Characterisation of APS-1 Experimental Models Is Crucial for Development of Novel Therapies.

Autoimmune polyglandular syndrome type 1 (APS-1) is an inherited autosomal disorder. The most common clinical features of the disease include adrenocortical failure, hypoparathyroidism (HP), and chronic mucocutaneous candidiasis (CMC). APS-1 is caused by mutations in the autoimmune regulator (AIRE) gene. AIRE is a transcriptional factor involved in the regulation of thousands of genes in the thymus. It facilitates central tolerance by promoting the ectopic expression of tissue-specific antigens (TSAs) in medullary thymic epithelial cells (mTECs), leading to the deletion of self-reactive thymocytes. Several Aire-deficient mice were developed separately, on different backgrounds; seven published Aire knockout mice show a variety of phenotypes depending on the strain used to generate the experimental model. The first Aire-deficient mice were generated on a "black 6" background almost 20 years ago. The model showed mild phenotype with relatively modest penetrance compared to models generated on BALBc or NOD backgrounds. The generation of all these experimental models is crucial for development and testing new therapeutics as well as reading the response to treatments.

AAV9-mediated AIRE gene delivery clears circulating antibodies and tissue T-cell infiltration in a mouse model of autoimmune polyglandular syndrome type-1.

OBJECTIVES: Autoimmune polyglandular syndrome type-1 (APS-1) is a monogenic recessive disorder characterised by multiple endocrine abnormalities, chronic mucocutaneous candidiasis and high titres of serum autoantibodies. To date, no curative treatment is available; current therapies manage the symptoms rather than treating the cause and have major side effects. APS-1 is caused by mutations in the autoimmune regulator (AIRE) gene. AIRE mediates central tolerance by directing the ectopic expression of tissue-specific antigens (TSAs) in medullary thymic epithelial cells, causing the deletion of self-reactive thymocytes. Therefore, loss-of-function mutations in AIRE result in a multisystem autoimmune disease. Because of the monogenic aetiology of APS-1 and availability of an APS-1 mouse model, we have explored the option of restoring functional AIRE using adeno-associated virus serotype 9 (AAV9). METHODS: The efficacy of AAV9-AIRE (AAV9 carrying AIRE cDNA) gene therapy was assessed in an APS-1 mouse model. We performed intrathymic injection of AAV9-AIRE into APS-1 mouse model using ultrasound imaging technique to accurately locating the thymus. We evaluated the efficiency of this approach alongside measures of autoimmunity and histology of target tissues. RESULTS: Intrathymic injection of AAV9-AIRE demonstrated high transduction efficiency and restored AIRE expression in the thymus. AIRE gene delivery led to a significant increase in TSA expression, and importantly a significant reduction of serum autoantibodies in treated versus control mice, which fell to near-undetectable levels by 4 weeks post-treatment. Furthermore, histological analysis of treated animals showed near-normal tissue morphology with no lymphocytic infiltrations, a hallmark of untreated Aire-deficient mice. CONCLUSION: This study has demonstrated the feasibility of AAV9-AIRE as a vehicle for gene therapy for APS-1.

Identification of single nucleotide variants in the Moroccan population by whole-genome sequencing.

BACKGROUND: Large-scale human sequencing projects have described around a hundred-million single nucleotide variants (SNVs). These studies have predominately involved individuals with European ancestry despite the fact that genetic diversity is expected to be highest in Africa where Homo sapiens evolved and has maintained a large population for the longest time. The African Genome Variation Project examined several African populations but these were all located south of the Sahara. Morocco is on the northwest coast of Africa and mostly lies north of the Sahara, which makes it very attractive for studying genetic diversity. The ancestry of present-day Moroccans is unknown and may be substantially different from Africans found South of the Sahara desert, Recent genomic data of Taforalt individuals in Eastern Morocco revealed 15,000-year-old modern humans and suggested that North African individuals may be genetically distinct from previously studied African populations. RESULTS: We present SNVs discovered by whole genome sequencing (WGS) of three Moroccans. From a total of 5.9 million SNVs detected, over 200,000 were not identified by 1000G and were not in the extensive gnomAD database. We summarise the SNVs by genomic position, type of sequence gene context and effect on proteins encoded by the sequence. Analysis of the overall genomic information of the Moroccan individuals to individuals from 1000G supports the Moroccan population being distinct from both sub-Saharan African and European populations. CONCLUSIONS: We conclude that Moroccan samples are genetically distinct and lie in the middle of the previously observed cline between populations of European and African ancestry. WGS of Moroccan individuals can identify a large number of novel SNVs and aid in functional characterisation of the genome.

