Biallelic NUDT2 variants defective in mRNA decapping cause a neurodevelopmental disease.

Dysfunctional RNA processing caused by genetic defects in RNA processing enzymes has a profound impact on the nervous system, resulting in neurodevelopmental conditions. We characterized a recessive neurological disorder in 18 children and young adults from 10 independent families typified by intellectual disability, motor developmental delay and gait disturbance. In some patients peripheral neuropathy, corpus callosum abnormalities and progressive basal ganglia deposits were present. The disorder is associated with rare variants in NUDT2, a mRNA decapping and Ap4A hydrolysing enzyme, including novel missense and in-frame deletion variants. We show that these NUDT2 variants lead to a marked loss of enzymatic activity, strongly implicating loss of NUDT2 function as the cause of the disorder. NUDT2-deficient patient fibroblasts exhibit a markedly altered transcriptome, accompanied by changes in mRNA half-life and stability. Amongst the most up-regulated mRNAs in NUDT2-deficient cells, we identified host response and interferon-responsive genes. Importantly, add-back experiments using an Ap4A hydrolase defective in mRNA decapping highlighted loss of NUDT2 decapping as the activity implicated in altered mRNA homeostasis. Our results confirm that reduction or loss of NUDT2 hydrolase activity is associated with a neurological disease, highlighting the importance of a physiologically balanced mRNA processing machinery for neuronal development and homeostasis.

Mutations in TAF8 cause a neurodegenerative disorder.

TAF8 is part of the transcription factor II D complex, composed of the TATA-binding protein and 13 TATA-binding protein-associated factors (TAFs). Transcription factor II D is the first general transcription factor recruited at promoters to assemble the RNA polymerase II preinitiation complex. So far disorders related to variants in 5 of the 13 subunits of human transcription factor II D have been described. Recently, a child with a homozygous c.781-1G>A mutation in TAF8 has been reported. Here we describe seven further patients with mutations in TAF8 and thereby confirm the TAF8 related disorder. In two sibling patients, we identified two novel compound heterozygous TAF8 splice site mutations, c.45+4A > G and c.489G>A, which cause aberrant splicing as well as reduced expression and mislocalization of TAF8. In five further patients, the previously described c.781-1G > A mutation was present on both alleles. The clinical phenotype associated with the different TAF8 mutations is characterized by severe psychomotor retardation with almost absent development, feeding problems, microcephaly, growth retardation, spasticity and epilepsy. Cerebral imaging showed hypomyelination, a thin corpus callosum and brain atrophy. Moreover, repeated imaging in the sibling pair demonstrated progressive cerebral and cerebellar atrophy. Consistently, reduced N-acetylaspartate, a marker of neuronal viability, was observed on magnetic resonance spectroscopy. Further review of the literature shows that mutations causing a reduced expression of transcription factor II D subunits have an overlapping phenotype of microcephaly, developmental delay and intellectual disability. Although transcription factor II D plays an important role in RNA polymerase II transcription in all cells and tissues, the symptoms associated with such defects are almost exclusively neurological. This might indicate a specific vulnerability of neuronal tissue to widespread deregulation of gene expression as also seen in Rett syndrome or Cornelia de Lange syndrome.

Evaluation of Novel Enhancer Compounds in Gentamicin-Mediated Readthrough of Nonsense Mutations in Rett Syndrome.

Rett syndrome (RTT), a severe X-linked neurodevelopmental disorder, is primarily caused by mutations in the methyl CpG binding protein 2 gene (MECP2). Over 35% RTT patients carry nonsense mutation in MECP2, making it a suitable candidate disease for nonsense suppression therapy. In our previous study, gentamicin was found to induce readthrough of MECP2 nonsense mutations with modest efficiency. Given the recent discovery of readthrough enhancers, CDX compounds, we herein evaluated the potentiation effect of CDX5-1, CDX5-288, and CDX6-180 on gentamicin-mediated readthrough efficiency in transfected HeLa cell lines bearing the four most common MECP2 nonsense mutations. We showed that all three CDX compounds potentiated gentamicin-mediated readthrough and increased full-length MeCP2 protein levels in cells expressing the R168X, R255X, R270X, and R294X nonsense mutations. Among all three CDX compounds, CDX5-288 was the most potent enhancer and enabled the use of reduced doses of gentamicin, thus mitigating the toxicity. Furthermore, we successfully demonstrated the upregulation of full-length Mecp2 protein expression in fibroblasts derived from Mecp2R255X/Y mice through combinatorial treatment. Taken together, findings demonstrate the feasibility of this combinatorial approach to nonsense suppression therapy for a subset of RTT patients.

Temporal profile of lymphocyte counts and relationship with infections with fingolimod therapy in paediatric patients with multiple sclerosis: Results from the PARADIGMS study.

