Biomarker Studies in Multiple Sclerosis: From Proteins to Noncoding RNAs

Multiple sclerosis (MS) is a neuroimmunological disorder characterized by central nervous system demyelination, axonal injury and loss. Considering the complexity of its aetiopathogenesis, early diagnosis of MS and individualized management are challenging in clinical practice. As the pathophysiologic and pharmacological indicators, studies on biomarkers in MS are useful for early prediction and diagnosis, monitoring of disease activity and predicting treatment response. In this review, we will summarize recent development of biomarker studies in MS from protein molecules to noncoding RNAs.     

A Risk Score for Predicting Multiple Sclerosis

ObjectiveMultiple sclerosis (MS) develops as a result of environmental influences on the genetically susceptible. Siblings of people with MS have an increased risk of both MS and demonstrating asymptomatic changes in keeping with MS. We set out to develop an MS risk score integrating both genetic and environmental risk factors. We used this score to identify siblings at extremes of MS risk and attempted to validate the score using brain MRI.Methods78 probands with MS, 121 of their unaffected siblings and 103 healthy controls were studied. Personal history was taken, and serological and genetic analysis using the illumina immunochip was performed. Odds ratios for MS associated with each risk factor were derived from existing literature, and the log values of the odds ratios from each of the risk factors were combined in an additive model to provide an overall score. Scores were initially calculated using log odds ratio from the HLA-DRB1*1501 allele only, secondly using data from all MS-associated SNPs identified in the 2011 GWAS. Subjects with extreme risk scores underwent validation studies. MRI was performed on selected individuals.ResultsThere was a significant difference in the both risk scores between people with MS, their unaffected siblings and healthy controls (p<0.0005). Unaffected siblings had a risk score intermediate to people with MS and controls (p<0.0005). The best performing risk score generated an AUC of 0.82 (95%CI 0.75–0.88).InterpretationsThe risk score demonstrates an AUC on the threshold for clinical utility. Our score enables the identification of a high-risk sibling group to inform pre-symptomatic longitudinal studies.     

Serum Mercury Level and Multiple Sclerosis

Exposure to heavy metals has been associated to a higher incidence of multiple sclerosis. In this work, we present a possible relationship between serum mercury levels and development of multiple sclerosis in Isfahan, the third largest city in Iran. Seventy-four patients affected by multiple sclerosis were retrieved from multiple sclerosis (MS) clinic in Isfahan, Iran. By matching sex and age, 74 healthy volunteers were chosen as control group. Blood samples were collected and serum mercury content was determined. Serum mercury level in MS patients was significantly higher than controls (9.6 ± 10.17 vs. 5.7 ± 8.6, P = 0.037). Concerning all MS patients, serum mercury value was significantly higher than the mercury concentration founded in control subjects {odd ratio: 2.39 (CI, 1.96–2.94), P = 0.00}. Serum mercury level is higher in MS patients with odd ratio equal to 2.39 compared with healthy individuals. It may reveal that high mercury levels in serum might help MS development in susceptible individuals. More studies with larger sample size are needed to confirm this hypothesis.     

Oral Contraceptives and Multiple Sclerosis/Clinically Isolated Syndrome Susceptibility

Background

The incidence of multiple sclerosis (MS) is rising in women.

Objective

To determine whether the use of combined oral contraceptives (COCs) are associated with MS risk and whether this varies by progestin content.

Methods

We conducted a nested case-control study of females ages 14–48 years with incident MS or clinically isolated syndrome (CIS) 2008–2011 from the membership of Kaiser Permanente Southern California. Controls were matched on age, race/ethnicity and membership characteristics. COC use up to ten years prior to symptom onset was obtained from the complete electronic health record.

Results

We identified 400 women with incident MS/CIS and 3904 matched controls. Forty- percent of cases and 32% of controls had used COCs prior to symptom onset. The use of COCs was associated with a slightly increased risk of MS/CIS (adjusted OR = 1.52, 95%CI = 1.21–1.91; p<0.001). This risk did not vary by duration of COC use. The association varied by progestin content being more pronounced for levenorgestrol (adjusted OR = 1.75, 95%CI = 1.29–2.37; p<0.001) than norethindrone (adjusted OR = 1.57, 95%CI = 1.16–2.12; p = 0.003) and absent for the newest progestin, drospirenone (p = 0.95).

