Journal Club

PRESENTER: DR SARATH KRISHNAN MP

Article of Interest
25-06-2023 DR SARATH KRISHNAN M P/JR3/BCHEM/AIIMSR 2

Journal: Eye
Impact factor-4.456(2022 Journal citation reports)
Official journal of “The ROYAL COLLEGE of OPHTHALMOLOGISTS”
Index-Scopus/Medline(PubMed)/Google Scholar
ISSN: 0950-222X, and eISSN: 1476-5454.
Subscription/Open access
Editor-in-Chief: Prof. Sobha Sivaprasad
Moorfields Eye Hospital and University College
London, UK

Level of evidence
*Evidence pyramid. Source: The University of Alabama at Birmingham, available from: https://guides.library.uab.edu/ebd/evidencestrength
Our Article of
Interest

Topic of Research-Keratoconus

Topic of Research-Keratoconus
Exact Pathophysiology: Not properly known
Theories
1. Immunological factors
2. Genetic factors
3. Environmental factors
4. Inflammatory mediators (Cytokines)

Background of the study
Thyroxin was found to cause biochemical changes in the corneal stroma as a
result of thyroxin–receptor interaction
El-Massry A, Doheim MF, Iqbal M, Fawzy O, Said OM, Yousif MO, et al. Association between keratoconus
and thyroid gland dysfunction: a casecontrol study. J Refract Surg. 2020;36:253–7.
Vitamin D enhances VDR and activates autophagic lysosomal clearance in
oxidatively damaged human corneal epithelial cells.
Shivakumar S, Rohit S, Ghosh A, Jeyabalan N. Vitamin D enhances the autophagic lysosomal clearance in
oxidatively stressed human corneal epithelial cells: a therapeutic intervention for keratoconus. Investig
Ophthalmol Vis Sci. 2019;60:2819.

Research Question???
Is there any association between vitamin D level, VDR
polymorphism, and thyroid disorders in KC patients ?

Subjects and Methods
Study design: Prospective, Observational, Cross sectional study
Place of Study: Mansoura ophthalmic center, Mansoura University, Egypt
Time period: March 2021 to September 2021
Sample size calculation: https://www.openepi.com/SampleSize/SSMean.htm
Study Sample size: 177 keratoconus patients + 85 healthy controls

Diagnosis of keratoconus: Slit lamp
Biomicroscopy
Slit Lamp examination: Flat (K1), Steep
(K2), and maximum (Kmax) simulated
keratometric readings, and corneal
pachymetry

CASES-INCLUSION CRITERIA
Slit lamb examination confirmed
cases of Keratoconus
CASES-EXCLUSION CRITERIA
Ophthalmic pathology other than KC
KC with known thyroid disease
KC with known systemic disorders
KC patients who didn't provide
informed consent

CONTROLS-INCLUSION CRITERIA
Healthy individuals with Normal
cornea
CONTROLS-EXCLUSION CRITERIA
With known thyroid disease
With any systemic illness
Healthy individuals who didn't
provide informed consent

Research Algorithm
5ml Venous
blood
3ml on Plain
Vial
Serum
FT3,FT4 &
TSH
Serum
25HCC
2ml on EDTA
vial
PCR-RFLP

Laboratory investigations-Hormonal Study
Hormonal assessment: All cases and controls
FT3; FT4; TSH: ELISA (CTK Biotech (San Diego, CA,USA)).
Interpretation of TFT
Euthyroidism FT3,FT4 & TSH-N/L
Subclinical Hypothyroidism High TSH(FT3 &FT4-N/L)
Overt Hypothyroidism High TSH (Low FT3 and/or FT4)
Subclinical Hyperthyroidism Low TSH (FT3&FT4-N/L)
Overt Hyperthyroidism Low TSH (High FT3 and/or FT4)

