Dear Editor, Frontal fibrosing alopecia (FFA) is an inflammatory primary scarring alopecia of uncertain aetiology that represents a variant of lichen planopilaris.1 It predominantly, although not exclusively, affects postmenopausal women.2 Its pathogenesis is characterized by immune-mediated follicular destruction at the level of the hair bulge, which leads to a clinical phenotype of progressive frontotemporal hair and eyebrow loss that is often preceded by widespread body-hair loss.2 Histologically, a lichenoid inflammatory infiltrate surrounds the isthmus and infundibulum of the hair follicle, and this progresses to follicular scarring and dropout in advanced disease.2 We recently completed the first genome-wide association study (GWAS) in FFA coupled with transcriptomic and metabolomic analyses, which have provided important insights into its pathogenesis.3 We have conducted and herein present a descriptive cross-sectional study of the clinical phenotype in women from the FFA UK GWAS Cohort. Ethical approval was obtained from the Northampton NRES Committee, UK (REC 15/EM/0273). Patients with a formal diagnosis of FFA made by a consultant dermatologist from 20 secondary care dermatology departments across the UK were eligible for inclusion. A diagnosis of FFA was made based on clinical criteria, with histological confirmation if required.2 Each patient was assessed for multiple clinical variables based on a standardized pro forma. Analysis was limited to female participants of Eurasian ancestry in line with our previous GWAS.3 Statistical analysis was descriptive and exploratory, estimating frequencies and measures of centrality and spread, and participants for whom data were missing for a given variable were excluded from the analysis. All analyses were conducted using Stata version 15 (StataCorp, College Station, TX, USA). Phenotypic data were available for 711 UK women with FFA among a total of 1044 participants in the GWAS cohort. Their median age was 66 (interquartile range 59–72) and the median duration of scalp hair loss was 7 years (interquartile range 5–10). In 485 of 663 (73·2%) participants with available data, frontotemporal hairline recession occurred following menopause. Other clinical characteristics and comorbidities are summarized in Table 1. Perifollicular erythema was present in 77·3% and hyperkeratosis in 26·0% of participants. In addition to frontotemporal recession, concomitant occipital recession was noted in 26·0%. Eyebrow loss was noted in 90·6% and eyelash loss in 44·5%. Limb hair loss was also documented in 77·5% and most commonly affected both arms and legs, while concomitant axillary or pubic hair loss was reported in 67·0%. Concurrent multifocal involvement suggesting coexistence of classic lichen planopilaris (14·7%) and nail changes of any type (23·7%) were noted in a smaller proportion of participants. Other forms of lichen planus were seen in 9·5% of participants, with oral (5·1%) and vulval disease (3·5%) being most prevalent. Only 44·0% of our cohort were prescribed a medication relevant to their FFA. The most frequent treatment was hydroxychloroquine (24·0%), with other treatments such as topical corticosteroids (16·6%), oral tetracycline antibiotics (10·1%), topical calcineurin inhibitors (3·8%), intralesional steroids (1·7%) and oral corticosteroids (1·3%) being less common. Use of systemic immunosuppressant or antiproliferative agents including retinoids, mycophenolate mofetil, ciclosporin and methotrexate was rare (2·1%). In keeping with the immune-mediated pathogenesis of FFA, 20·7% of participants reported at least one comorbid autoimmune disease (Table 1). The most common was autoimmune thyroid disease (12·9%), followed by coeliac disease (1·5%) and pernicious anaemia (1·2%). As hormonal aberrations have been implicated in the pathogenesis of FFA, we also examined whether certain endocrine disorders were prevalent in this cohort.2 A history of oestrogen deficiency secondary to oophorectomy or primary ovarian insufficiency was present in 5·6% of women, while 2·3% reported exposure to selective oestrogen receptor modulators (tamoxifen or clomiphene). With regard to exogenous hormone use, the oral contraceptive pill was used for > 6 months by 71·2% of women. In summary, this descriptive study outlines the clinical characteristics and treatment modalities in a cohort of 711 women with FFA, recapitulating findings described by other international studies.4 Analysis of comorbidities revealed that autoimmune disease, thyroid hormone abnormalities and oestrogen deficiency were more prevalent than in the general population, while the frequency of oral contraceptive use was similar.5-7 These findings accord with other epidemiological studies and the results of our genetic investigation, which implicated causal genetic variation related to antigen presentation and hormone or xenobiotic metabolism in FFA pathogenesis.3, 8 This investigation is limited by its cross-sectional design, the absence of a control group, and missing data for certain clinical features. Therefore, further detailed clinical and experimental investigations are required to dissect the roles of autoimmunity and hormone metabolism in FFA pathogenesis. We thank the study participants for their help. We thank the NIHR Rare Genetic Disease Research Consortium Agreement Team, especially Gillian Borthwick, for their help with setting up multiple UK research participating sites. We thank Emma Stell and the numerous research assistants and nurses, especially Teena Mackenzie, Sophie Devine, Ruth Joslyn, Sonia Baryschpolec, Anne Thomson, Pauline Buchanan and Caroline White for their help with recruitment. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Alopecia areata (AA) is a hair loss disorder resulting from an autoimmune reaction against hair follicles. T-helper 1 cells are a major contributor to this disorder, but little is known about the role of T-regulatory cells (Tregs) in AA. Here, we analysed the distribution of circulating Treg subsets in twenty AA patients with active hair loss and fifteen healthy subjects by flow cytometry. The Treg suppressor HLA-DR+ subpopulation was significantly reduced in the patients (P<0.001) and there were significantly fewer cells expressing CD39 among the CD4+CD25+Foxp3+ Treg subpopulation in patients (P=0.001). FOXP3 CD39 Treg cells were also reduced in hair follicles; by 75% in non-lesional skin and 90% in lesional skin, when compared to control healthy skin. To further characterise Treg cells in AA; Tregs (CD4+CD25+FOXP3+) were investigated for their TCRβ sequence. PCR products analysed by Next Generation Sequencing techniques, showed that all frequent public clonotypes in AA Tregs were also present in controls at relatively similar frequencies, excepting two public clonotypes: CATSRDEGGLDEKLFF (V15 D1 J1-4) and CASRDGTGPSNYGYTF (V2 D1 J1-2), which were exclusively present in controls. This suggests that these Treg clonotypes may have a protective effect and that they may be an exciting subject for future therapeutic applications.