Autosomal-dominant hypotrichosis with woolly hair: Novel gene locus on chromosome 4q35.1-q35.2.

Hypotrichosis simplex (HS) with and without woolly hair (WH) comprises a group of rare, monogenic disorders of hair loss. Patients present with a diffuse loss of scalp and/or body hair, which usually begins in early childhood and progresses into adulthood. Some of the patients also show hair that is tightly curled. Approximately 10 genes for autosomal recessive and autosomal dominant forms of HS have been identified in the last decade, among them five genes for the dominant form. We collected blood and buccal samples from 17 individuals of a large British family with HS and WH. After having sequenced all known dominant genes for HS in this family without the identification of any disease causing mutation, we performed a genome-wide scan, using the HumanLinkage-24 BeadChip, followed by a classical linkage analysis; and whole exome-sequencing (WES). Evidence for linkage was found for a region on chromosome 4q35.1-q35.2 with a maximum LOD score of 3.61. WES led to the identification of a mutation in the gene SORBS2, encoding sorbin and SH3 domain containing 2. Unfortunately, we could not find an additional mutation in any other patient/family with HS; and in cell culture, we could not observe any difference between cloned wildtype and mutant SORBS2 using western blotting and immunofluorescence analyses. Therefore, at present, SORBS2 cannot be considered a definite disease gene for this phenotype. However, the locus on chromosome 4q is a robust and novel finding for hypotrichosis with woolly hair. Further fine mapping and sequencing efforts are therefore warranted in order to confirm SORBS2 as a plausible HS disease gene.

Correction: Alopecia areata patients show deficiency of FOXP3+CD39+ T regulatory cells and clonotypic restriction of Treg TCRß-chain, which highlights the immunopathological aspect of the disease.

[This corrects the article DOI: 10.1371/journal.pone.0210308.].

Alopecia areata patients show deficiency of FOXP3+CD39+ T regulatory cells and clonotypic restriction of Treg TCRß-chain, which highlights the immunopathological aspect of the disease.

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 TCRß 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.

Epigallocatechin-3 Gallate Inhibits STAT-1/JAK2/IRF-1/HLA-DR/HLA-B and Reduces CD8 MKG2D Lymphocytes of Alopecia Areata Patients.

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.

Whole Genome Sequencing in an Acrodermatitis Enteropathica Family from the Middle East.

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.

Meta-Analysis of Autoimmune Regulator-Regulated Genes in Human and Murine Models: A Novel Human Model Provides Insights on the Role of Autoimmune Regulator in Regulating STAT1 and STAT1-Regulated Genes.

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 AIREhi, 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 1A3AIREhi 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 AIREhi with STAT1 shRNA did not induce any significant variation in the expression of 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.

A Novel Homozygous p.L539F Mutation Identified in PINK1 Gene in a Moroccan Patient with Parkinsonism.

Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease. Ten of fifteen causative genes linked to familial forms of PD have been reported to cause autosomal recessive forms. Among them, mutations in the PTEN-induced kinase 1 (PINK1) gene were shown to be responsible for a phenotype characterized by early onset, good response to levodopa, and a benign course. Using chromosomal microarray analysis and Sanger sequencing, we identified a homozygous G/C substitution in a 58-year-old Moroccan man diagnosed with recessive inherited Parkinson's disease. This G-to-C transition occurred at position 1617 leading to an amino acid change L/F at position 539 located in highly conserved motif in the C terminal sequence of PINK1. Interestingly, the c.1617G>C substitution is absent in 192 ethnically matched control chromosomes. Our findings have shown that the p.L539F is a novel mutation located in the C terminal sequence of the PINK1 protein that could be pathogenic and responsible for a clinical phenotype resembling idiopathic Parkinson's disease with rapid progression and early cognitive impairment.

The AIRE -230Y Polymorphism Affects AIRE Transcriptional Activity: Potential Influence on AIRE Function in the Thymus.

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.

Investigation of four novel male androgenetic alopecia susceptibility loci: no association with female pattern hair loss.

Female pattern hair loss (FPHL) is a common hair loss disorder in women and has a complex mode of inheritance. The etiopathogenesis of FPHL is largely unknown; however, it is hypothesized that FPHL and male pattern baldness [androgenetic alopecia (AGA)] share common genetic susceptibility alleles. Our recent findings indicate that the major AGA locus, an X-chromosome region containing the androgen receptor and the ectodysplasin A2 receptor (EDA2R) genes, may represent a common genetic factor underlying both early-onset FPHL and AGA. This gives further support for the widespread assumption of shared susceptibility loci for FPHL and AGA. However, we could not demonstrate association of further AGA risk loci, including 20p11, 1p36.22, 2q37.3, 7p21.1, 7q11.22, 17q21.31, and 18q21.1, with FPHL. Interestingly, a recent study identified four novel AGA risk loci in chromosomal regions 2q35, 3q25.1, 5q33.3, and 12p12.1. In particular, the 2q35 locus and its gene WNT10A point to an as-yet unknown involvement of the WNT signaling pathway in AGA. We hypothesized that the novel loci and thus also the WNT signaling may have a role in the etiopathogenesis of FPHL and therefore examined the role of these novel AGA risk loci in our FPHL samples comprising 440 German and 145 UK affected patients, 500 German unselected controls (blood donors), and 179 UK supercontrols. Patients and controls were genotyped for the top two single nucleotide polymorphisms at each of the four AGA loci. However, none of the genotyped variants displayed any significant association. In conclusion, the results of this study provide no support for the hypothesis that the novel AGA loci influence susceptibility to FPHL.

Selected variants of the melanocortin 4 receptor gene (MC4R) do not confer susceptibility to female pattern hair loss.

Female pattern hair loss (FPHL) is a common hair loss disorder in women with a complex mode of inheritance. Its etiopathogenesis is poorly understood. Widespread assumptions of overlapping susceptibility variants between FPHL and male pattern baldness (androgenetic alopecia) and a crucial role of androgens or distinct sexual steroid hormones in the development of FPHL could neither be clearly demonstrated nor completely excluded at the molecular level up to date. Interestingly, recent studies suggested an association of metabolic syndrome-including obesity, hyperlipidaemia, hypertension and diabetes mellitus type 2 or abnormally high fasting blood glucose-with FPHL. Of note, mutations in the melanocortin 4 receptor gene (MC4R) have been identified in patients with morbid obesity. Interestingly, this neuropeptide receptor has been detected amongst others in the dermal papilla of the hair follicle. As almost half of our FPHL patients of German origin present with adipositas and/or obesity, we hypothesized as to whether FPHL could be associated with variants of the MC4R gene. Thus, we genotyped a total of six variants from MC4R in our case-control sample comprising 245 UK patients of German and UK origin. However, based on our present study none of the genotyped MC4R variants displayed any significant association, neither in the overall UK and German samples nor in any subgroup analyses. In summary, these results do not point to an involvement of MC4R in FPHL.

CLN6 p.I154del mutation causing late infantile neuronal ceroid lipofuscinosis in a large consanguineous Moroccan family.

