|Year : 2021 | Volume
| Issue : 1 | Page : 29-33
Copy number variation of the NCF1 gene in patients of acne vulgaris
Mohammed A. M. Shoeb1, Ola A Bakry1, Shaimaa E Soliman2, Lamiaa K Shehata3
1 Department of Dermatology, Andrology, and STDs, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Molecular Biology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Dermatology, Andrology and STDs, Nasser General Hospital, Al Kheimah, Al Qalyubia Governorate, Egypt
|Date of Submission||06-Apr-2019|
|Date of Decision||12-May-2019|
|Date of Acceptance||20-May-2019|
|Date of Web Publication||27-Mar-2021|
Lamiaa K Shehata
Department of Dermatology, Andrology and STDs, Nasser General Hospital, Al Kheimah, Al Qalyubia Governorate
Source of Support: None, Conflict of Interest: None
The objective of this study was to evaluate the influence of neutrophil cytosolic factor 1 gene copy number variation (CNV) on acne vulgaris in a sample of Egyptian population to explore whether this CNV affects disease occurrence or increases its risk.
Acne vulgaris is a common skin condition that affects the majority of the population during adolescence. Acne is characterized by a variable combination of comedones, pustules, inflammation, and scarring. Oxidative stress has been considered to play a role in acne. Neutrophil cytosolic factor 1 (NCF1) is a component of NADPH oxidase enzyme, which is responsible for the production of reactive oxygen species from phagocytes. It is reported that increased or decreased copy numbers of NCF1 gene affects many diseases such as rheumatoid arthritis and systemic lupus erythematosus.
Patients and methods
This study included 50 patients divided into two groups: 25 acne patients and 25 apparently healthy controls. Both groups were investigated for NCF1 CNV by quantitative real-time PCR.
We found a statistically significant increased CNV of the NCF1 gene in the acne group compared with the control group (P = 0.028).
Our study suggests that an increased number of copies of the NCF1 gene may be a predisposing factor for acne vulgaris development. Moreover, decreased NCF1 copy numbers may be protective for acne.
Keywords: acne, copy number variation, neutrophil cytosolic factor 1, oxidative stress, reactive oxygen species
|How to cite this article:|
Shoeb MA, Bakry OA, Soliman SE, Shehata LK. Copy number variation of the NCF1 gene in patients of acne vulgaris. Menoufia Med J 2021;34:29-33
|How to cite this URL:|
Shoeb MA, Bakry OA, Soliman SE, Shehata LK. Copy number variation of the NCF1 gene in patients of acne vulgaris. Menoufia Med J [serial online] 2021 [cited 2021 May 8];34:29-33. Available from: http://www.mmj.eg.net/text.asp?2021/34/1/29/311995
| Introduction|| |
Acne vulgaris is a distressing condition of the pilosebaceous unit characterized by the formation of open and closed comedones, papules, nodules, pustules and cysts . Acne consistently represents one of the most prevalent skin conditions in the general population, estimated to affect up to 80% of individuals at some point between the ages of 11 and 30 years . Until now, the etiology of acne vulgaris has been uncertain . Genetic and environmental factors play an important role in the etiology and pathogenesis of AV . Propionibacterium acnes, which is a gram-positive anaerobic bacterium, has the main role in the development of inflammation in acne. Chemotactic substances released from the bacteria attract polymorph nuclear leukocytes to the site of inflammation. Those cells are activated locally to produce inflammatory cytokines such as tumor necrosis factor α, interleukin (IL) 1β, and IL-8 . After phagocytosis of the bacteria, the attracted neutrophils are thought to release lysosomal enzymes and produce reactive oxygen species (ROS) that can damage the follicular epithelium. ROS are involved in that process, as the production of hydrogen peroxide is increased in neutrophils from acne patients . Oxidative stress is the result of an increased number of ROS and an impaired antioxidant system. It has been suggested that oxidative stress plays a role in the pathogenesis of acne vulgaris . The neutrophil cytosolic factor 1 (NCF1) protein is an essential component of the phagocytic NADPH oxidase complex type 2 (the NOX2 complex). Upon phosphorylation in the cytosol, the NCF1 complexes with NCF2 (p67phox) and NCF4 (p40phox), adheres to the membrane and forms the NOX2 complex together with the cytochrome complex of NOX2/CYBB/gp91phox and CYBA/p22phox and, subsequently, transports electrons from NADPH to oxygen, resulting in a variety of ROS . NCF1 gene, which is located on chromosome 7 at location 11.23, encodes the NCF1 protein. In healthy individuals, the NCF1 gene locus contains one NCF1 gene and two pseudo-NCF1 (Ψ-NCF1) genes, one on either side of NCF1 . Copy number variation (CNV), namely regions of the genome that can be either deleted or duplicated in a variable way, has emerged as an important source of genetic variance in the human genome . Genomic studies suggested that gene duplication occurred frequently and in variable numbers during the recent history of human populations, which has led to de-novo formations of CNV. Presumably, because of positive selection, genes encoding certain protein categories are particularly enriched in CNVs, such as those involved in processes related to environmental responses. In this process, duplicated genes are thought to be the 'successful' copies; pseudogenes are those 'unsuccessful' duplicates retained in the genome . The aim of this study was to evaluate the influence of NCF1 gene CNV on acne vulgaris in a sample of Egyptian population to explore whether this CNV affects disease occurrence or increases its risk.
| Patients and methods|| |
This case–control study was carried out on 50 participants (25 patients with acne) and (25 apparently healthy persons as the control group). The patients were attendants of the outpatients' clinic of the Dermatology Department, Menoufia University Hospital, during the period spanning from June 2018 to November 2018, whereas the controls were recruited from healthy volunteers of the blood bank. They were classified into two groups. Group I (patient group) included 25 patients with acne vulgaris, comprising 14 female patients and 11 male patients; their ages ranged between 15 and 29 years. Group II (control group) included 25 age-matched and sex-matched healthy individuals as controls, comprising 14 female individuals and 11 male individuals. Their ages ranged between 15 and 29 years. A written consent was taken from all patients and controls. This work was approved by the ethics committee of our university. Patients below 12 and above 30 years, female patients with pregnancy or lactation, patients suffering from active malignancy, patients with cosmetic-induced acne, presence of concomitant inflammatory disease such as infections and autoimmune disorders, immunocompromised patients, patients with diabetes mellitus or familial hypercholesterolemia, obesity, smoking habit, recent major surgical operations, and drug intake including vitamins and anti-inflammatory drugs for the last 3 months before sample intake were excluded from this study.
Acne severity is determined by scoring of acne lesions by Global Acne Grading System. This system divides the face, chest and back into six areas (forehead, each cheek, nose, chin and chest and back) and assigns a factor to each area on the basis of size .
Three milliliters of venous blood were taken from each patient into an EDTA-containing tube to prevent blood coagulation. All collected samples were stored at −30°C until the time of assay.
Copy number assay by real-time PCR procedure starts with genomic DNA purification. After quality control, the DNA is mixed with the ready-to-use qBiomarker SYBR Mastermix (QIAGEN Inc., Germantown, MD, USA). The mixture is aliquoted into each well of the array plate containing preloaded gene-specific primers.
After real-time PCR, the copy number profile of a particular sample is determined using the ΔΔCt method, measuring the difference in ΔCt values (target gene Ct–reference assay Ct) between a test sample genome and a reference genome (cat. no. 337812; Qiagen, Hilden, Germany).
