HLA-B gene methylation and expression in Behçet's syndrome: a potential role of epigenetics in the pathogenesis.

OBJECTIVES: The HLA-B51 locus has the strongest association with Behçet's syndrome (BS). The presence of a CpG island in the HLA-B gene led us to examine the role of epigenetic regulation in BS. METHODS: HLA-B51 genotyping was performed via sequence-specific PCR in 15 index familial BS cases, 17 affected relatives, 26 unaffected relatives, 46 sporadic BS cases, and 41 healthy controls. HLA-B methylation level was determined using the Zymo OneStep qMethyl kit, and HLA-B51 mRNA level was assessed by quantitative real-time PCR in 14 index familial BS cases, 15 affected relatives, 15 unaffected relatives, 11 sporadic BS cases, and 10 healthy controls. RESULTS: HLA-B51 carrier ratio was 13/15 in index familial cases, 13/17 in affected relatives, 22/26 in unaffected relatives, 8/25 in healthy controls, and 35/47 in sporadic BS cases. HLA-B51 expression level in HLA-B51+ BS cases was 2.2-fold higher than in their unaffected relatives (p=0.0149) and 1.3-fold higher than in healthy controls (p=0.0188), while sporadic BS cases had a 2.7-fold higher level than healthy controls (p=0.0487). HLA-B promoter methylation was significantly lower in HLA-B51+ familial BS cases than in unaffected relatives (0.4-fold, p=0.01), affected relatives (0.36-fold, p=0.0219), and healthy controls (0.34-fold, p=0.0371) and slightly lower in HLA-B51+ sporadic BS cases than in healthy controls (0.71-fold, p=0.2347). There was an inverse correlation between HLA-B promoter methylation and HLA-B51 expression in HLA-B51+ sporadic BS cases (p=0.0164). CONCLUSIONS: This study indicates epigenetic involvement associated with the HLA-B51 locus in BS, both in familial and sporadic cases. Further studies with larger sample sizes are needed to confirm our results.

Detection of a rare variant in PSTPIP1 through three generations in a family with an initial diagnosis of FMF/MKD-overlapping phenotype.

OBJECTIVE: The presence of FMF cases without MEFV (MEFV innate immunity regulator, pyrin) pathogenic variants led us to search for other genes' involvement in the disease development. Here, we describe the presence of genetic heterogeneity in a three-generation family with an FMF/mevalonate kinase deficiency (MKD)-overlapping phenotype without MEFV/MVK (mevalonate kinase) pathogenic variants. METHOD: Targeted sequencing revealed a rare, fully penetrant variant in PSTPIP1 (p.Arg228Cys, rs781341816). Computational stability analyses of PSTPIP1 protein were performed. PSTPIP1-pyrin protein interaction was examined by immunoprecipitation and immunoblotting in peripheral blood mononuclear cells (PBMCs) of patients and healthy controls. PBMCs were cultured, and inflammation was induced by LPS+ATP treatment, followed by protein level measurements of caspase-1, IL1ß, pyrin and PSTPIP1 in cell lysates and mature caspase-1 and mature IL1ß in supernatants. RESULTS: The conserved, rare (GnomAD, 0.000028) PSTPIP1 p.Arg228Cys variant, previously reported in ClinVar as a variant with uncertain significance, showed complete penetrance in the family presenting an autosomal dominant pattern. Computational analyses showed a potentially destabilizing effect of the variant on PSTPIP1 protein. Accordingly, PSTPIP1-pyrin interaction was increased in patients harboring the variant, which resulted in elevated levels of mature caspase-1 and IL1ß in the inflammation-induced patient samples. CONCLUSIONS: Unlike previously described cases with pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA)-associated PSTPIP1 variants, our patients with the p.Arg228Cys variant presented with an FMF/MKD-overlapping phenotype. As additional data on the genetic heterogeneity in the variable clinical spectrum of autoinflammatory syndromes, we suggest that the p.Arg228Cys variant in PSTPIP1 is related to inflammation responses through strong PSTPIP1-pyrin interaction and pyrin inflammasome activation.

Genome-wide analysis of schizophrenia and multiple sclerosis identifies shared genomic loci with mixed direction of effects.

