Recognising the spectrum of scleromyositis: HEp-2 ANA patterns allow identification of a novel clinical subset with anti-SMN autoantibodies.

OBJECTIVE: To describe systemic sclerosis (SSc) with myopathy in patients without classic SSc-specific and SSc-overlap autoantibodies (aAbs), referred to as seronegative scleromyositis. METHODS: Twenty patients with seronegative scleromyositis diagnosed by expert opinion were analysed retrospectively for SSc features at myositis diagnosis and follow-up, and stratified based on HEp-2 nuclear patterns by indirect immunofluorescence (IIF) according to International Consensus of Autoantibody Patterns. Specificities were analysed by protein A-assisted immunoprecipitation. Myopathy was considered an organ involvement of SSc. RESULTS: SSc sine scleroderma was a frequent presentation (45%) at myositis diagnosis. Myositis was the most common first non-Raynaud manifestation of SSc (55%). Lower oesophagal dysmotility was present in 10 of 11 (91%) investigated patients. At follow-up, 80% of the patients met the American College of Rheumatology/EULAR SSc classification criteria. Two-thirds of patients had a positive HEp-2 IIF nuclear pattern (all with titers ≥1/320), defining three novel scleromyositis subsets. First, antinuclear antibody (ANA)-negative scleromyositis was associated with interstitial lung disease (ILD) and renal crisis. Second, a speckled pattern uncovered multiple rare SSc-specific aAbs. Third, the nuclear dots pattern was associated with aAbs to survival of motor neuron (SMN) complex and a novel scleromyositis subset characteriszed by calcinosis but infrequent ILD and renal crisis. CONCLUSIONS: SSc skin involvement is often absent in early seronegative scleromyositis. ANA positivity, Raynaud phenomenon, SSc-type capillaroscopy and/or lower oesophagal dysmotility may be clues for scleromyositis. Using HEp-2 IIF patterns, three novel clinicoserological subsets of scleromyositis emerged, notably (1) ANA-negative, (2) ANA-positive with a speckled pattern and (3) ANA-positive with nuclear dots and anti-SMN aAbs.

The gemin2-binding site on SMN protein: accessibility to antibody.

Reduced levels of SMN (survival-of-motor-neurons) protein are the cause of spinal muscular atrophy, an inherited disorder characterised by loss of motor neurons in early childhood. SMN associates with more than eight other proteins to form an RNA-binding complex involved in assembly of the spliceosome. Two monoclonal antibodies (mAbs), MANSMA1 and MANSMA12, have been widely-used in studies of SMN function and their precise binding sites on SMN have now been identified using a phage-displayed peptide library. The amino-acid residues in SMN required for antibody binding are the same as the five most important contact residues for interaction with gemin2. MANSMA12 immuno-precipitated SMN and gemin2 from HeLa cell extracts as efficiently as mAbs against other SMN epitopes or against gemin2. We explain this by showing that SMN exists as large multimeric complexes. This SMN epitope is highly-conserved and identical in human and mouse. To explain the vigorous immune response when mice are immunised with recombinant SMN alone, we suggest this region is masked by gemin2, or a related protein, throughout development, preventing its recognition as a "self-antigen". The epitope for a third mAb, MANSMA3, has been located to eight amino-acids in the proline-rich domain of SMN.

Implication of the SMN complex in the biogenesis and steady state level of the signal recognition particle.

Spinal muscular atrophy is a severe motor neuron disease caused by reduced levels of the ubiquitous Survival of MotoNeurons (SMN) protein. SMN is part of a complex that is essential for spliceosomal UsnRNP biogenesis. Signal recognition particle (SRP) is a ribonucleoprotein particle crucial for co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum. SRP biogenesis is a nucleo-cytoplasmic multistep process in which the protein components, except SRP54, assemble with 7S RNA in the nucleolus. Then, SRP54 is incorporated after export of the pre-particle into the cytoplasm. The assembly factors necessary for SRP biogenesis remain to be identified. Here, we show that 7S RNA binds to purified SMN complexes in vitro and that SMN complexes associate with SRP in cellular extracts. We identified the RNA determinants required. Moreover, we report a specific reduction of 7S RNA levels in the spinal cord of SMN-deficient mice, and in a Schizosaccharomyces pombe strain carrying a temperature-degron allele of SMN. Additionally, microinjected antibodies directed against SMN or Gemin2 interfere with the association of SRP54 with 7S RNA in Xenopus laevis oocytes. Our data show that reduced levels of the SMN protein lead to defect in SRP steady-state level and describe the SMN complex as the first identified cellular factor required for SRP biogenesis.

