Genome-wide association study of subfoveal choroidal thickness in a longitudinal cohort of older adults

doi:10.21203/rs.3.rs-4134234/v1

Download PDF

Article

Genome-wide association study of subfoveal choroidal thickness in a longitudinal cohort of older adults

https://doi.org/10.21203/rs.3.rs-4134234/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 09 Oct, 2024

Read the published version in Scientific Reports →

You are reading this latest preprint version

Purpose

To identify genetic influences on subfoveal choroidal thickness using a genome-wide association study (GWAS).

Methods

We recruited 300 participants from the population-based Korean Longitudinal Study on Health and Aging (KLoSHA) and Korean Longitudinal Study on Cognitive Aging and Dementia (KLOSCAD) cohort studies and 500 participants from the Bundang age-related macular degeneration (AMD) cohort study dataset. We conducted a GWAS on older adult populations in the KLoSHA and KLOSCAD cohorts. Single nucleotide polymorphisms (SNPs) associated with choroidal thickness were identified with P values < 1.0 × 10^− 4 in both the right and left eyes, followed by validation using the Bundang AMD cohort dataset. This association was further confirmed by a functional in vitro study using human umbilical vein endothelial cells (HUVECs).

Results

The ages of the cohort participants in the discovery and validation datasets were 73.5 ± 3.3 and 71.3 ± 7.9 years, respectively. In the discovery dataset, three SNPs (rs1916762, rs7587019, and rs13320098) were significantly associated with choroidal thickness in both eyes. This association was confirmed for rs1916762 (genotypes GG, GA, and AA) and rs7587019 (genotypes GG, GA, and AA), but not for rs13320098. The mean choroidal thickness decreased by 56.7 µm (AA, 73.8%) and 31.1 µm (GA, 85.6%) compared with that of the GG genotype of rs1916762, and by 55.4 µm (AA, 74.2%) and 28.2 µm (GA, 86.7%) compared with that of the GG genotype of rs7587019. The SNPs rs1916762 and rs7587019 were located close to the FAM124B gene near its cis-regulatory region. Moreover, FAM124B was highly expressed in vascular endothelial cells. In vitro HUVEC experiments showed that the inhibition of FAM124B was associated with decreased vascular endothelial proliferation, suggesting a potential mechanism of choroidal thinning.

Conclusions

FAM124B was identified as a susceptibility gene affecting subfoveal choroidal thickness in older adults. This gene may be involved in mechanisms underlying retinal diseases associated with altered choroidal thickness, such as age-related macular degeneration.

Health sciences/Signs and symptoms/Eye manifestations

Biological sciences/Genetics

choroidal thickness

GWAS

CUL3

FAM124B

HUVEC

Choroidal vascular structure and its alterations are associated with various retinal diseases, such as age-related macular degeneration (AMD), polypoidal choroidal vasculopathy (PCV), and central serous chorioretinopathy (CSC).^[1–4] The development of the enhanced depth imaging (EDI) technique of spectral-domain optical coherence tomography (SD-OCT) has enabled clinicians to discern details of choroidal structure and accurately measure its thickness.^{[5, 6]} Using the SD-OCT EDI mode, a subfoveal choroidal thickness (SFCT) analysis demonstrated that PCV presented with thickening of the choroid, whereas exudative AMD showed choroidal thinning.^[7] Similarly, as clinicians continue to focus on the choroidal layer in retinal diseases, the term “pachychoroid” has been introduced to describe a spectrum of diseases characterized by clinically significant choroidal thickening with dilated large choroidal vessels and overlying choriocapillaris.^[8]

Recent genome-wide association studies (GWAS) have identified susceptibility genes for AMD, PCV, and CSC. Notably, single nucleotide polymorphism (SNP) rs1061170 in the complement factor H (CFH) gene located on chromosome 1q31 was reported in 2005.^[9–11] Subsequently, numerous SNPs and genes, including ABCA1, APOE, ARMS2/HTRA1, B3FALTL, CFI, C2, C3, COL4A3, GATA5, LIPC, MMP9, TIMP3, TNFRSF10A, and VIPR2, were discovered using GWAS.^[12–19] Furthermore, efforts to correlate susceptibility genes (genotypes) and clinical phenotypes have been increasing. Mori et al. reported a shared genetic susceptibility between PCV and macular neovascularization secondary to CSC.^[17] Thee et al. from the EYE-RISK consortium analyzed AMD features and macular thickness following the harmonization of genetic data.^[19] Their findings suggested that risk variants at ARMS2/HTRA1 exhibit an increased risk of late AMD progression, with phenotypes resembling the complement pathway variants. Since retinal and choroidal morphological changes are linked to disease patterns and visual outcomes, exploring the potential association between genetic discoveries and structural phenotypes is crucial. Changes in choroidal thickness are a critical feature of retinal diseases, including AMD, and are likely to be involved in their pathogenesis. However, no GWAS has targeted choroidal thickness.

Therefore, in this study, we investigated genetic influences affecting the SFCT using a GWAS in an older adult population-based cohort, identified susceptibility genes affecting choroidal structure, and validated the association using an AMD cohort. Additionally, we conducted a functional experiment using human umbilical vein endothelial cells (HUVECs) to investigate the in vitro effects of the altered expression of associated genes.

