New insight of human-IgH 3'regulatory regions in immunoglobulins switch.

BACKGROUND: Several studies in animal models have demonstrated the role of the 3' Regulatory Region (3'RR) in the B cell maturation in mammals. In healthy humans, the concentration of each class of circulating immunoglobulins (Igs) has stable but different levels, due to several control mechanisms that also involve a duplicated version of the 3'RR on the chromosome 14 (chr14). The classes' equilibrium can be altered during infections and in other pathological conditions. MATERIAL AND METHODS: We studied the concentrations of IgA, IgM, IgG classes and IgG subclasses in a cohort of 1235 people having immunoglobulin concentrations within normal range to determine the presence of any correlation between the Igs serum concentrations, age and ratio among Ig classes and IgG subclasses in healthy humans. Furthermore, we assessed the concentrations of IgE and the allelic frequency of 3'RR1 hs1.2 enhancer in a group of 115 subjects with high levels of circulating IgE due to acute exacerbation of allergic asthma and in a control group of 118 healthy subjects. RESULTS: In both children and adult subjects, the concentrations of the four IgG subclasses decreased from IgG1 to IgG4. Furthermore, the 3'RR1 enhancer hs1.2 alleles contribute to the control of the IgG subclasses levels, but it does not affect the IgE levels. CONCLUSION: The 3'RR1 controls IgG and IgE through different mechanisms, only in the IgG case involving the hs1.2 alleles. Thus, considering the IgH constant genes loci on the chromosome 14 and the multiple steps of switch that rearrange the whole region, we found that in humans the classes of Igs are modulated by mechanisms involving a complex interaction and transition between 3'RR1 and 3'RR2, also in physiological conditions.

Concerted variation of the 3' regulatory region of Ig heavy chain and Gm haplotypes across human continental populations.

OBJECTIVES: The 3' regulatory region of the immunoglobulin heavy chain gene (IGH) includes the HS1.2 enhancer displaying length polymorphism with four known variants. The goal of the research was to provide an overview of this variability and of its evolutionary significance across human populations. MATERIALS AND METHODS: We compiled published and original data on HS1.2 polymorphism in 3,100 subjects from 26 human populations. Moreover, we imputed the haplotypic arrangement of the HS1.2 region in the 1000 Genomes Project (1KGP). In this dataset, imputation could also be obtained for the G1m-G3m allotype by virtue of the precise correspondence between serological types and amino acid (and DNA) substitutions in IGHG1 and IGHG3. RESULTS: HS1.2 variant frequencies displayed similar patterns of continental partitioning as those reported in the literature for the physically neighboring IGHG1-IGHG3 system. The 1KGP data revealed that linkage disequilibrium (LD) can explain the spread of joint HS1.2-IGHG1-IGHG3 associations across continents and within continental populations, with stronger LD out of Africa and the features of an evolutionarily stable genomic block with differential expression in lymphoblastoid cell lines. DISCUSSION: Strong population structuring involves at least the entire 70 kb genomic region here considered, due to the tight LD which maintained HS1.2, IGHG1, and IGHG3 in nonrandom arrangements. This might be key to better understand the evolutionary path of the entire genomic region driven by immune response capabilities, during the formation of continental gene pools.

Liraglutide and cardiovascular outcomes in a real world type 2 diabetes cohort.

