Anna M. Pietroboni

Decreased circulating miRNA levels in patients with primary progressive multiple sclerosis

Emerging evidence underlines the importance of micro(mi)RNAs in the pathogenesis of multiple sclerosis (MS). Free-circulating miRNAs were investigated in serum from MS patients compared to controls. Statistically significant decreased levels of miR-15b, miR-23a and miR-223 were observed in MS patients (p < 0.05). Results were validated and replicated in two further independent MS populations. A direct correlation between miRNA levels and the EDSS score was determined in PPMS (p < 0.007). The generalized trend toward miRNA down-regulation could result in over-expression of target genes involved in disease pathogenesis. Circulating miRNA profiling could thus represent a new avenue to identify easily detectable disease biomarkers.

Phenotypic Heterogeneity of the GRN Asp22fs Mutation in a Large Italian Kindred

The Asp22fs(g.63_64insC) mutation in progranulin gene (GRN) has been so far reported in one patient who developed frontotemporal dementia (FTD) at the age of 65. Here, we describe the clinical heterogeneity associated with the GRN Asp22fs mutation in a large Italian family. Clinical and instrumental workup of two symptomatic carriers in two generations has been carried out, together with genetic analysis of probands and of nine asymptomatic family members. The first proband was a 47-year old male clinically diagnosed with FTD. Family history was positive and suggestive of an autosomal dominant pattern of inheritance. Evaluation of plasma GRN levels was consistent with the presence of a mutation in its encoding gene, that was demonstrated by sequencing [Asp22fs(g.63_64insC)]. Brain MRI showed multiple T2 and FLAIR hyperintense areas in the frontal lobe white matter and right hemisphere cortical atrophy. The second proband was his 79 year old uncle, presenting with mild cognitive impairment. Brain MRI showed small T2 hyperintense lesions and widespread cortical atrophy. Cerebrospinal fluid amyloid-β, tau, and phosphotau protein levels were in both cases in the range of normality. Additional nine asymptomatic family members were studied. This familys description expands the spectrum of clinical presentations of frontotemporal lobar degeneration caused by GRN mutations, suggesting that the diagnosis could be missed in some individuals with an atypical presentation, and points up the importance of GRN plasma level evaluation.

Expression and Genetic Analysis of MicroRNAs Involved in Multiple Sclerosis

Evidence underlines the importance of microRNAs (miRNAs) in the pathogenesis of multiple sclerosis (MS). Based on the fact that miRNAs are present in human biological fluids, we previously showed that miR-223, miR-23a and miR-15b levels were downregulated in the sera of MS patients versus controls. Here, the expression levels of these candidate miRNAs were determined in peripheral blood mononuclear cells (PBMCs) and the serum of MS patients, in addition to three genotyped single nucleotide polymorphisms (SNPs). Mapping in the genomic regions of miR-223, miR-23a and miR-15b genes, 399 cases and 420 controls were tested. Expression levels of miR-223 and miR-23a were altered in PBMCs from MS patients versus controls. Conversely, there were no differences in the expression levels of miR-15b. A significantly decreased genotypic frequency of miR-223 rs1044165 T/T genotype was observed in MS patients. Moreover, the allelic frequency of miR-23a rs3745453 C allele was significantly increased in patients versus controls. In contrast, there were no differences in the distribution of miR-15b SNP. In conclusion, our results suggest that miR-223 and miR-23a could play a role in the pathogenesis of MS. Moreover, miR-223 rs1044165 polymorphism likely acts as a protective factor, while miR-23a rs3745453 variant seems to act as a risk factor for MS.

Recognition of viral and self-antigens by TH1 and TH1/TH17 central memory cells in patients with multiple sclerosis reveals distinct roles in immune surveillance and relapses

