Association between NF-kB polymorphism and age-related macular degeneration in a high-altitude population.

OBJECTIVE: To investigate the association between the nuclear factor kappa B (NF-kB) gene polymorphism and age-related macular degeneration (AMD) in a high-altitude population.
METHODS: Fifty-five patients with AMD and 57 control subjects were recruited from the Qinghai Provincial People's Hospital, China. Genomic DNA was extracted from the blood sample of each participant. Four NF-kB polymorphisms (rs3774959, rs3774932, rs3774937, and rs230526) were genotyped using a MassARRAY system. The genotype and allele frequencies were compared between the case and control groups using the chi-squared test or Fisher's exact test.
RESULTS: There was no significant difference in sex, age, hypertension, diabetes, blood lipid level or smoking and drinking status between the AMD and control groups (P > 0.05). The genotype distributions of four NF-kB polymorphisms were in accordance with Hardy-Weinberg equilibrium in the control group (P > 0.05). The frequencies of genotype AA of rs3774932 and genotype CC of rs3774937 were nominally significantly higher in the AMD group than in the control group (P = 0.046 and 0.023, respectively), although these associations did not survive the Bonferroni correction (corrected P > 0.05). Genotype distributions of rs3774959 and rs230526 were not significantly different between the two groups (P = 0.08 and 0.16, respectively). No significant difference in the allele frequencies of the four polymorphisms was found between the AMD and control groups (P > 0.05).
CONCLUSIONS: Genotype AA of rs3774932 and genotype CC of rs3774937 in NF-kB might be risk factors for AMD.

Introduction

Age-related macular degeneration (AMD) is one of the most common irreversible blinding eye diseases, which seriously affects patients’ quality of life. The disease affects tens of millions of people all over the world [1]. In China, the prevalence of AMD ranged from 2.44% in people aged 45–49 years to 18.98% in people aged 85–89 years [2]. The development of AMD is related to many factors, such as age, sex, race, eating habits, obesity, and sun exposure, but the specific etiology is not clear [3]. Among various factors affecting the pathogenesis of AMD, mitochondrial damage in the retinal pigment epithelium (RPE) is an important cause of RPE dysfunction [4]. Studies have shown that mitochondrial damage in the RPE is closely related to inflammation, autophagy, and apoptosis [5-7]. In recent years, results from many studies have suggested that genetic factors may play an important role in the development of AMD. Studies have shown that the heritability of AMD can be as high as 71% [8], which indicates that genetic factors are closely related to the development of AMD. Over 50% of the heritability of AMD has been explained by two major genes (CFH and ARMS2/HTRA1), making it one of the most well-defined genetically complex disorders [9]. A recent genome-wide association study (GWAS) on 43,566 subjects identified 52 independently associated variants spanning 34 loci for AMD [10].

The nuclear factor kappa B (NF-kB) family of transcription factors plays a pivotal role in regulating inflammatory response, immune function, and malignant transformation [11]. In addition, NF-kB affects the expression of genes for cell [6] differentiation, proliferation, and survival in almost all multicellular organisms [12]. There is also a significant correlation between NF-kB and autophagy [13]. NF-kB is composed of a group of homodimer and heterodimer protein complexes, and p50/p65/p53 heterodimer complex is the most common complex [14]. Results from studies have suggested that the NF-kB gene plays a role in the occurrence of lung cancer, colorectal cancer, breast cancer, and other cancers [15-18]. Some studies have shown that the NF-kB gene has strong correlation with inflammation and autophagy, while inflammation and autophagy are important pathogenesis of AMD [19-23]. A recent genome-wide meta-analysis identified novel loci associated with AMD, including C4BPA-CD55, ZNF385B, ZBTB38, and NFKB1 [24].

However, researchers have not investigated the association between the NF-kB gene and the AMD risk in a Chinese population. In the present study, four single nucleotide polymorphisms (SNPs) of NF-kB (rs3774959, rs3774932, rs3774937, and rs230526) were selected for genotyping and their associations with the risk of AMD were analyzed in a high-altitude Chinese population.

