The autoimmune risk gene ZMIZ1 is a vitamin D responsive marker of a molecular phenotype of multiple sclerosis.

Multiple Sclerosis (MS) is a neurological condition driven in part by immune cells from the peripheral circulation, the targets for current successful therapies. The autoimmune and MS risk gene ZMIZ1 is underexpressed in blood in people with MS. We show that, from three independent sets of transcriptomic data, expression of ZMIZ1 is tightly correlated with that of hundreds of other genes. Further we show expression is partially heritable (heritability 0.26), relatively stable over time, predominantly in plasmacytoid dendritic cells and non-classical monocytes, and that levels of ZMIZ1 protein expression are reduced in MS. ZMIZ1 gene expression is increased in response to calcipotriol (1,25 Vitamin D3) (p < 0.0003) and associated with Epstein Barr Virus (EBV) EBNA-1 antibody titre (p < 0.004). MS therapies fingolimod and dimethyl fumarate altered blood ZMIZ1 gene expression compared to untreated MS. The phenotype indicates susceptibility to MS, and may correspond with clinical response and represent a novel clinical target.

Cistromic and genetic evidence that the vitamin D receptor mediates susceptibility to latitude-dependent autoimmune diseases.

The vitamin D receptor (VDR) is a ligand-activated transcription factor that regulates gene expression in many cell types, including immune cells. It requires binding of 1,25 dihydroxy vitamin D3 (1,25D3) for activation. Many autoimmune diseases show latitude-dependent prevalence and/or association with vitamin D deficiency, and vitamin D supplementation is commonly used in their clinical management. 1,25D3 is regulated by genes associated with the risk of autoimmune diseases and predominantly expressed in myeloid cells. We determined the VDR cistrome in monocytes and monocyte-derived inflammatory (DC1) and tolerogenic dendritic cells (DC2). VDR motifs were highly overrepresented in ChIP-Seq peaks in stimulated monocyte (40%), DC1 (21%) and DC2 (47%), P

IFNL3/4 genotype is associated with altered immune cell populations in peripheral blood in chronic hepatitis C infection.

Single-nucleotide polymorphisms near the interferon lambda 3 (IFNL3) gene predict outcomes to infection and anti-viral treatment in hepatitis C virus (HCV) infection. To identify IFNL3 genotype effects on peripheral blood, we collected phenotype data on 400 patients with genotype 1 chronic hepatitis C (CHC). The IFNL3 responder genotype predicted significantly lower white blood cells (WBCs), as well as lower absolute numbers of monocytes, neutrophils and lymphocytes for both rs8099917 and rs12979860. We sought to define the WBC subsets driving this association using flow cytometry of 67 untreated CHC individuals. Genotype-associated differences were seen in the ratio of CD4CD45RO+ to CD4CD45RO-; CD8CD45RO+ to CD8CD45RO-, NK CD56 dim to bright and monocyte numbers and percentages. Whole blood expression levels of IFNL3, IFNLR1 (interferon lambda receptor 1), IFNLR1-mem (a membrane-associated receptor), IFNLR1-sol (a truncated soluble receptor), MxA and T- and NK (natural killer) cell transcription factors TBX21, GATA3, RORC, FOXP3 and EOMES in two subjects were also determined. CHC patients demonstrated endogenous IFN activation with higher levels of MxA, IFNLR1, IFNLR1-mem and IFNLR1-sol, and IFNL3 genotype-associated differences in transcription factors. Taken together, these data provide evidence of an IFNL3 genotype association with differences in monocyte, T- and NK cell levels in the peripheral blood of patients with CHC. This could underpin genotype associations with spontaneous and treatment-induced HCV clearance and hepatic necroinflammation.

Impact of common risk factors of fibrosis progression in chronic hepatitis C.

