Selma Emre, MD

Melasma is an acquired hyperpigmentation disorder characterized by irregular brown macules and patches that usually appear on sun-exposed areas of the skin. The term melasma originates from the Greek word melas meaning black.¹ Facial melasma is divided into 2 groups according to its clinical distribution: centrofacial lesions are located in the center of the face (eg, the glabellar, frontal, nasal, zygomatic, upper lip, chin areas), and peripheral lesions manifest on the frontotemporal, preauricular, and mandibular regions.^1,2 There is debate on the categorization of zygomatic (or malar) melasma; some researchers argue it should be categorized independent of other areas, while others include malar melasma in the centrofacial group because of its frequent association with the centrofacial type, especially with glabellar lesions.² Mandibular melasma is rare and occurs mostly in postmenopausal women after intense sun exposure.^1,2 Although the etiopathogenesis of the disease is not clearly known, increased melanogenesis, extracellular matrix alterations, inflammation, and angiogenesis are assumed to play a role.³ Various risk factors such as genetic predisposition, UV radiation (UVR) exposure, pregnancy, thyroid dysfunction, and exogenous hormones (eg, oral contraceptives, hormone replacement therapy) have been identified; phototoxic drugs, anticonvulsants, and some cosmetics also have been implicated.^4,5 Exposure to UVR is thought to be the main triggering environmental factor by inducing both melanin production and oxidative stress.⁵ However, it also has been shown that visible light can induce hyperpigmentation in darker skin types.⁶

The presence of oxidative stress in melasma recently has become an intriguing topic of interest. First, the presence of oxidative stress in the etiopathogenesis of melasma was thought to be based on the effectiveness of antioxidants in treatment. A few studies also have confirmed the presence of oxidative stress in melasma.^7-10 Classically, oxidative stress can be described as a disturbance in the balance between oxidants and antioxidants. Reactive oxygen species (ROS) are highly reactive molecules due to the unpaired electrons in their structure. Although ROS are present at low levels in physiologic conditions and are involved in critical physiologic events, they damage cellular components such as fat, protein, and nucleic acid at high concentrations.⁵

Dynamic thiol/disulfide homeostasis is one of the most important markers of oxidative stress in biological systems. Thiols are organic compounds containing a sulfhydryl (-SH) group. Thiols are considered highly potent antioxidants because they reduce unstable free radicals by donating electrons. They are the first antioxidants to be depleted in an oxidative environment.^11,12 In case of oxidative stress, they transform into reversible forms called disulfide bridges between 2 thiol groups. Disulfide bridges can be reduced back to thiol groups, which is how dynamic thiol/disulfide homeostasis is maintained. Dynamic thiol/disulfide homeostasis is responsible for cellular events such as antioxidant defense, signal transduction, regulation of enzyme function, and apoptosis.^11,12

The aim of this study was to evaluate the presence of oxidative stress in melasma by comparing dynamic thiol/disulfide homeostasis in patients with melasma compared with age- and sex-matched healthy controls.

Materials and Methods

Participants and Eligibility Criteria—We conducted a prospective study in a tertiary-care hospital (Ankara Bilkent City Hospital [Ankara, Turkey]) of patients with melasma who were followed from October 2021 to October 2022 compared with age- and sex-matched healthy volunteers. Ethics committee approval was obtained from Ankara Bilkent City Hospital before the study (E2-21-881)(13.10.2021). Written informed consent was obtained from all participants, and all were older than 18 years. Patients were excluded if there was the presence of any systemic disease or dermatologic disease other than melasma; smoking or alcohol use; any use of vitamins, food supplements, or any medication in the last 3 months; or pregnancy.

Melasma Severity—The modified melasma area and severity index (mMASI) score was used to determine the severity of melasma. The score is calculated from assessments of the darkness of the pigmentation and the percentage of affected area on the face. The mMASI score is the sum of the darkness score (D); area score (A); and separate fixed coefficients for the forehead, as well as the right malar, left malar, and chin regions.¹³ The mMASI score, with a range of 0 to 24, is a reliable and objective marker in the calculation of melasma severity.⁴

Biochemical Analysis of Samples—The 6-cc peripheral fasting venous blood samples obtained from the study participants were centrifuged at 1500 g for 10 minutes, and the separated sera were stored in a freezer at −80 °C until the time of analysis. When the study was completed, the disulfide and thiol values were analyzed. Serum native and total thiol concentrations indicating thiol/disulfide homeostasis were calculated by a new fully automatic colorimetric method developed by Erel and Neselioglu.¹⁴ Using this method, short disulfide bonds are first reduced with sodium borohydride solution to form free-functional thiol groups, and then the unused sodium borohydride is removed using formaldehyde. Finally, all thiol groups are reacted with 5,5’-dithiobis-(2-nitrobenzoic) acid (Ellman reagent), and all thiol groups are detected after reaction with 5,5’-dithiobis-(2-nitrobenzoic) acid. When a disulfide bond (−S−S−) is reduced, 2 thiol groups are formed. For this reason, half of the difference between total thiol (-SH + the amount of thiol formed by the reduction of disulfides) and native thiol (-SH) corresponds to the dynamic disulfide amount (total thiol − native thiol/2).¹⁴

Statistical Analysis—Statistical analysis was performed using SPSS software (version 24.0). Descriptive statistics were presented as numbers and percentages for categorical variables, and numerical variables were presented as mean, SD, median, minimum, maximum, 25th quartile, and 75th quartile. The conformity of the variables to normal distribution was examined using visual (histograms and probability plots) and analytical methods (Kolmogorov-Smirnov/Shapiro-Wilk tests). In pairwise group comparisons for numerical variables, a Mann-Whitney U test was used when normal distribution was not met, and a t test was used when normal distribution was met. The statistical significance level was accepted as P<.05.

Results

Our study included 67 patients with melasma and 41 healthy age- and sex-matched controls. Of the participants with melasma, 60 (89.5%) were female and 7 (10.5%) were male. The control group was similar to the melasma group in terms of sex (87.8% female vs 12.2% male [P=.59]). The mean age (SD) was 33.1 (6.7) years in the melasma group and 31.9 (6.7) years in the control group. Age was similar across both groups (P=.41). All participants were of Asian race, and Fitzpatrick skin types (types II–IV) were similar across both groups.

Fifty-four (80.6%) participants had centrofacial melasma and 13 (19.4%) had mixed-type melasma. The mMASI score ranged from 3 to 20; the mean (SD) mMASI score was 11.28 (3.2). Disease duration ranged from 2 to 72 months; the mean (SD) disease duration was 12.26 (6.3) months. The demographics and clinical characteristics of the study group are shown in eTable 1.

eTable 2 provides a summary of disulfide, native thiol, and total thiol levels, as well as disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol ratios in the study population. Disulfide/native thiol and disulfide/total thiol ratios were higher in melasma patients (Figure 1), whereas the native thiol/total thiol ratio was higher in the control group (P=.025, P=.025, and P=.026, respectively).

All correlations between age, disease duration, and mMASI scores and disulfide, native thiol, and total thiol levels, as well as disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol ratios, are summarized in eTable 3. No significant correlation was observed between age and disease duration and disulfide, native thiol, and total thiol levels or disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol ratios.

We independently assessed whether Fitzpatrick skin types II, III, and IV exhibited distinct levels of oxidative stress in clinical melasma. There were no significant correlations with Fitzpatrick skin type (disulfide/native thiol, P=.25; disulfide/total thiol, P=.19). We further evaluated if the thiol/disulfide parameters were correlated with duration of melasma by dividing the melasma patients into 3 groups (<6 months [n=12], 6–18 months [n=32], >18 months [n=23]), but there was not any significant correlation (disulfide/native thiol, P=.15; disulfide/total thiol, P=.15). We also divided our patients into 3 groups according to age (<27 years [n=14], 27–36 years [n=33], >36 years [n=20]). There was no correlation of the parameters with age (disulfide/native thiol, P=.15; disulfide/total thiol, P=.14).

There was a positive correlation between mMASI score and disulfide, native thiol, and total thiol levels and disulfide/native thiol and disulfide/total thiol ratios, as well as a negative correlation between mMASI score and native thiol/total thiol ratio. The correlations between mMASI scores and disulfide/native thiol and disulfide/total thiol ratios are shown in Figure 2 and eTable 3.

Comment

Melasma is a common condition that may cause psychosocial problems in affected patients and negatively affect quality of life.¹ It occurs in all races but is more common in individuals with darker skin types (eg, Fitzpatrick skin types III and IV). Although melasma is more common in women during reproductive years (50%–70%), it also has been observed in 10% to 30% of men.⁵

Treatment options include topical bleaching agents, chemical peels, and laser therapy, as well as discontinuation of medications that may potentially trigger melasma; use of broad-spectrum sunscreens also is recommended.⁴ Vitamins A, C, and E, as well as niacinamide, are used in the treatment of melasma, especially for their antioxidant properties. The key role of antioxidants in the treatment of melasma supports the importance of oxidative stress in the pathogenesis.⁷ Melasma often is challenging to treat, particularly the mixed or dermal types, due to their stubborn nature. This condition poses a considerable therapeutic challenge for dermatologists.⁴

kocradrupathedriphuhatriwebripudrudrimechametehosothahapedaslihebulalakathapuretumod

pimifrumafricrijeuamostoshimubarocrobiwegospogotrikesheclouoswivuvuhonucohasuthujestaspimocradifrutrekuthubrebrocrospichukalifrahalaceputhalacruswospahapheswi

Oxidative stress and oxidant-antioxidant imbalance previously have been studied in various diseases, but research investigating the presence of oxidative stress in melasma are limited.^7-10 Exposure of the skin to polluted air and intense UVR, as well as some food by-products, cosmetics, and drugs (eg, oral contraceptives), can directly or indirectly cause ROS production in the skin. Reactive oxygen species are thought to be involved in the pathophysiology of melasma by affecting apoptotic pathways and causing cell proliferation. The intermediate heme pathway has pro-oxidant effects and produces ROS and metabolites such as redox-active quinines. Exposure to UVR leads to the generation of ROS, highlighting the role of oxidative stress in the onset of melasma. ⁵