Methods

Study participants

Fifty-five patients with AMD were and 57 control participants were recruited from the Qinghai Provincial People’s Hospital, China from December 2016 to December 2019. This study was conducted in accordance with the tenets of the Declaration of Helsinki and has been approved by the local hospital ethics committee (approval number 2017–21). Written informed consent was obtained from all participants.

According to the Preferred Practice Pattern Guidelines: Age-related Macular Degeneration [25] and the “Chinese clinical diagnosis and treatment pathway for age-related macular degeneration” [26], AMD was diagnosed when the presence of one or more of the following criteria was met: 1) medium-sized hyaline warts (>63 μm in diameter); 2) RPE abnormalities such as hypopigmentation, pigment proliferation, migration, and metaplasia; and 3) any of the following characteristics: retinal pigment epithelium map atrophy, choroidal neovascularization (exudative), polypoid choroidal vasculopathy, and retinal hemangioma hyperplasia.

The inclusion criteria for AMD patients were as follows: 1) long-term (>20 years) residence at altitudes >2,000 m; 2) age >40 years; and 3) diagnosed with AMD based on the diagnostic criteria. The exclusion criteria for AMD patients were as follows: 1) late vitelliform macular degeneration; 2) choroidal neovascularization with high myopia; 3) Stargardt disease; 4) retinal vascular occlusion; 5) chorioretinitis; 6) diabetic retinopathy; 7) hypertensive retinopathy; and 8) other eye diseases.

All control participants were >40 years of age and diagnosis of AMD and other fundus diseases had been ruled out by fundus examination. Participants with other diseases such as high myopia and glaucoma were also excluded. In addition, those with serious systemic diseases such as hypertension, diabetes, renal insufficiency, blood diseases, and benign or malignant tumors were excluded.

Sample collection and DNA extraction

Two milliliters of peripheral blood was collected from all participants. After anticoagulant treatment, the samples were frozen at −8°C before use. Genomic DNA was extracted using xxx. DNA quality was assessed using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

SNP genotyping

Single nucleotide polymorphisms (SNPs) were genotyped using a MassARRAY system (Sequenom, San Diego, CA, USA). Single-base extension primers were designed and synthesized by the Sangon Biotech (Shanghai, China). The sequences of the primers are listed in Table 1.

Table 1

Sequences of the PCR primers used in this study.

SNP	Forward primer	Reverse primer	Amplicon (bp)
rs3774959	5’-AGTAACACCACATAGGCAGTAACG-3’	5’-TGACTGATGAGATACTGGGGCTA-3’	184
rs3774932	5’-TCTAGCAGAATCCCACAACTGAATA-3’	5’-ATTCCAAGTCTCCATTAATCGTACA-3’	332
rs3774937	5’-CATAATGCATGAGGTCTACTTCTTC-3’	5’-AACTGCTTCAATACCTCTGTTCATG-3’	242
rs230526	5’-GCTTGGCAGCAGCAATTTAA-3’	5’-TACGGCAACAAAGGTACATACAA-3’	307

PCR, polymerase chain reaction; SNP, single nucleotide polymorphism.

The PCR reaction was performed using a MassARRAY mass spectrometer (Sequenom, San Diego, CA). Genotypes were called using the MassARRAY RS1000 software (Sequenom).

Statistical analysis

SPSS statistical software (version 25.0; IBM, Armonk, NY, USA) was used for statistical analysis. The numerical data were expressed as mean ± standard deviation and compared between the case and control groups using the Student’s t test. The categorical data were expressed as n (%) and compared between the two groups using the chi-squared test. Hardy-Weinberg equilibrium was tested for genotype distributions using the chi-squared test. Genotype and allele frequencies were compared between the case and control groups using the chi-squared test or Fisher’s exact test. We corrected for multiple tests using the Bonferroni method. A P < 0.05 was considered statistically significant. Power analysis was performed using the Genetic Power Calculator [27].