OBJECTIVE: The natural course of chronic hepatitis C varies widely. To improve the profiling of patients at risk of developing advanced liver disease, we assessed the relative contribution of factors for liver fibrosis progression in hepatitis C. DESIGN: We analysed 1461 patients with chronic hepatitis C with an estimated date of infection and at least one liver biopsy. Risk factors for accelerated fibrosis progression rate (FPR), defined as ≥ 0.13 Metavir fibrosis units per year, were identified by logistic regression. Examined factors included age at infection, sex, route of infection, HCV genotype, body mass index (BMI), significant alcohol drinking (≥ 20 g/day for ≥ 5 years), HIV coinfection and diabetes. In a subgroup of 575 patients, we assessed the impact of single nucleotide polymorphisms previously associated with fibrosis progression in genome-wide association studies. Results were expressed as attributable fraction (AF) of risk for accelerated FPR. RESULTS: Age at infection (AF 28.7%), sex (AF 8.2%), route of infection (AF 16.5%) and HCV genotype (AF 7.9%) contributed to accelerated FPR in the Swiss Hepatitis C Cohort Study, whereas significant alcohol drinking, anti-HIV, diabetes and BMI did not. In genotyped patients, variants at rs9380516 (TULP1), rs738409 (PNPLA3), rs4374383 (MERTK) (AF 19.2%) and rs910049 (major histocompatibility complex region) significantly added to the risk of accelerated FPR. Results were replicated in three additional independent cohorts, and a meta-analysis confirmed the role of age at infection, sex, route of infection, HCV genotype, rs738409, rs4374383 and rs910049 in accelerating FPR. CONCLUSIONS: Most factors accelerating liver fibrosis progression in chronic hepatitis C are unmodifiable.

Hepatic metallothionein expression in chronic hepatitis C virus infection is IFNL3 genotype-dependent.

The IFNL3 genotype predicts the clearance of hepatitis C virus (HCV), spontaneously and with interferon (IFN)-based therapy. The responder genotype is associated with lower expression of interferon stimulated genes (ISGs) in liver biopsies from chronic hepatitis C patients. However, ISGs represent many interacting molecular pathways, and we hypothesised that the IFNL3 genotype may produce a characteristic pattern of ISG expression explaining the effect of genotype on viral clearance. For the first time, we identified an association between a cluster of ISGs, the metallothioneins (MTs) and IFNL3 genotype. Importantly, MTs were significantly upregulated (in contrast to most other ISGs) in HCV-infected liver biopsies of rs8099917 responders. An association between lower fibrosis scores and higher MT levels was demonstrated underlying clinical relevance of this association. As expected, overall ISGs were significantly downregulated in biopsies from subjects with the IFNL3 rs8099917 responder genotype (P=2.38 × 10(-7)). Peripheral blood analysis revealed paradoxical and not previously described findings with upregulation of ISGs seen in the responder genotype (P=1.00 × 10(-4)). The higher MT expression in responders may contribute to their improved viral clearance and MT-inducing agents may be useful adjuncts to therapy for HCV. Upregulation of immune cell ISGs in responders may also contribute to the IFNL3 genotype effect.

CCR5-Δ32 genotype does not improve predictive value of IL28B polymorphisms for treatment response in chronic HCV infection.

IL28B polymorphisms strongly predict spontaneous and treatment-induced clearance of hepatitis C virus (HCV) infection. A recent study proposed a 32-base pair deletion in the CC-chemokine receptor 5 (CCR5) gene (CCR5-Δ32) interacting with the IL28B polymorphisms to influence spontaneous HCV clearance. The aim of this study was to clarify the role of CCR5-Δ32 in treatment-induced clearance of chronic hepatitis C (CHC). A cross-sectional cohort of 813 Caucasian patients with CHC genotype 1 (365 responders and 448 non-responders) who had received standard of care dual therapy with interferon (IFN)-α and ribavirin (RBV) was genotyped for the CCR5-Δ32 and IL28B polymorphisms to examine their interaction with respect to treatment response. CCR5-Δ32 did not influence treatment-induced recovery to IFN-α/RBV in CHC, and did not improve prediction of sustained virological response in the context of the IL28B polymorphisms in a multivariate model. CCR5-Δ32 homozygotes were significantly more frequent in those with CHC than healthy controls in the European cohorts (2.9% vs 0.4%, P<0.0001), but not in Australians of European ancestry. In conclusion, CCR5-Δ32 does not influence treatment response in the context of IL28B polymorphisms. Although CCR5-Δ32 may affect viral clearance within closely controlled geographical and genetic environments, we found no effect in larger cohorts treated with dual therapy.

