To determine the role of complement in the disease pathology of multifocal motor neuropathy (MMN), we investigated complement activation, and inhibition, on binding of MMN patient-derived ...immunoglobulin M (IgM) antibodies in an induced pluripotent stem cell (iPSC)-derived motor neuron (MN) model for MMN.
iPSC-derived MNs were characterized for the expression of complement receptors and membrane-bound regulators, for the binding of circulating IgM anti-GM1 from patients with MMN, and for subsequent fixation of C4 and C3 on incubation with fresh serum. The potency of ARGX-117, a novel inhibitory monoclonal antibody targeting C2, to inhibit fixation of complement was assessed.
iPSC-derived MNs moderately express the complement regulatory proteins CD46 and CD55 and strongly expressed CD59. Furthermore, MNs express C3aR, C5aR, and complement receptor 1. IgM anti-GM1 antibodies in serum from patients with MMN bind to MNs and induce C3 and C4 fixation on incubation with fresh serum. ARGX-117 inhibits complement activation downstream of C4 induced by patient-derived anti-GM1 antibodies bound to MNs.
Binding of IgM antibodies from patients with MMN to iPSC-derived MNs induces complement activation. By expressing complement regulatory proteins, particularly CD59, MNs are protected against complement-mediated lysis. Yet, because of expressing C3aR, the function of these cells may be affected by complement activation upstream of membrane attack complex formation. ARGX-117 inhibits complement activation upstream of C3 in this disease model for MMN and therefore represents an intervention strategy to prevent harmful effects of complement in MMN.
To assess the association between copy number (CN) variation in the survival motor neuron (
) locus and multifocal motor neuropathy (MMN), progressive muscular atrophy (PMA), and primary lateral ...sclerosis (PLS) susceptibility and to determine the association of
and
CN with MMN, PMA, and PLS disease course.
In this monocenter study, we used multiplex ligation-dependent probe amplification to determine
and
CN in Dutch patients with MMN, PMA, and PLS and controls. We stratified clinical parameters for
and
CN. We analyzed
and
exons 1-6, intron 6, and exon 8 CN to study the genetic architecture of
duplications.
and
CN were determined in 132 patients with MMN, 150 patients with PMA, 104 patients with PLS, and 956 control subjects. MMN and PLS were not associated with CN variation in
or
. By contrast, patients with PMA more often than controls carried
duplications (≥3
copies, 12.0% vs 5.0%, odds ratio 2.69 (1.43-4.91),
0.0020).
and
CN status was not associated with MMN, PLS, or PMA disease course. In case of
exon 7 duplications, exons 1-6, exon 8, and introns 6 and 7 were also duplicated, suggesting full
duplications.
duplications are associated with PMA, but not with PLS and MMN.
duplications in PMA are balanced duplications. The results of this study highlight the primary effect of altered
CN on lower motor neurons.
Abstract
Clinical severity and treatment response vary significantly between patients with spinal muscular atrophy. The approval of therapies and the emergence of neonatal screening programmes ...urgently require a more detailed understanding of the genetic variants that underlie this clinical heterogeneity. We systematically investigated genetic variation other than SMN2 copy number in the SMN locus. Data were collected through our single-centre, population-based study on spinal muscular atrophy in the Netherlands, including 286 children and adults with spinal muscular atrophy Types 1–4, including 56 patients from 25 families with multiple siblings with spinal muscular atrophy. We combined multiplex ligation-dependent probe amplification, Sanger sequencing, multiplexed targeted resequencing and digital droplet polymerase chain reaction to determine sequence and expression variation in the SMN locus. SMN1, SMN2 and NAIP gene copy number were determined by multiplex ligation-dependent probe amplification. SMN2 gene variant analysis was performed using Sanger sequencing and RNA expression analysis of SMN by droplet digital polymerase chain reaction. We identified SMN1–SMN2 hybrid genes in 10% of spinal muscular atrophy patients, including partial gene deletions, duplications or conversions within SMN1 and SMN2 genes. This indicates that SMN2 copies can vary structurally between patients, implicating an important novel level of genetic variability in spinal muscular atrophy. Sequence analysis revealed six exonic and four intronic SMN2 variants, which were associated with disease severity in individual cases. There are no indications that NAIP1 gene copy number or sequence variants add value in addition to SMN2 copies in predicting the clinical phenotype in individual patients with spinal muscular atrophy. Importantly, 95% of spinal muscular atrophy siblings in our study had equal SMN2 copy numbers and structural changes (e.g. hybrid genes), but 60% presented with a different spinal muscular atrophy type, indicating the likely presence of further inter- and intragenic variabilities inside as well as outside the SMN locus. SMN2 gene copies can be structurally different, resulting in inter- and intra-individual differences in the composition of SMN1 and SMN2 gene copies. This adds another layer of complexity to the genetics that underlie spinal muscular atrophy and should be considered in current genetic diagnosis and counselling practices.