Frontal fibrosing alopecia (FFA) is a recently described inflammatory and scarring type of hair loss affecting almost exclusively women. Despite a dramatic recent increase in incidence the aetiopathogenesis of FFA remains unknown. We undertake genome-wide association studies in females from a UK cohort, comprising 844 cases and 3,760 controls, a Spanish cohort of 172 cases and 385 controls, and perform statistical meta-analysis. We observe genome-wide significant association with FFA at four genomic loci: 2p22.2, 6p21.1, 8q24.22 and 15q2.1. Within the 6p21.1 locus, fine-mapping indicates that the association is driven by the HLA-B*07:02 allele. At 2p22.1, we implicate a putative causal missense variant in CYP1B1, encoding the homonymous xenobiotic- and hormone-processing enzyme. Transcriptomic analysis of affected scalp tissue highlights overrepresentation of transcripts encoding components of innate and adaptive immune response pathways. These findings provide insight into disease pathogenesis and characterise FFA as a genetically predisposed immuno-inflammatory disorder driven by HLA-B*07:02.

Autoimmune regulator (AIRE) regulates promiscuous expression of tissue-restricted antigens in medullary epithelial cells (mTEC) of the thymus. To understand the diverse effects of AIRE, it is crucial to elucidate the molecular mechanisms underlying the process of AIRE-regulated gene expression. In this study, we generated a recombinant AIRE expression variant of the TEC 1A3 human cell line, TEC 1A3 AIRE(hi), to determine genes targeted by AIRE, and using microarray analysis, we identified 482 genes showing significant differential expression (P < 0.05; false discovery rate <5%), with 353 upregulated and 129 downregulated by AIRE expression. Microarray data were validated by quantitative PCR, confirming the differential expression of 12 known AIRE-regulated genes. Comparison of AIRE-dependent differential expression in our cell line model with murine datasets identified 447 conserved genes with a number of transcription regulatory interactions, forming several key nodes, including STAT1, which had over 30 interactions with other AIRE-regulated genes. As STAT1 mutations cause dominant chronic mucocutaneous candidiasis and decreased STAT1 levels in monocytes of autoimmune polyglandular syndrome 1 (APS-1) patients, it was important to further characterize AIRE-STAT1 interactions. TEC 1A3AIRE(hi) were treated with the STAT1 phosphorylation inhibitors fludarabine and LLL3 showed that phosphorylated STAT1 (p-STAT1) was not responsible for any of the observed differential expression. Moreover, treatment of TEC 1A3 AIRE(hi) with STAT1 shRNA did not induce any significant variation in the expression Cof unphosphorylated STAT1 (U-STAT1) downstream genes, suggesting that these genes were directly regulated by AIRE but not via U-STAT1. The novel model system we have developed provides potential opportunities for further analysis of the pathogenesis of (APS-1) and the wider roles of the AIRE gene.