Autoantibodies to survival of motor neuron complex in patients with polymyositis: immunoprecipitation of D, E, F, and G proteins without other components of small nuclear ribonucleoproteins.

OBJECTIVE: Autoantibodies in the systemic rheumatic diseases are clinically useful biomarkers of the diagnosis or of certain clinical characteristics. An unusual pattern of immunoprecipitation, in which the D, E, F, and G proteins of small nuclear RNPs (snRNP) but without other components of the snRNP, was noticed at the autoantibody screening. The purpose of this study was to examine the target antigens and clinical manifestations associated with this specificity. METHODS: Autoantibodies in sera from 1,966 American patients (including 434 with systemic lupus erythematosus, 121 with scleroderma, 86 with polymyositis/dermatomyositis [PM/DM]) and 248 Italian patients with autoimmune diseases were screened by immunoprecipitation of (35) S-methionine-labeled cell extracts. Sera with which D, E, F, and G proteins of snRNP was immunoprecipitated, but without the other snRNP proteins, were further examined by analysis of RNA components by immunoprecipitation (silver staining), Western blotting using survival of motor neuron (SMN) complex, and immunofluorescence. RESULTS: Three sera that immunoprecipitated D, E, F, and G proteins without other components (U1-70K, A, B'/B, C) of the snRNP were found. Four additional proteins (130 kd, 120 kd, 38 kd, and 33 kd) were also commonly immunoprecipitated. The target antigen was identified as SMN complex (Gemin 3, Gemin 4, SMN, and Gemin 2, respectively), which plays a critical role in the assembly of snRNP. In immunofluorescence analyses, all 3 sera showed nuclear dots (Cajal bodies) and cytoplasmic staining. Only 1 serum was weakly positive on Western blotting of SMN, suggesting that these sera mainly recognize native molecule or quaternary structure. All 3 patients were white women with PM, an interesting finding, since deletion or mutation of SMN is known to cause spinal muscular atrophy. CONCLUSION: SMN complex was identified as a new Cajal body autoantigen recognized by sera from white patients with PM. The biologic and clinical significance of anti-SMN autoantibodies will need to be clarified.

Detection of human survival motor neuron (SMN) protein in mice containing the SMN2 transgene: applicability to preclinical therapy development for spinal muscular atrophy.

Spinal muscular atrophy (SMA), the leading genetic cause of infant death results from loss of spinal motor neurons causing atrophy of skeletal muscle. SMA is caused by loss of the Survival Motor Neuron 1 (SMN1) gene, however, an identically coding gene called SMN2 is retained, but is alternatively spliced to produce approximately 90% truncated protein. Most SMA translational and preclinical drug development has relied on the use of SMA mice to determine changes in SMN protein levels. However, the SMA mouse models are relatively severe and analysis of SMN-inducing compounds is confounded by the early mortality of these animals. An antibody that could detect SMN protein on a Smn background could circumvent this limitation and allow unaffected, heterozygous animals to be examined. Here we describe the generation and characterization of a monoclonal anti-SMN antibody, 4F11, which specifically recognizes human SMN protein. 4F11 detects SMN (human) but not native Smn (mouse) protein in SMN2 transgenic mice and in SMA cell lines. We demonstrate the feasibility of using 4F11 to detect changes in SMN2-derived SMN protein in SMA patient fibroblasts and in healthy SMN2 transgenic mice. This antibody is, therefore, an excellent tool for examining SMN2-inducing therapeutics in patient cells as well as in transgenic mice.