BACKGROUND: Reduction in absolute lymphocyte count (ALC) is expected with fingolimod treatment. OBJECTIVE: To evaluate the effect of fingolimod 0.5 mg versus intramuscular interferon ß-1a (30 µg) on ALC and its relationship with infections in paediatric-onset multiple sclerosis (POMS) up to 4 years. METHODS: We assessed ALC at baseline, monthly till 3 months, and every 3 months (core phase) and with variable periodicity (extension phase) of Phase 3 PARADIGMS study (N = 215). Incidence rates (IRs) of infection-related adverse events (infAEs)/100 patient-years were analysed by on-study nadir ALC. RESULTS: With fingolimod, ALC rapidly reduced to 29.9%-34.4% of baseline values within 2 weeks and remained stable thereafter; no relevant changes observed with interferon. IRs of infAEs were 67.6 with fingolimod and 61.8 with interferon; IR ratios with respect to interferon, overall: 1.09, by nadir ALC 0.2-0.4 × 109/L: 1.13 and >0.4 × 109/L: 0.91. Three patients had a single episode of ALC <0.2 × 109/L (core phase). No opportunistic infections were observed and infection risk did not increase during the extension phase. CONCLUSION: In paediatric patients, the overall incidence of infections was comparable between fingolimod and interferon. No association was observed between nadir ALC and infections in POMS, although sample size may have been too small to rule an association.

Consistent control of disease activity with fingolimod versus IFN ß-1a in paediatric-onset multiple sclerosis: further insights from PARADIGMS.

BACKGROUND: In PARADIGMS, a double-blind phase III trial in 215 paediatric patients with multiple sclerosis (MS) (10 to <18 years), fingolimod administered for up to 2 years significantly reduced the annualised relapse rate (ARR) and rate of new/newly enlarged T2 (n/neT2) lesions compared with interferon (IFN) ß-1a. OBJECTIVES: To investigate (1) differences between treatment groups across subpopulations (treatment-naïve, younger/prepubertal patients); (2) disability progression. METHODS: ARRs at 10, 11 and 12 years were estimated based on predefined modelling extrapolations. Changes in Expanded Disability Status Scale (EDSS), and in 3 month (3M) and 6 month (6M) confirmed disability progression (CDP) were evaluated post hoc. RESULTS: In the treatment-naïve subpopulation, fingolimod reduced ARR and n/neT2 lesions by 85.8% and 53.4%, respectively versus INF ß-1a (both p<0.001), compared with 81.9% and 52.6% in the overall population. Model-based ARR reductions in younger patients (≤12 years) were 91.9%-94.6%. Twice as many IFN ß-1a-treated than fingolimod-treated patients had worse EDSS scores at study end (20.6% vs 10.5%, p=0.043). Risk reductions in 3M-CDP and 6M-CDP were 77.2% (p=0.007) and 80.2% (p=0.040), respectively. CONCLUSIONS: Fingolimod in paediatric MS was associated with consistent control of disease activity versus IFN ß-1a (including treatment-naïve and younger patients) and resulted in less disability progression for up to 2 years. TRIAL REGISTRATION NUMBER: NCT01892722.

Therapy of highly active pediatric multiple sclerosis.

OBJECTIVE: Study aims were to determine the frequency of highly active disease in pediatric multiple sclerosis (MS), the response to natalizumab (NTZ) and fingolimod (FTY) treatment, and the impact of current treatment modalities on the clinical course. METHODS: Retrospective single-center study in the German Center for MS in Childhood and Adolescence. RESULTS: Of 144 patients with first MS manifestation between 2011 and 2015, 41.6% fulfilled the criteria for highly active MS. In total, 55 patients treated with NTZ and 23 with FTY demonstrated a significant reduction in relapse rate (NTZ: 95.2%, FTY: 75%), new T2 lesions (NTZ: 97%, FTY: 81%), and contrast-enhancing lesions (NTZ: 97%, FTY: 93%). However, seven patients switched from NTZ to FTY experienced an increase in disease activity. Comparing pediatric MS patients treated in 2005 with those treated in 2015 showed a 46% reduction in relapse rate and a 44% reduction in mean Expanded Disability Status Scale (EDSS). CONCLUSION: The rate of highly active disease among pediatric MS patients is high; more than 40% in our cohort. Response to NTZ and FTY treatment is similar if not better than observed in adults. Current treatment modalities including earlier treatment initiation and the introduction of NTZ and FTY have significantly improved the clinical course of pediatric MS.

Extensive acute axonal damage in pediatric multiple sclerosis lesions.

