Polymorphism of FAS and FAS Ligand Genes in Alopecia Areata: A Case-control Study in Egyptian Population.

BACKGROUND: Alopecia areata (AA) is a common dermatologic disease with suspected autoimmune etiology. Tumor necrosis factor superfamily member 6 or CD95 (FAS) and FAS ligand (FASL) are proapoptotic proteins. The relationship between apoptosis and autoimmunity is well recognized. Inflammatory T cells in AA are cytotoxic and possess FAS/FASL antigens. AIM: This study aims to investigate the association between FAS-670 A/G and FASL-124 A/G gene polymorphisms and AA to clarify if these polymorphisms influence disease occurrence or increase disease risk.
MATERIALS AND METHODS: A case-control study was conducted on sixty patients with AA, and 40 age- and sex-matched healthy subjects, as a control group. Disease severity was assessed by severity of alopecia tool (SALT) Score. FAS 670A/G and FASL 124A/G gene polymorphisms were investigated by the restriction fragment length polymorphism polymerase chain reaction.
RESULTS: For FAS gene, G/G genotype was significantly higher in cases than in control group with odds ratio 5.1. G allele was more prevalent among patient group with odds ratio 1.75. For FASL gene, A/G genotype was significantly higher in cases than in control group with odds ratio 4.53. G allele was more prevalent among patient group with odds ratio 1.88. GG genotype of FAS was significantly associated with longer disease duration (P =0.001), recurrent attacks (P =0.01), higher SALT score (P =0.009), alopecia universalis (P =0.002), and severe disease (P =0.006).
CONCLUSION: FAS and FASL gene polymorphisms are associated with AA. Further large-scale studies on different ethnicities are required for more clarification of their role in disease development. Therapeutic modalities based on their inhibition could be promising in the treatment of a common disease like AA.

What was known?

FAS/FASL gene polymorphisms are associated with AA.

FAS 670 GG and FASL 124 AG genotypes are associated with increased disease risk.

Introduction

Alopecia areata (AA) is a common dermatosis with nonscarring patches of alopecia on any hair-bearing area of the body.[1] Its etiology is not exactly known. Clinical and experimental studies have pointed to autoimmune involvement, targeting immune privilege sites of hair follicles.[2]

AA almost exclusively attacks anagen hair follicles.[3] Inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1 β, interferon-γ [IFN-γ]) stimulate premature follicular involution through the induction of apoptosis followed by hair loss.[45]

In catagen and telogen, the target autoantigens are not expressed. Therefore, hair follicle is not attacked by immune system and can reenter a new cycle.[6]

FAS is one of the members of the TNF receptor superfamily.[7] It is a transmembrane receptor expressed in brain, heart, kidney, liver, pancreas, thymus, and lymphoid tissues. It belongs to the death receptor family [89] and acts as the target of cell death-inducing antibodies.[10] The human FAS gene, located at chromosome 10q24.1, consists of nine exons and eight introns.[11]

FAS ligand (FASL) is a homotrimeric type II transmembrane protein belonging to TNF superfamily.[12] The human FASL gene is located on chromosome 1q23, consists of four exons spanning, 8 kb, and encodes 281 amino acids.[13]

Engagement of the FASL triggers programmed cell death, which is important in apoptosis mediated by cytotoxic T cells and natural killer cells.[14]

Inflammatory T cells in AA are cytotoxic and possess granzyme B and FAS/FASL cytotoxic mechanisms.[15] Hair follicle epithelium also expresses FAS.[16]

Apoptotic cell death may initiate and propagate autoimmune diseases as it may provide self-antigens or represent the target of the immune response.[17]

It was postulated that polymorphisms of FAS and FASL genes reduce the ability of cells to undergo apoptosis [18] and/or induce exposure of abnormal amounts of apoptosis-related antigens.[19]

The aim of the present work was to investigate the association between FAS-670 A/G and FASL-124 A/G gene polymorphisms and AA in Egyptian population to clarify if these polymorphisms influenced disease occurrence or increased disease risk.

