A Compound Heterozygous Mutation in Calpain 1 Identifies a New Genetic Cause for Spinal Muscular Atrophy Type 4 (SMA4).

Spinal Muscular Atrophy (SMA) is a heterogeneous group of neuromuscular diseases characterized by degeneration of anterior horn cells of the spinal cord, leading to muscular atrophy and weakness. Although the major cause of SMA is autosomal recessive exon deletions or loss-of-function mutations of survival motor neuron 1 (SMN1) gene, next generation sequencing technologies are increasing the genetic heterogeneity of SMA. SMA type 4 (SMA4) is an adult onset, less severe form of SMA for which genetic and pathogenic causes remain elusive.Whole exome sequencing in a 30-year-old brother and sister with SMA4 identified a compound heterozygous mutation (p. G492R/p. F610C) in calpain-1 (CAPN1). Mutations in CAPN1 have been previously associated with cerebellar ataxia and hereditary spastic paraplegia. Using skin fibroblasts from a patient bearing the p. G492R/p. F610C mutation, we demonstrate reduced levels of CAPN1 protein and protease activity. Functional characterization of the SMA4 fibroblasts revealed no changes in SMN protein levels and subcellular distribution. Additional cellular pathways associated with SMA remain unaffected in the patient fibroblasts, highlighting the tissue specificity of CAPN1 dysfunction in SMA4 pathophysiology. This study provides genetic and functional evidence of CAPN1 as a novel gene for the SMA4 phenotype and expands the phenotype of CAPN1 mutation disorders.

Patient autoantibodies deplete postsynaptic muscle-specific kinase leading to disassembly of the ACh receptor scaffold and myasthenia gravis in mice.

The postsynaptic muscle-specific kinase (MuSK) coordinates formation of the neuromuscular junction (NMJ) during embryonic development. Here we have studied the effects of MuSK autoantibodies upon the NMJ in adult mice. Daily injections of IgG from four MuSK autoantibody-positive myasthenia gravis patients (MuSK IgG; 45 mg day(1)i.p. for 14 days) caused reductions in postsynaptic ACh receptor (AChR) packing as assessed by fluorescence resonance energy transfer (FRET). IgG from the patients with the highest titres of MuSK autoantibodies caused large (51-73%) reductions in postsynaptic MuSK staining (cf. control mice; P < 0.01) and muscle weakness. Among mice injected for 14 days with control and MuSK patient IgGs, the residual level of MuSK correlated with the degree of impairment of postsynaptic AChR packing. However, the loss of postsynaptic MuSK preceded this impairment of postsynaptic AChR. When added to cultured C2 muscle cells the MuSK autoantibodies caused tyrosine phosphorylation of MuSK and the AChR beta-subunit, and internalization of MuSK from the plasma membrane. The results suggest a pathogenic mechanism in which MuSK autoantibodies rapidly deplete MuSK from the postsynaptic membrane leading to progressive dispersal of postsynaptic AChRs. Moreover, maintenance of postsynaptic AChR packing at the adult NMJ would appear to depend upon physical engagement of MuSK with the AChR scaffold, notwithstanding activation of the MuSK-rapsyn system of AChR clustering.

Proof of genetic heterogeneity in X-linked Charcot-Marie-Tooth disease.

OBJECTIVE: To characterize a large family with X-linked Charcot-Marie-Tooth (CMT) neuropathy without mutations in the gap junction protein B1 (GJB1) gene, which has an unusual phenotype that is different in some aspects from classic CMTX1. METHODS: We tested CMT families consistent with X-linked inheritance for GJB1 mutations. We compared the largest family (CMT623) without GJB1 mutation and with linkage excluding the CMTX1 locus to CMTX1 and normal individuals. RESULTS: Only 51% of probable X-linked CMT families had mutations in GJB1. Family CMT623 shows linkage to Xq26.3-q27.1 (lod score z = 6.58), a region within the previously identified locus for CMTX3, Xq26-q28. Unlike CMTX1, affected males in family CMT623 report pain and paraesthesia before the onset of sensory loss, and women are usually asymptomatic. As in CMTX1, affected males have widely ranging intermediate motor conduction velocities. The coding regions of 14 positional candidate genes within the narrowed CMTX3 locus have been excluded for a pathogenic role in the disease. CONCLUSION: This study is the first to confirm the CMTX3 locus and to refine the genetic interval to a 5.7-Mb region flanked by the markers DXS1041 and DXS8106. GJB1 mutation-negative forms of X-linked CMT, such as CMTX3, may account for a significant proportion of X-linked CMT.