Background Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that is caused by autoreactive T cells and associated with viral infections. However, the phenotype of pathogenic T cells in peripheral blood remains to be defined, and how viruses promote MS is debated. Objective We aimed to identify and characterize potentially pathogenic autoreactive T cells, as well as protective antiviral T cells, in patients with MS. Methods We analyzed CD4+ helper T‐cell subsets from peripheral blood or cerebrospinal fluid for cytokine production, gene expression, plasticity, homing potentials, and their reactivity to self‐antigens and viral antigens in healthy subjects and patients with MS. Moreover, we monitored their frequencies in untreated and fingolimod‐ or natalizumab‐treated patients with MS. Results TH1/TH17 central memory (TH1/TH17CM) cells were selectively increased in peripheral blood of patients with relapsing‐remitting MS with a high disease score. TH1/TH17CM cells were closely related to conventional TH17 cells but had more pathogenic features. In particular, they could shuttle between lymph nodes and the CNS and produced encephalitogenic cytokines. The cerebrospinal fluid of patients with active MS was enriched for CXCL10 and contained mainly CXCR3‐expressing TH1 and TH1/TH17 subsets. However, while TH1 cells responded consistently to viruses, TH1/TH17CM cells reacted strongly with John Cunningham virus in healthy subjects but responded instead to myelin‐derived self‐antigens in patients with MS. Fingolimod and natalizumab therapies efficiently targeted autoreactive TH1/TH17CM cells but also blocked virus‐specific TH1 cells. Conclusions We propose that autoreactive TH1/TH17CM cells expand in patients with MS and promote relapses after bystander recruitment to the CNS, whereas TH1 cells perform immune surveillance. Thus the selective targeting of TH1/TH17 cells could inhibit relapses without causing John Cunningham virus–dependent progressive multifocal encephalomyelitis.

Cerebrospinal fluid progranulin levels in patients with different multiple sclerosis subtypes

Progranulin has recently attracted attention due to the discovery of mutations in its encoding gene (GRN) in several cases of frontotemporal lobar degeneration, but also for a possible role in inflammatory processes. In adult central nervous system, GRN mRNA is expressed in forebrain, olfactory bulbs and spinal cord. Progranulin cerebrospinal fluid (CSF) levels were evaluated in 55 patients with multiple sclerosis (MS) as well as in 35 subjects with non-inflammatory neurological diseases (NIND), 7 individuals with other inflammatory neurological disease (OIND) and 8 controls (CON), matched for ethnic background, gender and age. No statistically significant differences were found in patients compared with either NIND, OIND or CON (P>0.05), even stratifying according to disease subtype or gender. A positive correlation between progranulin CSF levels and age was observed in patients (rho=0.29, P=0.03). According to these data, progranulin does not likely play a major role in the pathogenesis of MS.

C9ORF72 repeat expansion not detected in patients with multiple sclerosis

A hexanucleotide repeat expansion in the chromosome 9 Open Reading Frame 72 gene (C9ORF72) has recently been reported to be cause of familial amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Nevertheless, in the last few years this mutation has been found to be associated with heterogeneous phenotypes, including multiple sclerosis (MS) in concurrence with amyotrophic lateral sclerosis. In this study, we sought to evaluate the presence of the C9ORF72 repeat expansion in a cohort consisting of 314 patients with MS and 222 control subjects. No pathogenic expansion was found in MS and control populations, suggesting that C9ORF72 does not play a major role in MS pathogenesis.

A 66-year-old patient with vanishing white matter disease due to the p.Ala87Val EIF2B3 mutation

Vanishing white matter (VWM; OMIM # 603896) is one of the most prevalent inherited childhood leukoencephalopathies. It has, however, become evident that VWM has a wider clinical spectrum, with age at onset inversely related to clinical severity. Many affected women experience a combination of leukoencephalopathy and primary amenorrhea or premature ovarian failure, a condition named ovarioleukodystrophy. Mutations in any of the genes encoding the 5 subunits of the Eukaryotic Initiation Factor 2B gene (EIF2B1, 2, 3, 4, and 5) can independently cause VWM.1

Effect of fingolimod treatment on circulating miR-15b, miR23a and miR-223 levels in patients with multiple sclerosis

MicroRNAs (miRNAs) have recently found to be dysregulated in serum from multiple sclerosis (MS) patients. Cell free circulating miR-15b, -23a and 223 levels were analyzed by Real Time PCR in a cohort consisting of 30 serum samples from Relapsing Remitting MS patients at baseline (T0) and after three, six, nine and twelve months (T1, T2, T3, T4) after starting the treatment. A down-regulation of miRNA levels in patients at T0 compared with controls was present (p<0.001). MiRNA levels slightly increased at T1 and this trend reached the statistical significance at T2 vs T0 and remains stable at T3 and T4. Our preliminary results suggest that aberrant levels of circulating miRNAs are recovered in fingolimod treated MS patients. Circulating miRNAs profiling could thus represent an easy detectable biomarker of disease and response to treatment.

Sciatic endometriosis presenting as periodic (catamenial) sciatic radiculopathy.