The Institutional Review Board (IRB) of the Seoul National University Bundang Hospital (SNUBH) approved this study, which adhered to the principles of the Declaration of Helsinki (IRB No. B-1812/510 − 107). Written informed consent was obtained from all the participants.

Discovery Dataset

We used the two population-based longitudinal cohort studies, which enrolled Korean elderly adults aged more than 60 years: the Korean Longitudinal Study on Health and Aging (KLoSHA) and Korean Longitudinal Study on Cognitive Aging and Dementia (KLOSCAD).^{[20, 21]} Overall, 300 participants were recruited for genetic studies regarding the development of ophthalmic and cognitive disease between September 2010 and September 2011. All participants underwent comprehensive baseline ophthalmic examinations, including best-corrected visual acuity, intraocular pressure, auto kerato-refractometry, optical biometry, axial length calculation (IOL Master; Carl-Zeiss Meditec, Dublin, CA, USA), and spectral domain optical coherence tomography (SD-OCT; Heidelberg Engineering, Heidelberg, Germany). Two independent retinal specialists (H.M.K. and Y.J.P.) manually measured the SFCT using the EDI mode, and the average of repeated measurements was analyzed. The SFCT was measured as the vertical perpendicular distance from the innermost hyperreflective line of the chorioretinal interface to the hyperreflective line of Bruch’s membrane. The left and right eyes were analyzed separately in the dataset used in this study. We excluded patients with significant myopia (axial length exceeding 26 mm), high intraocular pressure (greater than 21 mmHg), self-reported glaucoma history, and combined ocular pathologies, including age-related macular degeneration, epiretinal membrane, diabetic macular edema, and diabetic retinopathy found on OCT infrared imaging, which may affect subfoveal choroidal thickness.

Validation Dataset

For the validation dataset, we used the SNUBH AMD cohort.^{[22, 23]} This cohort included Korean adults aged more than 50 years who were initially diagnosed with non-exudative or exudative AMD in the presence of multiple drusen bodies on the macula, with or without pigmentation changes. A total of 500 participants were enrolled, and detailed ophthalmic assessments were performed, including color fundus photography and SD-OCT (Heidelberg Engineering, Heidelberg, Germany). Similar to the study dataset, two independent retinal specialists (H.M.K. and Y.J.P.) manually measured the subfoveal choroidal thickness, and the average of repeated measurements was analyzed. Similarly, we excluded patients with significant myopia, high intraocular pressure, self-reported glaucoma history, and combined ocular pathologies other than AMD.

GWAS analysis

GWAS genotyping was performed using the Illumina Human OmniExpress or Human Hap610-Quad bead chips. For replication, genotyping was performed using the MassArray platform (Sequenom) and TaqMan allelic discrimination probes (Applied Biosystems). A GWAS was conducted on choroidal thickness data from 300 participants (left and right eyes). For the left eye, 300 participants were analyzed after excluding 23 patients without data on choroidal thickness. For the right eye, 300 participants were analyzed, excluding 28 patients without data on choroidal thickness. The analysis was performed using the PLINK software, selecting variants based on genotyping mutations in more than 95% of samples, Hardy-Weinberg 10^− 6, and mutations present in more than 1% of all samples. An association study analyzed the choroidal thickness of the right and left eyes. Among the 590,000 SNPs examined, those with P < 10^− 4 in both the left and right eyes were extracted.

In silico analysis using open data source

The captured Hi-C data in the GM12878 cell line were visualized using the 3D Genome Browser from the YUE Laboratory (http://3dgenome.fsm.northwestern.edu/view.php). GTEx (https://gtexportal.org/home/) was used to identify interacting genes regulated by candidate SNPs and to analyze target gene expression using bulk RNA sequencing data across multiple tissues. The Protein Atlas (https://www.proteinatlas.org/) and Expression Atlas (https://www.ebi.ac.uk/gxa/home) were used to analyze cell type-specific RNA and gene expression data at the single-cell level. Single-cell-specific patterns were visualized by modifying the Tabula Sapiens data (https://tabula-sapiens-portal.ds.czbiohub.org/).

HUVEC experiment

Cell culture

HUVECs (cat no. 4453, Sartorius, Germany) were purchased from Sartorius and used in the study. The cells were cultured in a cell incubator at a temperature of 37°C and with 5% CO₂. Endothelial Cell Growth Medium 2 (EGM-2, cat no. C-22011, PromoCell, Germany) supplemented with 1% penicillin/streptomycin (cat no. L0018, BioWest, France) was used as the culture medium. The medium was replaced every 48 h, and the cells were passaged upon reaching 70–80% confluence. Cells passaged between two and five times were used in the experiments.

Small interfering ribonucleic acid (siRNA) Transfection

FAM124B (Human) 3 unique 27-mer siRNA duplexes (cat no. SR312644, OriGene, USA) were purchased from OriGene and used in the study. The sequences of the siRNAs were as follows: 5’-AUGUUCUAGAAAUGGAGUACUGACC-3’, 5’-GCAGUUUAAGGUUCAAGAGAUCGGC-3’, and 5’-GGCUUGACCAUCAUAAAUUCUGAAC-3’. For transfection, Lipofectamine 3000 (cat no. L3000001, Invitrogen, USA) was purchased and the experiments were conducted according to the manufacturer's instructions. Approximately 3–4 × 10⁴ cells were seeded per well in an 8-well Lab-Tek II Chamber Slides (cat no. 154534, Thermo Scientific, USA). When the cells reached 70–90% confluence under conditions of 37°C and 5% CO₂, Lipofectamine 3000 reagent (0.15 and 0.3 µL) and Opti-MEM medium (cat no. 31985070, Gibco, USA) were mixed according to the manufacturer's instructions, along with 0.2 µg siRNA, 0.4 µL P3000 reagent, and 10 µL Opti-MEM medium in a 1:1 ratio. The mixture was then incubated at room temperature for 15 min. The resulting mixture was added to each well and incubated at 37°C and 5% CO₂ for 48 h. Immunofluorescence was performed 48 h after siRNA treatment, with the timing of the siRNA treatment as the reference point.