Conclusions

Our findings should be interpreted cautiously. While the use of some combination oral contraceptives may contribute to the rising incidence of MS in women, an unmeasured confounder associated with the modern woman’s lifestyle is a more likely explanation for this weak association.     

Iron Is a Sensitive Biomarker for Inflammation in Multiple Sclerosis Lesions

MRI phase imaging in multiple sclerosis (MS) patients and in autopsy tissue have demonstrated the presence of iron depositions in white matter lesions.The accumulation of iron in some but not all lesions suggests a specific, potentially disease-relevant process, however; its pathophysiological significance remains unknown.Here, we explore the role of lesional iron in multiple sclerosis using multiple approaches: immunohistochemical examination of autoptic MS tissue, an in vitro model of iron-uptake in human cultured macrophages and ultra-highfield phase imaging of highly active and of secondary progressive MS patients.Using Perls'' stain and immunohistochemistry, iron was detected in MS tissue sections predominantly in non-phagocytosing macrophages/microglia at the edge of established, demyelinated lesions. Moreover, iron-containing macrophages but not myelin-laden macrophages expressed markers of proinflammatory (M1) polarization.Similarly, in human macrophage cultures, iron was preferentially taken up by non-phagocytosing, M1-polarized macrophages and induced M1 (super) polarization. Iron uptake was minimal in myelin-laden macrophages and active myelin phagocytosis led to depletion of intracellular iron.Finally, we demonstrated in MS patients using GRE phase imaging with ultra-highfield MRI that phase hypointense lesions were significantly more prevalent in patients with active relapsing than with secondary progressive MS.Taken together, our data provide a basis to interpret iron-sensitive GRE phase imaging in MS patients: iron is present in non-phagocytosing, M1-polarized microglia/macrophages at the rim of chronic active white matter demyelinating lesions. Phase imaging may therefore visualize specific, chronic proinflammatory activity in established MS lesions and thus provide important clinical information on disease status and treatment efficacy in MS patients.     

Diagnostic and Prognostic Value of the Cerebrospinal Fluid Concentration of Immunoglobulin Free Light Chains in Clinically Isolated Syndrome with Conversion to Multiple Sclerosis

Background and objective

In this study, we evaluated the diagnostic and prognostic significance of cerebrospinal fluid free light chains (CSF FLC) at the time of clinically isolated syndrome (CIS).

Methods

We compared FLC-parameters at the moment of CIS in patients with conversion to multiple sclerosis (MS) after 2 years (CIS-MS), patients who remained stable both clinically and radiologically after 2 years (CIS-nonMS), patients with non-inflammatory neurologic diseases (NIND) as a comparison group and patients with other inflammatory neurologic diseases (IND) with intrathecal oligoclonal bands (OCB) synthesis. ROC-analysis was conducted to define FLC-assay characteristics and cut-off values. We also compared FLC-concentrations in CIS patients to determine their OCB-status. A correlation analysis was performed between FLC-concentrations and the expanded disability scale score (EDSS), annualized relapse rate (ARR) and MRI-activity (i.e., number of new and gadolinium-enhancing (Gd+) lesions) in patients.

Results

The levels of kappa-FLC (k-FLC_CSF) and lambda-FLC (λ-FLC_CSF) as well as kappa- and lambda-quotients (Q-k and Q-λ) were elevated in CIS-MS compared to the CIS-nonMS and NIND groups. These levels did not differ significantly when compared with the IND group. We identified several patients with high k-FLC_CSF and λ-FLC_CSF in OCB-negative CIS and IND groups. The level of k-FLC_CSF production was significantly higher in OCB-positive patients in the CIS-MS group compared to the CIS-nonMS group. The concentrations of k-FLC_CSF and Q-k in the CIS-MS group showed significant correlation with the level of EDSS after 2 years (k-FLC: r = 0.4477,p = 0.0016; Q-k: r = 0.4621, p = 0.0016). λ-FLC_CSF and Q-λ inversely correlated with the number of Gd+ lesions (CSF λ-FLC: r = -0.3698, p = 0.0223; Q-λ: r = -0.4527, p = 0.0056).