Results

Demographic and clinical features of patients with AMD and controls

The AMD group consisted of 28 males and 27 females, with an average age of 66.5 (±11.8) years. The control group had 29 males and 28 females, with an average age of 65.5 (±11.5) years. There was no significant difference in sex, age, hypertension, diabetes, blood lipid level, or smoking or drinking status between the AMD group and the control group (P > 0.05; Table 2).

Table 2

Demographic and clinical features of patients with AMD and controls.

Feature	AMD (n = 55)	Controls (n = 57)	P
Female (%)	27 (49.1)	28 (49.1)	0.99
Age (years)	66.5 ± 11.8	65.5 ± 11.5	0.13
Hypertension (%)	21 (38.2)	19 (33.3)	0.69
Diabetes (%)	7 (12.7)	9 (15.8)	0.98
Hyperlipidemia (%)	9 (16.4)	7 (12.3)	0.54
Smoking (%)	4 (7.3)	6 (10.5)	0.55
Drinking (%)	4 (7.3)	6 (10.5)	0.55

AMD, age-related macular degeneration.

Genotype distributions of NF-kB gene polymorphisms in patients with AMD and controls

Genotype distributions of all four SNPs followed Hardy-Weinberg equilibrium in the control group (P > 0.05). The frequencies of genotype AA of rs3774932 and genotype CC of rs3774937 were nominally significantly higher in the AMD group than in the control group (P = 0.046 and 0.023, respectively), although these associations did not survive the Bonferroni correction (corrected P > 0.05). Genotype distributions of rs3774959 and rs230526 were not significantly different between the two groups (P = 0.08 and 0.16, respectively; Table 3).

Table 3

Genotype distributions of NF-kB gene polymorphisms in patients with AMD and controls.

SNP	Allele	Group	Genotype, n (%)			P
	(1/2)		1/1	1/2	2/2
rs3774959	G/A	AMD	22 (0.400)	21 (0.382)	12 (0.218)	0.08
		Control	26 (0.456)	27 (0.474)	4 (0.070)
rs3774932	A/G	AMD	21 (0.382)	19 (0.345)	15 (0.273)	0.046
		Control	14 (0.246)	33 (0.579)	10 (0.175)
rs3774937	T/C	AMD	29 (0.527)	15 (0.273)	11 (0.200)	0.023
		Control	28 (0.491)	26 (0.456)	3 (0.053)
rs230526	G/A	AMD	19 (0.345)	23 (0.418)	13 (0.236)	0.16
		Control	20 (0.351)	31 (0.544)	6 (0.105)

AMD, age-related macular degeneration; SNP, single nucleotide polymorphism.

Allele distributions of NF-kB gene polymorphisms in patients with AMD and controls

There was no significant difference in allele frequencies of four polymorphisms between the AMD and control groups (P > 0.05; Table 4).

Table 4

Genotype distributions of NF-kB gene polymorphisms in patients with AMD and controls.

SNP	Allele	AMD group	Control group	P	OR	95% CI
rs3774959	A	45 (0.409)	35 (0.307)	0.11	1.56	[0.90, 2.71]
	G	65 (0.591)	79 (0.693)
rs3774932	A	61 (0.555)	61 (0.535)	0.77	1.08	[0.64, 1.83]
	G	49 (0.445)	53 (0.465)
rs3774937	C	37 (0.336)	32 (0.281)	0.37	1.30	[0.74, 2.29]
	T	73 (0.664)	82 (0.719)
rs230526	A	49 (0.445)	43 (0.377)	0.30	1.33	[0.78, 2.26]
	G	61 (0.555)	71 (0.623)

AMD, age-related macular degeneration; CI, confidence interval; OR, odds ratio; SNP, single nucleotide polymorphism.

Discussion

AMD is a complex, highly inherited multifactorial disease caused by the interaction of genetic and environmental risk factors [28]. In this study, we compared the frequencies of NF-kB gene polymorphisms between AMD cases and controls to explore the correlation between the NF-kB gene and AMD. The allele frequencies of the four SNPs analyzed in this study are comparable to the corresponding allele frequencies in East Asians reported in the public gnomAD database [29] (Table 4). Our results showed that the frequencies of genotype AA of rs3774932 and genotype CC of rs3774937 were nominally significantly higher in the AMD group than in the control group (Table 3), although these associations did not survive the Bonferroni correction (corrected P > 0.05). These findings suggest that individuals carrying genotype AA of rs3774932 or genotype CC of rs3774937 may have a higher risk of developing AMD.

