Membrane Proteome-Wide Screening of Autoantibodies in CIDP Using Human Cell Microarray Technology.

BACKGROUND AND OBJECTIVES: Autoantibody discovery in complex autoimmune diseases is challenging. Diverse successful antigen identification strategies are available, but, so far, have often been unsuccessful, especially in the discovery of protein antigens in which conformational and post-translational modification are critical. Our study assesses the utility of a human membrane and secreted protein microarray technology to detect autoantibodies in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). METHODS: A cell microarray consisting of human embryonic kidney-293 cells expressing >5,000 human proteins was used. First, a validation step was performed with 4 serum samples from patients with autoimmune nodopathy (AN) to assess the ability of this technology to detect circulating known autoantibodies. The ability of the cell microarray technology to discover novel IgG autoantibodies was assessed incubating the array with 8 CIDP serum samples. Identified autoantibodies were subsequently validated using cell-based assays (CBAs), ELISA, and/or tissue immunohistochemistry and analyzed in a cohort of CIDP and AN (n = 96) and control (n = 100) samples. RESULTS: Serum anti-contactin-1 and anti-neurofascin-155 were detected by the human cell microarray technology. Nine potentially relevant antigens were found in patients with CIDP without other detectable antibodies; confirmation was possible in six of them: ephrin type-A receptor 7 (EPHA7); potassium-transporting ATPase alpha chain 1 and subunit beta (ATP4A/4B); leukemia-inhibitory factor (LIF); and interferon lambda 1, 2, and 3 (IFNL1, IFNL2, IFNL3). Anti-ATP4A/4B and anti-EPHA7 antibodies were detected in patients and controls and considered unrelated to CIDP. Both anti-LIF and anti-IFNL antibodies were found in the same 2 patients and were not detected in any control. Both patients showed the same staining pattern against myelinating fibers of peripheral nerve tissue and of myelinating neuron-Schwann cell cocultures. Clinically relevant correlations could not be established for anti-LIF and anti-IFNL3 antibodies. DISCUSSION: Our work demonstrates the utility of human cell microarray technology to detect known and discover unknown autoantibodies in human serum samples. Despite potential CIDP-associated autoantibodies (anti-LIF and anti-IFNL3) being identified, their clinical and pathogenic relevance needs to be elucidated in bigger cohorts.

Excellent response to anti-CD38 therapy with daratumumab in a patient with severe refractory CANOMAD.

BACKGROUND: Intravenous immunoglobulin (IVIG) and rituximab are considered the first-line and second-line treatments for Chronic Ataxic Neuropathy and Ophthalmoplegia with IgM-paraprotein, cold Agglutinins, and anti-Disialosyl antibodies (CANOMAD), with an overall clinical response around 50%. New anti-CD38 daratumumab, targeting long-lived plasma cells, has been reported as a promising therapy for treatment-refractory antibody-mediated disorders. We report the first case of a severe refractory CANOMAD, successfully treated with daratumumab. METHODS: A patient in their 70s with severe relapsing CANOMAD, refractory to IVIG, steroids, rituximab and ibrutinib developed severe tetraparesis and respiratory failure. Plasma exchange (PE) improved motor and ventilatory function; however, after 6 weeks, patient remained PE dependent. Intravenous daratumumab was initiated at 16 mg/kg weekly for 3 weeks, every 2 weeks for the second and third month, and monthly afterwards. RESULTS: After 3 weeks of starting daratumumab, PE was discontinued and, since then, the patient evolved to complete recovery. Antidisialosyl antibody titres decreased after PE and remained stable during daratumumab. Serum neurofilament light-chain levels were elevated in the exacerbation phase and normalised after daratumumab. The patient remains in clinical remission under monthly daratumumab, 12 months after initiation. CONCLUSIONS: The first patient with aggressive treatment-refractory CANOMAD treated with daratumumab provides proof-of-principle evidence that daratumumab may be an effective treatment in IgM-related neuropathies.