Laboratory investigations-Hormonal Study
25HCC: ELISA (MyBioSource (San Diego, CA, USA)).
Interpretation of 25HCC: 2012 American Endocrine Society guidelines
Sufficiency >30ng/ml
Insufficiency 21-30ng/ml
Deficiency <20ng/ml

Laboratory investigations-Genetic Study
(PCR)
VDR gene polymorphism: Taq I (rs731236); Apa I (rs7975232); Bsm I
(rs1544410)
DNA isolation: Gene Jet gene DNA Purification kits -Thermo Scientific (Rath
Business Park, Dublin, Ireland)
Amplification of Genomic DNA- PCR (Thermocycler)
Validation: National center for Biotechnology information
VDR polymorphisms FP RP
Taq I (rs731236) 5′-CGGGGAGTATGAAGGACAAA-3′ 5′-CCATCTCTCAGGCTCC AAAG-3′
Apa I (rs7975232) 5′-CTAGGTCTGGATCCTAAATGCA-3′ 5′-TTAGGTTGGACAGGAGAGAGAA-3′
Bsm I (rs1544410) 5′-CTAGGTCTGG ATCCTAAATGCA-3′ 5′-TTAGGTTGGACAGGA GAGAGAA-3′

(PCR)
PCR master mix 25uL
FP 25pmoL
RP 25pmoL
Genomic DNA 10mcg
NFW 18uL
Total 50uL
Initial denaturation 95°C for 3min
Denaturation 95°C for 30sec
Annealing Taq I
(rs731236)
61°C for 30sec
Annealing Apa I
(rs7975232)
72°C for 30sec
Annealing Bsm I
(rs1544410)
58.5°C for 30sec
Extension 72°C for 30sec
Final extension 72°C for 5min
30
Cycles

(RFLP)
Restriction enzymes
DNA fragments: Visualised using 2% agarose gel electrophoresis in UV light
PCR products of Taq I (rs731236) Taq I (Thermo scientific)
PCR products of Apa I (rs7975232) Apa I (Thermo scientific)
PCR products of Bsm I (rs1544410) Bsm I (Thermo scientific)

Agarose gel electrophoresis
showing the PCR-RFLP of VDR
genotypes.
A) Apa I genotypes. Lanes 3, 4,
6, 7, and 9 represent Aa
genotype. Lanes 5, 10, and
12 represent AA genotype.
Lanes 8 and 11 represent aa
genotype.
B) Taq I genotypes: Lanes 1, 4,
and 5 represent Tt
genotype. Lanes 2, 6, 7, and
9 represent TT genotype.
Lane 3 represents tt
genotype.
C) Bsm I genotypes: Lanes 1,
2, 3, 5, 6,8, 9, 10, and 12
represent BB genotype.
Lanes 4 and 11 represent Bb
genotype. Lane 7 represents
bb genotype.

Statistical Analysis
SPSS program for Windows (SPSS, Inc, Chicago, IL) version 22.
Normal distribution: Kolmogorov-Smirnov test
?What is Kolmogorov-Smirnov test???????
?Any other method for normality testing????

Quantitative data: NOT NORMALLY DISTRIBUTED- MEDIAN (IQR)
Qualitative data: NUMBER & PERCENTAGES
Median comparison: MANN-WHITNEY U TEST
???Other tests for median comparison

Chi-square test or Fisher’s exact test : Association between categorical
variables.
Chi-square test : Hardy–Weinberg equilibrium
Odds ratio (OR) and 95% confidence interval (CI)
A logistic regression model – Backward stepwise method

Results
Study Analysis
Cases Controls
Hormonal and
genetic analysis
Hormonal and
genetic analysis
Comparison of demographic and lab data of cases and
controls
VDR genotypes and gene variant alleles in KC Vs Control group
Vit D levels
among VDR
genotypes in
KC patients
Log
Regression
analysis in
KC group

Table 1
Demographic,
topographic, and
laboratory data of
studied subjects.