Silico analyses suggest that SNP rs3774932 could change the Nkx2 and SIX5 motifs of the NF-kB protein, while SNP rs3774937 could change the DMRT1, LUN-1, and YY1 motifs of the NF-kB protein [30]. Functional studies such as the ChIP assay to identify the binding activity of these genotypes or luciferase reporter assay to test the function of these polymorphisms, especially the NF-kB binding site activity in genotype AA of rs3774932 or genotype CC of rs3774937, would be helpful to further elucidate the potential effects of these genotypes on gene transcriptional regulation and expression, consequently affecting the NF-kB pathway activation and/or susceptibility to AMD pathology.

This study had several limitations. First, the power of this study to detect the association between the NF-kB gene polymorphisms and AMD was limited due to small sample sizes. We estimated that this study had a power of 0.78 to detect the association at OR = 2.0 and a prevalence of 7.11% for people aged 65–69 years [2]. Therefore, the negative associations we observed after correcting for multiple tests in this study did not disprove potential real associations between the NF-kB gene polymorphisms and AMD. Second, the use of 40 years of age as an inclusion criterion for AMD cases and controls may have compromised the representativeness of the samples in this study. Although AMD can be diagnosed as early as 35 years, most AMD cases are diagnosed at age 60 years and older [2]. In addition, since control participants younger than 60 years may develop AMD in their later life, the age cut-off may have introduced selection bias in this study, resulting in lower power to detect real associations. Third, even though one of the strengths of this study is that all patients with AMD were long-term residents (>20 years) at a high altitude (over 2,000 m), which is a unique population that has been under-represented in genetic studies of AMD, the lack of comparison to non-high-altitude patients in this study made it impossible to conclude that the risk alleles are associated with AMD in the high-altitude population alone.

In conclusion, the results of this study suggest that the AA genotype at rs3774932 and the CC genotype at rs3774937 in the NF-kB gene may be risk factors for the development of AMD. This is the first study that indicates an association between these two NF-kB variants and the risk of AMD. Whether this association exists in non-Chinese populations is worth further study.

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4 Mar 2021

PONE-D-20-40186

Association between NF-kB polymorphism and Age-related macular degeneration in high altitude populationAssociation between NF-kB polymorphism and A ge-related macular degeneration in high altitude population

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Reviewer #1: Yin et al examined 55 patients and 57 controls from the Qinghai Provicial People’s Hospital and genotyped them for four SNPs in NFK-B. The paper claims a risk association with NFK-B for risk of AMD in a population that had lived more than 20 years in a high altitude climate (>2000m). The study is interesting but there are some major issues.

1) The usual age of diagnosis of AMD is >60. However, here >age 40 was used. Could the authors please clarify?

2) The sample size is extremely small (slightly over 100 people), and therefore conclusions regarding age, gender, or other differentiation is extremely hard to ascertain, including association of NFK-B to AMD. Could the authors provide please how they were able to have the power to draw the conclusions they did?

3) No comparison to non-high altitude patients was obtained, even a larger study to evaluate the NFK-B polymorphism to see how common the alleles are in the regular population- which means that this study cannot come to the conclusion as to whether or not the alternate allele is associated with AMD in the high-altitude population alone. The reader does not even know how common the alternate alleles are in the general population through examining databases like GnomeAD or ExAC. Could the authors please clarify?

4) The p-values are extremely high (0.04) to declare an association.

5) The paper language needs to be edited along with spelling and grammar, to improve the clarity for the reader.

Reviewer #2: Association between NF-kB polymorphism and Age-related macular degeneration in high altitude population

The authors have intended to investigate the association between NF-kB gene polymorphism and age-related macular degeneration in high altitude population. The study concludes that AA genotype at rs3774932 and CC genotype at rs3774937 in NF-kB gene could be the risk genotypes for AMD. However, this reviewer has few major concerns to be addressed by the authors in order to enhance the quality of the manuscript.

