Sabrina W. Yum

Acute Disseminated Encephalomyelitis in Children: Discordant Neurologic and Neuroimaging Abnormalities and Response to Plasmapheresis

Objectives. To describe our experience with acute disseminated encephalomyelitis (ADEM), focusing on (1) the relationship between clinical course and MRI findings and (2) the response to plasmapheresis in a subgroup of patients. Methods. A retrospective record review was conducted of 13 children who were admitted as inpatients with the diagnosis of ADEM during the period 1998–2003. Results. Diagnosis was established by clinical signs and symptoms, cerebrospinal fluid changes and multifocal involvement of deep gray and white matter based on MRI. Initial therapy was high-dose methylprednisolone and intravenous immunoglobulin in 12 patients. One child improved spontaneously. Six of 12 children did not improve with corticosteroid treatment. All 6 had an acute progressive course neurologically, and 5 of them also showed a delay in the onset of neuroimaging changes, eventually developing lesions in the deep gray matter and brainstem. This latter group received 5 sessions of plasmapheresis and recovered over the course of several months with varying degrees of residual neurologic deficits. Conclusions. Presentation of ADEM with delayed development of MRI lesions in deep gray matter and brainstem may herald a prolonged clinical course and lack of response to glucocorticoid therapy. Plasmapheresis might be an effective therapeutic intervention in these patients. The role of plasmapheresis versus corticosteroids and intravenous immunoglobulin as a primary treatment of ADEM needs to be investigated further.

CMT subtypes and disease burden in patients enrolled in the Inherited Neuropathies Consortium natural history study: a cross-sectional analysis.

Background The international Inherited Neuropathy Consortium (INC) was created with the goal of obtaining much needed natural history data for patients with Charcot-Marie-Tooth (CMT) disease. We analysed clinical and genetic data from patients in the INC to determine the distribution of CMT subtypes and the clinical impairment associated with them. Methods We analysed data from 1652 patients evaluated at 13 INC centres. The distribution of CMT subtypes and pathogenic genetic mutations were determined. The disease burden of all the mutations was assessed by the CMT Neuropathy Score (CMTNS) and CMT Examination Score (CMTES). Results 997 of the 1652 patients (60.4%) received a genetic diagnosis. The most common CMT subtypes were CMT1A/PMP22 duplication, CMT1X/GJB1 mutation, CMT2A/MFN2 mutation, CMT1B/MPZ mutation, and hereditary neuropathy with liability to pressure palsy/PMP22 deletion. These five subtypes of CMT accounted for 89.2% of all genetically confirmed mutations. Mean CMTNS for some but not all subtypes were similar to those previously reported. Conclusions Our findings confirm that large numbers of patients with a representative variety of CMT subtypes have been enrolled and that the frequency of achieving a molecular diagnosis and distribution of the CMT subtypes reflects those previously reported. Measures of severity are similar, though not identical, to results from smaller series. This study confirms that it is possible to assess patients in a uniform way between international centres, which is critical for the planned natural history study and future clinical trials. These data will provide a representative baseline for longitudinal studies of CMT. Clinical trial registration ID number NCT01193075.

Central Nervous System Dysfunction in a Mouse Model of FA2H Deficiency

Fatty acid 2‐hydroxylase (FA2H) is responsible for the synthesis of myelin galactolipids containing hydroxy fatty acid (hFA) as the N‐acyl chain. Mutations in the FA2H gene cause leukodystrophy, spastic paraplegia, and neurodegeneration with brain iron accumulation. Using the Cre‐lox system, we developed two types of mouse mutants, Fa2h−/− mice (Fa2h deleted in all cells by germline deletion) and Fa2hflox/flox Cnp1‐Cre mice (Fa2h deleted only in oligodendrocytes and Schwann cells). We found significant demyelination, profound axonal loss, and abnormally enlarged axons in the CNS of Fa2h−/− mice at 12 months of age, while structure and function of peripheral nerves were largely unaffected. Fa2h−/− mice also exhibited histological and functional disruption in the cerebellum at 12 months of age. In a time course study, significant deterioration of cerebellar function was first detected at 7 months of age. Further behavioral assessments in water T‐maze and Morris water maze tasks revealed significant deficits in spatial learning and memory at 4 months of age. These data suggest that various regions of the CNS are functionally compromised in young adult Fa2h−/− mice. The cerebellar deficits in 12‐month‐old Fa2hflox/flox Cnp1‐Cre mice were indistinguishable from Fa2h−/− mice, indicating that these phenotypes likely stem from the lack of myelin hFA‐galactolipids. In contrast, Fa2hflox/flox Cnp1‐Cre mice did not show reduced performance in water maze tasks, indicating that oligodendrocytes are not involved in the learning and memory deficits found in Fa2h−/− mice. These findings provide the first evidence that FA2H has an important function outside of oligodendrocytes in the CNS.