Anti-beta2-glycoprotein I autoantibodies require an antigen density threshold, consistent with divalent binding.

Autoantibodies binding beta2-glycoprotein I (B2GPI) are an important finding in the antiphospholipid syndrome. While antibodies from mice or rabbits immunized with B2GPI readily bind B2GPI coated on a polystyrene microwell plate, anti-B2GPI autoantibodies only do so when using a modified microwell plate with a negatively charged surface. This study demonstrates that, for the detection of anti-B2GPI autoantibodies in an ELISA using modified plates, an antigen coating concentration threshold exists, such that minimal or no binding occurs below a certain coating concentration of antigen, even though antigen is easily demonstrable on the plate. This is consistent with the hypothesis that autoantibodies require divalent binding to B2GPI for detection, as sufficient antigen density for two protein molecules to be sufficiently close to enable divalent binding would only be expected to occur at higher coating concentrations. Several mutant forms of B2GPI developed for epitope determination experiments are shown to have decreased binding to microtitre plates compared to wild-type. If wild-type and mutants are assayed for antibody binding near the threshold a significant diminution in binding to mutants occurs that is the result of inadequate binding to the plate, but could be misinterpreted as the result of interruption of an epitope by the mutation.

Detection of 'antiphospholipid' antibodies: a single chromogenic assay of thrombin generation sensitively detects lupus anticoagulants, anticardiolipin antibodies, plus antibodies binding beta(2)-glycoprotein I and prothrombin.

The diagnosis of the antiphospholipid syndrome (APS) requires both a typical clinical event plus a persistently positive test in an assay for either anticardiolipin (aCL) antibodies or a lupus anticoagulant (LA). Enzyme linked immunosorbent assays (ELISA) specific for autoantibodies against beta(2)-glycoprotein I (beta(2)GPI) or prothrombin are also used, but none of the tests are adequately sensitive or specific. A chromogenic assay was developed that measures the effect of test antibody or plasma samples on in vitro thrombin formation. It is able to detect both LA and beta(2)GPI-dependent aCL antibodies and may have greater specificity for APS than currently available tests. Using this method various monoclonal antibodies (MoAbs) were examined, from mice immunized with beta(2)GPI, mice with a spontaneous animal model of APS, and from three humans with APS. Plasma and affinity purified antibodies from patients with APS and control groups were also examined. Thrombin inhibition was more sensitive to perturbation by MoAbs than a combination of tests for LA (P < 0.05) and at lower antibody concentrations (12.5 microg/ml versus 100 microg/ml). There was a significant correlation between inhibition of thrombin generation and the level of MoAb reactivity to beta(2)GPI (r = 0.90; P < 0.001) but not to CL (r = 0.06; P = 0.76). Plasma and affinity purified antibodies from patients with APS also inhibited thrombin generation, and significantly more so than patients with aPL from causes other than APS. APS patient samples showed thrombin inhibition in the presence of anti-beta(2)GPI or antiprothrombin antibodies. All MoAbs binding beta(2)GPI showed inhibition of thrombin generation, while MoAbs binding domain I of beta(2)GPI had more LA effect.

Mast cells/basophils in the peripheral blood of allergic individuals who are HIV-1 susceptible due to their surface expression of CD4 and the chemokine receptors CCR3, CCR5, and CXCR4.

A population of metachromatic cells with mast cell (MC) and basophil features was identified recently in the peripheral blood of patients with several allergic disorders. This study now shows that these metachromatic cells express on their surface the high-affinity IgE receptor (FcepsilonRI), CD4, and the chemokine receptors CCR3, CCR5, and CXCR4, but not the T-cell surface protein CD3 and the monocyte/macrophage surface protein CD68. This population of MCs/basophils can be maintained ex vivo for at least 2 weeks, and a comparable population of cells can be generated in vitro from nongranulated hematopoietic CD3(-)/CD4(+)/CD117(-) progenitors. Both populations of MCs/basophils are susceptible to an M-tropic strain of human immunodeficiency virus 1 (HIV-1). Finally, many patients with acquired immunodeficiency syndrome have HIV-1-infected MCs/basophils in their peripheral blood. Although it is well known that HIV-1 can infect CD4(+) T cells and monocytes, this finding is the first example of a human MC or basophil shown to be susceptible to the retrovirus. (Blood. 2001;97:3484-3490)

Reversible delayed leukoencephalopathy following intravenous heroin overdose.