Table 2
Distribution of VDR
genotypes and gene
variant alleles in KC
patients versus
control group.

Table 3
Comparison of
Vitamin D level
among studied
VDR genotypes in
KC patients.

Table 4
Logistic regression
analysis in
keratoconus group.

Discussion
Higher incidence of thyroid dysfunction  Hypothyroidism in KC patients
Karabulut et al.: Patients with Grave’s disease have significantly altered corneal
biomechanical properties
Gatzioufas et al.: Hypothyroidism may induce corneal topographical and
biomechanical changes or even exacerbate KC

Discussion
Significant decrease in serum vitamin D level in KC patients than the control
group
Akkaya and Ulusoy : Lower serum vitamin D level in KC patients when
compared to the healthy controls  inflammatory and immunological nature of
the KC.
Ghanavati et al.: Inflammatory, apoptotic, or oxidative mechanisms may cause
ocular surface affection due to vitamin D deficiency.
Evaluating serum vitamin D levels in KC patients at the onset and the follow-up
examinations may predict the disease course

Discussion
Taq I (rs731236) VDR polymorphism: tt genotype had a higher risk to develop
KC[OR=3.45] as t allele was more prevalent in KC patients [OR=1.94]
T allele was shown to have a protective effect against the disease
Assessment of vitamin D level:
TT genotype: Higher serum Vit-D level
Tt genotype: Insufficient serum Vit-D level
tt genotype: Deficient serum Vit-D level

Discussion
TSH, T4,hypothyroidism, insufficient and deficient vitamin D levels were
significantly associated with keratoconus.

Limitations of the study
Didn’t correlate the vitamin D level to keratoconus grade and the state of
progression.
Assessment of parathyroid hormone.
Future studies are needed to assess this relationship and to confirm the effect
of vitamin D supplementation on keratoconus stability.

Conclusion
Known Before???
The study adds:
Thyroid dysfunction and vitamin D deficiency are significantly associated with
keratoconus
Vitamin D receptor polymorphism is associated with keratoconus
development
Taq I (rs731236) VDR polymorphism: tt genotype had a higher risk to develop
KC[OR=3.45] as t allele was more prevalent in KC patients [OR=1.94]

Critical appraisal
Names of VDR polymorphisms not mentioned in the introduction part
The authors mentioned the study as prospective, but in this cross-sectional
study, no prospective follow-up was done.
Ideally, the number of controls should be equal or greater compared to the
number of case subjects, which is not followed in this study.
Sample size calculation is done through online software, but the reference
article, power and alpha error were not mentioned.

Critical appraisal
PCR-RFLP: The size of the PCR product, digestion product, and DNA ladder used
were not mentioned.
Genotype analysis: Failed to mention which is wild and which is a mutant
variety properly.
In Table 3, they compared three medians, which will not be possible with
Mann-Whitney, so the use of Kruskal-Wallis one-way ANOVA was not
mentioned.

Enhancing the QUAlity and Transparency
Of health Research

Strengthening The Reporting of OBservational
studies in Epidemiology (STROBE)
Title and abstract
(a) Indicate the study’s design with a commonly used
term in the title or the abstract
(b) Provide in the abstract an informative and
balanced summary of what was done and what
was found
Yes(Both are mentioned)
Introduction
Background/rationale
Objectives
Yes(Both mentioned)
Methods
Study design
Setting
Participants
Variables
Yes
Yes
Yes
Yes

Strengthening The Reporting of OBservational
studies in Epidemiology (STROBE)
Data sources/ measurement
Bias
Study size
Quantitative variables
Statistical methods
Yes
No
Incomplete calculation
Yes
Incomplete
Results
Participants
Descriptive data
Outcome data
Main results
Other analyses
Yes
Yes
Yes
Yes
Yes
Discussion
Key results
Limitations
Interpretation
Generalizability
Yes
Yes
Yes
No
Funding Mentioned

Thank you & Have a Nice day

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Editor's Notes