The role of SNPs in the α-chain of the IL-7R gene in CD4+ T-cell recovery in HIV-infected African patients receiving suppressive cART.

We previously found an association between faster CD4+ T-cell recovery in HIV-infected patients receiving combination antiretroviral therapy (cART) and interleukin-7 receptor-α (IL-7Rα) haplotype-2 in a predominantly Caucasian cohort. This study aims to determine whether this association was also significant in Africans. Patients were recruited from the Uganda AIDS Rural Treatment Outcomes (UARTO) cohort (n=352). We used survival analysis and linear mixed modelling (LMM) to determine factors associated with CD4 T-cell recovery. Eight IL-7Rα single-nucleotide polymorphisms (SNPs) were genotyped in both Africans and Caucasians (n=57). Soluble (s)IL-7Rα levels were measured by ELISA. In UARTO, IL-7Rα haplotype-2 was associated with slower CD4 T-cell recovery following cART by using survival analysis (P=0.020) and no association was found with LMM (P=0.958). The tagging-SNP for IL-7Rα haplotype-2 (rs6897932) was associated with decreased sIL-7Rα (P<0.001). The haplotypes for the IL-7Rα were significantly different in Africans and Caucasians. Using IL-7Rα genotypes we found slower CD4 T-cell recovery in UARTO patients was still associated with rs6897932 (P=0.009) and rs3194051 was associated with faster CD4 T-cell recovery (P=0.006). Unlike Caucasians, we did not demonstrate a significant association between IL-7Rα haplotype 2 and faster CD4 T-cell recovery in Africans. The IL-7Rα SNPs associated with CD4 T-cell recovery following cART differ in African and Caucasian cohorts.

Haplotypes of the interleukin 7 receptor alpha gene are correlated with altered expression in whole blood cells in multiple sclerosis.

IL7 regulates T cell survival, differentiation and proliferation. The alpha chain of its receptor, CD127, is polymorphic, and its haplotypes are associated with recovery from transplantation and with the autoimmune disease multiple sclerosis (MS), especially primary progressive MS (PPMS). We demonstrate that two CD127 haplotypes are highly associated with the proportion of the mRNA encoding the soluble isoform of CD127 (P

The elusive multiple self-healing squamous epithelioma (MSSE) gene: further mapping, analysis of candidates, and loss of heterozygosity.

The MSSE gene predisposes to multiple invasive but self-healing skin tumours (multiple self-healing epitheliomata). MSSE was previously mapped to chromosome 9q22-q31 and a shared haplotype in affected families suggested a founder mutation. We have refined the MSSE critical region (<1 cM, <1 Mb) between the zinc-finger gene ZNF169 and the Fanconi anaemia gene FANCC. By genetic mapping we have excluded ZNF169 and FANCC as well as PTCH (PATCHED) and TGFBR1 (transforming growth factor beta receptor type-1) genes. The CDC14B cell cycle phosphatase gene also lies in the region but screening of the complete coding region revealed no mutation in MSSE patients. Somatic cell hybrids created by haploid conversion of an MSSE patient's cells enabled screening of the MSSE chromosome 9 and showed no CDC14B deletion or mutation that abrogates CDC14B mRNA expression. Thus, CDC14B is unlikely to be the MSSE gene. We also report the first molecular analysis of MSSE tumours showing loss of heterozygosity of the MSSE region, with loss of the normal allele, providing the first evidence that MSSE is a tumour suppressor gene.

Hereditary hepatic and systemic amyloidosis caused by a new deletion/insertion mutation in the apolipoprotein AI gene.