Immunofluorescence

The siRNA-treated HUVECs were fixed with a 4% paraformaldehyde solution at room temperature for 5 min and subsequently with 100% methanol at -20°C for 5 min. After two washes with phosphate-buffered saline (PBS), the fixed cells were blocked using a blocking buffer (5% fetal bovine serum (FBS), 5% goat serum, 0.1% Triton-X 100, 0.02% sodium azide in PBS) at room temperature for 10 min. To detect FAM124B protein, an anti-FAM124B antibody (cat no. 21313-1-AP, Proteintech, Germany) was used, followed by Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (cat no. A-11008, Invitrogen, USA). To detect Ki67, an anti-Ki67 antibody (cat no. STJ119231, St John’s Laboratory, UK) was used, followed by Alexa Fluor 594 goat anti-rabbit IgG secondary antibody (cat no. A-11012, Invitrogen, USA). The samples were observed under a fluorescence microscope (Axio imager M2, Carl Zeiss, Germany).

Cell migration and invasion assay

One million (1 × 10⁶) HUVECs were seeded into each well of a six-well tissue culture plate containing 2 ml of M199 medium supplemented with 20% FBS and antibiotics. The cells were left to grow overnight to achieve nearly full coverage of the well. To inhibit cell proliferation, 5 µg/mL of mitomycin C (100 µL from a stock of 100 µg/mL) was added to the medium 2 h before creating a scratch. An aseptic P-200 pipette tip was used to create a controlled scratch wound by drawing a line across the center of each well to simulate an artificial wound in the cell layer. Both the control and FAM124B siRNA knockdown transfections were performed immediately after the scratch was created. The plate was subsequently placed in a CO₂ incubator for a 24-h period after transfection. The cells were then rinsed with PBS, stained using a 0.5% crystal violet solution, observed, photographed, and quantified using an inverted microscope. The migrated area was calculated by subtracting the area without cells at each time point from the area without cells at 0 h, and then dividing this area by the area without cells at 0 h.

In total, 300 and 500 participants met the inclusion criteria for the KLoSHA/KLOSCAD discovery and Bundang AMD validation cohorts, respectively. The participants’ ages at the first visit in the cohort dataset were 73.5 ± 3.3 years and 71.3 ± 7.9 years, respectively. The sex ratios (male:female) were 153:147 (51%:49%) and 296:204 (59%:41%), respectively. The GWAS analysis revealed three SNPs that had the greatest evidence of association with subfoveal choroidal thickness: rs1916762 at chromosome 2 (intergenic, 24 kb from FAM124B and 44 kb from CUL3, left eye P = 6.55 × 10^− 5, right eye P = 9.13 × 10^− 6); rs7587019 at chromosome 2 (intergenic, 37 kb from FAM124B and 30 kb from CUL3, left eye P = 3.80 × 10^− 5, right eye P = 6.65 × 10^− 5); and rs13320098 at chromosome 3 (intergenic, 81 kb from MIR466 and 288 kb from STT3B, left eye P = 2.24 × 10^− 5, right eye P = 8.68 × 10^− 6) (Table 1).

Table 1

GWAS results of SNPs with the greatest evidence of association for the subfoveal choroidal thickness
Chromosome	2	2	3
SNP	rs1916762	rs7587019	rs13320098
Base pair	225290792	225304551	31285081
Reference Allele	G	G	T
Alternative Allele	A	A	C
P-value (Left eye)	6.55 x 10^− 5	3.80 x 10^− 5	2.24 x 10^− 5
P-value (Right eye)	9.13 x 10^− 6	6.65E x 10^− 5	8.68 x 10^− 6
Gene (Intergenic)	24kb from FAM124B / 44kb from CUL3	37kb from FAM124B / 30kb from CUL3	81kb from MIR466 / 288kb from STT3B

The SFCT was independently measured by two retinal specialists using SD-OCT. The discovery and validation cohort datasets were then classified into three genotypes: reference/reference alleles, reference/alternative alleles, and alternative/alternative alleles (Tables 1 and 2). The average age and sex proportions did not differ significantly among the three genotypes in each cohort dataset (Table 2). The SFCT was significantly different between the rs1916752 and rs7587019 genotypes in both the discovery and validation datasets (P < 0.001). Meanwhile, the SFCT in the rs13320098 SNP was significantly different among the genotypes in the discovery cohort (P < 0.001), but not in the validation cohort (P = 0.149) (Table 2). No significant difference in the SFCT was observed between the left and right eyes within each genotype in the study cohort. Figure 1 summarizes the association between the SFCT and the genotypes of the three SNPs. Post-hoc analyses revealed that choroidal thickness significantly differed between the rs1916762 and rs7587019 genotypes. However, there was no significant difference in choroidal thickness between the TT and TC genotypes of rs13320098 in the validation cohort (Fig. 1): rs1916762 GG 226.0 ± 35.0 µm (100.0%), GA 173.0 ± 31.5 µm (76.5%), and AA 147.4 ± 33.3 µm (65.2%); rs7587019 GG 226.6 ± 36.0 µm (100.0%), GA 172.4 ± 31.3 µm (76.1%), and AA 149.5 ± 34.6 µm (65.9%); and rs13320098 TT 169.1 ± 41.6 µm (100.0%), TC 171.1 ± 38.4 µm (101.1%), and CC 149.2 ± 43.2 µm (88.2%) (Table 2).