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BACKGROUND:Alopecia areata (AA) is associated with Interferon- γ (IFN-γ) mediated T-lymphocyte dysfunction and increased circulating Interleukine-17 (IL-17) levels. Epigallocatechin-3-gallate (EGCG) specifically inhibits IFN-γ pathways and unlike Janus Kinase 1 and 2 (JAK1/JAK2) inhibitors (tofacitinib, ruxolitinib), EGCG is safer, more cost-effective, and is a topically active agent. Our objective is to test the mode of action of EGCG in vitro and ex vivo using HaCat, Jurkat cell lines, and peripheral blood mononuclear cells (PBMCs) of AA patients and healthy controls (HCs), respectively.METHODS:distribution of T helper cells (Th1, Th17), and cytotoxic cells (CD8) in PBMCs isolated from 30 AA patients and 30 HCs was investigated by flowcytomterty. In vitro treatment of HaCat and Jurkat cells with 40 μm EGCG for 48 h was performed to measure the level of phosphorylation of signal transducer and activator of transcription protein STAT1, and replicated in ex vivo model using PBMCs of AA patients.RESULTS:Interestingly, 40 μm EGCG is capable of completely inhibiting phosphorylation of STAT1 after 48 h in HaCat and Jurkat cells and ex vivo in PBMCs of AA patients. Based on QPCR data, the action of EGCG on p-STAT1 seems to be mediated via downregulation of the expression of JAK2 but not JAK1 leading to the inhibition of human leukocyte antigens (HLA-DR and HLA-B) expression probably via IRF-1. On the other hand, AA patients have significantly increased levels of Th1, Th17, and CD8 cells and the production of IFN-γ and IL-17 by PBMCs in AA patients was significantly higher compared to HC; p = 0.008 and p = 0.006, respectively. Total numbers of CD8+ cells were not significantly different between treated and untreated samples. However, CD8+ cells with positive Natural killer group 2 member D (NKG2D) transmembrane receptor (CD8+ NKG2D+ subset) was significantly reduced when PBMCs were treated with 20 μm EGCG for 48 h.CONCLUSION:These results suggest that EGCG has a synergistic action that inhibits expression of HLA-DR and HLA-B molecules via the IFN-γ pathway to maintain immune privilege in HF; also it reduces CD8+ NKG2D+ subset.

BACKGROUNDA substantial number of melanoma patients develop local or metastatic recurrence, and early detection of these is vital to maximise benefit from new therapies such as inhibitors of BRAF and MEK, or immune checkpoints. This study explored the use of novel DNA copy-number profiles in circulating cell-free DNA (cfDNA) as a potential biomarker of active disease and survival.PATIENTS AND METHODSMelanoma patients were recruited from oncology and dermatology clinics in Sheffield, UK, and cfDNA was isolated from stored blood plasma. Using low-coverage whole-genome sequencing, we created copy-number profiles from cfDNA from 83 melanoma patients, 44 of whom had active disease. We used scoring algorithms to summarize copy-number aberrations and investigated their utility in multivariable logistic and Cox regression analyses.RESULTSThe copy-number aberration score (CNAS) was a good discriminator of active disease (odds ratio, 3.1; 95% CI, 1.5-6.2; P = 0.002), and CNAS above or below the 75th percentile remained a significant discriminator in multivariable analysis for active disease (P = 0.019, with area under ROC curve of 0.90). Additionally, mortality was higher in those with CNASs above the 75th percentile than in those with lower scores (HR, 3.4; 95% CI, 1.5-7.9; P = 0.005), adjusting for stage of disease, disease status (active or resected), BRAF status, and cfDNA concentration.CONCLUSIONSThis study demonstrates the potential of a de novo approach utilizing copy-number profiling of cfDNA as a biomarker of active disease and survival in melanoma. Longitudinal analysis of copy-number profiles as an early marker of relapsed disease is warranted.