Common genetic variants identified in genome-wide association studies (GWAS) show varying degrees of genetic pleiotropy across complex human disorders. Clinical studies of schizophrenia (SCZ) suggest that in addition to neuropsychiatric symptoms, patients with SCZ also show variable immune dysregulation. Epidemiological studies of multiple sclerosis (MS), an autoimmune, neurodegenerative disorder of the central nervous system, suggest that in addition to the manifestation of neuroinflammatory complications, patients with MS may also show co-occurring neuropsychiatric symptoms with disease progression. In this study, we analyzed the largest available GWAS datasets for SCZ (N = 161,405) and MS (N = 41,505) using Gaussian causal mixture modeling (MiXeR) and conditional/conjunctional false discovery rate (condFDR) frameworks to explore and quantify the shared genetic architecture of these two complex disorders at common variant level. Despite detecting only a negligible genetic correlation (rG = 0.057), we observe polygenic overlap between SCZ and MS, and a substantial genetic enrichment in SCZ conditional on associations with MS, and vice versa. By leveraging this cross-disorder enrichment, we identified 36 loci jointly associated with SCZ and MS at conjunctional FDR < 0.05 with mixed direction of effects. Follow-up functional analysis of the shared loci implicates candidate genes and biological processes involved in immune response and B-cell receptor signaling pathways. In conclusion, this study demonstrates the presence of polygenic overlap between SCZ and MS in the absence of a genetic correlation and provides new insights into the shared genetic architecture of these two disorders at the common variant level.

Peripheral blood mononuclear cell proteome profile in Behçet's syndrome.

Behçet's syndrome (BS) is a systemic inflammatory disorder with unknown etiology. Investigation of proteome profiles of disease specific cells facilitates our understanding of the processes and related molecular pathways, especially in disorders like BS with complex inheritance pattern and clinical heterogeneity. In the current study, we evaluated the peripheral blood mononuclear cells (PBMCs) proteome of 59 patients with BS (33 in active and 26 in inactive phases) and of 28 healthy controls using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Differentially expressed protein spots with at least twofold and/or statistically significant change (p ≤ 0.05) between active BS vs inactive BS, and also active BS vs healthy controls were identified by mass spectrometry (MALDI-TOF/TOF). Bioinformatic analyses revealed 16 differentially expressed proteins (12 of them in active vs inactive BS comparison, whereas 11 of them for active BS vs healthy control comparison) belonging to glycolysis, cytoskeleton organization, protein folding, and regulation of blood coagulation pathways. Stathmin (active BS vs inactive BS; fourfold, active BS vs healthy control; 4.7-fold) and WD repeat-containing protein-1 (active BS vs inactive BS; 2.7-fold, active BS vs healthy control; 2.7-fold), which are cytoskeleton-related proteins, were found to be lower in active patients compared to inactive patients and healthy control. Decreased levels of calreticulin (active BS vs inactive BS; 1.29-fold) and heat shock 70 kDa protein 8 (active BS vs healthy control; 1.5-fold) which are involved in protein folding and endoplasmic reticulum (ER) stress process, were observed in patients with active phase of BS. Down-regulation of protein folding and ER stress process proteins in BS patients may further support the involvement of ER stress in BS.

Other autoinflammatory disease genes in an FMF-prevalent population: a homozygous MVK mutation and a novel heterozygous TNFRSF1A mutation in two different Turkish families with clinical FMF.

OBJECTIVES: No MEFV mutations are detected in approximately 10% of the patients with clinical FMF in populations where the disease is highly prevalent. Causative mutations were searched in other genes in two such families with "MEFV negative clinical FMF". METHODS: Father and daughter of family A had attacks of fever, abdominal pain and AA amyloidosis. The two sibs of family B complained of febrile episodes with abdominal pain and arthritis. The patients were clinically investigated. Exome analysis in the daughter in family A and linkage analysis and candidate gene sequencing for the members of family B were performed. All patients were re-evaluated in the light of the genetic findings. RESULTS: In the daughter in family A, filtering of the exome file for variants in 25 autoimmune/inflammatory disease-related genes revealed two heterozygous missense variants in TNFRSF1A, novel p.Cys72Phe and frequent p.Arg121Gln. In family B, novel, homozygous missense p.Cys161Arg in MVK was identified. A clinical re-evaluation of the patients revealed a phenotype consistent with FMF rather than TRAPS in family A and an overlap of FMF with HIDS in family B. CONCLUSIONS: In high risk populations of FMF a proportion of patients without MEFV mutations may carry causative mutations in other genes, and the clinical findings may not be fully consistent with the phenotype expected of the mutation identified but rather resemble FMF or an overlap syndrome.