In any cutaneous disease in which oxidative stress plays a role, oxidant and antioxidant levels may be expected to vary both locally and systemically; however, measurement of oxidative stress markers in serum instead of skin is technically and economically more advantageous.⁸ Firstly, serum collection is less invasive and technically simpler than skin biopsies. Drawing blood is a routine procedure that requires minimal specialized equipment and training compared to the extraction and processing of skin samples. Secondly, analyzing serum samples generally is less expensive than processing skin tissue.⁸

In our study, we evaluated dynamic thiol/disulfide homeostasis in serum to investigate the presence of oxidative stress in the setting of melasma. Functional sulfhydryl (-SH) groups in thiols act as substrates for antioxidant enzymes and as free-radical scavengers. They constitute one of the most powerful defense systems against the unwanted effects of ROS. Thiols, which become the main target of ROS under oxidative stress, oxidize with oxidant molecules and form disulfide bridges.¹⁵

Thiol/disulfide homeostasis has been studied many times in dermatologic diseases,^16-19 and the results obtained from these studies are heterogenous depending on the extent of oxidative damage. It has been shown that thiol/disulfide homeostasis plays a role in oxidative stress in conditions such as psoriasis,¹⁷ seborrheic dermatitis,¹¹ atopic dermatitits,¹⁸ and rosacea.¹⁹ In our study, disulfide/native thiol and disulfide/total thiol levels were significantly higher (both P=.025) in the melasma group compared with the control group, which indicates that the thiol/disulfide balance in patients with melasma is shifted to disulfide formation and thiols are oxidized to disulfide bonds in the presence of oxidative stress.

Seçkin et al⁷ evaluated the role of oxidative stress in the pathogenesis of melasma and found that the serum levels of the antioxidants superoxide dismutase and glutathione peroxidase were significantly higher in the patient group compared with the control group (both P<.001). They also found that the levels of nitric oxide (another antioxidant) were increased in the patient group and the levels of protein carbonyl (an oxidative metabolite) were significantly lower (both P<.001). These findings indicated that free-radical damage may be involved in the pathogenesis of melasma.⁷

In a study of 75 patients with melasma, serum levels of the antioxidants melatonin and catalase were significantly (P<.001 and P=.001, respectively) lower in the melasma group compared with the control group, while serum levels of the oxidants protein carbonyl and nitric oxide were significantly higher (P=.002 and P=.001, respectively). No significant correlation was found between oxidative stress parameters and melasma severity.⁸

Choubey et al⁹ found that serum malondialdehyde (an end product of lipid peroxidation), superoxide dismutase, and glutathione peroxidase levels were significantly higher in the melasma group (n=50) compared with the control group (n=50)(all P<.001). In addition, a significant positive correlation (correlation coefficient, +0.307; P<.05) was found between serum malondialdehyde levels and melasma severity. The mean age (SD) of the patients was 32.22 (6.377) years, and the female (n=41) to male (n=9) ratio was 4.55:1. The most common melasma pattern was centrofacial, followed by malar.⁹

In a study with 50 melasma patients and 50 controls, Rahimi et al¹⁰ examined bilirubin and uric acid levels, which are major extracellular antioxidants. The mean age (SD) at disease onset was 32.6 (6.7) years, and the mean MASI score (SD) was 18.1 (9). Serum bilirubin levels were found to be higher in the melasma group than in the control group and were correlated with disease severity. No significant difference in uric acid levels was found between the groups, and no correlation was found between MASI score and bilirubin and uric acid levels.¹⁰

In our study, the melasma group was similar to those in other reportsin the literature regarding gender distribution, mean age, and melasma pattern.^7-10 Additionally, the correlation of mMASI score with disulfide/native thiol and disulfide/total thiol values in the melasma group suggested that oxidative stress also is correlated with melasma severity.

Thiol-based treatments such as n-acetyl cysteine, which contains a thiol compound, may be helpful in melasma.²⁰ In a double-blind, placebo-controlled study, topical n-acetyl cysteine combined with hydroquinone 2% was used in 10 female patients with melasma. Mild to strong bleaching of the skin was observed in 90% (9/10) of the patients.²¹ Systemic use of n-acetyl cysteine in melasma also may be a potential research topic.

Major limitations of our study were the small sample size and lack of measurement of oxidative stress parameters in the skin concurrently with serum.

Conclusion

In our study, the presence of oxidative stress in melasma was demonstrated by evaluating thiol/disulfide homeostasis—one of the strongest markers of oxidative stress. Oxidative stress also correlated with melasma disease severity in our analysis. The data obtained in this study may contribute to understanding the etiopathogenesis of melasma and may open new horizons in treatment; however, more comprehensive studies should be conducted to support our findings.

gowribriphosaslaswarucusparucrocubiseruclajowrothageclumaslabotenochukanethepronechaphijiuuclakiwruslaclusleshistospasophojephu

modrawespospuvuravachacrojogistewowospuwuviphohastos

ph

Melasma is an acquired hyperpigmentation disorder characterized by irregular brown macules and patches that usually appear on sun-exposed areas of the skin. The term melasma originates from the Greek word melas meaning black.¹ Facial melasma is divided into 2 groups according to its clinical distribution: centrofacial lesions are located in the center of the face (eg, the glabellar, frontal, nasal, zygomatic, upper lip, chin areas), and peripheral lesions manifest on the frontotemporal, preauricular, and mandibular regions.^1,2 There is debate on the categorization of zygomatic (or malar) melasma; some researchers argue it should be categorized independent of other areas, while others include malar melasma in the centrofacial group because of its frequent association with the centrofacial type, especially with glabellar lesions.² Mandibular melasma is rare and occurs mostly in postmenopausal women after intense sun exposure.^1,2 Although the etiopathogenesis of the disease is not clearly known, increased melanogenesis, extracellular matrix alterations, inflammation, and angiogenesis are assumed to play a role.³ Various risk factors such as genetic predisposition, UV radiation (UVR) exposure, pregnancy, thyroid dysfunction, and exogenous hormones (eg, oral contraceptives, hormone replacement therapy) have been identified; phototoxic drugs, anticonvulsants, and some cosmetics also have been implicated.^4,5 Exposure to UVR is thought to be the main triggering environmental factor by inducing both melanin production and oxidative stress.⁵ However, it also has been shown that visible light can induce hyperpigmentation in darker skin types.⁶

The presence of oxidative stress in melasma recently has become an intriguing topic of interest. First, the presence of oxidative stress in the etiopathogenesis of melasma was thought to be based on the effectiveness of antioxidants in treatment. A few studies also have confirmed the presence of oxidative stress in melasma.^7-10 Classically, oxidative stress can be described as a disturbance in the balance between oxidants and antioxidants. Reactive oxygen species (ROS) are highly reactive molecules due to the unpaired electrons in their structure. Although ROS are present at low levels in physiologic conditions and are involved in critical physiologic events, they damage cellular components such as fat, protein, and nucleic acid at high concentrations.⁵

Dynamic thiol/disulfide homeostasis is one of the most important markers of oxidative stress in biological systems. Thiols are organic compounds containing a sulfhydryl (-SH) group. Thiols are considered highly potent antioxidants because they reduce unstable free radicals by donating electrons. They are the first antioxidants to be depleted in an oxidative environment.^11,12 In case of oxidative stress, they transform into reversible forms called disulfide bridges between 2 thiol groups. Disulfide bridges can be reduced back to thiol groups, which is how dynamic thiol/disulfide homeostasis is maintained. Dynamic thiol/disulfide homeostasis is responsible for cellular events such as antioxidant defense, signal transduction, regulation of enzyme function, and apoptosis.^11,12

The aim of this study was to evaluate the presence of oxidative stress in melasma by comparing dynamic thiol/disulfide homeostasis in patients with melasma compared with age- and sex-matched healthy controls.

Materials and Methods

Participants and Eligibility Criteria—We conducted a prospective study in a tertiary-care hospital (Ankara Bilkent City Hospital [Ankara, Turkey]) of patients with melasma who were followed from October 2021 to October 2022 compared with age- and sex-matched healthy volunteers. Ethics committee approval was obtained from Ankara Bilkent City Hospital before the study (E2-21-881)(13.10.2021). Written informed consent was obtained from all participants, and all were older than 18 years. Patients were excluded if there was the presence of any systemic disease or dermatologic disease other than melasma; smoking or alcohol use; any use of vitamins, food supplements, or any medication in the last 3 months; or pregnancy.

Melasma Severity—The modified melasma area and severity index (mMASI) score was used to determine the severity of melasma. The score is calculated from assessments of the darkness of the pigmentation and the percentage of affected area on the face. The mMASI score is the sum of the darkness score (D); area score (A); and separate fixed coefficients for the forehead, as well as the right malar, left malar, and chin regions.¹³ The mMASI score, with a range of 0 to 24, is a reliable and objective marker in the calculation of melasma severity.⁴

Biochemical Analysis of Samples—The 6-cc peripheral fasting venous blood samples obtained from the study participants were centrifuged at 1500 g for 10 minutes, and the separated sera were stored in a freezer at −80 °C until the time of analysis. When the study was completed, the disulfide and thiol values were analyzed. Serum native and total thiol concentrations indicating thiol/disulfide homeostasis were calculated by a new fully automatic colorimetric method developed by Erel and Neselioglu.¹⁴ Using this method, short disulfide bonds are first reduced with sodium borohydride solution to form free-functional thiol groups, and then the unused sodium borohydride is removed using formaldehyde. Finally, all thiol groups are reacted with 5,5’-dithiobis-(2-nitrobenzoic) acid (Ellman reagent), and all thiol groups are detected after reaction with 5,5’-dithiobis-(2-nitrobenzoic) acid. When a disulfide bond (−S−S−) is reduced, 2 thiol groups are formed. For this reason, half of the difference between total thiol (-SH + the amount of thiol formed by the reduction of disulfides) and native thiol (-SH) corresponds to the dynamic disulfide amount (total thiol − native thiol/2).¹⁴

Statistical Analysis—Statistical analysis was performed using SPSS software (version 24.0). Descriptive statistics were presented as numbers and percentages for categorical variables, and numerical variables were presented as mean, SD, median, minimum, maximum, 25th quartile, and 75th quartile. The conformity of the variables to normal distribution was examined using visual (histograms and probability plots) and analytical methods (Kolmogorov-Smirnov/Shapiro-Wilk tests). In pairwise group comparisons for numerical variables, a Mann-Whitney U test was used when normal distribution was not met, and a t test was used when normal distribution was met. The statistical significance level was accepted as P<.05.