• The ethical committee numbers are different on the ethic statement and in the manuscript.

Introduction: Overall, this section needs to be revised.

• The details in this section is not sufficient enough to substantiate the primary goal. I suggest including more information and citations relevant to Nf-KB gene and its association with AMD.

• The subheading “Subjects” could be under the methods section.

Methods: Overall, the method section needs a revision and more references could be added to enable the readers to follow without confusion.

• 1.2.1 - Provide sample size with more details.

• 1.2.2 - Provide reference and add more details.

• 1.2.3 - Provide the primers and probe details in the table format.

• 1.2.4 - The representative gel images could be added.

• 1.2.5 - very less details

• Did the author further confirm the genotypes/SNP of these four variants of NF-KB by PCR-RFLP?

• Besides, either the ChIP assay to identify the binding activity of this genotypes or luciferase reporter assay to test the function of these polymorphisms, especially the NF-κB binding site activity in AA and CC alleles, could have been attempted.

Results: Overall, the results are not presented well and need thorough revision.

• 2.3 - grou-----group

Discussion:

The current data obtained from this study was not sufficient enough to conclude. Also, the findings are not discussed well.

• The results should be discussed with respect to how these genetic variations (rs3774932 and rs3774937) might influence gene transcriptional regulation and expression, consequently affecting NF-κB pathway activation and/or the susceptibility to AMD pathology.

Figures &Tables:

Some of the details on table-3 and supplementary materials are provided in different language.

Please consider.

• The punctuations/ spacings throughout the manuscript should be rectified.

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9 Apr 2021

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Responses to Reviewer #1:

Reviewer #1: Yin et al examined 55 patients and 57 controls from the Qinghai Provincial People’s Hospital and genotyped them for four SNPs in NFK-B. The paper claims a risk association with NFK-B for risk of AMD in a population that had lived more than 20 years in a high-altitude climate (> 2000 m). The study is interesting but there are some major issues.

Response: Thank you for the opportunity to improve our manuscript.

1) The usual age of diagnosis of AMD is > 60. However, here > age 40 was used. Could the authors please clarify?

Response: We agree with the Reviewer that the use of ≥ 40 years of age as an inclusion criterion for AMD cases and controls may have compromised the representativeness of the samples in this study. Although AMD can be diagnosed as early as 35 years, most AMD cases are diagnosed at ≥ 60 years [Song P, Du Y, Chan KY, Theodoratou E, Rudan I. The national and subnational prevalence and burden of age-related macular degeneration in China. J Glob Health. 2017 Dec;7(2):020703.]. In addition, since control participants younger than 60 years may develop AMD later in life, our inclusion criterion of patients ≥ 40 years may have introduced selection bias in this study, resulting in lower power to detect real associations. We have added these statements to the Discussion.

2) The sample size is extremely small (slightly over 100 people), and therefore conclusions regarding age, gender, or other differentiation are extremely hard to ascertain, including association of NFK-B to AMD. Could the authors provide please how they were able to have the power to draw the conclusions they did?

Response: We agree with the Reviewer that the power of our study to detect the association between the NF-kB gene polymorphisms and AMD was limited due to the small sample size. We estimated that this study had the power of 78% to detect the association at OR = 2.0 and the prevalence at 7.11% for people aged 65–69 years [Song P, Du Y, Chan KY, Theodoratou E, Rudan I. The national and subnational prevalence and burden of age-related macular degeneration in China. J Glob Health. 2017 Dec;7(2):020703.]. Therefore, the negative associations we observed after correcting for multiple tests in this study did not disprove potential associations between the NF-kB gene polymorphisms and AMD. We have added these statements to the Discussion.

3) No comparison to non-high-altitude patients was obtained, even a larger study to evaluate the NFK-B polymorphism to see how common the alleles are in the regular population- which means that this study cannot come to the conclusion as to whether or not the alternate allele is associated with AMD in the high-altitude population alone. The reader does not even know how common the alternate alleles are in the general population through examining databases like GnomeAD or ExAC. Could the authors please clarify?