We report a Spanish family with autosomal-dominant non-neuropathic hereditary amyloidosis with a unique hepatic presentation and death from liver failure, usually by the sixth decade. The disease is caused by a previously unreported deletion/insertion mutation in exon 4 of the apolipoprotein AI (apoAI) gene encoding loss of residues 60-71 of normal mature apoAI and insertion at that position of two new residues, ValThr. Affected individuals are heterozygous for this mutation and have both normal apoAI and variant molecules bearing one extra positive charge, as predicted from the DNA sequence. The amyloid fibrils are composed exclusively of NH2-terminal fragments of the variant, ending mainly at positions corresponding to residues 83 and 92 in the mature wild-type sequence. Amyloid fibrils derived from the other three known amyloidogenic apoAI variants are also composed of similar NH2-terminal fragments. All known amyloidogenic apoAI variants carry one extra positive charge in this region, suggesting that it may be responsible for their enhanced amyloidogenicity. In addition to causing a new phenotype, this is the first deletion mutation to be described in association with hereditary amyloidosis and it significantly extends the value of the apoAI model for investigation of molecular mechanisms of amyloid fibrillogenesis.

Data characterizing the ZMIZ1 molecular phenotype of multiple sclerosis.

The data presented in this article are related to the research article entitled "The autoimmune risk gene ZMIZ1 is a vitamin D responsived marker of a molecular phenotype of multiple sclerosis" Fewings et al. (2017) [1]. Here we identify the set of genes correlated with ZMIZ1 in multiple cohorts, provide phenotypic details on those cohorts, and identify the genes negatively correlated with ZMIZ1 and the cells predominantly expressing those genes. We identify the metabolic pathways in which the molecular phenotype genes are over-represented. Finally, we present the flow cytometry gating strategy we have used to identify the immune cells from blood which are producing ZMIZ1 and RPS6.

Gene expression and genotyping studies implicate the interleukin 7 receptor in the pathogenesis of primary progressive multiple sclerosis.

Multiple sclerosis (MS) is an enigmatic disease of the central nervous system resulting in sclerotic plaques with the pathological hallmarks of demyelination and axonal damage, which can be directly or indirectly orchestrated by cells from the peripheral circulation. The majority of patients with MS follow a relapsing-remitting course in the early stages of the disease (RRMS) but most ultimately enter a secondary progressive phase (SPMS). About 10% of patients follow a primary progressive course from the onset (PPMS). We measured gene expression in whole blood of people with and without chronic progressive MS (CPMS), PPMS and SPMS, to discover genes which may be differentially expressed in peripheral blood in active disease, and so identify pathologically significant genes and pathways; and we investigated genetic differences in the promoters of dysregulated genes encoded in genomic regions associated with MS. If SPMS and PPMS were independently compared to the controls, there was little overlap in the set of most dysregulated genes. Ribosomal protein genes, whose expression is usually associated with cell proliferation and activation, were dramatically over-represented in the set of most down-regulated genes in PPMS compared to SPMS (P < 10(-4), chi(2)). The T cell proliferation gene IL7R (CD127) was also underexpressed in PPMS, but was up-regulated in SPMS compared to the controls. One interleukin 7 receptor (IL7R) promoter single nucleotide polymorphism (SNP), -504 C, was undertransmitted in PPMS trios (P = 0.05, TDT), and carriers of this allele were under-represented in PPMS cases from two independent patient cohorts (combined P = 0.006, FE). The four known IL7R promoter haplotypes were shown to have similar expression levels in healthy controls, but not in CPMS (P < 0.01, t test). These data support the hypothesis that PPMS has significant pathogenetic differences from SPMS, and that IL7R may be a useful therapeutic target in PPMS.

Clinical and biochemical outcome of hepatorenal transplantation for hereditary systemic amyloidosis associated with apolipoprotein AI Gly26Arg.

BACKGROUND: Treatment of systemic amyloidosis comprises measures to support failing organ function coupled with attempts to reduce the supply of the respective amyloid fibril precursor protein. Orthotopic hepatic transplantation is effective in familial amyloid polyneuropathy associated with variant transthyretin, because this protein is produced almost exclusively in the liver. Hepatic transplantation has not been performed in hereditary apolipoprotein AI (apoAI) amyloidosis, and the liver's contribution to plasma apoAI levels has not been determined in vivo. METHODS: A 57-year-old Irish man with hereditary systemic amyloidosis associated with apoAI Gly26Arg, which had led to end-stage renal failure and progressive liver dysfunction, underwent hepatorenal transplantation. His outcome was followed clinically and his amyloid deposits were monitored with serum amyloid P component scintigraphy. The proportion of variant apoAI in the plasma was estimated by quantitative isoelectric focusing before and after liver transplantation. RESULTS: Plasma levels of variant apoAI decreased by 50% after liver transplantation, and the patient was asymptomatic 2 years after surgery. Subclinical amyloid deposits that were present in his spleen and heart preoperatively have regressed and stabilized respectively. CONCLUSIONS: Orthotopic liver transplantation substantially reduces the supply of the amyloid fibril precursor protein in hereditary apoAI amyloidosis, and the excellent outcome in this patient probably reflects the balance between deposition and turnover of amyloid having been altered in favor of the latter. These findings support the use of liver transplantation in patients with hereditary apoAI amyloidosis who develop hepatic dysfunction.