In the last years, due to new regulatory guidelines requiring a stringent documentation of cardiovascular (CV) safety of novel drugs for type 2 diabetes, cardiovascular outcomes safety trials (CVOTs) are requested. CVOTs increase the knowledge about the safety profile of the new drugs, but they have intrinsic limits that make difficult their transferability to clinical practice. For this reason, real world evidence is considered an important complement to experimental data. Among the glucagon-like peptide-1 receptor agonists, liraglutide in the LEADER CVOT demonstrated superiority in reducing the risk of major CV events (MACEs) vs. placebo. We conducted an observational, retrospective, longitudinal study based on 307 patients with T2DM treated with liraglutide under routine clinical practice conditions. Real world impact of liraglutide on metabolic control, CV risk factors, hypoglycemia and CV events was assessed. Improvements during 36 months were found in HbA1c (-1.0%; p < 0.0001), fasting blood glucose (-17.6 mg/dL; p < 0.0001), body weight (-3.2 kg; p < 0.0001), waist circumference (-1.45 cm; p = 0.004), systolic blood pressure (-10.41 mmHg; p < 0.0001), diastolic blood pressure (-3.69 mmHg; p < 0.0001), total cholesterol (-7.96 mg/dL; p =0.008) and triglycerides (-20.60 mg/dl; p = 0.01). No severe hypoglycemia occurred. Incidence of MACEs in this cohort was lower than in the LEADER study (2.59 vs. 3.4 events per 100 person-years), confirming CV safety of liraglutide even in the real world. On the other hand, a higher incidence of CV event in patients with established CV disease was documented (8.1 events per 100 person-years), in spite of the use of liraglutide. In conclusion, 36-month durability and CV safety of liraglutide were documented in a real world cohort of T2DM patients, with sustained benefits on a large array of CV risk factors.

Association between Psoriasis and haplotypes of the IgH 3' Regulatory Region 1.

The association studies of several immune-diseases with the 3' Regulatory Region 1 (3'RR1) increased interest on the role that the region plays in the immune-regulation. The 3'RR1 is a polymorphic region on human chromosome 14q32, acting as a cis-regulative element on the Immunoglobulin constant-gene locus recently considered as super-enhancer. The human 3'RR1 share large sequences with its paralogous 3'RR2, at high level of similarity. Thus, a focused investigation was necessary to discriminate each one of the duplicated components of the two regions and its specific contribution to the immunologic phenotype. One of the duplicated elements is the hs1.2 enhancer. The 3'RR1 alleles of this enhancer were demonstrated to play a role in autoimmune diseases, including Psoriasis. We sequenced a specific region internal to the 3'RR1 in hs1.2 homozygous subjects, to detect SNPs associated to the main alleles of the enhancer. We identified two alternative nine-SNPs haplotypes strictly linked to the allele *1 and *2 of hs1.2, that could be used as markers to further investigate the region and associations to pathology. Finally, we identified two haplotypes, namely E2A1 and E2A2, that strongly support the hypothesis of a relevant effect of the rs35216181 in the onset of Psoriasis when the *2 allele is present.

Association between IgH enhancer hs1.2 and type 1 diabetes.

AIM: To investigate the association of alleles of the 3' immunoglobulin heavy-chain regulatory region 1 (3'RR-1) enhancer hs1.2 in patients with type 1 diabetes (T1D). METHODS: Eighty-one patients with T1D [among which 12 had concomitant coeliac disease (CD) and 25 an autoimmune thyroid disease (AITD)] were compared to 248 healthy individuals. All subjects were recruited from the same geographical area. Blood samples were collected from all patients and a nested PCR was performed to amplify the core of the 3'RR-1 and detect the alleles of the hs1.2 enhancer. RESULTS: Allele distribution in healthy individuals was significantly different when compared to that of patients with T1D (p < 0.01). Even greater differences were detected comparing allele distribution of patients with T1D alone versus those with concomitant CD, but not versus those with concomitant AITD. The frequency of *2 allele is increased by 23% in patients with T1D and CD. CONCLUSIONS: The present study establishes that the multiallelic hs1.2 enhancer of the 3'RR-1 is associated with T1D, with higher frequency when there is co-occurrence of CD. This evidence has been previously observed in other immune diseases.

Clobetasol and Halcinonide Act as Smoothened Agonists to Promote Myelin Gene Expression and RxRγ Receptor Activation.

