Nakajo-Nishimura syndrome (NNS) is a disorder that segregates in an autosomal recessive fashion. Symptoms include periodic fever, skin rash, partial lipomuscular atrophy, and joint contracture. Here, we report a mutation in the human proteasome subunit beta type 8 gene (PSMB8) that encodes the immunoproteasome subunit β5i in patients with NNS. This G201V mutation disrupts the β-sheet structure, protrudes from the loop that interfaces with the β4 subunit, and is in close proximity to the catalytic threonine residue. The β5i mutant is not efficiently incorporated during immunoproteasome biogenesis, resulting in reduced proteasome activity and accumulation of ubiquitinated and oxidized proteins within cells expressing immunoproteasomes. As a result, the level of interleukin (IL)-6 and IFN-γ inducible protein (IP)-10 in patient sera is markedly increased. Nuclear phosphorylated p38 and the secretion of IL-6 are increased in patient cells both in vitro and in vivo, which may account for the inflammatory response and periodic fever observed in these patients. These results show that a mutation within a proteasome subunit is the direct cause of a human disease and suggest that decreased proteasome activity can cause inflammation.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. Accumulating evidence has shown that 43kDa TAR-DNA-binding protein (TDP-43) is the disease protein in ALS and frontotemporal lobar degeneration. We previously reported a familial ALS with Bumina bodies and TDP-43-positive skein-like inclusions in the lower motor neurons; these findings are indistinguishable from those of sporadic ALS. In three affected individuals in two generations of one family, we found a single base-pair change from A to G at position 1028 in TDP-43, which resulted in a Gln-to-Arg substitution at position 343. Our findings provide a new insight into the molecular pathogenesis of ALS.

Choline acetyltransferase (ChAT; EC 2.3.1.6 ) catalyzes the reversible synthesis of acetylcholine (ACh) from acetyl CoA and choline at cholinergic synapses. Mutations in genes encoding ChAT affecting motility exist in Caenorhabditis elegans and Drosophila , but no CHAT mutations have been observed in humans to date. Here we report that mutations in CHAT cause a congenital myasthenic syndrome associated with frequently fatal episodes of apnea (CMS-EA). Studies of the neuromuscular junction in this disease show a stimulation-dependent decrease of the amplitude of the miniature endplate potential and no deficiency of the ACh receptor. These findings point to a defect in ACh resynthesis or vesicular filling and to CHAT as one of the candidate genes. Direct sequencing of CHAT reveals 10 recessive mutations in five patients with CMS-EA. One mutation (523insCC) is a frameshifting null mutation. Three mutations (I305T, R420C, and E441K) markedly reduce ChAT expression in COS cells. Kinetic studies of nine bacterially expressed ChAT mutants demonstrate that one mutant (E441K) lacks catalytic activity, and eight mutants (L210P, P211A, I305T, R420C, R482G, S498L, V506L, and R560H) have significantly impaired catalytic efficiencies.

Recently, 43-kDa TAR DNA-binding protein (TDP-43) was identified as a component of ubiquitinated inclusions (UIs) in sporadic amyotrophic lateral sclerosis (SALS). To clarify whether TDP-43 immunoreactivity is present in neuronal inclusions in familial ALS (FALS), we examined immunohistochemically the brains and spinal cords from four cases of FALS, two with Cu/Zn superoxide dismutase (SOD1) gene mutation and two without, together with three cases of SALS and three control subjects, using two antibodies, one polyclonal and one monoclonal, against TDP-43. Neuropathologically, the SOD1-related FALS cases were characterized by Lewy body-like hyaline inclusions (LBHIs) in the lower motor neurons. On the other hand, the SOD1-unrelated FALS cases showed degeneration restricted to the upper and lower motor neuron systems, with Bunina bodies (BBs) and UIs in the lower motor neurons, being indistinguishable from SALS. No cytoplasmic TDP-43 immunoreactivity was observed in the control subjects or SOD1-related FALS cases; LBHIs were ubiquitinated, but negative for TDP-43. UIs observed in the SALS and SOD1-unrelated FALS cases were clearly positive for TDP-43. BBs were negative for this protein. Interestingly, in these SALS and FALS cases, glial cells were also found to have cytoplasmic TDP-43-positive inclusions. These findings indicate that the histological and molecular pathology of SALS can occur as a phenotype of FALS without SOD1 mutation.

