Objective:

Charcot-Marie-Tooth (CMT) disease is the most common inherited neuromuscular disorder, affecting 1 in 2,500 individuals. Mitochondrial DNA (mtDNA) mutations are not generally considered within the differential diagnosis of patients with uncomplicated inherited neuropathy, despite the essential requirement of ATP for axonal function. We identified the mtDNA mutation m.9185T>C in MT-ATP6, encoding the ATP6 subunit of the mitochondrial ATP synthase (OXPHOS complex V), at homoplasmic levels in a family with mitochondrial disease in whom a severe motor axonal neuropathy was a striking feature. This led us to hypothesize that mutations in the 2 mtDNA complex V subunit encoding genes, MT-ATP6 and MT-ATP8, might be an unrecognized cause of isolated axonal CMT and distal hereditary motor neuropathy (dHMN).

Methods:

A total of 442 probands with CMT type 2 (CMT2) (270) and dHMN (172) were screened for MT-ATP6/8 mutations after exclusion of mutations in known CMT2/dHMN genes. Mutation load was quantified using restriction endonuclease analysis. Blue-native gel electrophoresis (BN-PAGE) was performed to analyze the effects of m.9185T>C on complex V structure and function.

Results:

Three further probands with CMT2 harbored the m.9185T>C mutation. Some relatives had been classified as having dHMN. Patients could be separated into 4 groups according to their mutant m.9185T>C levels. BN-PAGE demonstrated both impaired assembly and reduced activity of the complex V holoenzyme.

Conclusions:

We have shown that m.9185T>C in MT-ATP6 causes CMT2 in 1.1% of genetically undefined cases. This has important implications for diagnosis and genetic counseling. Recognition that mutations in MT-ATP6 cause CMT2 enhances current understanding of the pathogenic basis of axonal neuropathy.

Congenital myasthenic syndromes are a heterogeneous group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle weakness. We performed linkage analysis, whole-exome and whole-genome sequencing to determine the underlying defect in patients with an inherited limb-girdle pattern of myasthenic weakness. We identify ALG14 and ALG2 as novel genes in which mutations cause a congenital myasthenic syndrome. Through analogy with yeast, ALG14 is thought to form a multiglycosyltransferase complex with ALG13 and DPAGT1 that catalyses the first two committed steps of asparagine-linked protein glycosylation. We show that ALG14 is concentrated at the muscle motor endplates and small interfering RNA silencing of ALG14 results in reduced cell-surface expression of muscle acetylcholine receptor expressed in human embryonic kidney 293 cells. ALG2 is an alpha-1,3-mannosyltransferase that also catalyses early steps in the asparagine-linked glycosylation pathway. Mutations were identified in two kinships, with mutation ALG2p.Val68Gly found to severely reduce ALG2 expression both in patient muscle, and in cell cultures. Identification of DPAGT1, ALG14 and ALG2 mutations as a cause of congenital myasthenic syndrome underscores the importance of asparagine-linked protein glycosylation for proper functioning of the neuromuscular junction. These syndromes form part of the wider spectrum of congenital disorders of glycosylation caused by impaired asparagine-linked glycosylation. It is likely that further genes encoding components of this pathway will be associated with congenital myasthenic syndromes or impaired neuromuscular transmission as part of a more severe multisystem disorder. Our findings suggest that treatment with cholinesterase inhibitors may improve muscle function in many of the congenital disorders of glycosylation.

Multiple sclerosis is a chronic inflammatory neurological condition characterized by focal and diffuse neurodegeneration and demyelination throughout the central nervous system. Factors influencing the progression of pathology are poorly understood. One hypothesis is that anatomical connectivity influences the spread of neurodegeneration. This predicts that measures of neurodegeneration will correlate most strongly between interconnected structures. However, such patterns have been difficult to quantify through post-mortem neuropathology or in vivo scanning alone. In this study, we used the complementary approaches of whole brain post-mortem magnetic resonance imaging and quantitative histology to assess patterns of multiple sclerosis pathology. Two thalamo-cortical projection systems were considered based on their distinct neuroanatomy and their documented involvement in multiple sclerosis: lateral geniculate nucleus to primary visual cortex and mediodorsal nucleus of the thalamus to prefrontal cortex. Within the anatomically distinct thalamo-cortical projection systems, magnetic resonance imaging derived cortical thickness was correlated significantly with both a measure of myelination in the connected tract and a measure of connected thalamic nucleus cell density. Such correlations did not exist between these markers of neurodegeneration across different thalamo-cortical systems. Magnetic resonance imaging lesion analysis depicted clearly demarcated subcortical lesions impinging on the white matter tracts of interest; however, quantitation of the extent of lesion-tract overlap failed to demonstrate any appreciable association with the severity of markers of diffuse pathology within each thalamo-cortical projection system. Diffusion-weighted magnetic resonance imaging metrics in both white matter tracts were correlated significantly with a histologically derived measure of tract myelination. These data demonstrate for the first time the relevance of functional anatomical connectivity to the spread of multiple sclerosis pathology in a ‘tract-specific’ pattern. Furthermore, the persisting relationship between metrics from post-mortem diffusion-weighted magnetic resonance imaging and histological measures from fixed tissue further validates the potential of imaging for future neuropathological studies.

Background

Neuromyelitis optica (NMO) is a demyelinating disease of the central nervous system (CNS) of putative autoimmune aetiology. Early discrimination between multiple sclerosis (MS) and NMO is important, as optimum treatment for both diseases may differ considerably. Recently, using indirect immunofluorescence analysis, a new serum autoantibody (NMO-IgG) has been detected in NMO patients. The binding sites of this autoantibody were reported to colocalize with aquaporin 4 (AQP4) water channels. Thus we hypothesized that AQP4 antibodies in fact characterize NMO patients.