OBJECTIVE: Axonal damage occurs early in multiple sclerosis (MS) and contributes to the degree of clinical disability. Children with MS more often show disabling and polyfocal neurological symptoms at disease onset than adults with MS. Thus, axonal damage may differ between pediatric and adult MS patients. METHODS: We analyzed axonal pathology in archival brain biopsy and autopsy samples from 19 children with early MS. Lesions were classified according to demyelinating activity and presence of remyelination. Axonal density and extent of acute axonal damage were assessed using Bielschowsky silver impregnation and immunohistochemistry for amyloid precursor protein (APP), respectively. Axonal injury was correlated with the inflammatory infiltrate as well as clinical characteristics. Results were compared with data from adult MS patients. RESULTS: Acute axonal damage was most extensive in early active demyelinating (EA) lesions of pediatric patients and correlated positively with the Expanded Disability Status Scale at attack leading to biopsy/autopsy. Comparison with 12 adult patients showed a 50% increase in the extent of acute axonal damage in EA lesions from children compared to adults, with the highest number of APP-positive spheroids found prior to puberty. The extent of acute axonal damage correlated positively with the number of lesional macrophages. Axonal density was reduced in pediatric lesions irrespective of the demyelinating activity or the presence of remyelination. Axonal reduction was similar between children and adults. INTERPRETATION: Our results provide evidence for more pronounced acute axonal damage in inflammatory demyelinating lesions from children compared to adults.

Compound heterozygous variants in PGAP1 causing severe psychomotor retardation, brain atrophy, recurrent apneas and delayed myelination: a case report and literature review.

BACKGROUND: Mutations in proteins involved in the glycosylphosphatidylinositol anchor biosynthesis and remodeling pathway are associated with autosomal recessive forms of intellectual disability. Recently mutations in the PGAP1 gene that codes for PGAP1, a protein localized in the endoplasmic reticulum responsible for the first step of the remodeling of glycosylphosphatidylinositol was linked to a disorder characterized by psychomotor retardation and facial dysmorphism. Whole exome sequencing (WES) was performed in siblings with severely delayed myelination and psychomotor retardation. Mutations in PGAP1 were confirmed by Sanger sequencing and RNA analysis. A literature search was performed to describe the emerging phenotype of PGAP1 related disease. CASE PRESENTATION: WES resulted in the detection of two novel compound heterozygous mutations in PGAP1, one base pair insertion leading to a frame shift c.334_335InsA (p.A112fs) and a splice site mutation leading to exon skipping c.G1173C (p.L391L). A symptom not described in PGAP1 related disorder before but prominent in the siblings were recurrent apnea especially during sleep that persisted at least until age 2 years. Sequential cerebral MRI at age one and two year(s) respectively revealed frontal accentuated brain atrophy and significantly delayed myelination. CONCLUSION: We report siblings with two novel mutations in PGAP1. Other that the common symptoms related to PGAP1 mutations including non-progressive psychomotor retardation, neonatal feeding problems, microcephaly and brain atrophy these patients displayed severely delayed myelination and recurrent apneas thereby widing the clinical spectrum associated with such mutations.

Age-Dependent Seroprevalence of JCV Antibody in Children.

Progressive multifocal leukoencephalopathy (PML) is an opportunistic central nervous system infection, caused by the John Cunningham virus (JCV). PML may occur during treatment with immunosuppressive agents or monoclonal antibodies such as natalizumab. The JCV seroprevalence increases with age with a seropositivity of 60% in the adult human population. In this study, we analyzed sera from 109 pediatric multiple sclerosis (MS) patients (mean age 14 years) as well as sera from 162 patients with a wide range of suspected neurologic disorders (mean age 6.3 years). Our results showed a considerably lower seroprevalence for JCV in our pediatric cohort with 33.3% and equal distribution in both subgroups, compared with reported seropositivity in adult population. This could result in a lower risk for drug-induced PML in pediatric patients compared with adult patients and can influence the indication for natalizumab therapy in pediatric MS patients.

Mutations in SLC33A1 cause a lethal autosomal-recessive disorder with congenital cataracts, hearing loss, and low serum copper and ceruloplasmin.

Low copper and ceruloplasmin in serum are the diagnostic hallmarks for Menkes disease, Wilson disease, and aceruloplasminemia. We report on five patients from four unrelated families with these biochemical findings who presented with a lethal autosomal-recessive syndrome of congenital cataracts, hearing loss, and severe developmental delay. Cerebral MRI showed pronounced cerebellar hypoplasia and hypomyelination. Homozygosity mapping was performed and displayed a region of commonality among three families at chromosome 3q25. Deep sequencing and conventional sequencing disclosed homozygous or compound heterozygous mutations for all affected subjects in SLC33A1 encoding a highly conserved acetylCoA transporter (AT-1) required for acetylation of multiple gangliosides and glycoproteins. The mutations were found to cause reduced or absent AT-1 expression and abnormal intracellular localization of the protein. We also showed that AT-1 knockdown in HepG2 cells leads to reduced ceruloplasmin secretion, indicating that the low copper in serum is due to reduced ceruloplasmin levels and is not a sign of copper deficiency. The severity of the phenotype implies an essential role of AT-1 in proper posttranslational modification of numerous proteins, without which normal lens and brain development is interrupted. Furthermore, AT-1 defects are a new and important differential diagnosis in patients with low copper and ceruloplasmin in serum.