Dominant connexin26 mutants associated with human hearing loss have trans-dominant effects on connexin30

Dominant mutations in GJB2, the gene encoding the human gap junction protein connexin26 (Cx26), cause hearing loss. We investigated whether dominant Cx26 mutants interact directly with Cx30. HeLa cells stably expressing nine dominant Cx26 mutants, six associated with non-syndromic hearing loss (W44C, W44S, R143Q, D179N, R184Q and C202F) and three associated with hearing loss and palmoplantar keratoderma (G59A, R75Q and R75W), individually or together with Cx30, were analyzed by immunocytochemistry, co-immunoprecipitation, and functional assays (scrape-loading and/or fluorescence recovery after photobleaching). When expressed alone, all mutants formed gap junction plaques, but with impaired intercellular dye transfer. When expressed with Cx30, all mutants co-localized and co-immunoprecipitated with Cx30, indicating they likely co-assembled into heteromers. Furthermore, 8/9 Cx26 mutants inhibited the transfer of neurobiotin or calcein, indicating that these Cx26 mutants have trans-dominant effects on Cx30, an effect that may contribute to the pathogenesis of hearing loss.

Spectrum of Neuropathophysiology in Spinal Muscular Atrophy Type I

Abstract Neuropathologic findings within the central and peripheral nervous systems in patients with spinal muscular atrophy type I (SMA-I) were examined in relation to genetic, clinical, and electrophysiologic features. Five infants representing the full clinical spectrum of SMA-I were examined clinically for compound motor action potential amplitude and SMN2 gene copy number; morphologic analyses of postmortem central nervous system, neuromuscular junction, and muscle tissue samples were performed and SMN protein was assessed in muscle samples. The 2 clinically most severely affected patients had a single copy of the SMN2 gene; in addition to anterior horn cells, dorsal root ganglia, and thalamus, neuronal degeneration in them was widespread in the cerebral cortex, basal ganglia, pigmented nuclei, brainstem, and cerebellum. Two typical SMA-I patients and a milder case each had 2 copies of the SMN2 gene and more restricted neuropathologic abnormalities. Maturation of acetylcholine receptor subunits was delayed and the neuromuscular junctions were abnormally formed in the SMA-I patients. Thus, the neuropathologic findings in human SMA-I are similar to many findings in animal models; factors other than SMN2 copy number modify disease severity. We present a pathophysiologic model for SMA-I as a protein deficiency disease affecting a neuronal network with variable clinical thresholds. Because new treatment strategies improve survival of infants with SMA-I, a better understanding of these factors will guide future treatments.

Non-recurrent SEPT9 duplications cause hereditary neuralgic amyotrophy

Background Genomic copy number variants have been shown to be responsible for multiple genetic diseases. Recently, a duplication in septin 9 (SEPT9) was shown to be causal for hereditary neuralgic amyotrophy (HNA), an episodic peripheral neuropathy with autosomal dominant inheritance. This duplication was identified in 12 pedigrees that all shared a common founder haplotype. Methods and results Based on array comparative genomic hybridisation, we identified six additional heterogeneous tandem SEPT9 duplications in patients with HNA that did not possess the founder haplotype. Five of these novel duplications are intragenic and result in larger transcript and protein products, as demonstrated through reverse transcription-PCR and western blotting. One duplication spans the entire SEPT9 gene and does not generate aberrant transcripts and proteins. The breakpoints of all the duplications are unique and contain regions of microhomology ranging from 2 to 9 bp in size. The duplicated regions contain a conserved 645 bp exon within SEPT9 in which HNA-linked missense mutations have been previously identified, suggesting that the region encoded by this exon is important to the pathogenesis of HNA. Conclusions Together with the previously identified founder duplication, a total of seven heterogeneous SEPT9 duplications have been identified in this study as a causative factor of HNA. These duplications account for one third of the patients in our cohort, suggesting that duplications of various sizes within the SEPT9 gene are a common cause of HNA.