Results were collected, tabulated, and statistically analyzed by IBM SPSS software package, version 20.0 (IBM Corp., Armonk, New York, USA) Qualitative data were described using number and percentage. The Kolmogorov–Smirnov test was used to verify the normality of distribution. Quantitative data were described using range (minimum and maximum), mean, SD and median. Significance of the obtained results was judged at the 5% level.
| Results|| |
This study included 25 patients and 25 age-matched and sex-matched healthy controls. Cases included 11 male patients and 14 female patients complaining of acne vulgaris. Their ages ranged from 15 to 29 years with a mean ± SD of 22.08 ± 4.21 years. Disease severity ranged from mild, moderate, severe to very severe with a mean ± SD of 29.88 ± 6.75 score according to the Global Acne Grading System.
All cases and controls were subjected to copy number assay by quantitative real-time PCR.
NCF1 copy number was significantly increased in the patients' group compared with the control group (P = 0.028), and it increased the risk of occurrence of acne by 4.030-folds [Table 1], [Table 2] and [Figure 1].
|Table 1: Comparison between cases and controls according to neutrophil cytosolic factor 1 copy number (n=25)|
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|Table 2: Comparison between the two studied groups according to neutrophil cytosolic factor 1 copy number|
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|Figure 1: Comparison of neutrophil cytosolic factor 1 copy number between patients and control group.|
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There was a significant relation between NCF1 copy number and duration of acne disease (P = 0.030). However, there was no significant relationship between NCF1 copy number and age, age of onset and acne scoring of the patients (P = 0.799, 0.508, and 0.365, respectively) [Table 3].
|Table 3: Relation of neutrophil cytosolic factor 1 copy number and clinical data of the cases' group (n=25)|
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There was no significant correlation between the NCF1 gene expression and clinical data of acne patients, including age (r=−0.053, P = 0.802), age of onset (r = 0.301, P = 0.144), disease duration (r=−0.353, P = 0.084) and acne scoring (r=−0.087, P = 0.679) [Table 4].
|Table 4: Correlation between neutrophil cytosolic factor 1 expression and clinical data of the cases' group|
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| Discussion|| |
Acne vulgaris is a chronic inflammatory skin disease of the pilosebaceous unit . It is characterized by seborrhea, comedones, erythematous papules and pustules, less frequently by nodules, deep pustules or pseudocysts, and, in some cases, is accompanied by scarring . The traditional etiologic factors include increased sebum production, ductal hyperkeratosis, abnormality of the microbial flora within the pilosebaceous unit, and mediators of inflammation . The inflammatory mediators recruit neutrophils that release lysosomal enzymes and produce ROS . ROS lead to irritation and destruction of surrounding healthy tissues together with the follicular wall . The NCF1 protein is an essential component of the phagocytic NADPH oxidase complex type 2 (the NOX2 complex) . With other subunits of NOX2, it transports electrons from NADPH to oxygen, resulting in a variety of ROS. The human NCF1 gene, which encodes NCF1 protein, is located at 7q11.23 . CNV is a phenomenon in which regions of the genome can be either deleted or duplicated in a variable way. Impact of CNVs on gene expression, protein function and disease traits is largely unknown. However, increased copy numbers of a gene leads to an increase in its expression, which may indicate subsequent production of the encoded protein . A number of previous studies suggested a possible role of NCF1 CNV in influencing many autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, mannan psoriasis, collagen-induced arthritis and inflammatory bowel disease through affecting ROS production and, subsequently, oxidative stress .
In this study, it was found that there was a statistically significant increase of the copy number of the NCF1 gene in the patients' group compared with the control group. The NCF1 gene increased the risk of occurrence of AV by 4.030-folds. To the best of our knowledge, there are no published data on NCF1 CNV in AV. However, the obtained result could be explained by the increasing NCF1 gene expression leading to the upregulation of ROS production, subsequently, increasing acne predisposition.
There are many previous studies that have suggested a strong association between NCF1 copy number and many autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis. All those studies reported that increase in copy numbers of the NCF1 gene is protective against the development of the disease, and decreased copy numbers of the NCF1 gene increases the risk of disease development ,,.
In contrast to acne vulgaris, low ROS in chronic autoimmune diseases leads to increased disease severity. In addition, increased ROS production regulates chronic autoimmune inflammation instead of promoting it . This may explain why decrease in NCF1 copy numbers is associated with high predisposition to autoimmune diseases in contrast to AV, wherein increase in NCF1 copy numbers is associated with disease development.