Many causes of traumatic sciatic neuropathy have been described in the literature [1], but extrapelvic endometriosis of the gluteal region presenting as sciatica remains difficult to recognize. Here, we present the case of a 45-year-old woman with a history of lower back pain and lower limb stiffness in whom hip MRI showed multiple loci of endometriosis localized in both iliac muscles and in the right gluteus. The woman presented with a 15-day history of lower back pain and impaired ambulation. Neurological examination showed lower limb stiffness with left leg flexure and right leg hyperextension, right foot dorsiflexion deficit, positive Lasegue sign, gait disturbance and severe pain with apparent (L4)-L5-S1 distribution. An EMG and a spinal cord MRI scan had previously been performed without any pathological findings. Intravenously administered myorelaxants and NSAIDs were tried without any benefit. Her past medical history was consistent only with diabetes. Routine blood tests showed only mild anaemia and high cancer antigen-125 (CA-125) levels. Brain and spinal cord MRI, backbone CT, and total-body PET scans did not show any pathological findings. Orthopaedic examination showed groin pain, irradiating to the knee and worsened by hip movement. On MRI T2-weighted images both the iliac and psoas muscles and the gluteus maximus and minimus appeared markedly bright. In the iliac muscles two T2-hyperintense formations, without contrast enhancement but with a hypervascular rim, were evident; the right gluteus showed similar findings (Fig. 1a). On the 4th day of hospitalization her period started and the patient reported worsening pain and severe abdominal cramps. Gynaecological examination showed a 2-cm fibrotic nodule in the pouch of Douglas, suggestive of endometriosis. CA-125 was 49.3 IU/ml (cutoff 35 IU/ml). A cycle of leuprorelin (a gonadotropinreleasing hormone agonist which downregulates the secretion of gonadotropin luteinizing hormone and folliclestimulating hormone leading to hypogonadism) was started, and thereafter symptoms slowly resolved. A follow-up MRI scan performed 3 months later showed a reduction in size and number of the lesions; neurological examination was normal (Fig. 1b). In accordance with previous report [1], CA-125 was elevated in our patient. The marked T2 brightness in both the iliac and gluteus muscles seen on the MRI scan was consistent with oedema and hyperaemia or, as described previously [2], neurogenic muscular injury. This finding may be suggestive of an endometriosic lesion localized at or near the sciatic notch. The T2-hypentense formations were consistent with foci of endometriosis localized in the muscle tissues. The cyclicity of the symptoms and their regression after a cycle of leuprorelin further supports our hypothesis [3, 4]. Endometriosis-induced cyclic sciatica was firstly reported by Schlincke in 1946 [5]. Since then, approximately 60 cases have been described. Lesions of the ipsilateral sciatic nerve are reported as the primary cause of almost all the endometriosis-induced cyclic sciatica [6]. The commonest localization is the sciatic notch where fibrosis, organized haematoma and endometrial tissue L. Ghezzi (&) A. Arighi A. M. Pietroboni F. Jacini G. G. Fumagalli N. Bresolin D. Galimberti E. Scarpini Department of Neurological Sciences, ‘‘Dino Ferrari’’ Center, Fondazione Ca Granda, IRCCS Ospedale Maggiore Policlinico, University of Milan, Milan, Italy e-mail: lauraghezzi@me.com

CSF β-amyloid as a putative biomarker of disease progression in multiple sclerosis.

Background: Neurodegeneration plays a major role in determining disability in multiple sclerosis (MS) patients. Hence, there is increasing need to identify reliable biomarkers, which could serve as prognostic measure of disease progression. Objectives: To assess whether cerebrospinal fluid (CSF) tau and β-amyloid (Aβ) levels were altered in newly diagnosed MS patients and correlated with disability. Moreover, we investigated whether these CSF biomarkers associate with macroscopic brain tissue damage measures. Methods: CSF Aβ and tau levels were determined by enzyme-linked immunosorbent assay in CSF samples from 48 newly diagnosed MS patients, followed-up clinically for 3 years by recording their Expanded Disability Status Scale score at 6-month intervals, and 45 controls. All patients underwent magnetic resonance imaging at baseline and at the end of follow-up to quantify their lesion load (LL). Results: CSF Aβ levels were significantly reduced in patients compared to controls (p < 0.001). Lower CSF Aβ levels at baseline were a disability predictor at 3-year follow-up (p = 0.009). CSF tau levels correlated with T2- and T1-LL (p < 0.001). Conclusion: CSF Aβ reduction is a promising biomarker of neurodegeneration and may predict patients’ clinical outcome. Therefore, CSF Aβ should be considered as a potential biomarker of prognostic value.