SAA1 alleles as risk factors in reactive systemic AA amyloidosis.

SAA1 is the predominant isoform of acute phase human SAA deposited as AA amyloid fibrils in reactive systemic amyloidosis. It has recently been reported that in the Japanese population, in whom the SAA1 gamma allele occurs with a frequency of 37%, possession of, and especially, homozygosity for this allele is a significant risk factor for AA amyloidosis in adult patients with rheumatoid arthritis (RA). In contrast we report here that in a control sample of 95 healthy adult male Caucasians the SAA1 gamma allele occurs at the much lower frequency of 5.3% and that, among 41 patients with juvenile chronic arthritis (JCA) and AA amyloidosis, there was a highly significantly increased frequency of the SAA1 alpha allele (90.2%), and particularly homozygosity for this allele (80.5%), compared both to the healthy controls (75.8% and 57.9% respectively) and to 8 JCA cases without amyloid (56.3% and 12.5%). A similar trend with respect to frequency of the SAA1 alpha allele and homozygosity for it was observed among 26 adult Caucasian RA patients with AA amyloid and 26 such cases without amyloid, although it did not reach statistical significance. These results suggest that there is probably differential amyloidogenicity amongst the different SAA1 isoforms and indicate that homozygosity for SAA1 alpha and SAA1 gamma in the different populations is a significant risk factor for development of AA amyloidosis. In Caucasian patients with JCA, the presence of the homozygous SAA1 alpha genotype indicates high risk of amyloidosis and should encourage early and aggressive anti-inflammatory therapy to keep circulating SAA levels as low as possible.

Pyrin/marenostrin mutations in familial Mediterranean fever.

Familial Mediterranean fever (FMF) is an inherited inflammatory disease that is frequently complicated by reactive systemic (AA) amyloidosis. It is principally recognized in certain Mediterranean populations, and the diagnosis depends on clinical features. Four mutations strongly linked to FMF have lately been identified in a gene encoding a novel protein that has been named pyrin or marenostrin. We studied 27 consecutive patients of varied ethnic origin, including an English man, who had classical, probable or possible FMF. Pyrin/marenostrin genotypes were determined, and AA amyloidosis was sought using serum amyloid P component scintigraphy. Among the 23 patients with classical or probable FMF, 17 were homozygotes or compound heterozygotes for pyrin/marenostrin mutations, and in five, only single allele mutations were identified. Two new mutations, T6811 and delta M694, were discovered in addition to the four described previously. No mutations were identified in three of the four patients with possible FMF. Nine patients had AA amyloidosis, but this association was not restricted to any particular genotype. Most patients with FMF have mutations in both pyrin/marenostrin alleles, and genotyping at this locus is a valuable diagnostic test. Unidentified second mutations are likely to occur in FMF patients who have apparently solitary mutations, and therefore genotype results must be interpreted in conjunction with the clinical picture.

Prevalence and significance of the familial Mediterranean fever gene mutation encoding pyrin Q148.

