Indistinguishable gene expression between healthy eyes and eyes with unilateral exfoliative glaucoma

Figures & data

Figure 1 Photograph showing the right eye of a patient included in the study suffering from exfoliative glaucoma.

Notes: Exfoliation material is seen as a margin on the lens surface. The pupil has been dilated.

Table 1 Baseline characteristics of the included patients

Variables	Eyes with exfoliative glaucoma (n = 10)	Eyes without exfoliative glaucoma (n = 10)	p-value (Student’s t-test)
Mean visual acuity before operation	0.34	0.52	0.001
Mean IOP (mmHg)	21.2	16.3	0.0001
Visual fields MD (dB)	−9.9	−0.58	<0.0001
Visual fields VFI (%)	70.4	98.5	<0.0001
Mean RNFL thickness OCT (µm)	72.7	99.4	<0.0001
Cup-to-disc ratio (0–0.99)	0.78	0.55	<0.0001
Gonioscopy; anterior chamber angle (0–4)	2.7	1.8	0.0003
Gonioscopy: pigmentation (0–3)	2.4	0.7	<0.0001

Abbreviations: IOP, intraocular pressure; MD, mean deviation; VFI, visual field index; RNFL, retinal nerve fiber layer; OCT, optical coherence tomography.

Table 2 Gene expression in conjunctival biopsies comparing healthy individuals versus exfoliative glaucoma patients

Gene	Healthy	Exfoliative glaucoma	Test	p-value
LOXL-1 $(\overline{x}\pm \text{SD})$	10.22 ± 0.91	10.01 ± 0.79	Student’s t-test	0.61
NTF4 $(\overline{x}\pm \text{SD})$	9.27 ± 0.83	9.30 ± 0.92	Student’s t-test	0.94
OPT-N $(\overline{x}\pm \text{SD})$	3.47 ± 0.25	3.52 ± 0.30	Student’s t-test	0.73
WDR36 $(\overline{x}\pm \text{SD})$	5.14 ± 0.35	5.17 ± 0.29	Student’s t-test	0.86
CYP1B1 $(\overline{x}\pm \text{SD})$	6.98 ± 0.95	6.30 ± 1.30	Student’s t-test	0.23

Notes: Data analysis: 1) genes with more than 50% missing data were removed (hMYOC and hGSTM1); 2) technical replicates were averaged; 3) the data were corrected for genomic background with ValidPrime (did not change the Cq-values significantly, except for some samples in hNTF4); 5) the data were normalized to reference genes (PPIA and UBC); 6) each diseased eye was normalized to its corresponding healthy eye; 7) Log2 was applied to data.

Table 3 Gene expression in the lens capsule comparing healthy individuals versus glaucoma patients

Gene	Healthy	Exfoliative glaucoma	Test	p-value
LOXL-1 $(\overline{x}\pm \text{SD})$	7.01 ± 0.89	6.78 ± 0.38	Student’s t-test	0.42
NTF4 $(\overline{x}\pm \text{SD})$	11.94 ± 0.78	12.67 ± 1.05	Student’s t-test	0.15
OPT-N $(\overline{x}\pm \text{SD})$	2.97 ± 0.26	2.80 ± 0.37	Student’s t-test	0.22
WDR36 $(\overline{x}\pm \text{SD})$	5.58 ± 0.15	5.38 ± 0.43	Student’s t-test	0.16
CYP1B1 $(\overline{x}\pm \text{SD})$	4.79 ± 0.90	3.99 ± 0.97	Student’s t-test	0.04*
MYOC $(\overline{x}\pm \text{SD})$	5.94 ± 1.10	6.54 ± 0.91	Student’s t-test	0.16
GSTM1 $(\overline{x}\pm \text{SD})$	7.38 ± 4.94	7.84 ± 5.02	Mann–Whitney	0.52

Notes: Data analysis: 1) technical replicates were averaged; 2) the data were corrected for genomic background with ValidPrime (did not change the Cq-values significantly); 3) the data were normalized to reference genes (GAPDH and UBC); 4) each right eye was normalized to its corresponding left eye; 5) relative expression to the maximum was set (lowest expression was set to 1); 6) Log2 was applied to data.

* Comparison was significant.

Table S1 Genes of interest

Gene	FWD-primer	REV-primer
LOXL1	GTGGGGAGCGGAACGG	TGCTTGCACATAGTTGGGGT
MYOC	GGAGGTAGCAAGGCTGAGAA	CTGGAAGGCCAAAGTGTCCAA
OPTN	CATGGGGCGAGAACAAGTGA	GGGGCAGGAATGAATCGGAA
WDRD36	GGCTTTTAGTCCTGATGGTCGT	GAGGAGCCGAGTCCAACAAA
CYP1B1	AACGTACCGGCCACTATCAC	TGATCCAATTCTGCCTGCACT
GSTM1	CCTCCACCGTATATTTGAGCC	TGAGAACACAGGTCTTGGGA
NTF4	CTGCAAGGCTGATAACGCTG	TCAGATACCCAGTGCCTCCT

Abbreviations: FWD, forward; REV, reverse.

Table S2 TATAA human reference gene panel and ValidPrime

Gene	FWD-primer	REV-primer
ACTB	AGAAAATCTGGCACCACACC	AGAGGCGTACAGGGATAGCA
B2M	GGGTTTCATCCATCCGACA	ACACGGCAGGCATACTCATC
GAPDH	CAGTCAGCCGCATCTTCTTTT	TGGCAACAATATCCACTTTACCAG
GUSB	GATGGAAGAAGTGGTGCGTAG	TTGCTCACAAAGGTCACAGG
HPRT	TGACACTGGCAAAACAATGCA	GGTCCTTTTCACCAGCAAGCT
UBC	CGCAGTTCTTGTTTGTGGA	GGGTGGACTCTTTCTGGATG
PPIA	AGGTCCCAAAGACAGCAGAA	CACCACCCTGACACATAAACC
RPLP	TCAACATCTCCCCCTTCTCC	GCAGACAGACACTGGCAACA
18S	GACTCAACACGGGAAACCTC	AGACAAATCGCTCCACCAAC
TBP	TTCGGAGAGTTCTGGGATTG	ATCAGTGCCGTGGTTCGT
TUBB5	CTTCAGACCAGACAACTTCGTG	ACTCCTTCCTCACCACATCC
YWHAZ	AGACGGAAGGTGCTGAGAAA	TTGGTATGCTTGTTGTGACTGA
ValidPrime	AGCACATTTCTATTCTCCGT	TCTTGACCTTCTCTACCTCC

Abbreviations: FWD, forward; REV, reverse.

Table S3 Quantitative PCR setup

Program	Temperature (°C)	Time (s)	Cycles
Polymerase activation	95	60	1
Denaturation	95	5
Annealing	60	30	45
Extension	72	10
Melt curve	60–95	–	–

Table S4 Quantitative PCR reaction mix

Reagent	Volume (µL)
Primers (Fwd + Rev)	0.4
TATAA SYBR Grandmaster mix	5
H2O	2.5
cDNA	2
Total	10