Phenotypic Variability of Childhood Charcot-Marie-Tooth Disease

IMPORTANCE Disease severity of childhood Charcot-Marie-Tooth disease (CMT) has not been extensively characterized, either within or between types of CMT to date. OBJECTIVE To assess the variability of disease severity in a large cohort of children and adolescents with CMT. DESIGN, SETTING, AND PARTICIPANTS A cross-sectional study was conducted among 520 children and adolescents aged 3 to 20 years at 8 universities and hospitals involved in the Inherited Neuropathies Consortium between August 6, 2009, and July 31, 2014, in Australia, Italy, the United Kingdom, and the United States. Data analysis was conducted from August 1, 2014, to December 1, 2015. MAIN OUTCOMES AND MEASURES Scores on the Charcot-Marie-Tooth Disease Pediatric Scale (CMTPedS), a well-validated unidimensional clinical outcome measure to assess disease severity. This instrument includes 11 items assessing fine and gross motor function, sensation, and balance to produce a total score ranging from 0 (unaffected) to 44 (severely affected). RESULTS Among the 520 participants (274 males) aged 3 to 20 years, CMT type 1A (CMT1A) was the most prevalent type (252 [48.5%]), followed by CMT2A (31 [6.0%]), CMT1B (15 [2.9%]), CMT4C (13 [2.5%]), and CMTX1 (10 [1.9%]). Disease severity ranged from 1 to 44 points on the CMTPedS (mean [SD], 21.5 [8.9]), with ankle dorsiflexion strength and functional hand dexterity test being most affected. Participants with CMT1B (mean [SD] CMTPedS score, 24.0 [7.4]), CMT2A (29.7 [7.1]), and CMT4C (29.8 [8.6]) were more severely affected than those with CMT1A (18.9 [7.7]) and CMTX1 (males: 15.3 [7.7]; females: 13.0 [3.6]) (P < .05). Scores on the CMTPedS tended to worsen principally during childhood (ages, 3-10 years) for participants with CMT4C and CMTX1 and predominantly during adolescence for those with CMT1B and CMT2A (ages, 11-20 years), while CMT1A worsened consistently throughout childhood and adolescence. For individual items, participants with CMT4C recorded more affected functional dexterity test scores than did those with all other types of CMT (P < .05). Participants with CMT1A and CMTX1 performed significantly better on the 9-hole peg test and balance test than did those with all other types of CMT (P < .05). Participants with CMT2A had the weakest grip strength (P < .05), while those with CMT2A and CMT4C exhibited the weakest ankle plantarflexion and dorsiflexion strength, as well as the lowest long jump and 6-minute walk test distances (P < .05). Multiple regression modeling identified increasing age (r = 0.356, β = 0.617, P < .001) height (r = 0.251, β = 0.309, P = .002), self-reported foot pain (r = 0.162, β = .114, P = .009), and self-reported hand weakness (r = 0.243, β = 0.203, P < .001) as independent predictors of disease severity. CONCLUSIONS AND RELEVANCE These results highlight the phenotypic variability within CMT genotypes and mutation-specific manifestations between types. This study has identified distinct functional limitations and self-reported impairments to target in future therapeutic trials.

Connexin43 Expression Increases in the Epithelium and Stroma along the Colonic Neoplastic Progression Pathway: Implications for Its Oncogenic Role