One of the causes of permanent disability in chronic multiple sclerosis patients is the inability of oligodendrocyte progenitor cells (OPCs) to terminate their maturation program at lesions. To identify key regulators of myelin gene expression acting at the last stages of OPC maturation we developed a drug repositioning strategy based on the mouse immortalized oligodendrocyte (OL) cell line Oli-neu brought to the premyelination stage by stably expressing a key factor regulating the last stages of OL maturation. The Prestwick Chemical Library of 1,200 FDA-approved compound(s) was repositioned at three dosages based on the induction of Myelin Basic Protein (MBP) expression. Drug hits were further validated using dosage-dependent reproducibility tests and biochemical assays. The glucocorticoid class of compounds was the most highly represented and we found that they can be divided in three groups according to their efficacy on MBP up-regulation. Since target identification is crucial before bringing compounds to the clinic, we searched for common targets of the primary screen hits based on their known chemical-target interactomes, and the pathways predicted by top ranking compounds were validated using specific inhibitors. Two of the top ranking compounds, Halcinonide and Clobetasol, act as Smoothened (Smo) agonists to up-regulate myelin gene expression in the Oli-neuM cell line. Further, RxRγ activation is required for MBP expression upon Halcinonide and Clobetasol treatment. These data indicate Clobetasol and Halcinonide as potential promyelinating drugs and also provide a mechanistic understanding of their mode of action in the pathway leading to myelination in OPCs. Furthermore, our classification of glucocorticoids with respect to MBP expression provides important novel insights into their effects in the CNS and a rational criteria for their choice in combinatorial therapies in de-myelinating diseases.

The change in Ig regulation from children to adults disconnects the correlation with the 3'RR hs1.2 polymorphism.

BACKGROUND: In the immune system, the serum levels of immunoglobulin (Ig) increase gradually during ageing. Through B cell development, the Ig heavy chain expression is modulated by a regulatory region at the 3' of the constant alpha gene (3'RR), in single copy in rodents and, due to a large duplication, in two copies in apes. The human 3'RR1 and 3'RR2 are both characterized by three enhancers, the central of which, namely hs1.2, is highly polymorphic. Human hs1.2 has four different variants with unique binding sites for transcription factors (e.g. NF-kB and SP1) and shows variable allelic frequencies in populations with immune disorders. In previous works, we have reported that in several autoimmune diseases the *2 allele of hs1.2 is genetically associated to high level of IgM in peripheral blood. In subjects with altered levels of circulating Ig, an increased level was associated to *2 allele of hs1.2 and low levels corresponded to high frequency of *1 allele. RESULTS: We have correlated the allelic frequencies of hs1.2 with IgM, IgG and IgA serum concentrations in two cohorts of healthy people of different age and after three years follow-up in children homozygous for the allele. Here we show that when the expression levels of Ig in children are low and medium, the frequencies of *1 and *2 alleles are the same. Instead, when the Ig expression levels are high, there is a significantly higher frequency of the allele *2. The follow-up of children homozygous for *1 and *2 alleles showed that the increase or decrease of circulating Ig was not dependent on the number of circulating mature B cells. CONCLUSIONS: These data support the idea that under physiologic condition there is a switch of regulative pathways involved in the maturation of Ig during ageing. This mechanism is evidenced by hs1.2 variants that in children but not in adults participate to Ig production, coordinating the three class levels.

The functional VNTR of IGH enhancer HS1.2 associates with human longevity and interacts with TNFA promoter diplotype in a population of Central Italy.

The dysregulation of both immune and inflammatory responses occurring with aging is believed to substantially contribute to morbidity and mortality in humans. We have already reported the association of the functional Variable Number of Tandem Repeat (VNTR) at the Immunoglobulin heavy chain (IGH) enhancer HS1.2 with Immunoglobulin levels and with several autoimmune diseases. Herein we tested the association of the VNTR at the HS1.2 enhancer with human longevity, also evaluating the possible modulatory effect of TNFA promoter diplotype (rs361525/rs1800629). HS1.2 enhancer genotypes have been determined for 193 unrelated healthy individuals from Central Italy divided into two groups: Group 1 (18-84 yrs, mean age 56.8 ± 19.4) and Group 2 (85-100 yrs, mean age 93.0 ± 3.5). Homozygous subjects for 2 allele were significantly disadvantaged in reaching higher life-expectancy (OR=0.457, p=0.021). A significant interaction between TNFA promoter diplotype status, HS1.2 2/2 genotype and the two Groups was found (p=0.014). Of note, TNFA -308A allele seems to exert a protective effect in HS1.2 2/2 carriers. These results support the hypothesis of an important role of HS1.2 VNTR in the puzzle of the immune-system regulation, evidenced also by the potential interaction with TNFA. Moreover, the previous results showing the association of HS1.2 2 allele with inflammatory phenomena are consistent with the hypothesis that this allele is a detrimental factor in reaching advanced age.