A case with febrile attacks and vasculopathy associated with ADA2 and MEFV pathogenic variants.

Deficiency of adenosine deaminase 2 (DADA2), caused by recessive mutations in the adenosine deaminase 2 (ADA2) gene, results in cutaneous or systemic vasculitis with variable clinical manifestations. There is only one other case in literature carrying both ADA2 and MEFV gene pathogenic variants. Here we report the second case that carries both ADA2 and MEFV pathogenic variants, presenting with characteristic phenotypes of both familial Mediterranean fever (FMF) and DADA2. A male patient, currently 29 years old, was initially diagnosed with FMF and developed livedo reticularis and nodular dermal lesions compatible with cutaneous polyarteritis nodosa (PAN) a year after diagnosis. His family history revealed a brother 2 years older than himself who was diagnosed with PAN and died at age 22 because of gut perforation secondary to acute mesenteric ischaemia. ADA2 gene mutation analysis on chromosome 22q11.1 was positive, and the patient responded to colchicine and infliximab.

A two-step purification platform for efficient removal of Fab-related impurities: A case study for Ranibizumab.

Antibodies (mAbs) and antibody fragments (Fabs) constitute one of the largest and most rapidly expanding groups of protein pharmaceuticals. In particular, antibody fragments have certain advantages over mAbs in some therapeutic settings. However, due to their greater chemical diversity, they are more challenging to purify for large-scale production using a standard purification platform. Besides, the removal of Fab-related byproducts poses a difficult purification challenge. Alternative Fab purification platforms could expedite their commercialization and reduce the cost and time invested. Accordingly, we employed a strong cation exchanger using a pH-based, highly linear gradient elution mode following Protein L affinity purification and developed a robust two-step purification platform for an antibody fragment. The optimized pH gradient elution conditions were determined on the basis of purity level, yield, and the abundance of Fab-related impurities, particularly free light chain. The purified Fab molecule Ranibizumab possessed a high degree of similarity to its originator Lucentis. The developed purification platform highly intensified the process and provided successful clearance of formulated Fab- and process-related impurities (∼98 %) with an overall process recovery of 50 % and, thus, might be a new option for Fab purification for both academic and industrial purposes.

Translation of Cellular Senescence to Novel Therapeutics: Insights From Alternative Tools and Models.

Increasing chronological age is the greatest risk factor for human diseases. Cellular senescence (CS), which is characterized by permanent cell-cycle arrest, has recently emerged as a fundamental mechanism in developing aging-related pathologies. During the aging process, senescent cell accumulation results in senescence-associated secretory phenotype (SASP) which plays an essential role in tissue dysfunction. Although discovered very recently, senotherapeutic drugs have been already involved in clinical studies. This review gives a summary of the molecular mechanisms of CS and its role particularly in the development of cardiovascular diseases (CVD) as the leading cause of death. In addition, it addresses alternative research tools including the nonhuman and human models as well as computational techniques for the discovery of novel therapies. Finally, senotherapeutic approaches that are mainly classified as senolytics and senomorphics are discussed.

Investigating the role of common and rare variants in multiplex multiple sclerosis families reveals an increased burden of common risk variation.

Many multiple sclerosis (MS)-associated common risk variants as well as candidate low-frequency and rare variants have been identified; however, approximately half of MS heritability remains unexplained. We studied seven multiplex MS families, six of which with parental consanguinity, to identify genetic factors that increase MS risk. Candidate genomic regions were identified through linkage analysis and homozygosity mapping, and fully penetrant, rare, and low-frequency variants were detected by exome sequencing. Weighted sum score and polygenic risk score (PRS) analyses were conducted in MS families (24 affected, 17 unaffected), 23 sporadic MS cases, 63 individuals in 19 non-MS control families, and 1272 independent, ancestry-matched controls. We found that familial MS cases had a significantly higher common risk variation burden compared with population controls and control families. Sporadic MS cases tended to have a higher PRS compared with familial MS cases, suggesting the presence of a higher rare risk variation burden in the families. In line with this, score distributions among affected and unaffected family members within individual families showed that known susceptibility alleles can explain disease development in some high-risk multiplex families, while in others, additional genetic contributors increase MS risk.

