Vito A. G. Ricigliano

TET2 gene expression and 5-hydroxymethylcytosine level in multiple sclerosis peripheral blood cells

Aberrant DNA methylation can lead to genome destabilization and to deregulated gene expression. Recently, 5-hydroxymethylcytosine (5hmC), derived from oxidation of 5-methylcytosine (5mC) by the Ten-Eleven Translocation (TET) enzymes, has been detected. 5hmC is now considered as a new epigenetic DNA modification with relevant roles in cell homeostasis regulating DNA demethylation and transcription. Our aim was to investigate possible changes in the DNA methylation/demethylation machinery in MS. We assessed the expression of enzymes involved in DNA methylation/demethylation in peripheral blood mononuclear cells (PBMCs) from 40 subjects with MS and 40 matched healthy controls. We performed also, DNA methylation analysis of specific promoters and analysis of global levels of 5mC and 5hmC. We show that TET2 and DNMT1 expression is significantly down-regulated in MS PBMCs and it is associated with aberrant methylation of their promoters. Furthermore, 5hmC is decreased in MS PBMCs, probably as a result of the diminished TET2 level.

A "candidate-interactome" aggregate analysis of genome-wide association data in multiple sclerosis

Though difficult, the study of gene-environment interactions in multifactorial diseases is crucial for interpreting the relevance of non-heritable factors and prevents from overlooking genetic associations with small but measurable effects. We propose a “candidate interactome” (i.e. a group of genes whose products are known to physically interact with environmental factors that may be relevant for disease pathogenesis) analysis of genome-wide association data in multiple sclerosis. We looked for statistical enrichment of associations among interactomes that, at the current state of knowledge, may be representative of gene-environment interactions of potential, uncertain or unlikely relevance for multiple sclerosis pathogenesis: Epstein-Barr virus, human immunodeficiency virus, hepatitis B virus, hepatitis C virus, cytomegalovirus, HHV8-Kaposi sarcoma, H1N1-influenza, JC virus, human innate immunity interactome for type I interferon, autoimmune regulator, vitamin D receptor, aryl hydrocarbon receptor and a panel of proteins targeted by 70 innate immune-modulating viral open reading frames from 30 viral species. Interactomes were either obtained from the literature or were manually curated. The P values of all single nucleotide polymorphism mapping to a given interactome were obtained from the last genome-wide association study of the International Multiple Sclerosis Genetics Consortium & the Wellcome Trust Case Control Consortium, 2. The interaction between genotype and Epstein Barr virus emerges as relevant for multiple sclerosis etiology. However, in line with recent data on the coexistence of common and unique strategies used by viruses to perturb the human molecular system, also other viruses have a similar potential, though probably less relevant in epidemiological terms.

Epstein-Barr virus genetic variants are associated with multiple sclerosis

Objective: We analyzed the Epstein-Barr nuclear antigen 2 (EBNA2) gene, which contains the most variable region of the viral genome, in persons with multiple sclerosis (MS) and control subjects to verify whether virus genetic variants are involved in disease development. Methods: A seminested PCR approach and Sanger sequencing were used to analyze EBNA2 in 53 patients and 38 matched healthy donors (HDs). High-throughput sequencing by Illumina MiSeq was also applied in a subgroup of donors (17 patients and 17 HDs). Patients underwent gadolinium-enhanced MRI and human leucocyte antigen typing. Results: MS risk significantly correlated with an excess of 1.2 allele (odds ratio [OR] = 5.13; 95% confidence interval [CI] 1.84–14.32; p = 0.016) and underrepresentation of 1.3B allele (OR = 0.23; 95% CI 0.08–0.51; p = 0.0006). We identified new genetic variants, mostly 1.2 allele- and MS-associated (especially amino acid variation at position 245; OR = 9.4; 95% CI 1.19–78.72; p = 0.0123). In all cases, the consensus sequence from deep sequencing confirmed Sanger sequencing (including the cosegregation of newly identified variants with known EBNA2 alleles) and showed that the extent of genotype intraindividual variability was higher than expected: rare EBNA2 variants were detected in all HDs and patients with MS (range 1–17 and 3–19, respectively). EBNA2 variants did not seem to correlate with human leucocyte antigen typing or clinical/MRI features. Conclusions: Our study unveils a strong association between Epstein-Barr virus genomic variants and MS, reinforcing the idea that Epstein-Barr virus contributes to disease development.