HS1,2 Ig enhancer alleles association to AIDS progression in a pediatric cohort infected with a monophyletic HIV-strain.

Alteration in the humoral immune response has been observed during HIV infection. The polymorphisms of enhancer HS1,2, member of the 3(') regulatory region of the Ig heavy chain cluster, may play a role in the variation of the humoral response leading to pathological conditions. To assess the role of the HS1,2 polymorphic variants in the progression of AIDS, the HS1,2-A allelic frequencies were investigated in a cohort of HIV infected pediatric subjects from a nosocomial outbreak with a monophyletic strain of HIV. From a total group of 418 HIV infected children in the outbreak cohort, 42 nonprogressors and 31 progressors without bias due to antiretroviral therapy were evaluated. HS1,2 allele (∗)1 has been associated with nonprogressors (allelic frequency: 51.19% versus 33.87% in progressors, OR 0.5, and P = 0.0437), while allele (∗)2 has been associated with progression (allelic frequency: 48.39% versus 30.95% in nonprogressors, OR 2.1, and P = 0.0393). Further, only subjects carrying allele (∗)2 in absence of allele (∗)1, either in homozygous condition for allele (∗)2 [nonprogressors 2/42 (4.76%), Progressors 7/31 (22.58%), OR 5.8, and P = 0.0315] or in combination with other allelic variants [nonprogressors 7/42 (16.67%), Progressors 13/31 (41.93%), OR 3.61, and P = 0.0321], have been associated with HIV progression to AIDS. In conclusion, while the HS1,2 allele (∗)1 has a protective effect on HIV progression when present, allele (∗)2 is associated with progression toward AIDS when allele (∗)1 is absent.

Polymorphisms of the IgH enhancer HS1.2 and risk of systemic lupus erythematosus.

OBJECTIVE: To determine whether the allelic frequency variation of the HS1.2 enhancer of the immunoglobulin heavy chain (IgH) 3' regulatory region (3'RR-1) locus represents a risk factor for systemic lupus erythematosus (SLE) and to identify a possible functional difference in the two most frequent alleles (*1 and *2) in binding nuclear factor- κB (NF-κB) and Sp1. METHODS: The frequency of the enhancer HS1.2 alleles was determined in two cohorts of patients with SLE (n=293) and in 1185 controls. Electrophoretic mobility shift assays (EMSA) were carried out with B cell nuclear extracts with different probes of HS1.2 alleles *1 and *2 to map the consensus binding sites of the nuclear factors. A confirmatory cohort of 121 patients with SLE was also included. RESULTS: The frequency of allele *2 of the HS1.2 enhancer was significantly increased in patients with SLE compared with controls (OR 1.60, 95% CI 1.33 to 1.92, p<0.001). EMSA experiments showed the presence of the Sp1 binding site in both alleles whereas only allele *2 carried the consensus for the NF-κB factor. The presence versus absence of allele *2 in patients with SLE correlated with a higher concentration of IgM levels and with the expression of B cell activating factor receptor (BAFF-R). CONCLUSIONS: The increased frequency of allele *2 in patients with SLE identifies a new genetic risk factor for SLE. A possible biological effect of the polymorphism could be the difference observed in the localisation of an NF-κB binding site which is specific for allele *2 and absent in allele *1. These observations suggest a functional effect of the HS1.2 enhancer in this disease.