Conclusion

The concentration of CSF FLC predicts conversion to MS within 2 years following CIS. OCB-positive patients with an early conversion have a higher concentration of CSF-FLC. We have also shown a prognostic significance of k-FLC_CSF for future EDSS-progression.     

Serum N-Glycans: A New Diagnostic Biomarker for Light Chain Multiple Myeloma

The aim of this study was to evaluate the diagnostic and differential diagnostic power of serum N-glycans for light chain multiple myeloma (LCMM). A total of 167 cases of subjects, including 42 LCMM, 42 IgG myeloma, 41 IgA myeloma, and 42 healthy controls were recruited in this study. DNA sequencer-assisted fluorophore-assisted capillary electrophoresis (DSA-FACE) was applied to determine the quantitive abundance of serum N-glycans. The core fucosylated, bisecting and sialylated modifications were analyzed in serum of LCMM patients (n=20) and healthy controls (n=20) randomly selected from the same cohort by lectin blot. Moreover, serum sialic acid (SA) level was measured by enzymatic method. We found two N-glycan structures (NG1A2F, Peak3; NA2FB, Peak7) showed the optimum diagnostic efficacy with area under the ROC curve (AUC) 0.939 and 0.940 between LCMM and healthy control. The sensitivity and specificity of Peak3 were 88.1% and 92.9%, while Peak7 were 92.9% and 97.6%, respectively. The abundance of Peak3 could differentiate LCMM from IgG myeloma with AUC 0.899, sensitivity 100% and specificity 76.2%, and Peak7 could be used to differentiate LCMM from IgA myeloma with AUC 0.922, sensitivity 92.9% and specificity 82.9%. Serum SA level was significantly higher in patients with LCMM than that in healthy controls. Moreover, the decreased core fucosylation, bisecting and increased sialylation characters of serum glycoproteins were observed in patients with LCMM. We concluded that serum N-glycan could provide a simple, reliable and non-invasive biomarker for LCMM diagnosis and abnormal glycosylation might imply a new potential therapeutic target in LCMM.     

Cerebrospinal Fluid Immunoglobulin Kappa Light Chain in Clinically Isolated Syndrome and Multiple Sclerosis

Background

Oligoclonal bands (OCB) are the most widely used CSF test to support the diagnosis of MS and to predict conversion of clinically isolated syndrome (CIS) to multiple sclerosis (MS). Since OCB tests are based on non-quantitative and difficult to standardise techniques, measurement of immunoglobulin kappa free light chains (KFLC) may represent an easier to use quantitative test.

Methods

KFLC were measured in CSF and serum of 211 patients using ELISA. These include patients without any inflammatory central nervous system reaction (NIND, n = 77), MS (n = 20), viral CNS infections (V-CNS-I, n = 10), neuroborreliosis (NB, n = 17) and other bacterial CNS infections (B-CNS-I, n = 10). Furthermore a cohort of 77 patients with CIS, including 39 patients that remained CIS over follow-up of two years (CIS-CIS) and 38 patients that developed MS over the same follow-up time (CIS-MS).

Results

CSF-serum ratio of KFLC (Q KFLC) was elevated in all patients with MS, 86.8% of patients with CIS-MS and 61.5% of patients with CIS-CIS. It was significantly elevated in CIS with presence of OCB (p<0.001). Q KFLC significantly correlated with other CSF variables such as CSF leukocyte count (p<0.001, R = 0.46), CSF CXCL13 levels (p<0.001, R = 0.64) and also intrathecal IgG synthesis (p<0.001, R = 0.74) as determined by nephelometry and quotient diagram. OCB were detected in 66.7% of CIS-CIS and in 92.1% of CIS-MS.

Conclusions

Although the measurement of CSF KFLC is a rapid and quantitative easy to standardize tool, it is almost equal but not superior to OCB with regard to diagnostic sensitivity and specificity in patients with early MS.     

