI) protocol, and the third one we adapted from
the FBI protocol. According to the obtained DNA yield and purity, the third protocol was
adopted and DNA was isolated from the 60 samples.
SOD1 Gene Analysis: The optimized PCR and sequencing conditions for SOD1 gene
amplification were applied to screen 13 samples. The generated sequences were compared
to the SOD1 gene reference sequence (RefSeq GenBank accession number NG_008689.1)
and then compared to each other.
VSX1 PCR and Sequencing Conditions optimization: The corresponding optimization
procedure is still in progress.
Next Generation Sequencing: the analysis/data interpretation of 15 additional samples
through next generation sequencing is currently in progress.
‫ و التثي قثدمت فثي‬/‫ و‬،‫ قائمة بثاألوارق ال لميثة التثي نشثرت فثي المجث ت عات ال قثة‬-4
‫ نسخة طبق األول من األ وارق المنشورة في مج ت‬-2 ‫ اقليمية وعالمية‬،‫مثاتمارت علمية محلية‬
.‫ كما نشرت في تلك المج ت‬،‫محكمة‬
6
Posters Presentation
Graduate Students Open House, American University of Science and Technology, March
2013
Articles
The project has not been published in any peer review journal yet.
As shown in table 6, this project was presented for the CNRS according to a two years plan.
Unfortunately, only part of the grant was approved. We are currently looking for funds in
order to complete the study as outlined in the submitted grant proposal. We believe that upon
completing the project, the results will be published in a peer reviewed journal.
‫اإلستمارة‬
Administrative Information ‫الم‬
‫لومات االدارية‬
01-10-12
:‫المرجج‬
Project Title - (‫عنوان المشروع )عربي وأجنبي‬
‫تحليل التشوهات الجينية للقرنية المخروطية في الشعب اللبناني‬
Mutational analysis of Keratoconus in the Lebanese population
Principal Investigator - ‫الباح الرئيسي‬
‫رقم الهاتف‬
‫العنوان االلكتروني‬
‫العنوان‬
‫الوظيفية‬
‫المؤسسة‬
‫االسم‬
Telephone
e-mail
Address
Post
Institution
Name
+961 1 218716
gretta.abousleymane
Achrafieh,
Chairperson of
@gmail.com
Alfred
the Department University
Naccache
of Laboratory
of Science
Street
Science and
and
Technology
Technology
American
Gretta
Abou
Sleymane
Co-Workers - ‫الباحثون المشاركون‬
‫العنوان االلكتروني‬
e-mail
‫االسم‬
‫المؤسسة‬
Institution
7
Name
University of Leeds
Christopher Francis
Inglehearn
l.f.abifarraj@leeds.ac.uk
University of Leeds
Layal Abi Farraj
nmallah@aust.edu.lb
American University
of Science and
Technology
Narmeen Mallah
c.inglehearn@leeds.ac.uk
: Duration - ‫المدة الت اقدية للمشروع‬
1 year
Scientific Information
‫لمية‬
ّ ‫الم لومات ال‬
Objectives - ‫الهدف‬
This project was initiated in order to investigate the main genes involved in the pathogenesis of
Keratocouns (KC) by conducting the following:
1. Mutational screening for SOD1 (Superoxide Dismutase 1) gene by direct sequencing in Lebanese
patients and controls.
2. Mutational screening for VSX1 (Visual System Homeobox 1) gene by direct sequencing in
Lebanese patients and controls.
3. Selection of an additional candidate gene involved in KC through genome wide sequencing in
collaboration with the group of Professor Christopher Francis Inglehearn at the Ophthalmology
and Neuroscience Section of Leeds Institute of Molecular Medicine.
-‫النجاًاًت المحققة‬
Achievements
8
During one year of grant contract, the following achievements were made:
1.
Collection of 60 peripheral blood samples including:
-
56 samples belonging to 13 Lebanese families: 34 KC patients and 22 related controls
-
1 sporadic KC patient
-
3 unrelated controls
2.
Optimization of a DNA extraction protocol from peripheral blood
3.
DNA isolation from all collected samples.
4.
Sequencing all five exons of SOD1 gene in 13 samples and detecting a change in exon 5 (13915
T-A) in two patients belonging to the same family.
5.
Ongoing optimization for the PCR and sequencing conditions for VSX1 gene.
6.
Testing 15 samples by next generation sequencing.
Perspectives - ‫آفاق البح‬
This project is established in order to elucidate the main genes involved in the pathogenesis of KC in
Lebanese patients.
Publications & - ‫المنشوارت والمساهمات في الماتمارت‬
Communications
Poster presentation
Graduate Students Open House, American University of Science and Technology, March 2013
Abstract - ‫موج عن نتائج البح‬
ً
Keratoconus or KC [MIM 148300] is a non-inflammatory, progressive thinning of the cornea that
results in a conically shaped protrusion. A genetic predisposition to KC has been observed; accordingly,
numerous linkage analysis studies and candidate gene approaches were performed in an attempt to
determine the gene/(s) responsible for the disease. Several genes were reported to be associated with KC
including SOD1 and VSX1 which have been investigated in different populations. Therefore, we aimed
in this study to determine whether these genes are involved in the development of KC in the Lebanese
patients. We also aimed to identify additional candidate genes through next generation sequencing.
Experimentally, we selected the most suitable DNA extraction protocol in terms of DNA purity
and yield. Subsequently, 60 peripheral blood samples were collected from Lebanese males and females
belonging to different ethnical groups. Patients’ selection was based on clinical examination and in
collaboration with specialized ophthalmologists. Genomic DNA was successfully extracted from all
collected samples as revealed by spectrophotometric measurements.
9
PCR and sequencing conditions for the SOD1 gene’s exon–intron junctions and the whole coding
region which includes five exons had been already optimized by our team. 13 samples that include nine
familial KC patients and two related controls belonging to three Lebanese families, one sporadic KC
patient and an unrelated control were subject to SOD1 mutational screening. Patients and controls
sequences were compared to SOD1 gene reference sequence (RefSeq GenBank accession number
NG_008689.1) and then compared to each other. One heterozygous change was detected exclusively in
two patients belonging to the same family. Further analyses are being performed on additional samples in
order to understand the significance of this change in the Lebanese population.
PCR and sequencing conditions for VSX1 gene which comprises seven exons are currently being
optimized. This will be followed by mutational screening of this gene in the collected samples. Likewise,
15 samples are currently being analyzed by next generation sequencing.
Results of both candidate gene and global approaches represent the first step towards the
establishment of a testing protocol and most importantly the elucidatination of the molecular mechanisms
underlying KC pathogenesis.
‫توقيج الباح‬
10