Results

Our study included 67 patients with melasma and 41 healthy age- and sex-matched controls. Of the participants with melasma, 60 (89.5%) were female and 7 (10.5%) were male. The control group was similar to the melasma group in terms of sex (87.8% female vs 12.2% male [P=.59]). The mean age (SD) was 33.1 (6.7) years in the melasma group and 31.9 (6.7) years in the control group. Age was similar across both groups (P=.41). All participants were of Asian race, and Fitzpatrick skin types (types II–IV) were similar across both groups.

Fifty-four (80.6%) participants had centrofacial melasma and 13 (19.4%) had mixed-type melasma. The mMASI score ranged from 3 to 20; the mean (SD) mMASI score was 11.28 (3.2). Disease duration ranged from 2 to 72 months; the mean (SD) disease duration was 12.26 (6.3) months. The demographics and clinical characteristics of the study group are shown in eTable 1.

eTable 2 provides a summary of disulfide, native thiol, and total thiol levels, as well as disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol ratios in the study population. Disulfide/native thiol and disulfide/total thiol ratios were higher in melasma patients (Figure 1), whereas the native thiol/total thiol ratio was higher in the control group (P=.025, P=.025, and P=.026, respectively).

All correlations between age, disease duration, and mMASI scores and disulfide, native thiol, and total thiol levels, as well as disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol ratios, are summarized in eTable 3. No significant correlation was observed between age and disease duration and disulfide, native thiol, and total thiol levels or disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol ratios.

We independently assessed whether Fitzpatrick skin types II, III, and IV exhibited distinct levels of oxidative stress in clinical melasma. There were no significant correlations with Fitzpatrick skin type (disulfide/native thiol, P=.25; disulfide/total thiol, P=.19). We further evaluated if the thiol/disulfide parameters were correlated with duration of melasma by dividing the melasma patients into 3 groups (<6 months [n=12], 6–18 months [n=32], >18 months [n=23]), but there was not any significant correlation (disulfide/native thiol, P=.15; disulfide/total thiol, P=.15). We also divided our patients into 3 groups according to age (<27 years [n=14], 27–36 years [n=33], >36 years [n=20]). There was no correlation of the parameters with age (disulfide/native thiol, P=.15; disulfide/total thiol, P=.14).

There was a positive correlation between mMASI score and disulfide, native thiol, and total thiol levels and disulfide/native thiol and disulfide/total thiol ratios, as well as a negative correlation between mMASI score and native thiol/total thiol ratio. The correlations between mMASI scores and disulfide/native thiol and disulfide/total thiol ratios are shown in Figure 2 and eTable 3.

Comment

Melasma is a common condition that may cause psychosocial problems in affected patients and negatively affect quality of life.¹ It occurs in all races but is more common in individuals with darker skin types (eg, Fitzpatrick skin types III and IV). Although melasma is more common in women during reproductive years (50%–70%), it also has been observed in 10% to 30% of men.⁵

Treatment options include topical bleaching agents, chemical peels, and laser therapy, as well as discontinuation of medications that may potentially trigger melasma; use of broad-spectrum sunscreens also is recommended.⁴ Vitamins A, C, and E, as well as niacinamide, are used in the treatment of melasma, especially for their antioxidant properties. The key role of antioxidants in the treatment of melasma supports the importance of oxidative stress in the pathogenesis.⁷ Melasma often is challenging to treat, particularly the mixed or dermal types, due to their stubborn nature. This condition poses a considerable therapeutic challenge for dermatologists.⁴

kocradrupathedriphuhatriwebripudrudrimechametehosothahapedaslihebulalakathapuretumod

pimifrumafricrijeuamostoshimubarocrobiwegospogotrikesheclouoswivuvuhonucohasuthujestaspimocradifrutrekuthubrebrocrospichukalifrahalaceputhalacruswospahapheswi

Oxidative stress and oxidant-antioxidant imbalance previously have been studied in various diseases, but research investigating the presence of oxidative stress in melasma are limited.^7-10 Exposure of the skin to polluted air and intense UVR, as well as some food by-products, cosmetics, and drugs (eg, oral contraceptives), can directly or indirectly cause ROS production in the skin. Reactive oxygen species are thought to be involved in the pathophysiology of melasma by affecting apoptotic pathways and causing cell proliferation. The intermediate heme pathway has pro-oxidant effects and produces ROS and metabolites such as redox-active quinines. Exposure to UVR leads to the generation of ROS, highlighting the role of oxidative stress in the onset of melasma. ⁵

In any cutaneous disease in which oxidative stress plays a role, oxidant and antioxidant levels may be expected to vary both locally and systemically; however, measurement of oxidative stress markers in serum instead of skin is technically and economically more advantageous.⁸ Firstly, serum collection is less invasive and technically simpler than skin biopsies. Drawing blood is a routine procedure that requires minimal specialized equipment and training compared to the extraction and processing of skin samples. Secondly, analyzing serum samples generally is less expensive than processing skin tissue.⁸

In our study, we evaluated dynamic thiol/disulfide homeostasis in serum to investigate the presence of oxidative stress in the setting of melasma. Functional sulfhydryl (-SH) groups in thiols act as substrates for antioxidant enzymes and as free-radical scavengers. They constitute one of the most powerful defense systems against the unwanted effects of ROS. Thiols, which become the main target of ROS under oxidative stress, oxidize with oxidant molecules and form disulfide bridges.¹⁵

Thiol/disulfide homeostasis has been studied many times in dermatologic diseases,^16-19 and the results obtained from these studies are heterogenous depending on the extent of oxidative damage. It has been shown that thiol/disulfide homeostasis plays a role in oxidative stress in conditions such as psoriasis,¹⁷ seborrheic dermatitis,¹¹ atopic dermatitits,¹⁸ and rosacea.¹⁹ In our study, disulfide/native thiol and disulfide/total thiol levels were significantly higher (both P=.025) in the melasma group compared with the control group, which indicates that the thiol/disulfide balance in patients with melasma is shifted to disulfide formation and thiols are oxidized to disulfide bonds in the presence of oxidative stress.

Seçkin et al⁷ evaluated the role of oxidative stress in the pathogenesis of melasma and found that the serum levels of the antioxidants superoxide dismutase and glutathione peroxidase were significantly higher in the patient group compared with the control group (both P<.001). They also found that the levels of nitric oxide (another antioxidant) were increased in the patient group and the levels of protein carbonyl (an oxidative metabolite) were significantly lower (both P<.001). These findings indicated that free-radical damage may be involved in the pathogenesis of melasma.⁷

In a study of 75 patients with melasma, serum levels of the antioxidants melatonin and catalase were significantly (P<.001 and P=.001, respectively) lower in the melasma group compared with the control group, while serum levels of the oxidants protein carbonyl and nitric oxide were significantly higher (P=.002 and P=.001, respectively). No significant correlation was found between oxidative stress parameters and melasma severity.⁸

Choubey et al⁹ found that serum malondialdehyde (an end product of lipid peroxidation), superoxide dismutase, and glutathione peroxidase levels were significantly higher in the melasma group (n=50) compared with the control group (n=50)(all P<.001). In addition, a significant positive correlation (correlation coefficient, +0.307; P<.05) was found between serum malondialdehyde levels and melasma severity. The mean age (SD) of the patients was 32.22 (6.377) years, and the female (n=41) to male (n=9) ratio was 4.55:1. The most common melasma pattern was centrofacial, followed by malar.⁹

In a study with 50 melasma patients and 50 controls, Rahimi et al¹⁰ examined bilirubin and uric acid levels, which are major extracellular antioxidants. The mean age (SD) at disease onset was 32.6 (6.7) years, and the mean MASI score (SD) was 18.1 (9). Serum bilirubin levels were found to be higher in the melasma group than in the control group and were correlated with disease severity. No significant difference in uric acid levels was found between the groups, and no correlation was found between MASI score and bilirubin and uric acid levels.¹⁰

In our study, the melasma group was similar to those in other reportsin the literature regarding gender distribution, mean age, and melasma pattern.^7-10 Additionally, the correlation of mMASI score with disulfide/native thiol and disulfide/total thiol values in the melasma group suggested that oxidative stress also is correlated with melasma severity.

Thiol-based treatments such as n-acetyl cysteine, which contains a thiol compound, may be helpful in melasma.²⁰ In a double-blind, placebo-controlled study, topical n-acetyl cysteine combined with hydroquinone 2% was used in 10 female patients with melasma. Mild to strong bleaching of the skin was observed in 90% (9/10) of the patients.²¹ Systemic use of n-acetyl cysteine in melasma also may be a potential research topic.

Major limitations of our study were the small sample size and lack of measurement of oxidative stress parameters in the skin concurrently with serum.