Clinicopathological correlates in frontotemporal lobar degeneration: motor neuron disease spectrum.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease (MND) that shares a common clinical, genetic and pathologic spectrum with frontotemporal dementia (FTD). It is highly heterogeneous in its presentation and features. Up to 50% of patients with MND develop cognitive-behavioural symptoms during the course of the disease, meeting criteria for FTD in 10-15% of cases. In the absence of a precise biomarker, neuropathology is still a valuable tool to understand disease nosology, reach a definite diagnostic confirmation and help define specific subgroups of patients with common phenotypic, genetic and biomarker profiles. However, few neuropathological series have been published, and the frequency of FTLD in MND is difficult to estimate. In this work we describe a large clinicopathologic series of MND, analysing the frequency of concurrent FTLD changes and trying to define specific subgroups of patients based on their clinical, genetic and pathological characteristics. We performed an observational, retrospective, multi-centre case study. We included all cases meeting neuropathological criteria for MND from the Neurological Tissue Bank of the FRCB-IDIBAPS-Hospital Clínic Barcelona Biobank between 1994 and 2022, regardless of their last clinical diagnosis. While brain donation is encouraged in all patients, it is performed in very few, and representativeness of the cohort might not be precise for all patients with MND. We retrospectively reviewed clinical and neuropathological data, and describe the main clinical, genetic and pathogenic features, comparing neuropathologic groups between MND with and without FTLD changes and aiming to define specific subgroups. We included brain samples from 124 patients, 44 of whom (35.5%) had FTLD neuropathologic features (i.e. FTLD-MND). Pathologic TDP-43 aggregates were present in 93.6% of the cohort and were more extensive (higher Brettschneider stage) in those with concurrent FTLD (p < 0.001). Motor symptom onset was more frequent in the bulbar region in FTLD-MND cases than in those with isolated MND (p = 0.023), with no differences in survival. We observed a better clinicopathological correlation in the MND group than in the FTLD-MND group (93.8% vs 61.4%; p < 0.001). Pathogenic genetic variants were more common in the FTLD-MND group, especially C9orf72. We describe a frequency of FTLD of 35.5% in our series of neuropathologically confirmed cases of MND. The FTLD-MND spectrum is highly heterogeneous in all aspects, especially in patients with FTLD, in whom it is particularly difficult to define specific subgroups. In the absence of definite biomarkers, neuropathology remains a valuable tool for a definite diagnosis, increasing our knowledge in disease nosology.

Clinical Classification of Variants in the Valosin-Containing Protein Gene Associated With Multisystem Proteinopathy.

Background and Objectives: Pathogenic variants in the valosin-containing protein (VCP) gene cause a phenotypically heterogeneous disorder that includes myopathy, motor neuron disease, Paget disease of the bone, frontotemporal dementia, and parkinsonism termed multisystem proteinopathy. This hallmark pleiotropy makes the classification of novel VCP variants challenging. This retrospective study describes and assesses the effect of 19 novel or nonpreviously clinically characterized VCP variants identified in 28 patients (26 unrelated families) in the retrospective VCP International Multicenter Study. Methods: A 6-item clinical score was developed to evaluate the phenotypic level of evidence to support the pathogenicity of the novel variants. Each item is allocated a value, a score ranging from 0.5 to 5.5 points. A receiver-operating characteristic curve was used to identify a cutoff value of 3 to consider a variant as high likelihood disease associated. The scoring system results were confronted with results of in vitro ATPase activity assays and with in silico analysis. Results: All variants were missense, except for one small deletion-insertion, 18 led to amino acid changes within the N and D1 domains, and 13 increased the enzymatic activity. The clinical score coincided with the functional studies in 17 of 19 variants and with the in silico analysis in 12 of 19. For 12 variants, the 3 predictive tools agreed, and for 7 variants, the predictive tools disagreed. The pooled data supported the pathogenicity of 13 of 19 novel VCP variants identified in the study. Discussion: This study provides data to support pathogenicity of 14 of 19 novel VCP variants and provides guidance for clinicians in the evaluation of novel variants in the VCP gene.

Antibodies against the flotillin-1/2 complex in patients with multiple sclerosis.