Response: One of the strengths of this study is that all patients with AMD were long-term (> 20 years) residents at a high altitude (> 2,000 m), which is a unique population that is under-represented in genetic studies of AMD. However, we agree that the lack of comparison to non-high-altitude patients in this study made it impossible to conclude that the risk alleles are associated with AMD in the high-altitude population alone. The allele frequencies of the four SNPs analyzed in this study are comparable to the corresponding allele frequencies in East Asians reported in the public gnomAD database (Table 3). We have added these statements to the Discussion.

4) The p-values are extremely high (0.04) to declare an association.

Response: We agree that the associations we observed in this study were nominally significant and did not survive the Bonferroni correction (corrected P > 0.05). We have clarified this in the Results.

5) The paper language needs to be edited along with spelling and grammar, to improve the clarity for the reader.

Response: Thank you for the comments. We have improved our language by correcting spelling and grammar.

Responses to Reviewer #2:

Reviewer #2: Association between NF-kB polymorphism and age-related macular degeneration in a high-altitude population.

The authors have intended to investigate the association between NF-kB gene polymorphism and age-related macular degeneration in a high-altitude population. The study concludes that the AA genotype at rs3774932 and CC genotype at rs3774937 in NF-kB gene could be the risk genotypes for AMD. However, this reviewer has few major concerns to be addressed by the authors in order to enhance the quality of the manuscript.

• The ethical committee numbers are different on the ethics statement and in the manuscript.

Response: Thank you for pointing out this error. We have corrected it in the manuscript.

Introduction: Overall, this section needs to be revised.

Response: We have revised the Introduction.

• The details in this section are not sufficient enough to substantiate the primary goal. I suggest including more information and citations relevant to the Nf-KB gene and its association with AMD.

Response: We have added more information and citations relevant to the NF-kB gene and its association with AMD.

• The subheading “Subjects” could be under the methods section.

Response: We have revised the subheadings across the entire manuscript.

Methods: Overall, the methods section needs a revision and more references could be added to enable the readers to follow without confusion.

• 1.2.1 - Provide sample size with more details.

• 1.2.2 - Provide reference and add more details.

• 1.2.3 - Provide the primers and probe details in the table format.

• 1.2.4 - The representative gel images could be added.

• 1.2.5 - Very less details

Response: We have revised the Methods according to the Reviewer’s comments.

• Did the author further confirm the genotypes/SNP of these four variants of NF-KB by PCR-RFLP?

Response: We did not further confirm the genotypes of these four SNPs in NF-kB by PCR-RFLP. However, we used the well-known MassARRAY system to genotype the SNPs.

• Besides, either the ChIP assay to identify the binding activity of these genotypes or luciferase reporter assay to test the function of these polymorphisms, especially the NF-κB binding site activity in AA and CC alleles, could have been attempted.

Response: Thank you for the comments on functional studies. Those will be one of the major aims in our future studies.

Results: Overall, the results are not presented well and need thorough revision.

Response: We have thoroughly revised the Results section.

• 2.3 - grou-----group

Response: We have corrected this typo.

Discussion:

The current data obtained from this study was not sufficient enough to conclude. Also, the findings are not discussed well.

• The results should be discussed with respect to how these genetic variations (rs3774932 and rs3774937) might influence gene transcriptional regulation and expression, consequently affecting NF-κB pathway activation and/or the susceptibility to AMD pathology.

Response: We have revised the Discussion.

Figures &Tables:

Some of the details on table-3 and supplementary materials are provided in a different language.

Response: We have removed all non-English text from the manuscript.

Please consider.

• The punctuations/ spacings throughout the manuscript should be rectified.

Response: We have reformatted the entire manuscript.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

6 May 2021

Association between NF-kB polymorphism and age-related macular degeneration in a high-altitude population

PONE-D-20-40186R1

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Reviewers' comments:

14 May 2021

PONE-D-20-40186R1

Association between NF-kB polymorphism and age-related macular degeneration in a high-altitude population

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