Conclusion

In our study, the presence of oxidative stress in melasma was demonstrated by evaluating thiol/disulfide homeostasis—one of the strongest markers of oxidative stress. Oxidative stress also correlated with melasma disease severity in our analysis. The data obtained in this study may contribute to understanding the etiopathogenesis of melasma and may open new horizons in treatment; however, more comprehensive studies should be conducted to support our findings.

gowribriphosaslaswarucusparucrocubiseruclajowrothageclumaslabotenochukanethepronechaphijiuuclakiwruslaclusleshistospasophojephu

modrawespospuvuravachacrojogistewowospuwuviphohastos

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Melasma is an acquired hyperpigmentation disorder characterized by irregular brown macules and patches that usually appear on sun-exposed areas of the skin. The term melasma originates from the Greek word melas meaning black.¹ Facial melasma is divided into 2 groups according to its clinical distribution: centrofacial lesions are located in the center of the face (eg, the glabellar, frontal, nasal, zygomatic, upper lip, chin areas), and peripheral lesions manifest on the frontotemporal, preauricular, and mandibular regions.^1,2 There is debate on the categorization of zygomatic (or malar) melasma; some researchers argue it should be categorized independent of other areas, while others include malar melasma in the centrofacial group because of its frequent association with the centrofacial type, especially with glabellar lesions.² Mandibular melasma is rare and occurs mostly in postmenopausal women after intense sun exposure.^1,2 Although the etiopathogenesis of the disease is not clearly known, increased melanogenesis, extracellular matrix alterations, inflammation, and angiogenesis are assumed to play a role.³ Various risk factors such as genetic predisposition, UV radiation (UVR) exposure, pregnancy, thyroid dysfunction, and exogenous hormones (eg, oral contraceptives, hormone replacement therapy) have been identified; phototoxic drugs, anticonvulsants, and some cosmetics also have been implicated.^4,5 Exposure to UVR is thought to be the main triggering environmental factor by inducing both melanin production and oxidative stress.⁵ However, it also has been shown that visible light can induce hyperpigmentation in darker skin types.⁶

The presence of oxidative stress in melasma recently has become an intriguing topic of interest. First, the presence of oxidative stress in the etiopathogenesis of melasma was thought to be based on the effectiveness of antioxidants in treatment. A few studies also have confirmed the presence of oxidative stress in melasma.^7-10 Classically, oxidative stress can be described as a disturbance in the balance between oxidants and antioxidants. Reactive oxygen species (ROS) are highly reactive molecules due to the unpaired electrons in their structure. Although ROS are present at low levels in physiologic conditions and are involved in critical physiologic events, they damage cellular components such as fat, protein, and nucleic acid at high concentrations.⁵

Dynamic thiol/disulfide homeostasis is one of the most important markers of oxidative stress in biological systems. Thiols are organic compounds containing a sulfhydryl (-SH) group. Thiols are considered highly potent antioxidants because they reduce unstable free radicals by donating electrons. They are the first antioxidants to be depleted in an oxidative environment.^11,12 In case of oxidative stress, they transform into reversible forms called disulfide bridges between 2 thiol groups. Disulfide bridges can be reduced back to thiol groups, which is how dynamic thiol/disulfide homeostasis is maintained. Dynamic thiol/disulfide homeostasis is responsible for cellular events such as antioxidant defense, signal transduction, regulation of enzyme function, and apoptosis.^11,12

The aim of this study was to evaluate the presence of oxidative stress in melasma by comparing dynamic thiol/disulfide homeostasis in patients with melasma compared with age- and sex-matched healthy controls.

Materials and Methods

Participants and Eligibility Criteria—We conducted a prospective study in a tertiary-care hospital (Ankara Bilkent City Hospital [Ankara, Turkey]) of patients with melasma who were followed from October 2021 to October 2022 compared with age- and sex-matched healthy volunteers. Ethics committee approval was obtained from Ankara Bilkent City Hospital before the study (E2-21-881)(13.10.2021). Written informed consent was obtained from all participants, and all were older than 18 years. Patients were excluded if there was the presence of any systemic disease or dermatologic disease other than melasma; smoking or alcohol use; any use of vitamins, food supplements, or any medication in the last 3 months; or pregnancy.

Melasma Severity—The modified melasma area and severity index (mMASI) score was used to determine the severity of melasma. The score is calculated from assessments of the darkness of the pigmentation and the percentage of affected area on the face. The mMASI score is the sum of the darkness score (D); area score (A); and separate fixed coefficients for the forehead, as well as the right malar, left malar, and chin regions.¹³ The mMASI score, with a range of 0 to 24, is a reliable and objective marker in the calculation of melasma severity.⁴

Biochemical Analysis of Samples—The 6-cc peripheral fasting venous blood samples obtained from the study participants were centrifuged at 1500 g for 10 minutes, and the separated sera were stored in a freezer at −80 °C until the time of analysis. When the study was completed, the disulfide and thiol values were analyzed. Serum native and total thiol concentrations indicating thiol/disulfide homeostasis were calculated by a new fully automatic colorimetric method developed by Erel and Neselioglu.¹⁴ Using this method, short disulfide bonds are first reduced with sodium borohydride solution to form free-functional thiol groups, and then the unused sodium borohydride is removed using formaldehyde. Finally, all thiol groups are reacted with 5,5’-dithiobis-(2-nitrobenzoic) acid (Ellman reagent), and all thiol groups are detected after reaction with 5,5’-dithiobis-(2-nitrobenzoic) acid. When a disulfide bond (−S−S−) is reduced, 2 thiol groups are formed. For this reason, half of the difference between total thiol (-SH + the amount of thiol formed by the reduction of disulfides) and native thiol (-SH) corresponds to the dynamic disulfide amount (total thiol − native thiol/2).¹⁴

Statistical Analysis—Statistical analysis was performed using SPSS software (version 24.0). Descriptive statistics were presented as numbers and percentages for categorical variables, and numerical variables were presented as mean, SD, median, minimum, maximum, 25th quartile, and 75th quartile. The conformity of the variables to normal distribution was examined using visual (histograms and probability plots) and analytical methods (Kolmogorov-Smirnov/Shapiro-Wilk tests). In pairwise group comparisons for numerical variables, a Mann-Whitney U test was used when normal distribution was not met, and a t test was used when normal distribution was met. The statistical significance level was accepted as P<.05.

Results

Our study included 67 patients with melasma and 41 healthy age- and sex-matched controls. Of the participants with melasma, 60 (89.5%) were female and 7 (10.5%) were male. The control group was similar to the melasma group in terms of sex (87.8% female vs 12.2% male [P=.59]). The mean age (SD) was 33.1 (6.7) years in the melasma group and 31.9 (6.7) years in the control group. Age was similar across both groups (P=.41). All participants were of Asian race, and Fitzpatrick skin types (types II–IV) were similar across both groups.

Fifty-four (80.6%) participants had centrofacial melasma and 13 (19.4%) had mixed-type melasma. The mMASI score ranged from 3 to 20; the mean (SD) mMASI score was 11.28 (3.2). Disease duration ranged from 2 to 72 months; the mean (SD) disease duration was 12.26 (6.3) months. The demographics and clinical characteristics of the study group are shown in eTable 1.

eTable 2 provides a summary of disulfide, native thiol, and total thiol levels, as well as disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol ratios in the study population. Disulfide/native thiol and disulfide/total thiol ratios were higher in melasma patients (Figure 1), whereas the native thiol/total thiol ratio was higher in the control group (P=.025, P=.025, and P=.026, respectively).

All correlations between age, disease duration, and mMASI scores and disulfide, native thiol, and total thiol levels, as well as disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol ratios, are summarized in eTable 3. No significant correlation was observed between age and disease duration and disulfide, native thiol, and total thiol levels or disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol ratios.

We independently assessed whether Fitzpatrick skin types II, III, and IV exhibited distinct levels of oxidative stress in clinical melasma. There were no significant correlations with Fitzpatrick skin type (disulfide/native thiol, P=.25; disulfide/total thiol, P=.19). We further evaluated if the thiol/disulfide parameters were correlated with duration of melasma by dividing the melasma patients into 3 groups (<6 months [n=12], 6–18 months [n=32], >18 months [n=23]), but there was not any significant correlation (disulfide/native thiol, P=.15; disulfide/total thiol, P=.15). We also divided our patients into 3 groups according to age (<27 years [n=14], 27–36 years [n=33], >36 years [n=20]). There was no correlation of the parameters with age (disulfide/native thiol, P=.15; disulfide/total thiol, P=.14).

There was a positive correlation between mMASI score and disulfide, native thiol, and total thiol levels and disulfide/native thiol and disulfide/total thiol ratios, as well as a negative correlation between mMASI score and native thiol/total thiol ratio. The correlations between mMASI scores and disulfide/native thiol and disulfide/total thiol ratios are shown in Figure 2 and eTable 3.

Comment

Melasma is a common condition that may cause psychosocial problems in affected patients and negatively affect quality of life.¹ It occurs in all races but is more common in individuals with darker skin types (eg, Fitzpatrick skin types III and IV). Although melasma is more common in women during reproductive years (50%–70%), it also has been observed in 10% to 30% of men.⁵

Treatment options include topical bleaching agents, chemical peels, and laser therapy, as well as discontinuation of medications that may potentially trigger melasma; use of broad-spectrum sunscreens also is recommended.⁴ Vitamins A, C, and E, as well as niacinamide, are used in the treatment of melasma, especially for their antioxidant properties. The key role of antioxidants in the treatment of melasma supports the importance of oxidative stress in the pathogenesis.⁷ Melasma often is challenging to treat, particularly the mixed or dermal types, due to their stubborn nature. This condition poses a considerable therapeutic challenge for dermatologists.⁴

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Oxidative stress and oxidant-antioxidant imbalance previously have been studied in various diseases, but research investigating the presence of oxidative stress in melasma are limited.^7-10 Exposure of the skin to polluted air and intense UVR, as well as some food by-products, cosmetics, and drugs (eg, oral contraceptives), can directly or indirectly cause ROS production in the skin. Reactive oxygen species are thought to be involved in the pathophysiology of melasma by affecting apoptotic pathways and causing cell proliferation. The intermediate heme pathway has pro-oxidant effects and produces ROS and metabolites such as redox-active quinines. Exposure to UVR leads to the generation of ROS, highlighting the role of oxidative stress in the onset of melasma. ⁵

In any cutaneous disease in which oxidative stress plays a role, oxidant and antioxidant levels may be expected to vary both locally and systemically; however, measurement of oxidative stress markers in serum instead of skin is technically and economically more advantageous.⁸ Firstly, serum collection is less invasive and technically simpler than skin biopsies. Drawing blood is a routine procedure that requires minimal specialized equipment and training compared to the extraction and processing of skin samples. Secondly, analyzing serum samples generally is less expensive than processing skin tissue.⁸

In our study, we evaluated dynamic thiol/disulfide homeostasis in serum to investigate the presence of oxidative stress in the setting of melasma. Functional sulfhydryl (-SH) groups in thiols act as substrates for antioxidant enzymes and as free-radical scavengers. They constitute one of the most powerful defense systems against the unwanted effects of ROS. Thiols, which become the main target of ROS under oxidative stress, oxidize with oxidant molecules and form disulfide bridges.¹⁵

Thiol/disulfide homeostasis has been studied many times in dermatologic diseases,^16-19 and the results obtained from these studies are heterogenous depending on the extent of oxidative damage. It has been shown that thiol/disulfide homeostasis plays a role in oxidative stress in conditions such as psoriasis,¹⁷ seborrheic dermatitis,¹¹ atopic dermatitits,¹⁸ and rosacea.¹⁹ In our study, disulfide/native thiol and disulfide/total thiol levels were significantly higher (both P=.025) in the melasma group compared with the control group, which indicates that the thiol/disulfide balance in patients with melasma is shifted to disulfide formation and thiols are oxidized to disulfide bonds in the presence of oxidative stress.