Multiple sclerosis is a tissue-specific autoimmune disease of the central nervous system in which the antigen(s) remains elusive. Antibodies targeting the flotillin-1/2 complex have been described in 1-2% of the patients in a recent study. Other candidate antigens as anoctamin-2 or neurofascin-155 have been previously described in multiple sclerosis patients, although their clinical relevance remains uncertain. Our study aims to analyse the frequency and clinical relevance of antibodies against neurofascin-155, anoctamin-2 and flotillin-1/2 complex in multiple sclerosis. Serum (n = 252) and CSF (n = 50) samples from 282 multiple sclerosis patients were included in the study. The control group was composed of 260 serum samples (71 healthy donors and 189 with other neuroinflammatory disorders). Anti-flotillin-1/2, anti-anoctamin-2 and anti-neurofascin-155 antibodies were tested by cell-based assays using transfected cells. We identified six multiple sclerosis patients with antibodies against the flotillin-1/2 complex (2.1%) and one multiple sclerosis patient with antibodies against anoctamin-2 (0.35%). All multiple sclerosis patients were negative for anti-neurofascin-155 antibodies. Three of the anti-flotillin-1/2 positive patients showed anti-flotillin-1/2 positivity in other serum samples extracted at different moments of their disease. Immunoglobulin G subclasses of anti-flotillin-1/2 antibodies were predominantly one and three. We confirm that antibodies targeting the flotillin-1/2 complex are present in a subgroup of patients with multiple sclerosis. Further studies are needed to understand the clinical and pathological relevance of anti-flotillin-1/2 autoantibodies in multiple sclerosis.

A new homozygous missense variant in LMOD3 gene causing mild nemaline myopathy with prominent facial weakness.

Nemaline myopathy (NEM) type 10, caused by biallelic mutations in LMOD3, is a severe congenital myopathy clinically characterized by generalized hypotonia and muscle weakness, respiratory insufficiency, joint contractures, and bulbar weakness. Here, we describe a family with two adult patients presenting mild nemaline myopathy due to a novel homozygous missense variant in LMOD3. Both patients presented mild delayed motor milestones, frequent falls during infancy, prominent facial weakness and mild muscle weakness in the four limbs. Muscle biopsy showed mild myopathic changes and small nemaline bodies in a few fibers. A neuromuscular gene panel revealed a homozygous missense variant in LMOD3 that co-segregated with the disease in the family (NM_198271.4: c.1030C>T; p.Arg344Trp). The patients described here provide evidence of the phenotype-genotype correlation, suggesting that non-truncating variants in LMOD3 lead to milder phenotypes of NEM type 10.

Leptospirosis-induced acute acquired inflammatory neuropathy.

Leptospirosis is a zoonotic infection that can present with neurological manifestations. Although uncommon, it may affect the peripheral nervous system in the form of polyradiculoneuropathy. We report the case of a 30-year-old male who developed flaccid tetraparesis and multiple cranial neuropathies on the fourteenth day of admission to the intensive care unit for fever and multi-organ failure. We also review the existing literature about peripheral nerve damage in leptospirosis and present our hypothesis on the possible pathogenic mechanisms. Electrophysiological findings were consistent with acute demyelinating polyradiculoneuropathy and extensive blood tests were positive for leptospiral IgM and IgG antibodies. Treatment with plasmapheresis was begun, followed by intravenous immunoglobulin (IVIg), and the patient improved slowly. Our work adds to the evidence of leptospirosis infection as a cause of acute demyelinating polyneuropathy. The possibility that leptospirosis-polyradiculoneuropathy may be caused by an immune pathogenesis emphasizes the importance of identifying this entity because immunomodulatory therapy could play a vital role in the recovery process.

Genotype-phenotype correlations in valosin-containing protein disease: a retrospective muticentre study.