Analysis of MEFV exon methylation and expression patterns in familial Mediterranean fever.

BACKGROUND: MEFV mutations and decreased expression level of the gene are related to FMF pathology. DNA methylation at CpG islands is a well-known mechanism for transcriptional silencing. MEFV has a CpG island, spanning a part of the first intron and the whole of the second exon of the gene covering 998 bp region. Here, we tested the hypothesis that the MEFV transcript level in FMF patients correlates with its methylation level, and methylation, by allowing transcription silencing, has a role in FMF ethiopathogenesis. METHODS: The study group was composed of pediatric FMF patients (N = 51) and age-gender matched healthy controls (N = 21). The relative expression level of MEFV was assessed via quantitative real-time PCR (qRT-PCR) and bisulfite sequencing (BS) was performed to analyse the methylation level quantitatively. RESULTS: MEFV expression in FMF patients were decreased compared to healthy controls (P = 0.031). Methylation level of exon 2 of MEFV was found to be slightly higher in FMF patients compared to healthy controls (76% versus 74%) (P = 0.049). The expression level of the MEFV was negatively correlated with the methylation level of the CpG island in both FMF and healthy controls groups (cor = -0.29, P = 0.041) but more so in the FMF only group (cor = -0.36, P = 0.035). CONCLUSIONS: In this study, the relation between reduced MEFV expression level and FMF was confirmed. Observed slight increase in methylation in FMF patients, and correlation of methylation with expression might be indicative of its role in FMF, however a larger dataset is needed to confirm our preliminary findings.

Investigation of neuro-inflammatory parameters in a cuprizone induced mouse model of multiple sclerosis.

Cuprizone, copper chelator, treatment of mouse is a toxic model of multiple sclerosis (MS) in which oligodendrocyte death, demyelination and remyelination can be observed. Understanding T and B cell subset as well as their cytokines involved in MS pathogenesis still requires further scrutiny to better understand immune component of MS. The study presented here, aimed to evaluate relevant cytokines, lymphocytes, and gene expressions profiles during demyelination and remyelination in the cuprizone mouse model of MS. Eighty male C57BL/6J mice fed with 0.2% cuprizone for eight weeks. Cuprizone has been removed from the diet in the following eight weeks. Cuprizone treated and control mice sacrificed biweekly, and corpus callosum of the brain was investigated by staining. Lymphocyte cells of mice analyzed by flow cytometry with CD3e, CD11b, CD19, CD80, CD86, CD4, CD25 and FOXP3 antibodies. IFN-gamma, IL-1alpha, IL-2, IL-5, IL-6, IL-10, IL-17, TNF-alpha cytokines were analyzed in plasma samples. Neuregulin 1 (Nrg1), ciliary neurotrophic factor (Cntf) and C-X-C chemokine receptor type 4 (Cxcr4) gene expressions in corpus callosum sections of the mice brain were quantified. Histochemistry analysis showed that demyelination began at the fourth week of cuprizone administration and total demyelination occurred at the twelfth week in chronic model. Remyelination occurred at the fourth week of following withdrawal of cuprizone from diet. The level of mature and activated T cells, regulatory T cells, T helper cells and mature B cells increased during demyelination and decreased when cuprizone removed from diet. Further, both type 1 and type 2 cytokines together with the proinflammatory cytokines increased. The level of oligodendrocyte maturation and survival genes showed differential gene expression in parallel to that of demyelination and remyelination. In conclusion, for the first-time, involvement of both cellular immune response and antibody response as well as oligodendrocyte maturation and survival factors having role in demyelination and remyelination of cuprizone mouse model of MS have been shown.

LACC1 deficiency links juvenile arthritis with autophagy and metabolism in macrophages.