Seçkin et al⁷ evaluated the role of oxidative stress in the pathogenesis of melasma and found that the serum levels of the antioxidants superoxide dismutase and glutathione peroxidase were significantly higher in the patient group compared with the control group (both P<.001). They also found that the levels of nitric oxide (another antioxidant) were increased in the patient group and the levels of protein carbonyl (an oxidative metabolite) were significantly lower (both P<.001). These findings indicated that free-radical damage may be involved in the pathogenesis of melasma.⁷

In a study of 75 patients with melasma, serum levels of the antioxidants melatonin and catalase were significantly (P<.001 and P=.001, respectively) lower in the melasma group compared with the control group, while serum levels of the oxidants protein carbonyl and nitric oxide were significantly higher (P=.002 and P=.001, respectively). No significant correlation was found between oxidative stress parameters and melasma severity.⁸

Choubey et al⁹ found that serum malondialdehyde (an end product of lipid peroxidation), superoxide dismutase, and glutathione peroxidase levels were significantly higher in the melasma group (n=50) compared with the control group (n=50)(all P<.001). In addition, a significant positive correlation (correlation coefficient, +0.307; P<.05) was found between serum malondialdehyde levels and melasma severity. The mean age (SD) of the patients was 32.22 (6.377) years, and the female (n=41) to male (n=9) ratio was 4.55:1. The most common melasma pattern was centrofacial, followed by malar.⁹

In a study with 50 melasma patients and 50 controls, Rahimi et al¹⁰ examined bilirubin and uric acid levels, which are major extracellular antioxidants. The mean age (SD) at disease onset was 32.6 (6.7) years, and the mean MASI score (SD) was 18.1 (9). Serum bilirubin levels were found to be higher in the melasma group than in the control group and were correlated with disease severity. No significant difference in uric acid levels was found between the groups, and no correlation was found between MASI score and bilirubin and uric acid levels.¹⁰

In our study, the melasma group was similar to those in other reportsin the literature regarding gender distribution, mean age, and melasma pattern.^7-10 Additionally, the correlation of mMASI score with disulfide/native thiol and disulfide/total thiol values in the melasma group suggested that oxidative stress also is correlated with melasma severity.

Thiol-based treatments such as n-acetyl cysteine, which contains a thiol compound, may be helpful in melasma.²⁰ In a double-blind, placebo-controlled study, topical n-acetyl cysteine combined with hydroquinone 2% was used in 10 female patients with melasma. Mild to strong bleaching of the skin was observed in 90% (9/10) of the patients.²¹ Systemic use of n-acetyl cysteine in melasma also may be a potential research topic.

Major limitations of our study were the small sample size and lack of measurement of oxidative stress parameters in the skin concurrently with serum.

Conclusion

In our study, the presence of oxidative stress in melasma was demonstrated by evaluating thiol/disulfide homeostasis—one of the strongest markers of oxidative stress. Oxidative stress also correlated with melasma disease severity in our analysis. The data obtained in this study may contribute to understanding the etiopathogenesis of melasma and may open new horizons in treatment; however, more comprehensive studies should be conducted to support our findings.

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Alopecia areata (AA) presents as sudden, nonscarring, recurrent hair loss characterized by well-circumscribed hairless patches. Although AA may be observed on any hair-bearing areas of the body, the most commonly affected sites are the scalp, beard area, eyebrows, and eyelashes.¹ The incidence of AA is 1% to 2% in the general population and it is more common in males than females younger than 40 years.² Although the majority of patients present with self-limited and well-circumscribed hairless patches that resolve within 2 years, 7% to 10% display a chronic and severe prognosis.³

The etiopathogenesis of AA is not clearly understood, but its occurrence and progression can involve immune dysfunction, genetic predisposition, infections, and physical and psychological trauma.² Alopecia areata is observed to occur sporadically in most patients. Family history has been found in 3% to 42% of cases, but simultaneous occurrence of AA in family members is rare.⁴ In this case series, we present 4 cases of active AA lesions occurring simultaneously in a family who also had associated psychologic disorders.

Case Series

Patient 1 (Proband)

An 11-year-old boy presented with a 6-year history of ongoing AA with recurrent improvement and relapses on the scalp, eyebrows, and eyelashes. Various topical and oral medications had been prescribed by several outside dermatologists; however, these treatments provided minimal benefit and resulted in the recurrence of AA. Dermatologic examination revealed hair loss on the entire frontal, parietal, and temporal regions of the scalp, as well as half of the occipital region and one-third of the lateral side of the eyebrows (Figure 1). Psychological evaluation revealed introvert personality characteristics, lack of self-confidence, and signs of depression and anxiety.

ct098007030_e_fig1.png

Patient 2 (Proband’s Father)

A 38-year-old man presented with a 16-year history of recurrent loss and regrowth of hair on the scalp and beard area and white spots on the penis and arms. He previously had not undergone any treatments. Dermatologic examination revealed well-circumscribed, 1- to 4-cm, hairless patches on the occipital region of the scalp and in the beard area (Figure 2A) and multiple, 2- to 10-mm, vitiliginous lesions on both forearms (Figure 2B) and the penis. The patient had been unemployed for 6 months. Psychological evaluation revealed obsessive-compulsive disorder and obsessive-compulsive personality disorder.

ct098007030_e_fig2.png

Patient 3 (Proband’s Mother)

A 32-year-old woman presented with a 3-year history of chronic AA. She previously had not undergone any treatments. Dermatologic examination revealed 2 well-circumscribed, 3- to 4-cm patches of hair loss on the occipital and left temporal regions of the scalp (Figure 3). Psychological evaluation revealed obsessive-compulsive personality disorder and depression. The patient did not have any autoimmune diseases.

ct098007030_e_fig3.png

Patient 4 (Proband’s Sister)

A 10-year-old girl presented with a 6-year history of recurrent, self-limited AA on various areas of scalp. She previously had not undergone any treatments. Dermatologic examination revealed a 3-cm hairless patch on the occipital region of the scalp (Figure 4). Psychiatric evaluation revealed narcissistic personality disorder, anxiety, and lack of self-confidence.

ct098007030_e_fig4.png

Laboratory Evaluation and HLA Antigen DNA Typing

Laboratory testing including complete blood cell count; liver, kidney, and thyroid function; and vitamin B₁₂, zinc, folic acid, and fasting blood sugar levels were performed in all patients.

HLA antigen DNA typing was performed by polymerase chain reaction with sequence-specific primers in all patients after informed consent was obtained.

Clinical and laboratory examinations revealed no symptoms or findings of Epstein-Barr virus and cytomegalovirus infections, cicatricial alopecia, or connective tissue diseases in any of the patients. HLA antigen DNA typing revealed the following HLA alleles: B^*35/40, C^*04/15, DRB1^*08/10, and DQB1^*03/05 in patient 1; B^*04/13, C^*06/15, DRB1^*07/10, and DQB1^*02/05 in patient 2; B^*33/37, C^*04/06, DRB1^*08/15, and DQ^*06/06 in patient 3; B^*13/37, C^*06/06, DRB1^*07/15, and DQB1^*02/06 in patient 4.

Laboratory testing revealed vitamin B₁₂ deficiency in patient 2 and iron deficiency anemia in patient 3; all other laboratory tests were within reference range. Antithyroglobulin and antithyroid peroxidase autoantibodies were all negative. Clinical features and laboratory analyses for all patients are summarized in the Table.

Treatment

All patients were recommended psychiatric therapy and started on dermatologic treatments. Topical corticosteroids, intralesional triamcinolone acetonide (8 mg/mL) injections into areas of hair loss, 8 total sessions of cryotherapy administered at 3-week intervals, and minoxidil solution 2% were administered respectively to all 4 patients. Alopecia areata in patients 3 and 4 completely regressed; however, no benefit was observed in patients 1 and 2 after 1 year of treatment. Because there was no response to the prior interventions, patient 1 was started on treatment with cyclosporine 2.5 mg/kg twice daily. However, therapy was discontinued after 1 month and treatment with narrowband UVB (3 times per week for 7 months [total of 57 sessions]) and topical corticosteroids were initiated (Table). The patient partially benefited from these regimens and recurrence was observed during the course of the treatment.

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Although it was recommended that all 4 patients undergo psychiatric treatment and follow-up regularly with a psychiatrist, the patients declined. After approximately 1 year of dermatologic treatment, all 4 patients were lost to follow-up.

