Lowell L. Williams

Charcot‐Marie‐Tooth disease and related neuropathies: Mutation distribution and genotype‐phenotype correlation

Charcot‐Marie‐Tooth disease (CMT) is a genetically heterogeneous disorder that has been associated with alterations of several proteins: peripheral myelin protein 22, myelin protein zero, connexin 32, early growth response factor 2, periaxin, myotubularin related protein 2, N‐myc downstream regulated gene 1 product, neurofilament light chain, and kinesin 1B. To determine the frequency of mutations in these genes among patients with CMT or a related peripheral neuropathy, we identified 153 unrelated patients who enrolled prior to the availability of clinical testing, 79 had a 17p12 duplication (CMT1A duplication), 11 a connexin 32 mutation, 5 a myelin protein zero mutation, 5 a peripheral myelin protein 22 mutation, 1 an early growth response factor 2 mutation, 1 a periaxin mutation, 0 a myotubularin related protein 2 mutation, 1 a neurofilament light chain mutation, and 50 had no identifiable mutation; the N‐myc downstream regulated gene 1 and the kinesin 1B gene were not screened for mutations. In the process of screening the above cohort of patients as well as other patients for CMT‐causative mutations, we identified several previously unreported mutant alleles: two for connexin 32, three for myelin protein zero, and two for peripheral myelin protein 22. The peripheral myelin protein 22 mutation W28R was associated with CMT1 and profound deafness. One patient with a CMT2 clinical phenotype had three myelin protein zero mutations (I89N+V92M+I162M). Because one‐third of the mutations we report arose de novo and thereby caused chronic sporadic neuropathy, we conclude that molecular diagnosis is a necessary adjunct for clinical diagnosis and management of inherited and sporadic neuropathy.

Serum fatty acid proportions are altered during the year following acute Epstein-Barr virus infection

Because abnormal serum fatty acid (FA) proportions had been found at three months after infectious mononucleosis (IM) in a pilot study, serum total FA profiles of 20 normal college students were measured at monthly intervals for one year following an acute Epstein-Barr virus (EBV) infection. Below normal proportions of arachidonic acid and a reversal of the usual serum ratio of linoleic and oleic acids were maximal during the third month after acute IM. These FA abnormalities coincided with the symptom of increased physical malaise, despite apparent clinical recovery, common after IM. Persistence of low linoleic acid content beyond six months postinfection occurred in all seven students who showed continued clinical symptoms. Estimation of FA enzyme activities over the post-IM year suggested that FA elongation function was normal, but that FA desaturation enzyme activities were lower than normal, particularly early after EBV infection. An inability of the host to normalize the serum total linoleic/oleic acid ratio may parallel a delayed recovery from EBV infection and may offer insight into its pathogenesis.

Activated T cells in type I Charcot-Marie-Tooth disease: evidence for immunologic heterogeneity

Common recognized variability in the familial peripheral neuropathy, type I Charcot-Marie-Tooth disease (CMT I), led to an examination of cell-mediated immune responses in 23 CMT I patients. Increased numbers of activated T cells were found in the peripheral blood of 14 (61%) patients using fluorescent monoclonal Ta1 antibody as quantitated by flow cytometry. Altered immunoregulation was also suggested by increased levels of prostaglandin-mediated lymphocyte suppression. In the other nine CMT I patients, immune responses were normal. Lack of a relationship between Ta1 expression and CMT clinical symptoms, but with consistency within six CMT families, support the concept of immunologic heterogeneity in type I CMT with a possible genetic component.

Expression of Schwann cell and peripheral T-cell activation epitopes in hereditary motor and sensory neuropathy

To evaluate possible immune-mediated mechanisms in hereditary motor and sensory neuropathy (HMSN-I, Charcot-Marie-Tooth syndrome), we examined class II major histocompatibility complex antigen expression (MHC-II, HLA-DR) in Schwann cells and peripheral lymphocyte T-cell (Ta1, CD26) activation in five unrelated adults with HMSN-I. Evidence of increased activation expression was found in both compartments but the pattern did not suggest a general state of hyperimmunity or appear related to clinical characteristics of HMSN. Significantly increased CD26+ T-cell activation and greater than normal fluctuation of values occurred intermittently in sequential tests of eight HMSN patients and at single time points in 24 others. The combined data, consistent with repeated stimulations of an immune reaction under normal feedback control, suggest that HMSN-I expresses some characteristics also found in autoimmune polyneuropathies.

Altered membrane fatty acids of cultured human retinal pigment epithelium persistently infected with rubella virus may affect secondary cellular function

SummaryPersistent infection with rubella virus (RV) can alter secondary functions of host cells. Previously we had documented defective phagocytosis of latex beads by cultured human retinal pigment epithelial cells (RPE), persistently infected with M-33 RV (RPE/RV). Here, examining possible mechanisms for altered function, we reported significant differences between the total esterified fatty acids (FA) of RPE and RPE/RV membranes, measured by gas liquid chromatography. RPE/RV contained an increased proportion of saturated FA, particularly palmitic acid, with a presence of unusual chromatographic FA peaks co-eluting with odd-numbered long-chain carbon atom FA not normally found in human cells. Apical membrane microvilli, structures essential to phagocytic activity of RPE and RPE/RV, observed by scanning and transmission electron microscopy, were similar in number and appearance between uninfected RPE and RPE/RV cells before and after latex bead addition. However, RPE/RV microvilli, possibly reflecting altered membrane FA composition, engaged latex beads less effectively than uninfected RPE microvilli. In addition, microvilli remained abnormally distributed on RPE/RV cell surfaces at 48 h after latex addition. Thus, RV persistent infection may affect the cellular membrane fluidity and functional activity of human cells with increased saturated FA proportions and altered FA components of membrane phospholipids. These changes may participate in the defective phagocytosis of RPE/RV.