Materials and Methods

Studied population

This case–control study was conducted on 60 unrelated patients with AA, and 40 age- and sex-matched unrelated healthy subjects, who had no present, past, or family history of AA as a control group.

Cases were selected from the Dermatology Outpatient Clinic from February 2016 to December 2016.

Only patients with the characteristic symptomless smooth uninflamed patch (es) were included in the study. Patients with other causes of hair loss including those with concomitant pattern baldness were excluded from the study.

A written consent form approved by the Local Research Ethics Committee was taken from every participant before study inclusion. This was done in accordance with Helsinki declaration of 1975 (revised in 2000).

All selected patients were subjected to complete history taking, general and dermatological examinations. Clinical data describing patients' age, disease duration, onset, course, site of lesions, number of attacks, and family history of AA were collected. For cases with recurrent attacks, disease duration was calculated from the time of the first attack to the time of presentation. Cases were clinically assessed according to:

Kavak et al.[20]

Mild – The presence of three or less patches of alopecia with the widest diameter of three cm or less, or the disease is limited to eyelashes and eyebrows

Moderate – Existence of more than three patches of alopecia, or a patch greater than three cm at the widest diameter without alopecia totalis or alopecia universalis

Severe – Alopecia totalis or alopecia universalis

Ophiasis – Snake-shaped patches extending to the scalp border or loss of hair in the shape of a wave at the circumference of the head.

Severity of Alopecia Tool (SALT) scoring – Scalp (S): S0, no hair loss; S1, <25% hair loss; S2, 25–49% hair loss; S3, 50–74% hair loss; S4, 75–99% hair loss; and S5, 100% hair loss. Body (B): B0, no body hair loss; B1, some body hair loss; and B2, 100% body (excluding scalp) hair loss.[21]

Exclusion criteria

Subjects using topical, intralesional, or systemic agents, such as, steroids or immunosuppressives likely to cause hair regrowth within the past month and subjects who took phototherapy sessions or received blood transfusion, in the past 6 months before this study were excluded. Individuals with other dermatological diseases and/or with systemic autoimmune diseases were also excluded. Cases with causes of hair loss other than AA were excluded. Controls were selected from patient attendees or from those who had minor ailments that were unlikely to affect the immune system so as to intefere with the present evaluation.

Every case and control subject underwent detection of FAS 670A/G and FASLG124A/G gene polymorphisms by restriction fragment length polymorphism polymerase chain reaction (PCR).

DNA Extraction from the whole blood using the Zymo Research Quick-g DNA Mini PrepGenomic DNA purification kit, (USA) was done. DNA eluted in buffer AE was stored at −20°C for further PCR procedure.

Determination of FAS 670 A/G and FAS ligand 124 A/G gene polymorphisms

PCR for the FAS 670 A/G and FASL 124 A/G gene polymorphisms were carried out to a total volume of 25 μl, containing 10 μl of genomic DNA; 1 μl of each primer; 12.5 μl of Master Mix (Genecraft; Germany); (Stratagene; USA); and 1.5 μl of distilled water.[22]

FAS 670 A/G gene was analyzed using the following designed primers (Midland, Texas):

Forward: 5'-ATAGCTGGGGCTATGCGATT-3'

Reverse: 5'-CATTTGACTGGGCTGTCCAT-3'

PCR amplification of FAS 670 A/G gene using Applied Biosystems 2720 thermal cycler (Singapore) was done. PCR condition consisted of one cycle of amplification at 94°C for 2 min followed by 35 cycles at 94°C for 30 s; 62°C for 30 s; 72°C for 45 s; and one final cycle of extension at 72°C for 7 min. The amplification products were separated by electrophoresis through 3% agarose gel stained with and visualized on ethidium bromide with positive band at 193 bp [Figure 1a].