Table 2

Demographics and clinical characteristics of KLOSHA/KLOSCAD study cohort and AMD validation cohort
SNP / Gene	rs1916762 / FAM124B;CUL3
Cohort	KLOSHA/KLOSCAD study				AMD validation
Genotype	GG	GA	AA	P-value	GG	GA	AA	P-value
Number	122	136	42		208	229	63
Age (years)	73.3 ± 3.4	73.8 ± 3.1	73.0 ± 3.4	0.312	71.3 ± 7.8	71.5 ± 8.6	70.8 ± 7.9	0.810
Sex (M:F)	65 : 57	68 : 68	20 : 22	0.791	130 : 78	131 : 98	35 : 28	0.436
Subfoveal (µm) choroidal thickness
Left eye	220.7 ± 74.3	184.1 ± 66.9	162.6 ± 68.1	< 0.001	226.0 ± 35.0	173.0 ± 31.5	147.4 ± 33.3	< 0.001
Right eye	215.6 ± 62.2	184.3 ± 66.1	158.0 ± 58.7	< 0.001	226.0 ± 35.0	173.0 ± 31.5	147.4 ± 33.3	< 0.001
SNP / Gene	rs7587019 / FAM124B;CUL3
Cohort	KLOSHA/KLOSCAD study				AMD validation
Genotype	GG	GA	AA	P-value	GG	GA	AA	P-value
Number	130	131	39		216	224	60
Age (years)	73.5 ± 3.6	73.5 ± 3.4	73.4 ± 3.0	0.972	71.2 ± 8.1	71.6 ± 8.4	70.7 ± 7.8	0.693
Sex (M:F)	65 : 65	63 : 68	19 : 20	0.956	134 : 82	130 : 94	32 : 28	0.427
Subfoveal (µm) choroidal thickness
Left eye	217.5 ± 66.4	188.3 ± 65.6	158.6 ± 63.0	< 0.001	226.6 ± 36.0	172.4 ± 31.3	149.5 ± 34.6	< 0.001
Right eye	212.2 ± 59.1	185.0 ± 65.1	160.3 ± 60.1	< 0.001	226.6 ± 36.0	172.4 ± 31.3	149.5 ± 34.6	< 0.001
SNP / Gene	rs13320098 / MIR466;STT3B
Cohort	KLOSHA/KLOSCAD study				AMD validation
Genotype	TT	TC	CC	P-value	TT	TC	CC	P-value
Number	216	78	6		361	124	15
Age (years)	73.7 ± 3.4	73.3 ± 3.5	71.5 ± 4.2	0.219	71.2 ± 8.2	71.3 ± 8.3	74.6 ± 5.9	0.310
Sex (M:F)	99 : 117	41 : 37	3 : 3	0.208	207 : 154	82 : 42	7 : 8	0.138
Subfoveal (µm) choroidal thickness
Left eye	191.8 ± 73.4	162.1 ± 68.8	115.9 ± 25.1	< 0.001	169.1 ± 41.6	171.1 ± 38.4	149.2 ± 43.2	0.149
Right eye	188.9 ± 71.0	161.8 ± 62.9	94.9 ± 44.5	< 0.001	169.1 ± 41.6	171.1 ± 38.4	149.2 ± 43.2	0.149

Because the two SNPs were non-coding variants, we investigated their chromosomal locations and utilized capture Hi-C data from the female B-cell lymphoblastoid cell line (GM12878) to identify specific genes regulated by these two SNPs. The rs1916762 and rs7587019 SNPs are located upstream of FAM124B and downstream of CUL3 (Fig. 2A). Both SNPs were positioned close to FAM124B, near its cis-regulatory region. The captured Hi-C data revealed interactions between rs7587019 and the starting exons, including the promoter regions, of FAM124B and CUL3, suggesting that rs7587019 may play a role in regulating FAM124B and/or CUL3 expression. Moreover, single-tissue expression quantitative trait loci (eQTLs) for the two GTEx variants predicted interactions with FAM124B across multiple tissues. Additionally, these two variants were predicted to interact with a long non-coding RNA (pseudogene) located in and near the CUL3 gene (Table 3). Subsequently, we analyzed gene expression data, including bulk RNA sequencing, from the Protein Atlas to determine which gene among FAM124B and CUL3 is regulated by the two SNPs. While FAM124B is expressed in many tissues such as the breast, colon, heart muscle, and arteries, it is notably enriched in endothelial cells (Figs. 2B,C). Conversely, CUL3 is ubiquitously expressed in the cell nucleus and is particularly enriched in spermatids. Single-cell RNA sequencing from the Protein Atlas and Expression Atlas revealed that FAM124B is highly expressed in adipocytes and vascular and lymphatic endothelial cells. Single-cell RNA sequencing data from Tabula Sapiens revealed that FAM124B exhibited expression patterns in endothelial cells, erythroid progenitors, and mast cells, with a significant overlap observed in retinal vascular endothelial cells (Fig. 2D). Given the association of rs1916762 and rs7587019 with choroidal thickness, we propose that FAM124B, which is specifically expressed in vascular endothelial cells, is a target gene for the regulation of choroidal thickness.