Familial Mediterranean fever (FMF) is caused by more than 25 mutations in the gene MEFV, which encodes pyrin (marenostrin), a protein implicated in the regulation of neutrophil activity. Pyrin Q148, is one of the five most common variants in populations in which FMF typically occurs. Our identification of the pyrin Q148 allele in several patients from ethnic groups in which FMF is not classically recognized who had longstanding fevers or AA amyloidosis prompted us to study the prevalence of pyrin Q148 in healthy British, Indian and Chinese subjects. The gene frequency was also sought in 50 British Caucasian patients with inflammatory arthritis, 25 of whom had AA amyloidosis, five Punjabi Indians with AA amyloidosis complicating inflammatory arthritis, and seven British Caucasian patients with uncharacterized longstanding fever syndromes. The allele frequency for pyrin Q148 was 21%, 15% and 0%, respectively, among Punjabi Indian, Chinese and Caucasian British controls, and was significantly increased among the patients with AA amyloidosis and the patients with obscure fever syndromes (p<0.01). Pyrin Q148 is a polymorphism and occurs widely in global terms, and, although it may cause FMF when associated with certain other MEFV mutations, homozygosity for Q148 alone must usually be insufficient to produce FMF in the populations studied. The association of pyrin Q148 with AA amyloidosis and with obscure chronic inflammatory diseases suggests the variant may augment inflammation non-specifically, which might have been beneficial during evolution, but could potentially exacerbate many chronic inflammatory disorders.

Curative hepatorenal transplantation in systemic amyloidosis caused by the Glu526Val fibrinogen alpha-chain variant in an English family.

A 53-year-old English woman who had been thought to have systemic monoclonal immunoglobulin light chain (AL) amyloidosis was investigated further because of her unusually long 17-year history and a suggestion of renal disease in the family. She was found to have the Glu526Val fibrinogen alpha-chain variant that causes autosomal dominant hereditary systemic amyloidosis. This has not previously been described in a British family. The mutant gene was associated with the same haplotype as in all other reported cases, suggesting a common founder. The patient had already received a renal transplant, but the graft failed within 6 years due to amyloid deposition. Progressive hepatic amyloidosis eventually caused liver failure, although the function of other organs was well preserved. She therefore received hepatic and renal transplants to replace the failed organs and the hepatic source of the amyloidogenic variant fibrinogen. Three years later she is completely well and has no amyloid deposits identifiable by serum amyloid P component scintigraphy. This is the first detailed report of hepatic transplantation for liver failure caused by amyloidosis of any type. The substantial follow-up suggests that fibrinogen alpha-chain amyloidosis is one of the inherited metabolic diseases that can be cured by liver transplantation. The mutation underlying Glu526Val fibrinogen alpha-chain amyloidosis is incompletely penetrant and has a variable phenotype that can clinically mimic AL amyloidosis. Hereditary fibrinogen amyloidosis may be more prevalent than previously suspected and, since AL amyloid is sometimes a diagnosis of exclusion, genotyping for other amyloidogenic proteins is mandatory in all cases in which the amyloid fibrils cannot be positively identified as AL.

The genetic basis of autosomal dominant familial Mediterranean fever.

Familial Mediterranean fever (FMF) is classically an autosomal recessive periodic inflammatory disease occurring in Mediterranean and Middle Eastern populations. It is caused by mutations affecting both alleles of MEFV, a gene that encodes pyrin (marenostrin), an uncharacterized neutrophil protein. Occasional reports of autosomal dominant FMF have often been discounted, on the basis that asymptomatic FMF carriers are common in certain populations, and give rise to pseudo-dominant inheritance. We performed comprehensive MEFV genotyping in five families in whom FMF appeared to be inherited dominantly. Transmission proved to be pseudo-dominant in two cases, but true dominant inheritance of FMF with variable penetrance was supported by the genotyping results in the other three families. The disease in these cases was associated with heterozygosity for either pyrin DeltaM694 alone or the compound pyrin variant E148Q/M694I, the latter occurring in two unrelated families. Complete MEFV sequencing failed to identify any coding region abnormality in the other allele in any of these cases, and, in the largest kindred, single-allele disease transmission was further supported by analysis of silent single nucleotide polymorphisms, which proved that affected individuals had at least three different complementary alleles. Studies of two further unrelated British patients with FMF associated with simple heterozygosity for pyrin DeltaM694 were also consistent with autosomal dominant inheritance. The clinical features of dominantly inherited FMF were absolutely typical, including AA amyloidosis in a patient with pyrin DeltaM694. These findings extend the spectrum of FMF, and suggest that the methionine residue at position 694 makes a crucial contribution to pyrin's function, and that a 50% complement of normal pyrin activity does not prevent susceptibility to FMF.