Multiple Kernel Learning Captures a Systems-Level Functional Connectivity Biomarker Signature in Amyotrophic Lateral Sclerosis

There is significant clinical and prognostic heterogeneity in the neurodegenerative disorder amyotrophic lateral sclerosis (ALS), despite a common immunohistological signature. Consistent extra-motor as well as motor cerebral, spinal anterior horn and distal neuromuscular junction pathology supports the notion of ALS a system failure. Establishing a disease biomarker is a priority but a simplistic, coordinate-based approach to brain dysfunction using MRI is not tenable. Resting-state functional MRI reflects the organization of brain networks at the systems-level, and so changes in of motor functional connectivity were explored to determine their potential as the substrate for a biomarker signature. Intra- as well as inter-motor functional networks in the 0.03–0.06 Hz frequency band were derived from 40 patients and 30 healthy controls of similar age, and used as features for pattern detection, employing multiple kernel learning. This approach enabled an accurate classification of a group of patients that included a range of clinical sub-types. An average of 13 regions-of-interest were needed to reach peak discrimination. Subsequent analysis revealed that the alterations in motor functional connectivity were widespread, including regions not obviously clinically affected such as the cerebellum and basal ganglia. Complex network analysis showed that functional networks in ALS differ markedly in their topology, reflecting the underlying altered functional connectivity pattern seen in patients: 1) reduced connectivity of both the cortical and sub-cortical motor areas with non motor areas 2)reduced subcortical-cortical motor connectivity and 3) increased connectivity observed within sub-cortical motor networks. This type of analysis has potential to non-invasively define a biomarker signature at the systems-level. As the understanding of neurodegenerative disorders moves towards studying pre-symptomatic changes, there is potential for this type of approach to generate biomarkers for the testing of neuroprotective strategies.     

Biomarkers of Inflammation and Axonal Degeneration/Damage in Patients with Newly Diagnosed Multiple Sclerosis: Contributions of the Soluble CD163 CSF/Serum Ratio to a Biomarker Panel

Background

Expression of soluble CD163 (sCD163), a macrophage/microglia biomarker, is increased in inflammatory conditions, and sCD163 levels in the cerebrospinal fluid (CSF) have recently been shown to be elevated in patients with multiple sclerosis (MS): the sCD163 CSF/serum ratio was elevated in patients with relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and clinically isolated syndrome (CIS) compared with symptomatic controls.

Objective

To investigate the contributions of the sCD163 CSF/serum ratio to a biomarker panel focusing on inflammation and axonal degeneration in newly diagnosed MS; thus optimising a diagnostic biomarker panel for MS.

Methods

After a full MS diagnostic work-up, including collection of paired samples of CSF and serum, 125 patients were included in this study. Patients were divided into groups based on their diagnosis, and patients with normal clinical and paraclinical findings were defined as symptomatic controls. Serum and CSF levels, ratios, and indices of sCD163, CXCL13, osteopontin, neopterin, and CSF levels of neurofilament light polypeptide were determined by enzyme-linked immunosorbent assays (ELISAs). For sCD163 the results constitute a post-hoc analysis of already published data.

Results

All tested biomarkers, notably the sCD163 ratio, the CXCL13 ratio, the NEO ratio, the CSF level of NfL, the IgG index, and the serum level of OPN, were significantly correlated to RRMS, PPMS, and/or CIS. The individual biomarkers in single tests had a lower performance than the IgG index, however, their combined receiver operating characteristic (ROC) curve demonstrated excellent diagnostic discriminatory power.

Conclusion

The biomarker panel showed distinct profiles for each patient group and could be a valuable tool for clinical differentiation of MS subgroups. The combined ROC analysis showed that sCD163 contributes positively as a diagnostic marker to a panel of established MS biomarkers. Patients with PPMS were demonstrated to have significantly elevated levels of both inflammatory and degenerative markers.     

