Next-generation sequencing of the whole mitochondrial genome identifies functionally deleterious mutations in patients with multiple sclerosis.

Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system with genetics and environmental determinants. Studies focused on the neurogenetics of MS showed that mitochondrial DNA (mtDNA) mutations that can ultimately lead to mitochondrial dysfunction, alter brain energy metabolism and cause neurodegeneration. We analyzed the whole mitochondrial genome using next-generation sequencing (NGS) from 47 Saudi individuals, 23 patients with relapsing-remitting MS and 24 healthy controls to identify mtDNA disease-related mutations/variants. A large number of variants were detected in the D-loop and coding genes of mtDNA. While distinct unique variants were only present in patients or only occur in controls, a number of common variants were shared among the two groups. The prevalence of some common variants differed significantly between patients and controls, thus could be implicated in susceptibility to MS. Of the unique variants only present in the patients, 34 were missense mutations, located in different mtDNA-encoded genes. Seven of these mutations were not previously reported in MS, and predicted to be deleterious with considerable impacts on the functions and structures of encoded-proteins and may play a role in the pathogenesis of MS. These include two heteroplasmic mutations namely 10237T>C in MT-ND3 gene and 15884G>C in MT-CYB gene; and three homoplasmic mutations namely 9288A>G in MT-CO3 gene, 14484T>C in MT-ND6 gene, 15431G>A in MT-CYB gene, 8490T>C in MT-ATP8 gene and 5437C>T in MT-ND2 gene. Notably some patients harboured multiple mutations while other patients carried the same mutations. This study is the first to sequence the entire mitochondrial genome in MS patients in an Arab population. Our results expanded the mutational spectrum of mtDNA variants in MS and highlighted the efficiency of NGS in population-specific mtDNA variant discovery. Further investigations in a larger cohort are warranted to confirm the role of mtDNA MS.

Knocking down Israa, the Zmiz1 intron-nested gene, unveils interrelated T cell activation functions in mouse.

We previously reported Israa (immune-system-released activating agent), a novel gene nested in intron 6 of the mouse Zmiz1 gene. Zmiz1 is involved in several functions such as fertility and T cell development and its knockout leads to non-viable embryos. We also reported ISRAA's expression in lymphoid organs, particularly in the thymus CD3+ T cells during all developmental stages. In addition, we showed that ISRAA is a binding partner of Fyn and Elf-1 and regulates the expression of T cell activation-related genes in vitro. In this paper, we report the generation and characterization of an Israa -/- constitutive knockout mouse. The histological study shows that Israa -/- mice exhibit thymus and spleen hyperplasia. Israa -/- derived T cells showed increased proliferation compared to the wild-type mice T cells. Moreover, gene expression analysis revealed a set of differentially expressed genes in the knockout and wild-type animals during thymus development (mostly genes of T cell activation pathways). Immunological phenotyping of the thymocytes and splenocytes of Israa -/- showed no difference with those of the wild-type. Moreover, we observed that knocking out the Zmiz1 intron embedded Israa gene does not affect mice fertility, thus does not disturb this Zmiz1 function. The characterization of the Israa -/- mouse confirms the role ISRAA plays in the expression regulation of genes involved in T cell activation established in vitro. Taken together, our findings point toward a potential functional interrelation between the intron nested Israa gene and the Zmiz1 host gene in regulating T cell activation. This constitutively Israa -/- mice can be a good model to study T cell activation and to investigate the relationship between host and intron-nested genes.

Mitochondrial DNA Copy Number in Peripheral Blood as a Potential Non-invasive Biomarker for Multiple Sclerosis.

The impaired mitochondrial function has been implicated in the pathogenicity of multiple sclerosis (MS), a chronic inflammatory, demyelinating, and neurodegenerative disease of the CNS. Circulating mtDNA copy number in body fluids has been proposed as an indicator for several neurodegenerative diseases, and the altered cerebrospinal fluid mtDNA has been shown as a promising marker for MS. The aim of this study was to determine changes and biomarker potential of circulating mtDNA in peripheral blood in MS. The mtDNA copy number was quantified by real-time PCR in blood samples from 60 patients with relapsing-remitting MS (RRMS) and 64 healthy controls. The RRMS patients had significantly lower circulating mtDNA copy number compared to controls. Subgroup analysis with stratification of RRMS patients based on disease duration under or over 10 years revealed that the mtDNA copy number was significantly lower in the group with longer disease duration. A negative correlation was observed between mtDNA copy number and disease duration. The ROC curve analysis indicated a significant ability of mtDNA copy number to separate RRMS patients from controls with an AUC of 0.859. This is the first study to measure peripheral blood mtDNA copy number in MS patients. Current data suggest that the reduction in peripheral blood mtDNA copy number may be an early event in MS and correlate with the disease progression. The findings of this study indicate that circulating blood-based mtDNA copy number may be a potential non-invasive candidate biomarker for mitochondria-mediated neurodegeneration and MS. This can put forward the clinical applicability over other invasive markers.

Induction of dissociated cytokine profiles by ISRAA with selective critical involvement of ERK1/2 in its signaling functions.

The immune system-released activating agent (ISRAA) is an immune mediator activated as a result of a nerve stimulus initiated by immune challenge. We have previously demonstrated that ISRAA and tumor necrosis factor (TNF) receptor 1 (TNFR1) share an interspecies-conserved motif (72% homology) that induces the apoptosis and proliferation of human peripheral blood mononuclear cells (hPBMCs) in a dose-dependent manner. In the present study, cytokine profiles were examined in response to the stimulation of hPBMCs with ISRAA. Furthermore, the signaling pathways induced by ISRAA were mapped. The results revealed high measurable levels of TNF-α, interleukin (IL)-6, IL-8, IL-10 and interferon (IFN)-γ, but not IL-4, IL-17 (IL-17A) or transforming growth factor (TGF)-ß. The analysis of signaling pathways revealed the activation of extracellular-regulated protein kinase (ERK)1/2 as a downstream signal in the mitogen­activated protein kinase (MAPK) pathway during TNF­α and IL-6 production and apoptosis, but not during proliferation following stimulation with ISRAA by triggering the Fas-associated protein with death domain (FADD). STAT3 was found to be unphosphorylated in the ISRAA­stimulated hPBMCs, and STAT3 was ubiquitously expressed in unstimulated cells, suggesting that ISRAA has a protein inhibitor of activated STAT (PIAS)-like activity, by functioning as a negative regulator of the effects of STAT3 on the Janus kinase (JAK)/STAT pathway. The determination of the nature of cytokine responses together with the signaling pathways of cellular activity induced by ISRAA paves the way for the investigation of a potential target of ISRAA and for the development of novel therapeutic approaches for the treatment of immune-regulated disorders.