The neuronal ceroid-lipofuscinosis (NCL) are a heterogeneous group of neurodegenerative diseases characterized by the lysosomal accumulation of ceroid and lipofuscin with mitochondrial ATP synthase subunit C in various tissues. Clinical features include progressive mental and motor deterioration, myoclonus, seizure, visual failure and premature death. Ten CLN genes have been identified, among them CLN6 genes for which 55 disease-causing mutations have already been reported. The authors describe here a large consanguineous Moroccan family with three affected patients due to the p.I154del mutation that has been exclusively reported in Portuguese patients. This is the first published report of a genetic study in a Moroccan family with NCL. A relatively inexpensive CLN6 mutation screening should be considered first in Morocco as an initial diagnosis step when the disease course is consistent with late infantile neuronal ceroid-lipofuscinosis.

Identification of 15 new psoriasis susceptibility loci highlights the role of innate immunity.

To gain further insight into the genetic architecture of psoriasis, we conducted a meta-analysis of 3 genome-wide association studies (GWAS) and 2 independent data sets genotyped on the Immunochip, including 10,588 cases and 22,806 controls. We identified 15 new susceptibility loci, increasing to 36 the number associated with psoriasis in European individuals. We also identified, using conditional analyses, five independent signals within previously known loci. The newly identified loci shared with other autoimmune diseases include candidate genes with roles in regulating T-cell function (such as RUNX3, TAGAP and STAT3). Notably, they included candidate genes whose products are involved in innate host defense, including interferon-mediated antiviral responses (DDX58), macrophage activation (ZC3H12C) and nuclear factor (NF)-κB signaling (CARD14 and CARM1). These results portend a better understanding of shared and distinctive genetic determinants of immune-mediated inflammatory disorders and emphasize the importance of the skin in innate and acquired host defense.

Selected variants of the steroid-5-alpha-reductase isoforms SRD5A1 and SRD5A2 and the sex steroid hormone receptors ESR1, ESR2 and PGR: no association with female pattern hair loss identified.

Female pattern hair loss (FPHL) is a common disorder with a complex mode of inheritance. Although understanding of its etiopathogenesis is incomplete, an interaction between genetic and hormonal factors is assumed to be important. The involvement of an androgen-dependent pathway and sex steroid hormones is the most likely hypothesis. We therefore selected a total of 21 variants from the steroid-5-alpha-reductase isoforms SRD5A1 and SRD5A2, the sex steroid hormone receptors ESR1, ESR2 (oestrogen receptor) and PGR (progesterone receptor) and genotyped these in a case-control sample of 198 patients (145 UK; 53 German patients) and 329 controls (179 UK; 150 German). None of these variants showed any significant association, either in the overall UK and German samples or in the subgroup analyses. In summary, the present results, while based on a limited selection of gene variants, do not point to the involvement of SRD5A1, SRD5A2, ESR1, ESR2 or PGR in FPHL.

HLA-C, CSTA and DS12346 susceptibility alleles confer over 100-fold increased risk of developing psoriasis: evidence of gene interaction.

Psoriasis is an inflammatory skin disorder that exhibits multifactorial mode of inheritance. In addition to the well-known susceptibility locus PSORS1 many other loci have been shown to be implicated in the genetic predisposition for disease. However, interactions between loci have not been thoroughly explored. Thus, we measured the effect of potential interaction between human leukocyte antigen (HLA)-C, CSTA and D1S236 at PSORS1, PSORS4 and PSORS5, respectively, in the development of psoriasis. Analysis of 130 Caucasian psoriatic families showed that the risk to an HLA-Cw6 +ve individual who carries two copies of the risk allele at both the CSTA and D1S2346 is 105 times the risk to an HLA-Cw6 +ve individual who does not carry any risk alleles at the CSTA or D1S2346. This is the first demonstration of an interaction between risk alleles in three susceptibility loci suggesting possible functional interaction between genes in these loci, which might explain the complexity of the pathogenesis of psoriasis.