Serum Arsenic and Lipid Peroxidation Levels in Patients with Multiple Sclerosis

Oxidative stress plays a crucial role in the pathogenesis of multiple sclerosis (MS). Previous studies have shown that oxidative stress is one of the main underlying mechanisms of arsenic-induced cellular damage. The aim of this study was to assess the serum levels of arsenic and its relationship with lipid peroxidation in MS patients from Tabriz, as the third polluted city of Iran. The study population included 38 MS female patients and 38 age-matched healthy controls. Serum malondialdehyde (MDA) and arsenic levels were measured using thiobarbituric acid reactive substances (TBARS) assay and electrothermal atomic absorption spectrometry, respectively. The results showed that the arsenic (P?<?0.01) and MDA (P?=?0.03) levels were significantly higher in patients with MS than those in control. Moreover, serum levels of arsenic and MDA were positively correlated in MS patients. The elevated levels of serum arsenic might explain the increased oxidative stress in MS patients. We suggest that high arsenic levels in serum may lead to MS development, and therefore, exposure to this metal should be limited.     

A Further TWEAK to Multiple Sclerosis Pathophysiology

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a member of the TNF super family that controls many cellular activities including proliferation, migration, differentiation, apoptosis, and inflammation by binding to fibroblast growth factor-inducible 14 (Fn14), a highly inducible cell surface receptor. Recent studies have indicated that TWEAK–Fn14 axis signaling may contribute to chronic autoimmune diseases. TWEAK expression via microglia in cortical lesions, presence of TWEAK⁺ macrophages in inflamed leptomeninges, and absence of TWEAK/Fn14 expression in healthy brain implicates importance of this pathway in pathogenesis of multiple sclerosis lesions. TWEAK–Fn14 axis blockade has also shown promise in various multiple sclerosis animal models. Stimulation of the TWEAK/Fn14 pathway can result in activation of both canonical and noncanonical NF-κB signaling and could also stimulate mitogen-activated protein kinase (MAPK) signaling pathways. Here, we have reviewed evidence of the possible role of TWEAK–Fn14 axis in pathophysiology of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) via neuroinflammation, tissue remodeling, blood–brain barrier (BBB) disruption, neurodegeneration, and astrogliosis.     

Serum Fucosylated Haptoglobin as a Novel Diagnostic Biomarker for Predicting Hepatocyte Ballooning and Nonalcoholic Steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is a growing medical problem around the world. NAFLD patients with nonalcoholic steatohepatitis (NASH) can develop cirrhosis and hepatocellular carcinoma. The ability to distinguish NASH from simple steatosis would be of great clinical significance. Ballooning hepatocytes are characteristic of typical pathological NASH; here, the polarized secretion of proteins is disrupted due to destruction of the cytoskeleton. We previously reported that fucosylated glycoproteins are secreted into bile, but not into sera in normal liver. Therefore, we hypothesized that the fucosylation-based sorting machinery would be disrupted in ballooning hepatocytes, and serum fucosylated glycoproteins would increase in NASH patients. To confirm our hypothesis, we evaluated serum fucosylated haptoglobin (Fuc-Hpt) levels in biopsy-proven NAFLD patients (n = 126) using a lectin-antibody ELISA kit. Fuc-Hpt levels were significantly increased in NASH patients compared with non-NASH (NAFLD patients without NASH) patients. Interestingly, Fuc-Hpt levels showed a significant stepwise increase with increasing hepatocyte ballooning scores. Multiple logistic regression analysis showed that Fuc-Hpt levels were independent and significant determinants of the presence of ballooning hepatocytes. Moreover, Fuc-Hpt levels were useful in monitoring liver fibrosis staging. Next, to investigate the significance of serum Fuc-Hpt in a larger population, we measured Fuc-Hpt levels in ultrasound-diagnosed NAFLD subjects (n = 870) who received a medical health checkup. To evaluate NAFLD disease severity, we used the FIB-4 index (based on age, serum AST and ALT levels, and platelet counts). Fuc-Hpt levels increased stepwise with increasing FIB-4 index.

Conclusion

Measurement of serum Fuc-Hpt levels can distinguish NASH from non-NASH patients, and predict the presence of ballooning hepatocytes in NAFLD patients with sufficient accuracy. These results support the potential usefulness of measuring Fuc-Hpt levels in clinical practice.     