Figure 1

(a) FAS 670 A/G gene, lanes from 2 to 11 show the length of the polymerase chain reaction amplicon which is 193 bp. ladder 50 bp was used. (b) FAS 670 A/G gene polymorphism (Lanes 2, 6, and 8 indicate AA genotype. Lane 4, 5, 10, and 11 indicate AG genotype. Lanes 3, 7, and 9 indicate GG genotype). (c) FAS ligand 124 A/G gene, lanes from 2 to 11 show the length of the polymerase chain reaction amplicon which is 230 bp. Ladder 50 bp was used. (d) FAS ligand 124 A/G gene polymorphism (Lanes 3, 4 and 11 indicate AA genotype. Lane 2, 5, 7, 8, and 10 indicate AG genotype. Lanes 6 and 9 indicate GG genotype)

FAS 670 A/G genotyping using the restriction fragment length polymorphism technique

Fifteen μl of the PCR products of FAS gene were mixed with 1 μl (1 unit) of Fast Digest® ScrFI restriction enzyme (provided by Fermentas) with 6.5 μl nuclease-free water and 2.5 μl of 10X FastDigest® Buffer.[22]

The mixture was well mixed and incubated at 65°C for 30 min; and then, 10 μl of the product were loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The uncut fragment was 193 bp, and digestion products were 136 bp and 57 bp [Figure 1b].

FASL 124 A/G gene was analyzed using the following designed primers (Midland, Texas):

Forward: 5'-GCAGTTCAGACCTACATGATTAGGAT-3'

Reverse: 5'-CCAGATACAGACCTGTTAAATGGGC-3'

PCR amplification of FASL 124 A/G gene using Applied Biosystems 2720 thermal cycler (Singapore) was done. PCR condition consisted of one cycle of amplification at 95°C for 5 min followed by 35 cycles at 95°C for 50 s; 58°C for 50 s; 72°C for 50 s; and one final cycle of extension at 72°C for 5 min. The amplification products were separated by electrophoresis through 3% agarose gel stained with and visualized on ethidium bromide with positive band at 230 bp [Figure 1c].

FAS ligand 124 A/G genotyping using the restriction fragment length polymorphism technique

Fifteen μl of the PCR products of FASL 124 A/G gene were mixed with 1 μl (1 unit) of Fast Digest® fok1 restriction enzyme (provided by Fermentas) with 6.5 μl nuclease-free water and 2.5 μl of 10X FastDigest® Buffer.[22] The mixture was well mixed and incubated at 37°C for 60 min, then 10 μl of the product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The uncut fragment was 230 bp and digestion products were 180 bp and 50 bp [Figure 1d].

Statistical analysis

Data were collected, tabulated, and statistically analyzed using a personal computer with “(SPSS) version 11” (SPSS Inc., Chicago, IL, USA) program. Fisher's exact test was used for comparison of qualitative variables in 2×2 tables when expected cell count of more than 25% of cases was <5. Chi-square test (χ2) was used to study the association between two qualitative variables. Mann–Whitney U test was used for comparison between two groups not normally distributed having quantitative variables. Odds ratio was used to describe the probability. Differences were considered statistically significant at P <0.05.

Results

Clinical data of selected cases are summarized in Table 1.

Table 1

Clinical data of selected cases

FAS genotypes and FAS alleles in studied groups

FAS (A/A) genotype was more prevalent in control group than in AA patients. (G/G) genotype was significantly higher in cases than in control group with odds ratio 5.1. (A/G) genotype was present in 61.7% of cases and 57.5% of control with odds ratio 2.23 [Table 2].

Table 2

Prevalence of FAS and FAS ligand genotypes and alleles in studied groups

(A) allele was more prevalent in control than in the patient group. (G) allele was more prevalent in the patient group with odds ratio 1.75 [Table 2].

FAS ligand genotypes and FAS ligand alleles in studied groups

(A/A) genotype was more prevalent in control group than AA patients while (A/G) genotype was significantly higher in cases than control group with odds ratio 4.53. (G/G) genotype was present in 13.3% of cases and 15% of controls with odds ratio 5.1 [Table 2].

(A) allele was more prevalent in control than the patient group. (G) allele was more prevalent in the patient group with odds ratio 1.88 [Table 2].

Relationship between FAS genotypes and clinical data of studied patients

GG genotype was significantly associated with longer disease duration (P =0.001), recurrent attacks (P =0.01), higher SALT score (P =0.009), alopecia universalis (P =0.002), and severe disease (P =0.006) [Figure 2].