Clinical variability, variation in treatment response and the introduction of neonatal screening programmes urgently require a better understanding of the genetic background of spinal muscular atrophy. Here, we report that SMN2 copies are structurally different between and within patients and show that (partial) deletions, conversions and single-nucleotide variants correlate with clinical severity.
Graphical Abstract
Graphical Abstract
To investigate mutations in genes that are potential modifiers of spinal muscular atrophy (SMA) severity.
We performed a hypothesis-based search into the presence of variants in fused in sarcoma (
)
...transactive response DNA-binding protein 43 (
), plastin 3 (
), and profilin 2 (
) in a cohort of 153 patients with SMA types 1-4, including 19 families. Variants were detected with targeted next-generation sequencing and confirmed with Sanger sequencing. Functional effects of the identified variants were analyzed in silico and for PLS3, by analyzing expression levels in peripheral blood.
We identified 2 exonic variants in
exons 5 and 6 (p.R216C and p.S135N) in 2 unrelated patients, but clinical effects were not evident. We identified 8 intronic variants in
in 33 patients. Five
variants (c.1511+82T>C; c.748+130 G>A; c.367+182C>T; c.891-25T>C (rs145269469); c.1355+17A>G (rs150802596)) potentially alter exonic splice silencer or exonic splice enhancer sites. The variant c.367+182C>T, but not RNA expression levels, corresponded with a more severe phenotype in 1 family. However, this variant or level of PLS3 expression did not consistently correspond with a milder or more severe phenotype in other families or the overall cohort. We found 3 heterozygous, intronic variants in
and
with no correlation with clinical phenotype or effects on splicing.
and
sequence variants do not modify SMA severity at the population level. Specific variants in individual patients or families do not consistently correlate with disease severity.
Background and Aims
To further substantiate the role of antibody‐mediated complement activation in multifocal motor neuropathy (MMN) immunopathology, we investigated the distribution of promotor ...polymorphisms of genes encoding the membrane‐bound complement regulators CD46, CD55, and CD59 in patients with MMN and controls, and evaluated their association with disease course.
Methods
We used Sanger sequencing to genotype five common polymorphisms in the promotor regions of CD46, CD55, and CD59 in 133 patients with MMN and 380 controls. We correlated each polymorphism to clinical parameters.
Results
The genotype frequencies of rs28371582, a 21‐bp deletion in the CD55 promotor region, were altered in patients with MMN as compared to controls (p .009; Del/Del genotype 16.8% vs. 7.7%, p .005, odds ratio: 2.43 1.27–4.58), and patients carrying this deletion had a more favorable disease course (mean difference 0.26 Medical Research Council MRC points/year; 95% confidence interval CI: 0.040–0.490, p .019). The presence of CD59 rs141385724 was associated with less severe pre‐diagnostic disease course (mean difference 0.940 MRC point/year; 95% CI: 0.083–1.80, p .032).
Interpretation
MMN susceptibility is associated with a 21‐bp deletion in the CD55 promotor region (rs2871582), which is associated with lower CD55 expression. Patients carrying this deletion may have a more favorable long‐term disease outcome. Taken together, these results point out the relevance of the pre‐C5 level of the complement cascade in the inflammatory processes underlying MMN.
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