BACKGROUND: Valosin-containing protein (VCP) disease, caused by mutations in the VCP gene, results in myopathy, Paget's disease of bone (PBD) and frontotemporal dementia (FTD). Natural history and genotype-phenotype correlation data are limited. This study characterises patients with mutations in VCP gene and investigates genotype-phenotype correlations. METHODS: Descriptive retrospective international study collecting clinical and genetic data of patients with mutations in the VCP gene. RESULTS: Two hundred and fifty-five patients (70.0% males) were included in the study. Mean age was 56.8±9.6 years and mean age of onset 45.6±9.3 years. Mean diagnostic delay was 7.7±6 years. Symmetric lower limb weakness was reported in 50% at onset progressing to generalised muscle weakness. Other common symptoms were ventilatory insufficiency 40.3%, PDB 28.2%, dysautonomia 21.4% and FTD 14.3%. Fifty-seven genetic variants were identified, 18 of these no previously reported. c.464G>A (p.Arg155His) was the most frequent variant, identified in the 28%. Full time wheelchair users accounted for 19.1% with a median time from disease onset to been wheelchair user of 8.5 years. Variant c.463C>T (p.Arg155Cys) showed an earlier onset (37.8±7.6 year) and a higher frequency of axial and upper limb weakness, scapular winging and cognitive impairment. Forced vital capacity (FVC) below 50% was as risk factor for being full-time wheelchair user, while FVC <70% and being a full-time wheelchair user were associated with death. CONCLUSION: This study expands the knowledge on the phenotypic presentation, natural history, genotype-phenotype correlations and risk factors for disease progression of VCP disease and is useful to improve the care provided to patient with this complex disease.

Rituximab in myasthenia gravis: efficacy, associated infections and risk of induced hypogammaglobulinemia.

The aim of this study is to evaluate the long-term efficacy, safety, and impact on immunoglobulin G (IgG) levels of rituximab in patients with myasthenia gravis (MG). A retrospective, observational study of drug-refractory MG patients treated with rituximab was done. The MG Foundation of America postintervention status (MGFA-PIS) was used to evaluate clinical response. Serum IgG levels were determined at baseline and post-treatment. Hypogammaglobulinemia was defined as IgG<7g/L. Thirty patients were included, 12 with anti-MuSK and 18 with anti-AChR antibodies. Mean (SD) follow-up was 85.5 (48) months. All 12 MuSK+ patients but only six (33%) AChR+ patients achieved minimal manifestations or remission (p<0.01). Nine severe infections were observed in five patients (17%). One patient was diagnosed with progressive multifocal leukoencephalopathy. At baseline, two patients (2/24; 8%) had hypogammaglobulinemia. During follow-up, hypogammaglobulinemia was observed in 60% (3/5) of patients who developed an infection and in 33% (7/21) who did not. Two of these patients died of infection-related complications. This study supports the effectiveness of rituximab in patients with MG, especially those with anti-MuSK antibodies. Severe infections may appear after rituximab treatment and hypogammaglobulinemia might play a role on it. A standard protocol would be needed to closely monitor IgG levels in MG patients treated with rituximab.

Immune Response and Safety of SARS-CoV-2 mRNA-1273 Vaccine in Patients With Myasthenia Gravis.

BACKGROUND AND OBJECTIVES: Evidence regarding the safety and efficacy of messenger RNA (mRNA) vaccines in patients with myasthenia gravis (MG) after immunosuppressive therapies is scarce. Our aim is to determine whether the mRNA-1273 vaccine is safe and able to induce humoral and cellular responses in patients with MG. METHODS: We performed an observational, longitudinal, prospective study including 100 patients with MG of a referral center for MG in our country, conducted from April 2021 to November 2021 during the vaccination campaign. The mRNA-1273 vaccine was scheduled for all participants. Blood samples were collected before vaccination and 3 months after a second dose. Clinical changes in MG were measured using the MG activities of daily life score at baseline and 1 week after the first and second doses. A surveillance of all symptoms of coronavirus disease 2019 (COVID-19) was conducted throughout the study. Humoral and cellular immune responses after vaccination were assessed using a spike-antibody ELISA and interferon gamma release assay in plasma. The primary outcomes were clinically significant changes in MG symptoms after vaccination, adverse events (AEs), and seroconversion and T-cell immune response rates. RESULTS: Ninety-nine patients completed the full vaccination schedule, and 98 had 2 blood samples taken. A statistically significant worsening of symptoms was identified after the first and second doses of the mRNA-1273 vaccine, but this was not clinically relevant. Mild AEs occurred in 14 patients after the first dose and in 21 patients after the second dose. Eighty-seven patients developed a humoral response and 72 patients showed a T-cell response after vaccination. A combined therapy with prednisone and other immunosuppressive drugs correlated with a lower seroconversion ratio (OR = 5.97, 95% CI 1.46-24.09, p = 0.015) and a lower T-cell response ratio (OR = 2.83, 95% CI 1.13-7.13, p = 0.024). DISCUSSION: Our findings indicate that the mRNA vaccination against COVID-19 is safe in patients with MG and show no negative impact on the disease course. Patients achieved high humoral and cellular immune response levels. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that patients with MG receiving the mRNA-1273 vaccine did not show clinical worsening after vaccination and that most of the patients achieved high cellular or immune response levels.