Juvenile idiopathic arthritis is the most common chronic rheumatic disease in children, and its etiology remains poorly understood. Here, we explored four families with early-onset arthritis carrying homozygous loss-of-expression mutations in LACC1. To understand the link between LACC1 and inflammation, we performed a functional study of LACC1 in human immune cells. We showed that LACC1 was primarily expressed in macrophages upon mTOR signaling. We found that LACC1 deficiency had no obvious impact on inflammasome activation, type I interferon response, or NF-κB regulation. Using bimolecular fluorescence complementation and biochemical assays, we showed that autophagy-inducing proteins, RACK1 and AMPK, interacted with LACC1. Autophagy blockade in macrophages was associated with LACC1 cleavage and degradation. Moreover, LACC1 deficiency reduced autophagy flux in primary macrophages. This was associated with a defect in the accumulation of lipid droplets and mitochondrial respiration, suggesting that LACC1-dependent autophagy fuels macrophage bioenergetics metabolism. Altogether, LACC1 deficiency defines a novel form of genetically inherited juvenile arthritis associated with impaired autophagy in macrophages.

A twin study in Behçet's syndrome.

OBJECTIVES: Case reports on monozygotic (MZ) twins with Behçet's syndrome (BS) have been few and we are not aware of formal twin studies. We sought the frequency of MZ and dizygotic (DZ) twin births in BS and compared it to a healthy population sample from the same geography. We also looked for the concordance rate among the MZ and DZ twins. METHODS: 1705 (1039M/666F) patients attending a dedicated BS outpatient clinic and 7761 (3848M/3913F) medical school students were asked about having a MZ or DZ twin sibling. MZ and DZ twins thus identified among both patients and controls were individually seen at the clinic. In addition, HLA, DNA microsatellite markers and blood groups were typed to further confirm twin- ship. All twins were contacted 8 years later for new emergence of disease. RESULTS: There were 14 (0.82%) patients with BS and 120 (1.55%) controls who had a twin sibling (p=0.022). Of these, 8 (0.47%) patients with BS and 92 (1.19%) controls had a DZ twin sibling (p=0.009). MZ twin frequency was similar between BS patients (6/1705; 0.35%) and control population (28/7761; 0.36%). The pairwise concordance rate for BS was 2/6 (95% CI: -0.21-0.88) for MZ and 1/8 (95% CI: -0.17-0.42) for DZ twins (p=0.538). Genetic effects accounted for 41% of the phenotypic variance for BS among twins. After 8 years of follow-up, 4 of 6 MZ and 6 of 7 DZ twin pairs were still discordant. CONCLUSIONS: The frequency of MZ twin births in BS is not different than that in the general population while the DZ twins were seen less frequently among the BS patients. The concordances for BS were higher in MZ compared with DZ twins, suggesting genetic predisposition. On the other hand, the persistence of discordance after 8 years of follow up among the remaining MZ twins demands further research to understand non- genetic factors in causation of BS.

The prevalence of Behçet's syndrome, familial Mediterranean fever, HLA-B51 and MEFV gene mutations among ethnic Armenians living in Istanbul, Turkey.

OBJECTIVES: We investigated the prevalence of Behçet's syndrome (BS) among the ethnic Armenians in Istanbul using Familial Mediterranean Fever (FMF) as a comparator disease. We also studied HLA-B51 and MEFV mutations among a group of healthy Armenians and a non-Armenian population. METHODS: The prevalence study was conducted in 2 parts in the Armenian primary schools in Istanbul, using the enrolled students as index cases to study the core family. In Part I, a questionnaire seeking only whether either parent had previously been diagnosed as having BS or FMF by a physician was distributed to a total of 1873 index students registered at 10 schools. A total of 1380 parents filled in the questionnaire, yielding a response rate of 37% (1380 / 3746). In Part II, eight schools participated with a response rate of 83 % (1183/1428). Also, genomic DNA samples of 108 healthy (14 M/94 F) Armenians and 97 (45 M/ 52 F) non-Armenians, were studied for HLAB51 and MEFV gene mutations. RESULTS: In Part I, none of the parents turned out to have been diagnosed as BS, whereas a total of 12 / 1380 (870/105) had been diagnosed as FMF. In the second part the estimated prevalence of BS was 90 /105 and that of FMF was 760/ 105. HLA-B51 carrier rate was found to be similar between the Armenian (27%, 29/108) and the non-Armenian participants (19%, 18/97), (p=0.158). Overall carrier rate of MEFV gene mutations was significantly higher in the Armenian group (36% vs. 20%, p=0.015). CONCLUSIONS: The genetic load for FMF is considerably higher among the Armenians when compared to the load for BS among the same ethnic group. On the other hand, the rather low frequency of BS among the Armenians when compared to the frequency among the general population living in the same environment is further evidence for a genetic predisposition to BS. HLA- B51 does not seem to play a dominant role in the said predisposition. Finally, as we have used an unorthodox epidemiological methodology in data collection our results might need to be further verified by more conventional methods.