Comment

The etiopathogenesis of AA is unclear, but there is strong evidence suggesting that it is a T-cell–mediated autoimmune disease targeting the hair follicles. Common association of AA with autoimmune diseases such as vitiligo and thyroiditis support the immunological origin of the disease.³ In our case, patient 2 had AA along with vitiligo, but no associated autoimmune diseases (eg, vitiligo, diabetes mellitus, pernicious anemia, thyroid diseases) were noted in the other patients. Genetic and environmental factors are known to be influential as much as immune dysfunction in the etiology of AA.²

The presence of family history in 20% of patients supports the genetic predisposition of AA.⁴ In a genetic study by Martinez-Mir et al,⁵ susceptibility loci for AA were demonstrated on chromosomes 6, 10, 16, and 18. HLA antigen alleles, which provide predisposition to AA, have been investigated and associations with many different HLA antigens have been described for AA. In these studies, a relationship between AA and HLA class I antigens was not determined. Notable results mainly focused on HLA class II antigens.^6-8 Colombe et al⁷ and Marques Da Costa et al⁸ demonstrated that long-lasting alopecia totalis or alopecia universalis (AT/AU) patients had a strong relationship with HLA-DRB1^*1104; DRB1^*04/05 was reported to be the most frequent HLA group among all patients with AA.^6-10 In contrast, we did not detect these alleles in our patients. Colombe et al^7,11 noted that HLA-DQB1^*03 is a marker for both patch-type AA and AT/AU. Colombe et al¹⁰ showed that HLA-DQB1^*03 was present in more than 80% of patients (N=286) with long-lasting AA. Barahmani et al⁹ confirmed a strong association between HLA-DQB1^*0301, DRB1^*1104, and AT/AU. In our patients, we detected HLA-DQB1^*03/05 in patient 1 who had the earliest onset and most severe presentation of AA. In some studies, HLA-DRB1^*03 was found to be less frequent in patients with AA, and this allele was suggested to be a protective factor.^6,12 However, this allele was not detected in any of our patients.

The association of HLA alleles and AA has been investigated in Turkish patients with AA.^13-15 Akar et al¹³ and Kavak et al¹⁴ detected that the frequency of HLA-DQB1^*03 allele was remarkably higher in patients with AA than in healthy controls. These results were consistent with Colombe et al.¹⁰ On the other hand, Kavak et al¹⁴ reported that the frequency of HLA-DR16 was decreased in the patient group with AA. In another study, the frequency of HLA-B62 was increased in patients with AA compared to healthy controls.¹⁵ The HLA-DQB1^*03 allele was found to be associated with AA in only patient 1 in our case series, and HLA alleles were not commonly shared among the 4 patients. Additionally, lack of consanguinity between patients 2 and 3 (the parents) also suggested that genetic factors were not involved in our familial cases.

Blaumeiser et al¹⁶ reported a lifetime risk of 7.4% in parents and 7.1% in siblings of 206 AA patients; however, because these studies investigated the presence of AA in any given life period of the family members, their results do not reflect frequency of simultaneous AA presence within one family. In a literature search using PubMed, Google Scholar, and other national databases for the terms alopecia areata as well as family, sibling, concurrently, concomitant, co-existent, and simultaneously, only 2 cases involving a husband and wife and 1 case of 2 siblings who concurrently had AA have been previously reported.^17,18 Simultaneous presence of AA in more than 3 members of the same family is rare, and these cases have been observed in different generations and time periods.¹⁹ Among our patients, despite different age of onset and duration, AA was simultaneously present in the entire family.

Moreover, Rodriguez et al²⁰ reported that the concordance rate of AA in identical twins was 42% and dizygotic twins was 10%. Environmental factors and infections also have been implicated in the etiology of AA. Infections caused by viruses such as cytomegalovirus and Epstein-Barr virus have been thought to be potential triggering factors; however, no evidence has been found.^21,22 The clinical and laboratory examinations in our study did not reveal any presence and/or history of any known infectious disease, and there was no history of contact with water infected by acrylamide or a similar chemical.

Various life events and intense psychological stress may play an important role in triggering AA. Depression, hysteria, psychopathic deviance, psychasthenia, schizophrenia, anxiety, health concerns, bizarre thoughts, and family problems were found to be more frequent in patients with AA than healthy controls.²³ The most common psychological disorders associated with AA are generalized anxiety disorder, major depressive disorder, adjustment disorders, and phobias.^1,24 Ruiz-Doblado et al²⁵ determined the presence of psychiatric comorbidities in 66% (21/32) of AA cases. Chu et al²⁶ reported that the differences in ages of onset of AA revealed differences in psychiatric comorbidities. The risk for depression was higher in patients with AA younger than 20 years. An increased rate of anxiety was detected with patients with an onset of AA between the ages of 20 and 39 years. Obsessive-compulsive disorder and anxiety were more common in patients aged 40 to 59 years. Interestingly, the investigators also observed that approximately 50% of psychiatric disorders occurred prior to onset of AA.²⁶ One study showed higher rates of stressful life events in children than in controls.²⁷ Ghanizadeh²⁴ reported at least 1 psychiatric disorder in 78% (11/14) of children and adolescents with AA. In the same study, obsessive-compulsive disorder was found to be the second common condition following major depression in AA.²⁴

In our patients, psychiatric evaluations revealed obsessive-compulsive personality disorder in patients 2 and 3, depression in patient 3, and symptoms of anxiety with a lack of self-confidence in patients 1 and 4. Psychiatric disorders affecting the entire family may stem from unemployment of the father. Similar to the results noted in prior studies, depression, the most commonly associated psychiatric disorder of AA, was present in 2 of 4 patients. Obsessive-compulsive disorder, the second most common psychiatric disorder among AA patients, was present in patients 2 and 3. These results indicate that AA may be associated with shared stressful events and psychiatric disorders. Therefore, in addition to dermatologic treatment, it was recommended that all patients undergo psychiatric treatment and follow-up regularly with a psychiatrist; however, the patients declined. At the end of a 1-year treatment period and follow-up, resistance to therapy with minimal recovery followed by a rapid recurrence was determined in patients 1 and 2.

Conclusion

This report demonstrated that familial AA was strongly associated with psychological disorders that were detected in all patients. In our patients, HLA alleles did not seem to have a role in the development of familial AA. These results suggest that HLA was not associated with AA triggered by psychological stress. We believe that psychological disorders and stressful life events may play an important role in the occurrence of AA and lead to the development of resistance against treatment in familial and resistant AA cases.

Alopecia areata (AA) presents as sudden, nonscarring, recurrent hair loss characterized by well-circumscribed hairless patches. Although AA may be observed on any hair-bearing areas of the body, the most commonly affected sites are the scalp, beard area, eyebrows, and eyelashes.¹ The incidence of AA is 1% to 2% in the general population and it is more common in males than females younger than 40 years.² Although the majority of patients present with self-limited and well-circumscribed hairless patches that resolve within 2 years, 7% to 10% display a chronic and severe prognosis.³

The etiopathogenesis of AA is not clearly understood, but its occurrence and progression can involve immune dysfunction, genetic predisposition, infections, and physical and psychological trauma.² Alopecia areata is observed to occur sporadically in most patients. Family history has been found in 3% to 42% of cases, but simultaneous occurrence of AA in family members is rare.⁴ In this case series, we present 4 cases of active AA lesions occurring simultaneously in a family who also had associated psychologic disorders.

Case Series

Patient 1 (Proband)

An 11-year-old boy presented with a 6-year history of ongoing AA with recurrent improvement and relapses on the scalp, eyebrows, and eyelashes. Various topical and oral medications had been prescribed by several outside dermatologists; however, these treatments provided minimal benefit and resulted in the recurrence of AA. Dermatologic examination revealed hair loss on the entire frontal, parietal, and temporal regions of the scalp, as well as half of the occipital region and one-third of the lateral side of the eyebrows (Figure 1). Psychological evaluation revealed introvert personality characteristics, lack of self-confidence, and signs of depression and anxiety.

ct098007030_e_fig1.png

Patient 2 (Proband’s Father)

A 38-year-old man presented with a 16-year history of recurrent loss and regrowth of hair on the scalp and beard area and white spots on the penis and arms. He previously had not undergone any treatments. Dermatologic examination revealed well-circumscribed, 1- to 4-cm, hairless patches on the occipital region of the scalp and in the beard area (Figure 2A) and multiple, 2- to 10-mm, vitiliginous lesions on both forearms (Figure 2B) and the penis. The patient had been unemployed for 6 months. Psychological evaluation revealed obsessive-compulsive disorder and obsessive-compulsive personality disorder.

ct098007030_e_fig2.png

Patient 3 (Proband’s Mother)

A 32-year-old woman presented with a 3-year history of chronic AA. She previously had not undergone any treatments. Dermatologic examination revealed 2 well-circumscribed, 3- to 4-cm patches of hair loss on the occipital and left temporal regions of the scalp (Figure 3). Psychological evaluation revealed obsessive-compulsive personality disorder and depression. The patient did not have any autoimmune diseases.

ct098007030_e_fig3.png

Patient 4 (Proband’s Sister)

A 10-year-old girl presented with a 6-year history of recurrent, self-limited AA on various areas of scalp. She previously had not undergone any treatments. Dermatologic examination revealed a 3-cm hairless patch on the occipital region of the scalp (Figure 4). Psychiatric evaluation revealed narcissistic personality disorder, anxiety, and lack of self-confidence.

ct098007030_e_fig4.png

Laboratory Evaluation and HLA Antigen DNA Typing

Laboratory testing including complete blood cell count; liver, kidney, and thyroid function; and vitamin B₁₂, zinc, folic acid, and fasting blood sugar levels were performed in all patients.

HLA antigen DNA typing was performed by polymerase chain reaction with sequence-specific primers in all patients after informed consent was obtained.