Table 3

Single-Tissue eQTLs for rs1916762 and rs7587019
SNPs	Interacting gene	P-Value	NES	Tissue
rs1916762 (chr2_224426075_G_A)	FAM124B	2.1x10^− 9	-0.31	Lung
	FAM124B	2.8x10^− 8	-0.29	Thyroid
	AC073052.1	5.6x10^− 7	0.36	Colon - Transverse
	AC073052.1	5.9x10^− 7	0.25	Thyroid
	FAM124B	1.4x10^− 6	-0.29	Esophagus - Mucosa
	FAM124B	4.4x10^− 6	-0.17	Artery - Tibial
	AC073052.1	4.6x10^− 6	0.25	Nerve - Tibial
	AC073052.1	2.7x10^− 5	0.34	Spleen
	AC073052.1	3.8x10^− 5	0.24	Muscle - Skeletal
	AC073052.1	3.8x10^− 5	0.24	Adipose - Subcutaneous
	AC073052.1	7.1x10^− 5	0.32	Cells - Cultured fibroblasts
	AC073052.1	1.1x10^− 4	0.19	Esophagus - Mucosa
rs7587019 (chr2_224439834_G_A)	FAM124B	1.4x10^− 10	-0.35	Lung
	AC073052.1	1.9 x10^− 9	0.31	Thyroid
	FAM124B	2.4x10^− 9	-0.31	Thyroid
	AC073052.1	4.8x10^− 9	0.45	Colon - Transverse
	AC073052.1	7.5x10^− 9	0.36	Muscle - Skeletal
	FAM124B	5.2x10^− 8	-0.52	Adrenal Gland
	AC073052.1	4.4x10^− 7	0.3	Adipose - Subcutaneous
	AC073052.1	5.1x10^− 7	0.35	Esophagus - Gastroesophageal Junction
	AC073052.1	9.0x10^− 7	0.42	Spleen
	AC073052.1	1.4x10^− 6	0.4	Cells - Cultured fibroblasts
	AC073052.1	1.6x10^− 6	0.24	Esophagus - Mucosa
	FAM124B	1.6x10^− 6	-0.32	Stomach
	FAM124B	3.6x10^− 6	-0.17	Artery - Tibial
	FAM124B	3.9x10^− 6	-0.32	Heart - Atrial Appendage
	AC073052.1	7.4x10^− 6	0.21	Skin - Not Sun Exposed (Suprapubic)
	AC073052.1	1.0x10^− 5	0.26	Skin - Sun Exposed (Lower leg)
	AC073052.1	1.4x10^− 5	0.26	Nerve - Tibial
	AC073052.1	1.4x10^− 5	0.32	Stomach
	AC073052.1	1.5x10^− 5	0.19	Lung
	FAM124B	2.6x10^− 5	-0.26	Esophagus - Mucosa
	AC073052.1	3.8x10^− 5	0.33	Colon - Sigmoid
	FAM124B	3.9x10^− 5	-0.28	Colon - Sigmoid
	AC073052.1	8.8x10^− 5	0.26	Artery - Tibial
	AC073052.1	9.7x10^− 5	0.16	Whole Blood
The data was aligned to the GRCh38/h38 reference genome. NES, normalized effect size, is defined as the slope of the linear regression, and is computed as the effect of the alternative allele (ALT) relative to the reference allele (REF) in the human genome reference. AC073052.1 (ENST00000622296.1, chr2:224467803–224474500) is predicted as a long non-coding RNA (pseudogene) in and near the CUL3 gene.

To investigate the molecular expression and function of FAM124B in vascular endothelial cells, we used HUVECs, which showed high expression of FAM124B in the Protein Atlas. The fluorescence of FAM124B was well-localized in the cytoplasm around the nucleus (Fig. 3A). Depletion of FAM124B using siRNAs significantly reduced the expression of the proliferation marker Ki67 (Figs. 3B,C), suggesting that FAM124B enhances the proliferation of vascular endothelial cells. However, no significant differences in cell migration were observed between the two groups (Figs. 3D,E).

We investigated the genotype-phenotype associations for susceptibility genes and their genotypes in the population-based KLoSHA/KLOSCAD cohort, followed by validation in the Bundang AMD cohort. We revealed that the SNPs rs1916762 and rs7587019 between the FAM124B and CUL3 genes (intergenic) affected choroidal structure, and the SFCT was significantly related to the genotypes.

Genetic analysis of pachychoroid spectrum diseases presenting with thickened choroid and choroidal vascular hyperpermeability has been conducted.^[24] GWAS investigations have reported ARMS2/HTRA1, CFH, COL4A3, and B3GALTL as strong susceptibility genes for PCV and CSC.^{[16, 17, 19]} In our study with discovery and validation cohorts, the FAM124B and CUL3 genes were associated with the SFCT.