IFN-β and multiple sclerosis: From etiology to therapy and back

Several immunomodulatory treatments are currently available for relapsing-remitting forms of multiple sclerosis (RRMS). Interferon beta (IFN) was the first therapeutic intervention able to modify the course of the disease and it is still the most used first-line treatment in RRMS. Though two decades have passed since IFN-β was introduced in the management of MS, it remains a valid approach because of its good benefit/risk profile. This is witnessed by new efforts of pharmaceutical industry to improve this line: a PEGylated form of subcutaneous IFN-β 1a, (Plegridy(®)) with a longer half-life, has been recently approved in RRMS. This review will survey the various stages of the use of type I IFN in MS, with special attention to the effect of the treatment on the supposed viral etiologic factors associated to the disease. The antiviral activities of IFN (that initially prompted its use as immunomodulatory agent in MS), and the mounting evidences in favor of a viral etiology in MS, allowed us to outline a re-appraisal from etiology to therapy and back.

Twin studies in multiple sclerosis: A meta-estimation of heritability and environmentality.

Background: Most twin studies of multiple sclerosis (MS) are inconclusive regarding the impact of genes and environment on disease susceptibility. In particular, high uncertainty exists about whether shared environmental factors are aetiologically relevant. Objective: To disentangle, with a reasonable degree of confidence, the relative contributions of heritability and of shared and unique environmental components of MS susceptibility. Methods: We performed a meta-analysis of previous twin studies. After a MEDLINE search, we selected eight twin studies in France, UK, Canada, Denmark, North America, Italy, Finland and Sweden. We conducted a biometric multi-group analysis under the liability-threshold model, by taking account of the study-specific ascertainment strategies and the population-specific prevalence rates of MS. Results: The meta-analytic estimates of tetrachoric correlations were 0.71 (95% confidence interval (CI): 0.67–0.74) in monozygotic pairs and 0.46 (95% CI: 0.41–0.50) in dizygotic pairs. The biometric multi-group model provided meta-analytic estimates of 0.50 (95% CI: 0.39–0.61) for heritability, 0.21 (95% CI: 0.11–0.30) for shared environmental component and 0.29 (95% CI: 0.26–0.33) for unique environmental component. Conclusion: Our results support the continuing efforts to identify unknown genetic factors that fill the gap of ‘missing heritability’; moreover, a ‘missing environmentality’ deserves future investigations into the role of non-heritable components that act as both shared and individual-specific exposures.

A Mechanistic, Stochastic Model Helps Understand Multiple Sclerosis Course and Pathogenesis

Heritable and nonheritable factors play a role in multiple sclerosis, but their effect size appears too small, explaining relatively little about disease etiology. Assuming that the factors that trigger the onset of the disease are, to some extent, also those that generate its remissions and relapses, we attempted to model the erratic behaviour of the disease course as observed on a dataset containing the time series of relapses and remissions of 70 patients free of disease-modifying therapies. We show that relapses and remissions follow exponential decaying distributions, excluding periodic recurrences and confirming that relapses manifest randomly in time. It is found that a mechanistic model with a random forcing describes in a satisfactory manner the occurrence of relapses and remissions, and the differences in the length of time spent in each one of the two states. This model may describe how interactions between “soft” etiologic factors occasionally reach the disease threshold thanks to comparably small external random perturbations. The model offers a new context to rethink key problems such as “missing heritability” and “hidden environmental structure” in the etiology of complex traits.

Contribution of Genome-Wide Association Studies to Scientific Research: A Pragmatic Approach to Evaluate Their Impact

The factual value of genome-wide association studies (GWAS) for the understanding of multifactorial diseases is a matter of intense debate. Practical consequences for the development of more effective therapies do not seem to be around the corner. Here we propose a pragmatic and objective evaluation of how much new biology is arising from these studies, with particular attention to the information that can help prioritize therapeutic targets. We chose multiple sclerosis (MS) as a paradigm disease and assumed that, in pre-GWAS candidate-gene studies, the knowledge behind the choice of each gene reflected the understanding of the disease prior to the advent of GWAS. Importantly, this knowledge was based mainly on non-genetic, phenotypic grounds. We performed single-gene and pathway-oriented comparisons of old and new knowledge in MS by confronting an unbiased list of candidate genes in pre-GWAS association studies with those genes exceeding the genome-wide significance threshold in GWAS published from 2007 on. At the single gene level, the majority (94 out of 125) of GWAS-discovered variants had never been contemplated as plausible candidates in pre-GWAS association studies. The 31 genes that were present in both pre- and post-GWAS lists may be of particular interest in that they represent disease-associated variants whose pathogenetic relevance is supported at the phenotypic level (i.e. the phenotypic information that steered their selection as candidate genes in pre-GWAS association studies). As such they represent attractive therapeutic targets. Interestingly, our analysis shows that some of these variants are targets of pharmacologically active compounds, including drugs that are already registered for human use. Compared with the above single-gene analysis, at the pathway level GWAS results appear more coherent with previous knowledge, reinforcing some of the current views on MS pathogenesis and related therapeutic research. This study presents a pragmatic approach that helps interpret and exploit GWAS knowledge.