High prevalence of paraspinal muscle involvement in adults with McArdle disease.

INTRODUCTION/AIMS: Very few studies analyzing the pattern of muscle involvement in magnetic resonance imaging (MRI) of patients with McArdle disease have been reported to date. We aimed to examine the pattern of muscle fat replacement in patients with McArdle disease. METHODS: We performed a retrospective study including all patients with genetically confirmed McArdle disease followed in our center from January 2010 to March 2021. Clinical data were collected from the medical record. Whole-body MRI was performed as part of the diagnostic evaluation. The distribution of muscle fat replacement and its severity were analyzed. RESULTS: Nine patients were included. Median age at onset was 7 y (range, 5-58) and median age at the time when MRI was performed was 57.3 y (range, 37.2-72.8). At physical examination, four patients had permanent weakness: in three the weakness was limited to paraspinal muscles, whereas in one the weakness involved the paraspinal and proximal upper limb muscles. Muscle MRI showed abnormalities in six of the seven studied patients. In all of them, fat replacement of paravertebral muscles was found. Other muscles frequently affected were the tongue in three, subscapularis in three, and long head of biceps femoris and semimembranosus in two. DISCUSSION: Our findings suggest that paraspinal muscle involvement is common in McArdle disease and support the need to include this disease in the differential diagnosis of the causes of paraspinal muscle weakness. Involvement of the tongue and subscapularis are also frequent in McArdle disease.

Clinical and Laboratory Features in Anti-NF155 Autoimmune Nodopathy.

BACKGROUND AND OBJECTIVES: To study the clinical and laboratory features of antineurofascin-155 (NF155)-positive autoimmune nodopathy (AN). METHODS: Patients with anti-NF155 antibodies detected on routine immunologic testing were included. Clinical characteristics, treatment response, and functional scales (modified Rankin Scale [mRS] and Inflammatory Rasch-built Overall Disability Scale [I-RODS]) were retrospectively collected at baseline and at the follow-up. Autoantibody and neurofilament light (NfL) chain levels were analyzed at baseline and at the follow-up. RESULTS: Forty NF155+ patients with AN were included. Mean age at onset was 42.4 years. Patients presented with a progressive (75%), sensory motor (87.5%), and symmetric distal-predominant weakness in upper (97.2%) and lower extremities (94.5%), with tremor and ataxia (75%). Patients received a median of 3 (2-4) different treatments in 46 months of median follow-up. Response to IV immunoglobulin (86.8%) or steroids (72.2%) was poor in most patients, whereas 77.3% responded to rituximab. HLA-DRB1*15 was detected in 91.3% of patients. IgG4 anti-NF155 antibodies were predominant in all patients; anti-NF155 titers correlated with mRS within the same patient (r = 0.41, p = 0.004). Serum NfL (sNfL) levels were higher in anti-NF155+ AN than in healthy controls (36.47 vs 7.56 pg/mL, p < 0.001) and correlated with anti-NF155 titers (r = 0.43, p = 0.001), with I-RODS at baseline (r = -0.88, p < 0.001) and with maximum I-RODS achieved (r = -0.58, p = 0.01). Anti-NF155 titers and sNfL levels decreased in all rituximab-treated patients. DISCUSSION: Anti-NF155 AN presents a distinct clinical profile and good response to rituximab. Autoantibody titers and sNfL are useful to monitor disease status in these patients. The use of untagged-NF155 plasmids minimizes the detection of false anti-NF155+ cases. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that anti-NF155 antibodies associate with a specific phenotype and response to rituximab.