FAM124B is a protein-coding gene, and only a few studies have explored its role in human diseases. Li et al. demonstrated that FAM124B has a higher DNA methylation level in ER+/PR + breast cancer compared with ER-/PR- breast cancers.^[25] One GWAS revealed that the SNP rs1523921 (intergenic between CUL3 and FAM124B) is associated with anorexia nervosa.^[26] Another study reported that in patients with acute myeloid leukemia, FAM124B is associated with AML prognosis.^[27] At the molecular level, FAM124B was identified as a potential interacting partner of CHD7 and CHD8 (chromodomain helicase DNA binding domain) containing complex, and thus related to the pathogenesis of CHARGE syndrome and neurodevelopmental disorders.^[28]

To the best of our knowledge, the FAM124B gene has not been directly associated with ocular diseases. In our study, we observed a potential association between the FAM124B gene and choroidal structure. To further investigate this hypothesis, we conducted an experiment using HUVEC. The results revealed that siFAM124B significantly reduced the expression of the proliferation marker Ki67, indicating the potential role of the FAM124B gene in enhancing the proliferation of vascular endothelial cells in the choroid. Overall, it appears plausible that individuals with alternative/alternative alleles (AA) in CUL3 and FAM124B exhibit thinner subfoveal choroidal thickness than those with reference/reference alleles (GG), considering that the two SNPs (rs1916762 and rs7587019) function as FAM124B cis-regulatory elements both in silico and experimentally.

CUL3 (E3 ubiquitin ligase Cullin 3) regulates cellular protein composition by providing target recognition and specificity to the ubiquitin-dependent proteasomal degradation pathway.^[29] Moreover, CUL3 mutations cause familial hyperkalemic hypertension by affecting vascular tone and renal sodium transport.^[30] CUL3 ubiquitin ligase maintains normal cardiovascular and renal physiology, and thus regulates blood pressure.^[31] Furthermore, patients with diabetes and CUL3 dysfunction exhibit vasoconstriction by increased abundance of WNK3, RhoA/ROCK activity, and phosphodiesterase 5, thereby enhancing sodium reabsorption, leading to increased risk of diabetic nephropathy.^[32]

In clinical settings, previous studies have yielded conflicting findings regarding the association between choroidal thickness and systemic vascular diseases. Xu et al. highlighted that patients with diabetes mellitus presented with a slightly significant thickening of the subfoveal choroid, while those diagnosed with diabetic retinopathy were not characterized by choroidal thickness.^[33] Meanwhile, other studies did not reveal significant differences in the SFCT in cardiovascular patients.^[34–36] The Montrachet population-based study suggested that the SFCT is not an appropriate biomarker for cardiovascular diseases.^[37] In our study, we propose that the vascular proliferation of FAM124B, known to affect systemic vascular diseases, is likely to affect choroidal thickness. Moreover, choroidal thickness in neovascular AMD or geographical atrophy is known to be significantly reduced compared to normal individuals.^[38–40] Therefore, our study results are potentially useful for clinicians and researchers in targeting choroidal vascular proliferation as a mechanism of AMD treatment.

This study had certain limitations. We focused on SNPs with p-values < 1.0 × 10^− 4 identified in the GWAS, a threshold that is typically not accepted as a level of significance in standard GWAS investigations. However, we only selected SNPs that were significant in both the right and left eyes. Additionally, the discovery cohort consisted solely of individuals without retinal disease, whereas the validation cohort comprised patients with AMD. This distinction indicates that a direct comparison of the SFCTs between the discovery and validation datasets may not be perfectly aligned. Moreover, our investigation included an in vitro HUVEC experiment targeting the FAM124B gene, conducting siRNA transfection with the proliferation marker Ki67. Additional proliferation markers, such as CD34 and, if necessary, CUL3 siRNA transfection, could be employed for further in vitro studies to elucidate the role of both genes in choroidal thickness.

In conclusion, FAM124B has been identified as a potential contributor to subfoveal choroidal thickness. The genotypes of the identified SNPs may be linked to variations in subfoveal choroidal thickness. Further studies are warranted to investigate the effect of genetic factors on choroidal thickness.

Author contributions :

H.M.K and K.J. : conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript. M.K, Y.J.P, J.W.H., K.W.K., and S.L. : collection and/or assembly of data, data analysis and interpretation, S.J.W. : conception and design, financial support, administrative support, manuscript writing, final approval of manuscript.

Data availability statement (mandatory) : Raw data, functional analysis sources and descriptions will be provided upon request. Correspondence should be addressed to Se Joon Woo. (Tel: +82-31-787-7377, Fax: +82-31-787-4057, E-mail: [email protected])

Funding: This research was supported by the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. RS-2023-00248480) and a grant from the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea [grant No. HI09C1379 (A092077)].

The funding organizations played no role in the design or conduct of this study.

Competing Interests Statement : The authors declare no competing interests.

Ethics Declarations

I attest that the research included in this report was conducted in a manner consistent with the principles of research ethics, including those described in the Declaration of Helsinki and the Belmont Report. This research was conducted with the voluntary informed consent of all research participants, free of coercion or coercive circumstances, and received Institutional Review Board (IRB) approval consistent with the principles of research ethics and the legal requirements of the lead authors' jurisdiction(s).