In this study there was a significant relation between NCF1 copy number and duration of acne disease [Table 3]. This may add support to the possible role of increased NCF1 expression in acne pathogenesis through more production of ROS. This result is compatible with other studies that showed an association between NCF1 expression and chronicity of autoimmune diseases, not acute exacerbations .
| Conclusion|| |
According to the present results, we conclude that the statistically significant increase in NCF1 CNV in acne patients compared with controls indicates its role in predisposition to acne vulgaris, and it increases the risk of acne occurrence. In addition, it may increase disease duration, but it does not affect the clinical presentation of the disease. Further genetic studies including different populations and ethnicities are needed to expand and validate the present results.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Clayton RW, Göbel K, Niessen CM, Paus R, Steensel MAM. Homeostasis of the sebaceous gland and mechanisms of acne pathogenesis. Br J Dermatol 2019; 17
Lynn DD, Umari T, Dunnick CA, Dellavalle RP. The epidemiology of acne vulgaris in late adolescence. Adolesc Health Med Ther 2016; 7
Bhate K, Williams HC. What's new in acne? An analysis of systematic reviews published in 2011–2012. Clin Exp Dermatol 2014; 39
Chang HC, Lin MH, Huang YC. Association between circulating adipokines and acne vulgaris: a systematic review and meta-analysis. Australas J Dermatol 2019; 13
Grange PA, Raingeaud J, Morelle W, Marcelin A-G, Calvez V, Dupin N. Characterization of a Propionibacterium acnes
surface protein as a fibrinogen-binding protein. Sci Rep 2017; 7
Awad SM, Morsy H, Sayed AA, Mohamed NA, Ezzat GM, Noaman MM. Oxidative stress and psychiatric morbidity in patients with facial acne. J Cosmet Dermatol 2018; 17
Hultqvist M, Sareila O, Vilhardt F, Norin U, Olsson LM, Olofsson P, et al
. Positioning of a polymorphic quantitative trait nucleotide in the Ncf1 gene controlling oxidative burst response and arthritis severity in rats. Antioxid Redox Signal 2011; 14
Olsson LM, Johansson ÅC, Gullstrand B, Jönsen A, Saevarsdottir S, Rönnblom L, et al
. A single nucleotide polymorphism in the NCF1 gene leading to reduced oxidative burst is associated with systemic lupus erythematosus. Ann Rheum Dis 2017; 76
Olsson LM, Holmdahl R. Copy number variation in autoimmunity – importance hidden in complexity? Eur J Immunol 2012; 42
Korbel JO, Kim PM, Chen X, Urban AE, Weissman S, Snyder M, Gerstein MB. The current excitement about copy-number variation: how it relates to gene duplications and protein families. Curr Opin Struct Biol 2008; 18
Doshi A, Zaheer A, Stiller MJ. A comparison of current acne grading systems and proposal of a novel system. Int J Dermatol 1997; 36
Kistowska M, Meier B, Proust T, Feldmeyer L, Cozzio A, Kuendig T, et al
. Propionibacterium acnes
promotes Th17 and Th17/Th1 responses in acne patients. J Invest Dermatol 2015; 135
Zhao J, Ma J, DengY, Kelly JA, Kim K. A missense variant in NCF1 is associated with susceptibility to multiple autoimmune diseases. Nat Genet 2017; 49
Wei X, Li M, Wenzhi L, Tiffany A, Brunson W, Xiaohua T, et al
. Functional pseudogenes inhibit the superoxide production. Precis Med 2015; 1
Zhong J, Olsson LM, Urbonaviciute V, Yang M, Bäckdahl L, Holmdahl R. Association of NOX2 subunits genetic variants with autoimmune diseases. Free Radic Biol Med 2018; 125
[Table 1], [Table 2], [Table 3], [Table 4]