Figure 2

Relationship between FAS genotypes and (a) disease duration/weeks, (b) number of attacks, (c) severity of alopecia tool Score, (d) disease severity, and (e) disease subtypes in studied cases

Relationship between FAS ligand genotypes and clinical data of studied cases

No significant association was found between FASL genotypes and clinical data of studied cases (data not shown in table or figure).

Discussion

In the current work, GG genotype of FAS gene was significantly more prevalent in cases compared with controls. It increased AA risk by 5.1 fold. In addition, AG genotype was more prevalent in cases. It increases the risk of disease development by 2.23 fold.

Conflicting with our findings, Kalkan et al. observed that GG genotype of FAS-670 A/G polymorphism was found to be protective against AA in a Turkish population.[22]

Fan et al. studied the associations between FAS polymorphisms and the risk of AA in Chinese Han population. They found that a reduced risk of alopecia appeared to be associated with the FAS 670 AG when compared with the FAS 670 AA genotype.[23]

These conflicting results may be explained by different clinical criteria of selected population and different ethnic backgrounds.

In the current work, GG genotype of FAS gene was significantly associated with severe disease, long disease duration, recurrent attacks, and higher SALT score.

The substitution of G to A in the position-670 changes the IFN-γ activation site (GAS)[24] which is involved in IFN-γ and IFN-α signaling.[25]

GAS elements bind to homodimers of a phosphorylated form of STAT1. IFN-γ cause tyrosine phosphorylation of STAT1. Subsequently, phosphorylated STAT1 translocates into the nucleus where it induces transcription of GAS containing genes [26] including FAS gene.[2728293031]

FAS-670 G variant containing GAS could be affected by IFN-γ production and increase the transcription of FAS. This may result in different degrees of apoptosis. The risk of apoptosis increased additively with the number of G alleles.[32]

IFN-γ may deprive dermal papilla cells from their ability to maintain anagen growth, and this produces immune privilege collapse of normal human anagen hair follicles.[33] It acts as a potent catagen inducer in human scalp hair follicles.[34]

The present work showed that AG genotype of FASL gene was significantly more prevalent in cases compared with controls. It increases the risk of alopecia by 4.53 times. In addition, GG genotype was more prevalent in cases with odds ratio 5.1. G allele was significantly associated with patients with odds ratio 1.88.

Kalkan et al. reported that there was no difference between AA patients and controls regarding genotype and allele distribution of FASL gene 124 A/G polymorphism. However, they found that alopecia severity was increased by FASL gene 124 A/G polymorphism.[22]

Based on the demonstrated findings, modulation of FAS/FASL pathway may have therapeutic effect on AA. Inhibition of the FAS-FASL system might protect hair follicles from autoimmune injury. However, such modulation should be restricted to hair follicles, to avoid disturbing essential control mechanisms of lymphocyte homeostasis.[18]

Kuwahara et al. reported a therapeutic effect of antihuman FAS antibody on graft-versus-host disease model. Administration of anti-human FAS antibody decreased the level of FAS-positive lymphocytes and suppressed the development of skin disease.[35]

It is possible to restrict FASL expression by injection of replication-defective viral vectors locally. FASL gene transfer results in apoptosis without eliciting systemic toxicity.[36]

Conclusion

FAS/FASL gene polymorphisms are associated with AA. FAS 670 GG and FASL 124 AG genotypes are associated with increased disease risk. Further large-scale studies on different ethnic groups are required for more clarification of their role in disease development. Their interaction with disease triggering factors and environmental factors needs further research. The use of therapeutic modalities based on their inhibition could be promising in the treatment of a common disease like AA. As AA is multifactorial disease, other genetic studies including gene-gene and gene-environment interactions are needed for more clarification of disease pathogenesis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

What is new?

This is the first study to report an association between FAS-670 A/G and FASL-124 A/G gene polymorphism and AA in Egyptian population.

GG genotype of FAS is associated with longer disease duration, recurrent attacks, higher SALT score, alopecia universalis, and severe disease.