A 9.5-year-old boy with recurrent neurological manifestations and severe hypertension, treated initially for polyarteritis nodosa, was subsequently diagnosed with adenosine deaminase type 2 deficiency (DADA2) which responded to anti-TNF-α.

A 9.5-year-old boy was referred with a 2-year history of recurrent fever, myalgia, abdominal pain and various neurological manifestations associated with increased acute phase reactants and IgG level. During the recent episode, severe hypertension and right-sided hemiparesis developed and angiography demonstrated irregularities and stenosis in renal and mesenteric artery branches. Although these manifestations were consistent with polyarteritis nodosa (PAN), the consanguinity of his parents, a cousin with similar clinical features and early disease onset led to suspicion of deficiency of adenosine deaminase type 2 (DADA2) diseases. DADA2 was established by demonstration of decreased ADA2 enzyme activity and a homozygous G47R mutation in the CECR1 gene. The diagnosis of DADA2 is challenging because of the overlapping manifestations with PAN and other periodic fever syndromes. DADA2 should be considered in the differential diagnosis of PAN. Raised IgG levels (usually low in DADA2) should be sought in future cases.Abbreviations: CECR1, cat eye syndrome chromosome region candidate 1; DADA2, deficiency of adenosine deaminase type 2; MEFV, Mediterranean fever; PAN, polyarteritis nodosa.

Atypical phenotype of an old disease or typical phenotype of a new disease: deficiency of adenosine deaminase 2.

Çakan M, Aktay-Ayaz N, Karadag SG, Tahir-Turanli E, Stafstrom K, Bainter W, Geha RS, Chou J. Atypical phenotype of an old disease or typical phenotype of a new disease: deficiency of adenosine deaminase 2. Turk J Pediatr 2019; 61: 413-417. Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive autoinflammatory disorder caused by mutations in CECR1 (cat eye syndrome chromosome region, canditate 1) gene, which encodes the enzyme adenosine deaminase 2 necessary for endothelial cell survival and function. The diversity of the clinical phenotypes associated with DADA2 include polyarteritis nodosa-like vasculitic features, early-onset stroke, mild to severe immunodeficiency and cytopenias. The diagnosis of the disease may be difficult due to complex clinical phenotype. Herein, we present a case of DADA2 presenting with vasculitis, amarousis fugax, gastrointestinal bleeding and silent lacunar infarct successfully treated with etanercept.

CSF Proteomics Identifies Specific and Shared Pathways for Multiple Sclerosis Clinical Subtypes.

Multiple sclerosis (MS) is an immune-mediated, neuro-inflammatory, demyelinating and neurodegenerative disease of the central nervous system (CNS) with a heterogeneous clinical presentation and course. There is a remarkable phenotypic heterogeneity in MS, and the molecular mechanisms underlying it remain unknown. We aimed to investigate further the etiopathogenesis related molecular pathways in subclinical types of MS using proteomic and bioinformatics approaches in cerebrospinal fluids of patients with clinically isolated syndrome, relapsing remitting MS and progressive MS (n=179). Comparison of disease groups with controls revealed a total of 151 proteins that are differentially expressed in clinically different MS subtypes. KEGG analysis using PANOGA tool revealed the disease related pathways including aldosterone-regulated sodium reabsorption (p=8.02x10-5) which is important in the immune cell migration, renin-angiotensin (p=6.88x10-5) system that induces Th17 dependent immunity, notch signaling (p=1.83x10-10) pathway indicating the activated remyelination and vitamin digestion and absorption pathways (p=1.73x10-5). An emerging theme from our studies is that whilst all MS clinical forms share common biological pathways, there are also clinical subtypes specific and pathophysiology related pathways which may have further therapeutic implications.