Clinical and laboratory examinations revealed no symptoms or findings of Epstein-Barr virus and cytomegalovirus infections, cicatricial alopecia, or connective tissue diseases in any of the patients. HLA antigen DNA typing revealed the following HLA alleles: B^*35/40, C^*04/15, DRB1^*08/10, and DQB1^*03/05 in patient 1; B^*04/13, C^*06/15, DRB1^*07/10, and DQB1^*02/05 in patient 2; B^*33/37, C^*04/06, DRB1^*08/15, and DQ^*06/06 in patient 3; B^*13/37, C^*06/06, DRB1^*07/15, and DQB1^*02/06 in patient 4.

Laboratory testing revealed vitamin B₁₂ deficiency in patient 2 and iron deficiency anemia in patient 3; all other laboratory tests were within reference range. Antithyroglobulin and antithyroid peroxidase autoantibodies were all negative. Clinical features and laboratory analyses for all patients are summarized in the Table.

Treatment

All patients were recommended psychiatric therapy and started on dermatologic treatments. Topical corticosteroids, intralesional triamcinolone acetonide (8 mg/mL) injections into areas of hair loss, 8 total sessions of cryotherapy administered at 3-week intervals, and minoxidil solution 2% were administered respectively to all 4 patients. Alopecia areata in patients 3 and 4 completely regressed; however, no benefit was observed in patients 1 and 2 after 1 year of treatment. Because there was no response to the prior interventions, patient 1 was started on treatment with cyclosporine 2.5 mg/kg twice daily. However, therapy was discontinued after 1 month and treatment with narrowband UVB (3 times per week for 7 months [total of 57 sessions]) and topical corticosteroids were initiated (Table). The patient partially benefited from these regimens and recurrence was observed during the course of the treatment.

RTEmagicC_CT098007030_e_Table.JPG.jpg

Although it was recommended that all 4 patients undergo psychiatric treatment and follow-up regularly with a psychiatrist, the patients declined. After approximately 1 year of dermatologic treatment, all 4 patients were lost to follow-up.

Comment

The etiopathogenesis of AA is unclear, but there is strong evidence suggesting that it is a T-cell–mediated autoimmune disease targeting the hair follicles. Common association of AA with autoimmune diseases such as vitiligo and thyroiditis support the immunological origin of the disease.³ In our case, patient 2 had AA along with vitiligo, but no associated autoimmune diseases (eg, vitiligo, diabetes mellitus, pernicious anemia, thyroid diseases) were noted in the other patients. Genetic and environmental factors are known to be influential as much as immune dysfunction in the etiology of AA.²

The presence of family history in 20% of patients supports the genetic predisposition of AA.⁴ In a genetic study by Martinez-Mir et al,⁵ susceptibility loci for AA were demonstrated on chromosomes 6, 10, 16, and 18. HLA antigen alleles, which provide predisposition to AA, have been investigated and associations with many different HLA antigens have been described for AA. In these studies, a relationship between AA and HLA class I antigens was not determined. Notable results mainly focused on HLA class II antigens.^6-8 Colombe et al⁷ and Marques Da Costa et al⁸ demonstrated that long-lasting alopecia totalis or alopecia universalis (AT/AU) patients had a strong relationship with HLA-DRB1^*1104; DRB1^*04/05 was reported to be the most frequent HLA group among all patients with AA.^6-10 In contrast, we did not detect these alleles in our patients. Colombe et al^7,11 noted that HLA-DQB1^*03 is a marker for both patch-type AA and AT/AU. Colombe et al¹⁰ showed that HLA-DQB1^*03 was present in more than 80% of patients (N=286) with long-lasting AA. Barahmani et al⁹ confirmed a strong association between HLA-DQB1^*0301, DRB1^*1104, and AT/AU. In our patients, we detected HLA-DQB1^*03/05 in patient 1 who had the earliest onset and most severe presentation of AA. In some studies, HLA-DRB1^*03 was found to be less frequent in patients with AA, and this allele was suggested to be a protective factor.^6,12 However, this allele was not detected in any of our patients.

The association of HLA alleles and AA has been investigated in Turkish patients with AA.^13-15 Akar et al¹³ and Kavak et al¹⁴ detected that the frequency of HLA-DQB1^*03 allele was remarkably higher in patients with AA than in healthy controls. These results were consistent with Colombe et al.¹⁰ On the other hand, Kavak et al¹⁴ reported that the frequency of HLA-DR16 was decreased in the patient group with AA. In another study, the frequency of HLA-B62 was increased in patients with AA compared to healthy controls.¹⁵ The HLA-DQB1^*03 allele was found to be associated with AA in only patient 1 in our case series, and HLA alleles were not commonly shared among the 4 patients. Additionally, lack of consanguinity between patients 2 and 3 (the parents) also suggested that genetic factors were not involved in our familial cases.

Blaumeiser et al¹⁶ reported a lifetime risk of 7.4% in parents and 7.1% in siblings of 206 AA patients; however, because these studies investigated the presence of AA in any given life period of the family members, their results do not reflect frequency of simultaneous AA presence within one family. In a literature search using PubMed, Google Scholar, and other national databases for the terms alopecia areata as well as family, sibling, concurrently, concomitant, co-existent, and simultaneously, only 2 cases involving a husband and wife and 1 case of 2 siblings who concurrently had AA have been previously reported.^17,18 Simultaneous presence of AA in more than 3 members of the same family is rare, and these cases have been observed in different generations and time periods.¹⁹ Among our patients, despite different age of onset and duration, AA was simultaneously present in the entire family.

Moreover, Rodriguez et al²⁰ reported that the concordance rate of AA in identical twins was 42% and dizygotic twins was 10%. Environmental factors and infections also have been implicated in the etiology of AA. Infections caused by viruses such as cytomegalovirus and Epstein-Barr virus have been thought to be potential triggering factors; however, no evidence has been found.^21,22 The clinical and laboratory examinations in our study did not reveal any presence and/or history of any known infectious disease, and there was no history of contact with water infected by acrylamide or a similar chemical.

Various life events and intense psychological stress may play an important role in triggering AA. Depression, hysteria, psychopathic deviance, psychasthenia, schizophrenia, anxiety, health concerns, bizarre thoughts, and family problems were found to be more frequent in patients with AA than healthy controls.²³ The most common psychological disorders associated with AA are generalized anxiety disorder, major depressive disorder, adjustment disorders, and phobias.^1,24 Ruiz-Doblado et al²⁵ determined the presence of psychiatric comorbidities in 66% (21/32) of AA cases. Chu et al²⁶ reported that the differences in ages of onset of AA revealed differences in psychiatric comorbidities. The risk for depression was higher in patients with AA younger than 20 years. An increased rate of anxiety was detected with patients with an onset of AA between the ages of 20 and 39 years. Obsessive-compulsive disorder and anxiety were more common in patients aged 40 to 59 years. Interestingly, the investigators also observed that approximately 50% of psychiatric disorders occurred prior to onset of AA.²⁶ One study showed higher rates of stressful life events in children than in controls.²⁷ Ghanizadeh²⁴ reported at least 1 psychiatric disorder in 78% (11/14) of children and adolescents with AA. In the same study, obsessive-compulsive disorder was found to be the second common condition following major depression in AA.²⁴

In our patients, psychiatric evaluations revealed obsessive-compulsive personality disorder in patients 2 and 3, depression in patient 3, and symptoms of anxiety with a lack of self-confidence in patients 1 and 4. Psychiatric disorders affecting the entire family may stem from unemployment of the father. Similar to the results noted in prior studies, depression, the most commonly associated psychiatric disorder of AA, was present in 2 of 4 patients. Obsessive-compulsive disorder, the second most common psychiatric disorder among AA patients, was present in patients 2 and 3. These results indicate that AA may be associated with shared stressful events and psychiatric disorders. Therefore, in addition to dermatologic treatment, it was recommended that all patients undergo psychiatric treatment and follow-up regularly with a psychiatrist; however, the patients declined. At the end of a 1-year treatment period and follow-up, resistance to therapy with minimal recovery followed by a rapid recurrence was determined in patients 1 and 2.

Conclusion

This report demonstrated that familial AA was strongly associated with psychological disorders that were detected in all patients. In our patients, HLA alleles did not seem to have a role in the development of familial AA. These results suggest that HLA was not associated with AA triggered by psychological stress. We believe that psychological disorders and stressful life events may play an important role in the occurrence of AA and lead to the development of resistance against treatment in familial and resistant AA cases.

Alopecia areata (AA) presents as sudden, nonscarring, recurrent hair loss characterized by well-circumscribed hairless patches. Although AA may be observed on any hair-bearing areas of the body, the most commonly affected sites are the scalp, beard area, eyebrows, and eyelashes.¹ The incidence of AA is 1% to 2% in the general population and it is more common in males than females younger than 40 years.² Although the majority of patients present with self-limited and well-circumscribed hairless patches that resolve within 2 years, 7% to 10% display a chronic and severe prognosis.³

The etiopathogenesis of AA is not clearly understood, but its occurrence and progression can involve immune dysfunction, genetic predisposition, infections, and physical and psychological trauma.² Alopecia areata is observed to occur sporadically in most patients. Family history has been found in 3% to 42% of cases, but simultaneous occurrence of AA in family members is rare.⁴ In this case series, we present 4 cases of active AA lesions occurring simultaneously in a family who also had associated psychologic disorders.