Shared environmental effects on multiple sclerosis susceptibility: conflicting evidence from twin studies

Sir, We read with interest the paper by Westerlind et al. (2014) on the familial risk in multiple sclerosis. They applied an extended family design to estimate familial risks for multiple sclerosis, and used a twin approach to quantify the contributions of genetic and environmental factors in shaping individual susceptibility to the disease. The correlations in susceptibility of monozygotic (MZ) and dizygotic (DZ) twins (‘tetrachoric correlations’ under the ‘liability-threshold’ model, which provide information on magnitude of genetic and environmental components; Neale and Cardon, 1992) disconfirmed shared environmental effects, in favour of genetic and unshared (individual-specific) environmental influences in …

Noise in multiple sclerosis: unwanted and necessary.

As our knowledge about the etiology of multiple sclerosis (MS) increases, deterministic paradigms appear insufficient to describe the pathogenesis of the disease, and the impression is that stochastic phenomena (i.e. random events not necessarily resulting in disease in all individuals) may contribute to the development of MS. However, sources and mechanisms of stochastic behavior have not been investigated and there is no proposed framework to incorporate nondeterministic processes into disease biology. In this report, we will first describe analogies between physics of nonlinear systems and cell biology, showing how small‐scale random perturbations can impact on large‐scale phenomena, including cell function. We will then review growing and solid evidence showing that stochastic gene expression (or gene expression “noise”) can be a driver of phenotypic variation. Moreover, we will describe new methods that open unprecedented opportunities for the study of such phenomena in patients and the impact of this information on our understanding of MS course and therapy.

Screening for neurotropic viruses in cerebrospinal fluid of patients with multiple sclerosis and other neurological diseases.

The paper by Virtanen et al.1 strengthens the association of herpes viruses with multiple sclerosis (MS). The correlation between the presence of Epstein Barr virus (EBV)/Herpes Virus 6 (HHV-6) DNA in cerebrospinal fluid (CSF) and number of contrast enhancing lesions (CELs) on magnetic resonance imaging (MRI) is of interest and mirrors our results from a screening for neurotropic viruses in cerebrospinal fluid of patients with MS and other neurological diseases. We performed PCR for herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), cytomegalovirus (CMV), varicella zoster virus (VZV), HHV6, JC virus (JCV) and EBV on CSF samples from 159 patients who had had a lumbar puncture for diagnostic purposes. All CSF samples were analyzed using a commercial RHA CNS kit (Labo Bio-medical Products) for the detection of cell-free and cell-associated viral nucleic acids. Patients were then grouped according to their final diagnosis: 60 with MS (58 relapsing–remitting [RR] and two primary progressive [PP]; mean age 37±13 years, 40 females, 20 males), 48 with other inflammatory diseases (OIND; mean age 57±17 years, 23 females, 25 males), and 51 with other non-inflammatory diseases (NIND; mean age 56±18 years, 43 females, 8 males). Gadolinium-enhanced MRI was performed in all patients within 10 days of CSF sampling. In the MS group, 14 patients had a positive PCR for EBV, two for HSV1, three for HSV2 and one for HHV6. In OIND patients, 12 had positive PCR for EBV, two for HSV1, three for HSV2, four for CMV, one for JCV and four for HHV6. In NIND patients, two had positive PCR for EBV, one for HHV6 and one for VZV. The frequency of viral DNA in CSF was comparable among MS and OIND groups. A significant bias in the distribution of EBV was observed in MS and OIND groups with respect to NIND (p = 0.011 at Fisher’s exact test), while CMV infection was more frequent in OIND patients (p = 0.036 at Fisher’s exact test). In patients with MS we observed an excess of large T2 lesions at MRI in cases with EBV DNA in CSF: T2 lesions larger than 10 mm were present in six out 14 EBVpositive and five out 46 EBV-negative patients (p = 0.01 at Fisher’s exact test). This finding was not found in OIND patients; it was also independent of the cell count in CSF, which was higher in OIND than in MS patients (p = 0.02 at Mann–Whitney U test), but was comparable between EBV-positive and EBV-negative MS patients. Thus, the relationship between lesion size and EBV-positive PCR seems not to reflect a non-specific recruitment of leucocytes in the central nervous system in the context of amplified inflammation. The increased chance of detecting EBV DNA in MS patients with more active lesions is instead in accord with Jacobson’s group’s finding1 (at least as far as EBV is concerned) and supports evidence of intrathecal reactivation and virus-driven immunopathogenic response in MS.2,3