In skeletal muscle, acetylcholinesterase (AChE) exists in homomeric globular forms of type T catalytic subunits (ACHE T ) and heteromeric asymmetric forms composed of 1, 2, or 3 tetrameric ACHE T attached to a collagenic tail (ColQ). Asymmetric AChE is concentrated at the endplate (EP), where its collagenic tail anchors it into the basal lamina. The ACHE T gene has been cloned in humans; COLQ cDNA has been cloned in Torpedo and rodents but not in humans. In a disabling congenital myasthenic syndrome, EP AChE deficiency (EAD), the normal asymmetric species of AChE are absent from muscle. EAD could stem from a defect that prevents binding of ColQ to ACHE T or the insertion of ColQ into the basal lamina. In six EAD patients, we found no mutations in ACHE T . We therefore cloned human COLQ cDNA, determined the genomic structure and chromosomal localization of COLQ, and then searched for mutations in this gene. We identified six recessive truncation mutations of COLQ in six patients. Coexpression of each COLQ mutant with wild-type ACHE T in SV40-transformed monkey kidney fibroblast (COS) cells reveals that a mutation proximal to the ColQ attachment domain for ACHE T prevents association of ColQ with ACHE T ; mutations distal to the attachment domain generate a mutant Ϸ10.5S species of AChE composed of one ACHE T tetramer and a truncated ColQ strand. The Ϸ10.5S species lack part of the collagen domain and the entire C-terminal domain of ColQ, or they lack only the C-terminal domain, which is required for formation of the triple collagen helix, and this likely prevents their insertion into the basal lamina.

Congenital myasthenic syndromes (CMSs) stem from genetic defects in endplate (EP)-specific presynaptic, synaptic, and postsynaptic proteins. The postsynaptic CMSs identified to date stem from a deficiency or kinetic abnormality of the acetylcholine receptor (AChR). All CMSs with a kinetic abnormality of AChR, as well as many CMSs with a deficiency of AChR, have been traced to mutations in AChR-subunit genes. However, in a subset of patients with EP AChR deficiency, the genetic defect has remained elusive. Rapsyn, a 43-kDa postsynaptic protein, plays an essential role in the clustering of AChR at the EP. Seven tetratricopeptide repeats (TPRs) of rapsyn subserve self-association, a coiled-coil domain binds to AChR, and a RING-H2 domain associates with beta-dystroglycan and links rapsyn to the subsynaptic cytoskeleton. Rapsyn self-association precedes recruitment of AChR to rapsyn clusters. In four patients with EP AChR deficiency but with no mutations in AChR subunits, we identify three recessive rapsyn mutations: one patient carries L14P in TPR1 and N88K in TPR3; two are homozygous for N88K; and one carries N88K and 553ins5, which frameshifts in TPR5. EP studies in each case show decreased staining for rapsyn and AChR, as well as impaired postsynaptic morphological development. Expression studies in HEK cells indicate that none of the mutations hinders rapsyn self-association but that all three diminish coclustering of AChR with rapsyn.

In a myasthenic syndrome associated with fatigable generalized weakness and recurrent attacks of respiratory and bulbar paralysis since birth, nerve stimulation at physiologic rates rapidly decremented the compound muscle action potential. Intercostal muscle studies revealed no abnormality of the resting membrane potential, evoked quantal release, synaptic potentials, acetylcholine receptor channel kinetics, or endplate ultrastructure, but endplate potentials depolarizing the resting potential to ؊40 mV failed to excite action potentials. Pursuing this clue, we sequenced SCN4A encoding the skeletal muscle sodium channel (Na v1.4) and detected two heteroallelic mutations involving conserved residues not present in 400 normal alleles: S246L in the S4͞S5 cytoplasmic linker in domain I, and V1442E in the S3͞S4 extracellular linker in domain IV. The genetically engineered V1442E-Na channel expressed in HEK cells shows marked enhancement of fast inactivation close to the resting potential, and enhanced use-dependent inactivation on high-frequency stimulation; S246L is likely a benign polymorphism. The V1442E mutation in SCN4A defines a novel disease mechanism and a novel phenotype with myasthenic features.

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