Linoleate and Fatty-acid Patterns of Serum Lipids in Multiple Sclerosis and Other Diseases

The linoleic acid content of serum lipids was measured in 47 patients with multiple sclerosis, 29 patients with other neurological diseases, 35 patients with acute non-neurological illnesses, and 49 healthy control subjects. Reduced linoleic acid content of serum lipids was not specific to multiple sclerosis and occurred in all ill patients with acute non-neurological illness. The fatty-acid pattern of serum lipids in illness resembles that of essential fatty-acid deficiency. It seems that this pattern of reduced linoleic acid content with increased oleic, palmitic, and palmitoleic acid content may be a general phenomenon in ill patients.     

Clinically Significant Fatigue: Prevalence and Associated Factors in an International Sample of Adults with Multiple Sclerosis Recruited via the Internet

BackgroundFatigue contributes a significant burden of disease for people with multiple sclerosis (PwMS). Modifiable lifestyle factors have been recognized as having a role in a range of morbidity outcomes in PwMS. There is significant potential to prevent and treat fatigue in PwMS by addressing modifiable risk factors.ObjectivesTo explore the associations between clinically significant fatigue and demographic factors, clinical factors (health-related quality of life, disability and relapse rate) and modifiable lifestyle, disease-modifying drugs (DMD) and supplement use in a large international sample of PwMS.MethodsPwMS were recruited to the study via Web 2.0 platforms and completed a comprehensive survey measuring demographic, lifestyle and clinical characteristics, including health-related quality of life, disability, and relapse rate.ResultsOf 2469 participants with confirmed MS, 2138 (86.6%) completed a validated measure of clinically significant fatigue, the Fatigue Severity Scale. Participants were predominantly female from English speaking countries, with relatively high levels of education, and due to recruitment methods may have been highly pro-active about engaging in lifestyle management and self-help. Approximately two thirds of our sample (1402/2138; 65.6% (95% CI 63.7–67.7)) screened positive for clinically significant fatigue. Bivariate associations were present between clinically significant fatigue and several demographic, clinical, lifestyle, and medication variables. After controlling for level of disability and a range of stable socio-demographic variables, we found increased odds of fatigue associated with obesity, DMD use, poor diet, and reduced odds of fatigue with exercise, fish consumption, moderate alcohol use, and supplementation with vitamin D and flaxseed oil.ConclusionThis study supports strong and significant associations between clinically significant fatigue and modifiable lifestyle factors. Longitudinal follow-up of this sample may help clarify the contribution of reverse causation to our findings. Further research is required to explore these associations including randomized controlled trials of lifestyle interventions that may alleviate fatigue.     

Correlation of -475 IL-2 Promoter Gene Polymorphisms and the Levels of Serum IL-2 on the Risk of Multiple Sclerosis

Background:The aim of present study is to asset the IL-2 promoter gene (SNP -475) as a candidate gene for multiple sclerosis (MS) susceptibility.Methods:This study included 70 patients with relapsing – remitting multiple sclerosis (RRMS) and 50 healthy controls. Following the extraction of genomic DNA from peripheral blood, frequency of genotypes and alleles of SNP -475 was calculated using Restriction fragment length polymorphism-polymer chain reaction (RFLP-PCR) and then the results were analyzed statistically.Results:The results revealed the unusual ratio for the heterozygous (AT) was 1.6972 indicating that heterozygous patients were at higher risk of multiple sclerosis than wild homozygous (AA), and homomutant (TT). The results show protective role for - 475 IL-2 promoter among individuals with multiple sclerosis, (O.R: 0.4872; C.I. 95%: 0.1617- 1.4680) and (O.R: 0.9275; C.I. 95%: 0.2476 - 3.4745) for both AA and TT genotypes, respectively.Conclusion:Our results showed that in this population of Iraqi patients, the AT genotype / A allele of -475 IL-2 promoter gene SNP may include attributed factors for MS predisposition.Key Words: IL-2, Multiple sclerosis, PCR-RFLP SNP