We report a family from Tabuk, Saudi Arabia, previously screened for Acrodermatitis Enteropathica (AE), in which two siblings presented with typical features of acral dermatitis and a pustular eruption but differing severity. Affected members of our family carry a rare genetic variant, p.Gly512Trp in the SLC39A4 gene which encodes a zinc transporter; disease is thought to result from zinc deficiency. Similar mutations have been reported previously; however, the variable severity within cases carrying the p.Gly512Trp variant and in AE overall led us to hypothesise that additional genetic modifiers may be contributing to the disease phenotype. Therefore whole genome sequencing was carried out in five family members, for whom material was available to search for additional modifiers of AE; this included one individual with clinically diagnosed AE. We confirmed that the p.Gly512Trp change in SLC39A4 was the only candidate homozygous change which was sufficiently rare (ExAC allele frequency 1.178e-05) and predicted deleterious (CADD score 35) to be attributable as a fully penetrant cause of AE. To identify other genes which may carry relevant genetic variation, we reviewed the relevant literature and databases including Gene Ontology Consortium, GeneMANIA, GeneCards, and MalaCards to identify zinc transporter genes and possible interacting partners. The affected individual carried variants in RECQL4 and GPAA1 genes with ExAC allele frequency <0.01 and CADD score >10. p.Gly512Trp is highly likely to be the pathogenic variant in this family. This variant was previously detected in a Tunisian proband with perfect genotype-phenotype segregation suggestive of pathogenicity. Further research is required in this area due to small sample size, but attention should be given to RECQL4 and GPAA1 to understand their role in the skin disease.

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We did not perform a matched one-to-one comparison between FFA subjects and controls, we compared two groups with similar ages. The only continuous variable was age. The responses to the questions posed were categorical. We therefore maintain that a Fisher's exact test rather than a McNemar's test was an appropriate method for analyzing the results.

Background The autoimmune regulator (AIRE) is expressed in the thymus, particularly in thymic medullary epithelial cells (mTECs), and is required for the ectopic expression of a diverse range of peripheral tissue antigens by mTECs, facilitating their ability to perform negative selection of auto-reactive immature T-cells. The expression profile of peripheral tissue antigens is affected not only by AIRE deficiency but also with variation of AIRE activity in the thymus. Method and Results Therefore we screened 591bp upstream of the AIRE transcription start site including AIRE minimal promoter for single nucleotide polymorphism (SNPs) and identified two SNPs -655R (rs117557896) and -230Y (rs751032) respectively. To study the effect of these variations on AIRE promoter activity we generated a Flp-In host cell line which was stably transfected with a single copy of the reporter vector. Relative promoter activity was estimated by comparing the luciferase specific activity for lysates of the different reporter AIRE promoter-reporter gene constructs including AIRE-655G AIRE-230C, AIRE-655G AIRE-230T and AIRE-655A AIRE-230C. The analysis showed that the commonest haplotype AIRE-655G AIRE-230C has the highest luciferase specific activity (p<0.001). Whereas AIRE-655G AIRE-230T has a luciferase specific activity value that approaches null. Both AIRE promoter polymorphic sites have one allele that forms a CpG methylation site which we determined can be methylated in methylation assays using the M.SssI CpG methyltransferase. Conclusion AIRE-230Y is in a conserved region of the promoter and is adjacent to a predicted WT1 transcription factor binding site, suggesting that AIRE-230Y affects AIRE expression by influencing the binding of biochemical factors to this region. Our findings show that AIRE-655GAIRE-230T haplotype could dramatically alter AIRE transcription and so have an effect on the process of negative selection and affect susceptibility to autoimmune conditions.

The guidelines have been revised and updated in accordance with a predetermined scope, based on that used in the 2003 guidelines. Recommendations in these guidelines supersede those in the 2003 guidelines. The objectives of the guidelines are to provide up-to-date recommendations for the manage- ment of alopecia areata in adults and children and a summary of the evidence base.Rosalind.Woodworth@utas.edu.au