Case Series

Patient 1 (Proband)

An 11-year-old boy presented with a 6-year history of ongoing AA with recurrent improvement and relapses on the scalp, eyebrows, and eyelashes. Various topical and oral medications had been prescribed by several outside dermatologists; however, these treatments provided minimal benefit and resulted in the recurrence of AA. Dermatologic examination revealed hair loss on the entire frontal, parietal, and temporal regions of the scalp, as well as half of the occipital region and one-third of the lateral side of the eyebrows (Figure 1). Psychological evaluation revealed introvert personality characteristics, lack of self-confidence, and signs of depression and anxiety.

ct098007030_e_fig1.png

Patient 2 (Proband’s Father)

A 38-year-old man presented with a 16-year history of recurrent loss and regrowth of hair on the scalp and beard area and white spots on the penis and arms. He previously had not undergone any treatments. Dermatologic examination revealed well-circumscribed, 1- to 4-cm, hairless patches on the occipital region of the scalp and in the beard area (Figure 2A) and multiple, 2- to 10-mm, vitiliginous lesions on both forearms (Figure 2B) and the penis. The patient had been unemployed for 6 months. Psychological evaluation revealed obsessive-compulsive disorder and obsessive-compulsive personality disorder.

ct098007030_e_fig2.png

Patient 3 (Proband’s Mother)

A 32-year-old woman presented with a 3-year history of chronic AA. She previously had not undergone any treatments. Dermatologic examination revealed 2 well-circumscribed, 3- to 4-cm patches of hair loss on the occipital and left temporal regions of the scalp (Figure 3). Psychological evaluation revealed obsessive-compulsive personality disorder and depression. The patient did not have any autoimmune diseases.

ct098007030_e_fig3.png

Patient 4 (Proband’s Sister)

A 10-year-old girl presented with a 6-year history of recurrent, self-limited AA on various areas of scalp. She previously had not undergone any treatments. Dermatologic examination revealed a 3-cm hairless patch on the occipital region of the scalp (Figure 4). Psychiatric evaluation revealed narcissistic personality disorder, anxiety, and lack of self-confidence.

ct098007030_e_fig4.png

Laboratory Evaluation and HLA Antigen DNA Typing

Laboratory testing including complete blood cell count; liver, kidney, and thyroid function; and vitamin B₁₂, zinc, folic acid, and fasting blood sugar levels were performed in all patients.

HLA antigen DNA typing was performed by polymerase chain reaction with sequence-specific primers in all patients after informed consent was obtained.

Clinical and laboratory examinations revealed no symptoms or findings of Epstein-Barr virus and cytomegalovirus infections, cicatricial alopecia, or connective tissue diseases in any of the patients. HLA antigen DNA typing revealed the following HLA alleles: B^*35/40, C^*04/15, DRB1^*08/10, and DQB1^*03/05 in patient 1; B^*04/13, C^*06/15, DRB1^*07/10, and DQB1^*02/05 in patient 2; B^*33/37, C^*04/06, DRB1^*08/15, and DQ^*06/06 in patient 3; B^*13/37, C^*06/06, DRB1^*07/15, and DQB1^*02/06 in patient 4.

Laboratory testing revealed vitamin B₁₂ deficiency in patient 2 and iron deficiency anemia in patient 3; all other laboratory tests were within reference range. Antithyroglobulin and antithyroid peroxidase autoantibodies were all negative. Clinical features and laboratory analyses for all patients are summarized in the Table.

Treatment

All patients were recommended psychiatric therapy and started on dermatologic treatments. Topical corticosteroids, intralesional triamcinolone acetonide (8 mg/mL) injections into areas of hair loss, 8 total sessions of cryotherapy administered at 3-week intervals, and minoxidil solution 2% were administered respectively to all 4 patients. Alopecia areata in patients 3 and 4 completely regressed; however, no benefit was observed in patients 1 and 2 after 1 year of treatment. Because there was no response to the prior interventions, patient 1 was started on treatment with cyclosporine 2.5 mg/kg twice daily. However, therapy was discontinued after 1 month and treatment with narrowband UVB (3 times per week for 7 months [total of 57 sessions]) and topical corticosteroids were initiated (Table). The patient partially benefited from these regimens and recurrence was observed during the course of the treatment.

RTEmagicC_CT098007030_e_Table.JPG.jpg

Although it was recommended that all 4 patients undergo psychiatric treatment and follow-up regularly with a psychiatrist, the patients declined. After approximately 1 year of dermatologic treatment, all 4 patients were lost to follow-up.

Comment

The etiopathogenesis of AA is unclear, but there is strong evidence suggesting that it is a T-cell–mediated autoimmune disease targeting the hair follicles. Common association of AA with autoimmune diseases such as vitiligo and thyroiditis support the immunological origin of the disease.³ In our case, patient 2 had AA along with vitiligo, but no associated autoimmune diseases (eg, vitiligo, diabetes mellitus, pernicious anemia, thyroid diseases) were noted in the other patients. Genetic and environmental factors are known to be influential as much as immune dysfunction in the etiology of AA.²

The presence of family history in 20% of patients supports the genetic predisposition of AA.⁴ In a genetic study by Martinez-Mir et al,⁵ susceptibility loci for AA were demonstrated on chromosomes 6, 10, 16, and 18. HLA antigen alleles, which provide predisposition to AA, have been investigated and associations with many different HLA antigens have been described for AA. In these studies, a relationship between AA and HLA class I antigens was not determined. Notable results mainly focused on HLA class II antigens.^6-8 Colombe et al⁷ and Marques Da Costa et al⁸ demonstrated that long-lasting alopecia totalis or alopecia universalis (AT/AU) patients had a strong relationship with HLA-DRB1^*1104; DRB1^*04/05 was reported to be the most frequent HLA group among all patients with AA.^6-10 In contrast, we did not detect these alleles in our patients. Colombe et al^7,11 noted that HLA-DQB1^*03 is a marker for both patch-type AA and AT/AU. Colombe et al¹⁰ showed that HLA-DQB1^*03 was present in more than 80% of patients (N=286) with long-lasting AA. Barahmani et al⁹ confirmed a strong association between HLA-DQB1^*0301, DRB1^*1104, and AT/AU. In our patients, we detected HLA-DQB1^*03/05 in patient 1 who had the earliest onset and most severe presentation of AA. In some studies, HLA-DRB1^*03 was found to be less frequent in patients with AA, and this allele was suggested to be a protective factor.^6,12 However, this allele was not detected in any of our patients.

The association of HLA alleles and AA has been investigated in Turkish patients with AA.^13-15 Akar et al¹³ and Kavak et al¹⁴ detected that the frequency of HLA-DQB1^*03 allele was remarkably higher in patients with AA than in healthy controls. These results were consistent with Colombe et al.¹⁰ On the other hand, Kavak et al¹⁴ reported that the frequency of HLA-DR16 was decreased in the patient group with AA. In another study, the frequency of HLA-B62 was increased in patients with AA compared to healthy controls.¹⁵ The HLA-DQB1^*03 allele was found to be associated with AA in only patient 1 in our case series, and HLA alleles were not commonly shared among the 4 patients. Additionally, lack of consanguinity between patients 2 and 3 (the parents) also suggested that genetic factors were not involved in our familial cases.

Blaumeiser et al¹⁶ reported a lifetime risk of 7.4% in parents and 7.1% in siblings of 206 AA patients; however, because these studies investigated the presence of AA in any given life period of the family members, their results do not reflect frequency of simultaneous AA presence within one family. In a literature search using PubMed, Google Scholar, and other national databases for the terms alopecia areata as well as family, sibling, concurrently, concomitant, co-existent, and simultaneously, only 2 cases involving a husband and wife and 1 case of 2 siblings who concurrently had AA have been previously reported.^17,18 Simultaneous presence of AA in more than 3 members of the same family is rare, and these cases have been observed in different generations and time periods.¹⁹ Among our patients, despite different age of onset and duration, AA was simultaneously present in the entire family.

Moreover, Rodriguez et al²⁰ reported that the concordance rate of AA in identical twins was 42% and dizygotic twins was 10%. Environmental factors and infections also have been implicated in the etiology of AA. Infections caused by viruses such as cytomegalovirus and Epstein-Barr virus have been thought to be potential triggering factors; however, no evidence has been found.^21,22 The clinical and laboratory examinations in our study did not reveal any presence and/or history of any known infectious disease, and there was no history of contact with water infected by acrylamide or a similar chemical.

Various life events and intense psychological stress may play an important role in triggering AA. Depression, hysteria, psychopathic deviance, psychasthenia, schizophrenia, anxiety, health concerns, bizarre thoughts, and family problems were found to be more frequent in patients with AA than healthy controls.²³ The most common psychological disorders associated with AA are generalized anxiety disorder, major depressive disorder, adjustment disorders, and phobias.^1,24 Ruiz-Doblado et al²⁵ determined the presence of psychiatric comorbidities in 66% (21/32) of AA cases. Chu et al²⁶ reported that the differences in ages of onset of AA revealed differences in psychiatric comorbidities. The risk for depression was higher in patients with AA younger than 20 years. An increased rate of anxiety was detected with patients with an onset of AA between the ages of 20 and 39 years. Obsessive-compulsive disorder and anxiety were more common in patients aged 40 to 59 years. Interestingly, the investigators also observed that approximately 50% of psychiatric disorders occurred prior to onset of AA.²⁶ One study showed higher rates of stressful life events in children than in controls.²⁷ Ghanizadeh²⁴ reported at least 1 psychiatric disorder in 78% (11/14) of children and adolescents with AA. In the same study, obsessive-compulsive disorder was found to be the second common condition following major depression in AA.²⁴

In our patients, psychiatric evaluations revealed obsessive-compulsive personality disorder in patients 2 and 3, depression in patient 3, and symptoms of anxiety with a lack of self-confidence in patients 1 and 4. Psychiatric disorders affecting the entire family may stem from unemployment of the father. Similar to the results noted in prior studies, depression, the most commonly associated psychiatric disorder of AA, was present in 2 of 4 patients. Obsessive-compulsive disorder, the second most common psychiatric disorder among AA patients, was present in patients 2 and 3. These results indicate that AA may be associated with shared stressful events and psychiatric disorders. Therefore, in addition to dermatologic treatment, it was recommended that all patients undergo psychiatric treatment and follow-up regularly with a psychiatrist; however, the patients declined. At the end of a 1-year treatment period and follow-up, resistance to therapy with minimal recovery followed by a rapid recurrence was determined in patients 1 and 2.

Conclusion

This report demonstrated that familial AA was strongly associated with psychological disorders that were detected in all patients. In our patients, HLA alleles did not seem to have a role in the development of familial AA. These results suggest that HLA was not associated with AA triggered by psychological stress. We believe that psychological disorders and stressful life events may play an important role in the occurrence of AA and lead to the development of resistance against treatment in familial and resistant AA cases.