Ferrara, D. et al. En face enhanced-depth swept-source optical coherence tomography features of chronic central serous chorioretinopathy. Ophthalmology 121, 719–726 (2014). https://doi.org/10.1016/j.ophtha.2013.10.014
Pang, C. E. & Freund, K. B. Pachychoroid neovasculopathy. Retina 35, 1–9 (2015). https://doi.org/10.1097/IAE.0000000000000331
Pauleikhoff, D., Chen, J. C., Chisholm, I. H. & Bird, A. C. Choroidal perfusion abnormality with age-related Bruch's membrane change. Am J Ophthalmol 109, 211–217 (1990). https://doi.org/10.1016/s0002-9394(14)75989-6
Warrow, D. J., Hoang, Q. V. & Freund, K. B. Pachychoroid pigment epitheliopathy. Retina 33, 1659–1672 (2013). https://doi.org/10.1097/IAE.0b013e3182953df4
Margolis, R. & Spaide, R. F. A pilot study of enhanced depth imaging optical coherence tomography of the choroid in normal eyes. Am J Ophthalmol 147, 811–815 (2009). https://doi.org/10.1016/j.ajo.2008.12.008
Spaide, R. F., Koizumi, H. & Pozzoni, M. C. Enhanced depth imaging spectral-domain optical coherence tomography. Am J Ophthalmol 146, 496–500 (2008). https://doi.org/10.1016/j.ajo.2008.05.032
Chung, S. E., Kang, S. W., Lee, J. H. & Kim, Y. T. Choroidal thickness in polypoidal choroidal vasculopathy and exudative age-related macular degeneration. Ophthalmology 118, 840–845 (2011). https://doi.org/10.1016/j.ophtha.2010.09.012
Dansingani, K. K., Balaratnasingam, C., Naysan, J. & Freund, K. B. En Face Imaging of Pachychoroid Spectrum Disorders with Swept-Source Optical Coherence Tomography. Retina 36, 499–516 (2016). https://doi.org/10.1097/IAE.0000000000000742
Edwards, A. O. et al. Complement factor H polymorphism and age-related macular degeneration. Science 308, 421–424 (2005). https://doi.org/10.1126/science.1110189
Haines, J. L. et al. Complement factor H variant increases the risk of age-related macular degeneration. Science 308, 419–421 (2005). https://doi.org/10.1126/science.1110359
Klein, R. J. et al. Complement factor H polymorphism in age-related macular degeneration. Science 308, 385–389 (2005). https://doi.org/10.1126/science.1109557
Arakawa, S. et al. Genome-wide association study identifies two susceptibility loci for exudative age-related macular degeneration in the Japanese population. Nat Genet 43, 1001–1004 (2011). https://doi.org/10.1038/ng.938
Chen, L. J. Genetic Association of Age-Related Macular Degeneration and Polypoidal Choroidal Vasculopathy. Asia Pac J Ophthalmol (Phila) 9, 104–109 (2020). https://doi.org/10.1097/01.APO.0000656976.47696.7d
Chen, W. et al. Genetic variants near TIMP3 and high-density lipoprotein-associated loci influence susceptibility to age-related macular degeneration. Proc Natl Acad Sci U S A 107, 7401–7406 (2010). https://doi.org/10.1073/pnas.0912702107
Dewan, A. et al. HTRA1 promoter polymorphism in wet age-related macular degeneration. Science 314, 989–992 (2006). https://doi.org/10.1126/science.1133807
Hosoda, Y. et al. CFH and VIPR2 as susceptibility loci in choroidal thickness and pachychoroid disease central serous chorioretinopathy. Proc Natl Acad Sci U S A 115, 6261–6266 (2018). https://doi.org/10.1073/pnas.1802212115
Mori, Y. et al. Genome-wide Survival Analysis for Macular Neovascularization Development in Central Serous Chorioretinopathy Revealed Shared Genetic Susceptibility with Polypoidal Choroidal Vasculopathy. Ophthalmology 129, 1034–1042 (2022). https://doi.org/10.1016/j.ophtha.2022.04.018
Neale, B. M. et al. Genome-wide association study of advanced age-related macular degeneration identifies a role of the hepatic lipase gene (LIPC). Proc Natl Acad Sci U S A 107, 7395–7400 (2010). https://doi.org/10.1073/pnas.0912019107
Thee, E. F. et al. The Phenotypic Course of Age-Related Macular Degeneration for ARMS2/HTRA1: The EYE-RISK Consortium. Ophthalmology 129, 752–764 (2022). https://doi.org/10.1016/j.ophtha.2022.02.026
Han, J. W. et al. Overview of the Korean Longitudinal Study on Cognitive Aging and Dementia. Psychiatry Investig 15, 767–774 (2018). https://doi.org/10.30773/pi.2018.06.02
Kim, H. M. et al. Association Between Retinal Layer Thickness and Cognitive Decline in Older Adults. JAMA Ophthalmol 140, 683–690 (2022). https://doi.org/10.1001/jamaophthalmol.2022.1563
Joo, K., Mun, Y. S., Park, S. J., Park, K. H. & Woo, S. J. Ten-Year Progression From Intermediate to Exudative Age-Related Macular Degeneration and Risk Factors: Bundang AMD Cohort Study Report 1. Am J Ophthalmol 224, 228–237 (2021). https://doi.org/10.1016/j.ajo.2020.11.012
Ryoo, N. K. et al. Thickness of retina and choroid in the elderly population and its association with Complement Factor H polymorphism: KLoSHA Eye study. PLoS One 13, e0209276 (2018). https://doi.org/10.1371/journal.pone.0209276