Circulating cytotoxic immune components in dominant Charcot-Marie-Tooth syndrome.

Activated T cells, measured repeatedly in the demyelinating peripheral neuropathy, Charcot-Marie-Tooth syndrome (CMT: hereditary motor sensory neuropathy), might participate in myelin loss by a destructive inflammatory autoimmune process. To explore this possibility, plasma proportions of hydroxyleukotrienes, their fatty acid precursor, arachidonic acid, and lymphocyte epitopes associated with immune cell activation expression were measured in 18 adults with dominant, Type I CMT. Compared to age-matched normal controls, CMT I patients showed eicosanoid-linked immunoactivation by an elevated content of 12-hydroxy-eicosatetraenoic acid (12-HETE) in parallel with a decreased plasma percentage of its fatty acid precursor, arachidonic acid. CMT patients also had increased numbers of peripheral lymphocytes expressing activation-related epitopes, CD25+, CD26+, CD4+, and CD4/CD45RO+ primed memory cells, with enhanced CD8+ cytotoxic cells and soluble CD8 protein content. Therefore, endogenously stimulated CMT I lymphocytes include functional cytotoxic cells which appear to deplete the plasma fatty acid precursor of prostenoid agents during the secretion of potentially destructive cytokines.

Systemic immunostimulation after retinal laser treatment in retinitis pigmentosa

Systemic immunostimulation followed an experimental treatment trial of scatter argon laser photocoagulation directed to the retina of one eye of 10 patients with heredo-degenerative retinitis pigmentosa (RP). Significantly increased RP lymphocyte CD25, CD26, and CD4/CD26 activation epitope expressions over prelaser values and controls were found with a normalization of soluble interleukin-2 receptor secretion after laser treatment. Serum interferon-gamma was low both pre- and postlaser. Interestingly, when a panel of viral antibodies was tested, only those to rubella virus were elevated in the early postlaser period. The character of RP immunostimulation after laser-induced inflammation could be consistent with an antigenic stimulus from laser-released retinal proteins which might be of autoimmune or latent infectious origin. Enhanced immune responses may be a common but unrecognized sequellae of retinal laser.

Immune alterations associated with T lymphocyte activation and regulation in retinitis pigmentosa patients

Altered immunoregulation, suggested previously in the heredo-familial retinal degeneration, retinitis pigmentosa (RP), led us to examine cell-mediated immune responses in 58 RP patients who expressed either recessive or dominant inheritance. Increased absolute numbers of activated T-cells, quantitated by flow cytometry using the Ta1 epitope, were found in the peripheral blood of 60% of these RP patients. The unusual finding was equally distributed between dominant (16 of 25) and recessive (19 of 33) types of RP, suggesting an immune process present in both patterns of heredity. Additional altered lymphocyte immunoregulation was suggested in all RP by lymphocyte responses to stimulants modified by indomethacin or histamine in vitro. Although no clear association could be found between Tal expression and demographic factors including age, sex, years with RP symptoms, or percentage of life with RP disease, the significantly altered immunoregulatory responses in RP may be related to the pathogenesis of RP.

Persistently altered T cell immunity in high school students with the congenital rubella syndrome and profound hearing loss

Because there are frequent progressive and autoimmune complications in children born with the congenital rubella syndrome, we evaluated immunoregulation in eight profoundly deaf adolescents with congenital rubella syndrome who lived in a state school. Serum antiviral antibodies, expressions of peripheral lymphocyte epitopes and serum soluble interleukin 2 receptor (IL-2R) content were compared with those of 16 classmates with profound hearing loss of unknown cause and of 24 age-matched, hearing students from this area. Both deaf groups showed activated but impaired T lymphocyte function compared with normals. Rubella virus alteration of T cell function was suggested in congenital rubella syndrome students by elevated numbers of both CD4+ helper and CD25+ IL-2R cells with unusually low released soluble IL-2R content. In contrast in deaf classmates elevated CD25+ and CD16+ natural killer cell groups and soluble IL-2R content with low numbers of CD4+ helper cells and CD4+ populations were of unknown etiology. Defective immunoregulation of the congenitally deaf to pathogens inherent in their environment may lead to autoimmune and other complications.

Arachidonic fatty acid in red blood cell membranes, lymphocytes, and cultured skin fibroblasts of dominant Charcot-Marie-Tooth syndrome

Altered proportions of long-chain unsaturated n - 6 fatty acids (FA) in plasma and myelin of the heredodegenerative peripheral neuropathy, Charcot-Marie-Tooth syndrome (CMT), may implicate FA metabolism in the pathogenesis of CMT demyelination. A significant relationship between low plasma values of the polyunsaturated n - 6 FA, arachidonic acid, and an increased amount of prostaglandin-mediated immune responses had suggested the possibility of immune system metabolic usage of these n - 6 FA rather than an inherited FA enzyme defect. This relationship between peripheral immunostimulation and altered FA was documented repeatedly in CMT patients. Increased and cyclic lymphocyte activation expressions were measured at the same time as low amounts of plasma arachidonic acid, which is the FA precursor of prostaglandin immunoregulatory agents. An autoimmune process with possible enhanced formation of the n - 6 immune prostenoid agents in CMT had also been suggested by increased class II antigen expression on CMT sural nerves. Here, normal proportions of total FA in cultured CMT skin fibroblasts diminishes a notion of hereditary defects in CMT fatty acid metabolism. In addition, a significant depletion of the key arachidonic acid in lipid stores of CMT red blood cell membranes with elevated values of this FA in functional CMT lymphocytes, compared to controls, support the concept in CMT patients of a metabolic diversion of n - 6 FA, particularly arachidonic acid, from tissue stores for immunoregulatory prostenoid agent formation.