Connexins (Cxs) are critical for normal tissue development, differentiation, and cell proliferation. Normal expression and function of Cxs are considered to play a role in tumor suppression, but abnormal localization and abnormally increased expression of Cxs have been found in a variety of carcinomas. Of the Cx family, Cx43 is a most prevalent member and has been known as a downstream target of β-catenin, a key component of Wnt signaling pathway. We evaluated the expression of Cx43 in the colonic neoplasia progression sequence with additional attention to the stromal component. Resections of 50 colonic adenocarcinomas were stained immunohistochemically for Cx43 on paraffin embedded sections. Cx43 cytoplasmic expression increased progressively in the colonic adenocarcinoma sequence in both the epithelial [normal (4 ± 1), adenomatous (20 ± 2), cancerous (124 ± 10) (P < 0.01)], and stromal [normal (19 ± 1), cancerous (45 ± 4) (P < 0.01)] components. In the epithelial component, Cx43 was expressed lower in stage I adenocarcinomas (69 ± 12) compared to stage III/IV (158 ± 10, P < 0.01). Additionally, Cx43 was relatively increased in the adenocarcinoma at the invasive tumor front in all stages. Cx43 may play a critical role in the pathogenesis of colon cancer via gap junction or other gap junction independent mechanisms such as the Wnt/β-catenin pathway.

Genotype–phenotype characteristics and baseline natural history of heritable neuropathies caused by mutations in the MPZ gene

We aimed to characterize genotype-phenotype correlations and establish baseline clinical data for peripheral neuropathies caused by mutations in the myelin protein zero (MPZ) gene. MPZ mutations are the second leading cause of Charcot-Marie-Tooth disease type 1. Recent research makes clinical trials for patients with MPZ mutations a realistic possibility. However, the clinical severity varies with different mutations and natural history data on progression is sparse. We present cross-sectional data to begin to define the phenotypic spectrum and clinical baseline of patients with these mutations. A cohort of patients with MPZ gene mutations was identified in 13 centres of the Inherited Neuropathies Consortium - Rare Disease Clinical Research Consortium (INC-RDCRC) between 2009 and 2012 and at Wayne State University between 1996 and 2009. Patient phenotypes were quantified by the Charcot-Marie-Tooth disease neuropathy score version 1 or 2 and the Charcot-Marie-Tooth disease paediatric scale outcome instruments. Genetic testing was performed in all patients and/or in first- or second-degree relatives to document mutation in MPZ gene indicating diagnosis of Charcot-Marie-Tooth disease type 1B. There were 103 patients from 71 families with 47 different MPZ mutations with a mean age of 40 years (range 3-84 years). Patients and mutations were separated into infantile, childhood and adult-onset groups. The infantile onset group had higher Charcot-Marie-Tooth disease neuropathy score version 1 or 2 and slower nerve conductions than the other groups, and severity increased with age. Twenty-three patients had no family history of Charcot-Marie-Tooth disease. Sixty-one patients wore foot/ankle orthoses, 19 required walking assistance or support, and 10 required wheelchairs. There was hearing loss in 21 and scoliosis in 17. Forty-two patients did not begin walking until after 15 months of age. Half of the infantile onset patients then required ambulation aids or wheelchairs for ambulation. Our results demonstrate that virtually all MPZ mutations are associated with specific phenotypes. Early onset (infantile and childhood) phenotypes likely represent developmentally impaired myelination, whereas the adult-onset phenotype reflects axonal degeneration without antecedent demyelination. Data from this cohort of patients will provide the baseline data necessary for clinical trials of patients with Charcot-Marie-Tooth disease caused by MPZ gene mutations.

Dominant Cx26 mutants associated with hearing loss have dominant-negative effects on wild type Cx26

Mutations in GJB2, the gene encoding the human gap junction protein connexin26 (Cx26), cause either non-syndromic hearing loss or syndromes affecting both hearing and skin. We have investigated whether dominant Cx26 mutants can interact physically with wild type Cx26. HeLa cells stably expressing wild type Cx26 were transiently transfected to co-express nine individual dominant Cx26 mutants; six associated with non-syndromic hearing loss (W44C, W44S, R143Q, D179N, R184Q, and C202F) and three associated with hearing loss and palmoplantar keratoderma (G59A, R75Q, and R75W). All mutants co-localized and co-immunoprecipitated with wild type Cx26, indicating that they interact physically, likely by forming admixed heteromeric/heterotypic channels. Furthermore, all nine mutants inhibited the transfer of calcein in cells stably expressing Cx26, demonstrating that they each have dominant effects on wild type Cx26. Taken together, these results show that dominant-negative effects of these Cx26 mutants likely contribute to the pathogenesis of hearing loss.