Miyake, M. et al. Choroidal neovascularization in eyes with choroidal vascular hyperpermeability. Invest Ophthalmol Vis Sci 55, 3223–3230 (2014). https://doi.org/10.1167/iovs.14-14059
Li, L. et al. Estrogen and progesterone receptor status affect genome-wide DNA methylation profile in breast cancer. Hum Mol Genet 19, 4273–4277 (2010). https://doi.org/10.1093/hmg/ddq351
Boraska, V. et al. A genome-wide association study of anorexia nervosa. Mol Psychiatry 19, 1085–1094 (2014). https://doi.org/10.1038/mp.2013.187
Kuang, Y., Wang, Y., Cao, X., Peng, C. & Gao, H. New prognostic factors and scoring system for patients with acute myeloid leukemia. Oncol Lett 22, 823 (2021). https://doi.org/10.3892/ol.2021.13084
Batsukh, T. et al. Identification and characterization of FAM124B as a novel component of a CHD7 and CHD8 containing complex. PLoS One 7, e52640 (2012). https://doi.org/10.1371/journal.pone.0052640
De Rubeis, S. et al. Synaptic, transcriptional and chromatin genes disrupted in autism. Nature 515, 209–215 (2014). https://doi.org/10.1038/nature13772
Morandell, J. et al. Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development. Nat Commun 12, 3058 (2021). https://doi.org/10.1038/s41467-021-23123-x
Schumacher, F. R. et al. Characterisation of the Cullin-3 mutation that causes a severe form of familial hypertension and hyperkalaemia. EMBO Mol Med 7, 1285–1306 (2015). https://doi.org/10.15252/emmm.201505444
Abdel Khalek, W. et al. Severe Arterial Hypertension from Cullin 3 Mutations Is Caused by Both Renal and Vascular Effects. J Am Soc Nephrol 30, 811–823 (2019). https://doi.org/10.1681/ASN.2017121307
Xu, J. et al. Subfoveal choroidal thickness in diabetes and diabetic retinopathy. Ophthalmology 120, 2023–2028 (2013). https://doi.org/10.1016/j.ophtha.2013.03.009
Querques, G. et al. Enhanced depth imaging optical coherence tomography in type 2 diabetes. Invest Ophthalmol Vis Sci 53, 6017–6024 (2012). https://doi.org/10.1167/iovs.12-9692
Regatieri, C. V., Branchini, L., Carmody, J., Fujimoto, J. G. & Duker, J. S. Choroidal thickness in patients with diabetic retinopathy analyzed by spectral-domain optical coherence tomography. Retina 32, 563–568 (2012). https://doi.org/10.1097/IAE.0b013e31822f5678
Vujosevic, S., Martini, F., Cavarzeran, F., Pilotto, E. & Midena, E. Macular and peripapillary choroidal thickness in diabetic patients. Retina 32, 1781–1790 (2012). https://doi.org/10.1097/IAE.0b013e31825db73d
Arnould, L. et al. Subfoveal Choroidal Thickness, Cardiovascular History, and Risk Factors in the Elderly: The Montrachet Study. Invest Ophthalmol Vis Sci 60, 2431–2437 (2019). https://doi.org/10.1167/iovs.18-26488
Kim, J. H., Kim, J. R., Kang, S. W., Kim, S. J. & Ha, H. S. Thinner choroid and greater drusen extent in retinal angiomatous proliferation than in typical exudative age-related macular degeneration. Am J Ophthalmol 155, 743–749, 749 e741-742 (2013). https://doi.org/10.1016/j.ajo.2012.11.001
Manjunath, V., Goren, J., Fujimoto, J. G. & Duker, J. S. Analysis of choroidal thickness in age-related macular degeneration using spectral-domain optical coherence tomography. Am J Ophthalmol 152, 663–668 (2011). https://doi.org/10.1016/j.ajo.2011.03.008
Yamazaki, T., Koizumi, H., Yamagishi, T. & Kinoshita, S. Subfoveal choroidal thickness after ranibizumab therapy for neovascular age-related macular degeneration: 12-month results. Ophthalmology 119, 1621–1627 (2012). https://doi.org/10.1016/j.ophtha.2012.02.022

No competing interests reported.

Download PDF

Journal Publication

published 09 Oct, 2024

Read the published version in Scientific Reports →

Editorial decision: Revision requested
08 Jul, 2024
Reviews received at journal
01 Jul, 2024
Reviewers agreed at journal
22 Jun, 2024
Reviews received at journal
04 May, 2024
Reviewers agreed at journal
29 Apr, 2024
Reviewers invited by journal
25 Apr, 2024
Editor assigned by journal
25 Apr, 2024
Editor invited by journal
16 Apr, 2024
Submission checks completed at journal
16 Apr, 2024
First submitted to journal
20 Mar, 2024

You are reading this latest preprint version

Genome-wide association study of subfoveal choroidal thickness in a longitudinal cohort of older adults

Status:

Journal Publication

Version 1

Abstract

Purpose

Methods

Results

Conclusions

Figures

Introduction

Materials and Methods

Discovery Dataset

Validation Dataset

GWAS analysis

In silico analysis using open data source

HUVEC experiment

Cell culture

Immunofluorescence

Cell migration and invasion assay

Results

Discussion

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1