Methods and Findings

Based on these observations we cloned human water channel AQP4, expressed the protein in a eukaryotic transcription/translation system, and employed the recombinant AQP4 to establish a new radioimmunoprecipitation assay (RIPA). Indeed, application of this RIPA showed that antibodies against AQP4 exist in the majority of patients with NMO (n = 37; 21 positive) as well as in patients with isolated longitudinally extensive transverse myelitis (n = 6; six positive), corresponding to a sensitivity of 62.8% and a specificity of 98.3%. By contrast, AQP4 antibodies were virtually absent in 291 other participants, which included patients with MS (n = 144; four positive), patients with other inflammatory and noninflammatory neurological diseases (n = 73; one positive), patients with systemic autoimmune diseases (n = 45; 0 positive), and healthy participants (n = 29; 0 positive).

Conclusions

In the largest series reported so far to our knowledge, we quantified AQP4 antibodies in patients with NMO versus various other diseases, and showed that the aquaporin 4 water channel is a target antigen in a majority of patients with NMO. The newly developed assay represents a highly specific, observer-independent, and easily reproducible detection method facilitating clinically relevant discrimination between NMO, MS, and other inflammatory diseases.

A newly developed method to detect antibodies to the aquaporin 4 water channel can help discriminate between neuromyelitis optica, multiple sclerosis, and other inflammatory diseases.

Editors' Summary

Background.

Neuromyelitis optica (NMO or Devic syndrome) is a rare disease in which the immune system destroys the myelin (fatty material that insulates nerve fibers so that the body and the brain can communicate using electrical messages) in the optic nerve and spinal cord. Myelin destruction (demyelination) in these parts of the central nervous system (CNS) causes pain and swelling (inflammation) of the optic nerve (optic neuritis) and spinal cord (myelitis). The resultant disruption of communication along these nerves means that patients with NMO experience temporary or permanent blindness in one or both eyes that is preceded or followed by limb weakness or paralysis and loss of bladder and bowel control. These two sets of symptoms can occur many months apart and may happen once during a person's lifetime or recur at intervals. There is no cure for NMO, but corticosteroids or plasmapheresis reduce inflammation during acute attacks and, because NMO is an autoimmune disease (one in which the immune system attacks the body's own tissues instead of foreign organisms), long-term immunosuppression may prevent further attacks.

Why Was This Study Done?

There are many inflammatory/demyelinating diseases of the CNS with clinical symptoms similar to those of NMO. It is particularly hard to distinguish between NMO and multiple sclerosis, an autoimmune disease that involves widespread demyelination. Neurologists need to make a correct diagnosis before starting any treatment and usually use clinical examination and magnetic resonance imaging (to detect sites of inflammation) to help them in this task. Recently, however, a biomarker for NMO was identified. Many patients with NMO make autoantibodies (proteins that recognize a component of a person's own tissues) called NMO-IgGs. These recognize aquaporin 4 (AQP4), a protein that allows water to move through cell membranes. It is not known how often patients with NMO or other demyelinating diseases make antibodies to AQP4, so it is unclear whether testing for these antibodies would help in the diagnosis of NMO. In this study, the researchers have developed a new assay for antibodies to AQP4 and then quantified the antibodies in patients with NMO and other demyelinating diseases.

What Did the Researchers Do and Find?

The researchers made radioactively labeled AQP4 in a test tube, then incubated samples of this with serum (the liquid portion of blood), added small beads coated with protein A (a bacterial protein that binds to antibodies) and allowed the beads to settle. The amount of radioactivity attached to the beads indicates the amount of antibody to AQP4 in the original serum. The researchers used this radioimmunoprecipitation assay to measure antibodies to AQP4 in sera from 37 patients with NMO and from six with another neurological condition, longitudinally extensive transverse myelitis (LETM), which is characterized by large demyelinated lesions across the width of the spinal cord but no optic neuritis; these patients often develop NMO. They also measured antibodies to AQP4 in the sera of nearly 300 other people including patients with multiple sclerosis, other neurological conditions, various autoimmune diseases, and healthy individuals. Nearly two-thirds of the patients with NMO and all those with LETM made antibodies against AQP4; very few of the other study participants made these antibodies. In particular, only four of the 144 patients with multiple sclerosis made AQP4 antibodies.

What Do These Findings Mean?

These findings indicate that testing for antibodies to AQP4 could help neurologists distinguish between NMO and multiple sclerosis and between NMO and other demyelinating diseases of the CNS. In addition, the new radioimmunoprecipitation assay provides a standardized, high-throughput way to quantitatively test for these antibodies, whereas the indirect immune fluorescence assay for measurement of unspecific NMO-IgG is observer-dependent and nonquantitative. Although these findings need to be confirmed in more patients and the assay's reliability demonstrated in different settings, the measurement of antibodies to AQP4 by radioimmunoprecipitation may become a standard part of the differential diagnosis of NMO. Additional research will determine whether AQP4 is the only protein targeted by autoantibodies in NMO and whether this targeting is a critical part of the disease process.

Additional Information.

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040133.

US National Institute of Neurological Disorders and Stroke has information for patients who have neuromyelitis optica, transverse myelitis, and multiple sclerosis

The Transverse Myelitis Association offers information and useful links for patients and their carers about transverse myelitis and neuromyelitis optica (in several languages, including English and Spanish)

Mayo Clinic information for patients on Devic's syndrome

Medline Plus encyclopedia pages discuss autoimmune disorders (in English and Spanish)

A brief overview of aquaporins is available from the University of Miami

The American MS Society has information on MS