We present serial neuropsychological, magnetic resonance (MR) imaging and EEG changes in a case of widespread CNS myelinopathy due to intravenous heroin overdose complicated by a period of prolonged unconsciousness. Following recovery from the acute overdose, the subject had the delayed onset of akinetic mutism with urinary incontinence. Sequential formal neuro-psychological assessments over 9 months showed evolution from severe global cerebral dysfunction to moderate disturbance of frontal lobe function. Almost complete resolution of diffuse white matter signal changes, accompanied by the development of a degree of volume loss, was evident on serial MR imaging over the same period, and generalized arrhythmic delta-range slowing on the EEG evolved int o a near normal pattern.

Impaired thrombin generation in beta 2-glycoprotein I null mice.

Autoimmune antibodies to beta(2)-glycoprotein I (beta2GPI) have been proposed to be clinically relevant because of their strong association with thrombosis, miscarriage, and thrombocytopenia. By using a homologous recombination approach, beta2GPI-null mice were generated to begin to understand the physiologic and pathologic role of this prominent plasma protein in mammals. When beta2GPI heterozygotes on a 129/Sv/C57BL/6 mixed genetic background were intercrossed, only 8.9% of the resulting 336 offspring possessed both disrupted alleles. These data suggest that beta2GPI plays a beneficial role in implantation and/or fetal development in at least some mouse strains. Although those beta2GPI-null mice that were born appeared to be relatively normal anatomically and histologically, subsequent analysis revealed that they possessed an impaired in vitro ability to generate thrombin relative to wild type mice. Thus, beta2GPI also appears to play an important role in thrombin-mediated coagulation.

Epitope studies with anti-beta 2-glycoprotein I antibodies from autoantibody and immunized sources.

This paper examines the methodology of anti-beta(2)-glycoprotein I (beta(2)-GPI) epitope determination and provides further epitope studies using human sera containing anti-beta(2)-GPI autoantibodies. Studies in this field may be misleading as the antigen coating density using mutant forms of beta(2)-GPI may be below the threshold required for monogamous divalent binding by low affinity anti-beta(2)-GPI autoantibodies, while being easily detected by high affinity anti-beta(2)-GPI from immunized animals. The antigen density threshold effect is found in anti-beta(2)-GPI autoantibodies from humans and from monoclonal anti-beta(2)-GPI derived from mice with models of autoimmune disease. Anti-beta(2)-GPI from an autoimmune mouse and from 18/21 human sera did not bind above background levels to a domain-I-deleted mutant. In addition, single point mutations in domain I result in dramatic changes in the binding of many human sera containing anti-beta(2)-GPI. These findings support a conclusion that domain I of beta(2)-GPI contains significant epitopes for the anti-beta(2)-GPI antibodies found in the antiphospholipid syndrome.

Chronic ophthalmoplegia with anti-GQ1b antibody.

Anti-GQ1b antibodies are typically found in patients with the Miller Fisher syndrome, all of whom will have, by definition, acute ophthalmoplegia. The authors describe three patients with chronic ophthalmoplegia in the presence of persistently high titers of immunoglobulin G anti-GQ1b antibody detected in an ELISA, one of whom improved with immunotherapy. Anti-GQ1b antibodies may be associated with some cases of chronic ophthalmoplegia of unknown cause.

Identification of basophilic cells that express mast cell granule proteases in the peripheral blood of asthma, allergy, and drug-reactive patients.

Metachromatic cells in the peripheral blood of patients with asthma, allergy, or an allergic drug reaction were evaluated for their nuclear morphology, surface expression of the mast cell (MC) marker c-kit, surface expression of the basophil marker Bsp-1, and granule expression of MC proteases. Consistent with previous findings by others, Bsp-1+/metachromatic cells represented <1% of the cells in the peripheral blood of normal individuals. These cells generally contained segmented nuclei. Very little, if any, tryptase (Try), chymase (Chy), or carboxypeptidase A (CPA) was found in their granules, and very little, if any, c-kit was observed on their surfaces. The number of metachromatic cells increased in the peripheral blood of the three groups of patients. Like the basophils in normal individuals, most of these metachromatic cells contained segmented nuclei and expressed Bsp-1. However, in contrast to the basophils in normal individuals, many of the metachromatic cells in the three patient groups expressed c-kit, Try, Chy, and/or CPA. That the metachromatic cells in the blood of our patients have some features of MCs and some features of basophils suggests that human basophils and MCs are derived from a common progenitor. As assessed by the chloroacetate esterase cytochemical assay, the immunoreactive Chy in the peripheral blood of these patients is enzymatically active. Because MC proteases regulate numerous immunologic and other biologic systems, the expression of Try, Chy, and/or CPA in a peripheral blood-localized cell in an individual having asthma, allergy, or an allergic drug reaction has important clinical implications.