JC virus antibody status in a pediatric multiple sclerosis cohort: prevalence, conversion rate and influence on disease severity.

BACKGROUND: Because of the emergence of novel therapies for multiple sclerosis (MS) and the associated increased risk of progressive multifocal leukoencephalopathy, John Cunningham (JC) virus infection has become a focus of interest for neurologists. However, little is known about JC virus infection in pediatric MS to date. OBJECTIVE: We aimed to analyze the prevalence of anti-JC virus antibodies, the conversion rate and the influence of the anti-JC virus antibody status on the clinical course in a large pediatric MS cohort. METHODS: Anti-JC virus antibodies were analyzed in serum samples within six months of disease onset and during the course of the disease. Clinical data were extracted from a pediatric MS databank. RESULTS: A total of 51.6% of 256 patients were found to be positive for anti-JC virus antibodies at onset of disease. No correlation between antibody status and clinical course was seen. Analyzing 693 follow-up serum samples revealed high titer stability, and an annual conversion rate of 4.37% was seen. CONCLUSION: No evidence was found that seropositivity for anti-JC virus antibodies influences the clinical course. Surprisingly, seroprevalence for anti-JC virus antibodies was more than twice as high as anticipated in this age group, raising the question of whether the infection increases the risk of MS development.

Chemokine-mediated redirection of T cells constitutes a critical mechanism of glucocorticoid therapy in autoimmune CNS responses.

Glucocorticoids (GCs) are the standard therapy for treating multiple sclerosis (MS) patients suffering from an acute relapse. One of the main mechanisms of GC action is held to be the induction of T cell apoptosis leading to reduced lymphocyte infiltration into the CNS, yet our analysis of experimental autoimmune encephalomyelitis (EAE) in three different strains of genetically manipulated mice has revealed that the induction of T cell apoptosis is not essential for the therapeutic efficacy of GCs. Instead, we identified the redirection of T cell migration in response to chemokines as a new therapeutic principle of GC action. GCs inhibited the migration of T cells towards CCL19 while they enhanced their responsiveness towards CXCL12. Importantly, blocking CXCR4 signaling in vivo by applying Plerixafor(®) strongly impaired the capacity of GCs to interfere with EAE, as revealed by an aggravated disease course, more pronounced CNS infiltration and a more dispersed distribution of the infiltrating T cells throughout the parenchyma. Our observation that T cells lacking the GC receptor were refractory to CXCL12 further underscores the importance of this pathway for the treatment of EAE by GCs. Importantly, methylprednisolone pulse therapy strongly increased the capacity of peripheral blood T cells from MS patients of different subtypes to migrate towards CXCL12. This indicates that modulation of T cell migration is an important mechanistic principle responsible for the efficacy of high-dose GC therapy not only of EAE but also of MS.

Long term follow-up in GAMT deficiency - Correlation of therapy regimen, biochemical and in vivo brain proton MR spectroscopy data.

GAMT deficiency is a rare autosomal recessive disease within the group of cerebral creatine deficiency syndromes. Cerebral creatine depletion and accumulation of guanidinoacetate (GAA) lead to clinical presentation with intellectual disability, seizures, speech disturbances and movement disorders. Treatment consists of daily creatine supplementation to increase cerebral creatine, reduction of arginine intake and supplementation of ornithine for reduction of toxic GAA levels. This study represents the first long-term follow-up over a period of 14 years, with detailed clinical data, biochemical and multimodal neuroimaging findings. Developmental milestones, brain MRI, quantitative single voxel 1H magnetic resonance spectroscopy (MRS) and biochemical analyses were assessed. The results reveal insights into the dose dependent effects of creatine/ornithine supplementation and expand the phenotypic spectrum of GAMT deficiency. Of note, the creatine concentrations, which were regularly monitored over a long follow-up period, increased significantly over time, but did not reach age matched control ranges. Our patient is the second reported to show normal neurocognitive outcome after an initial delay, stressing the importance of early diagnosis and treatment initiation.

Acute disseminated encephalomyelitis followed by recurrent or monophasic optic neuritis in pediatric patients.

BACKGROUND: Some pediatric patients with inflammatory demyelinating central nervous system disorders cannot be classified under any of the established disease entities, making their treatment and prognosis difficult. OBJECTIVE: The objective of this study is to characterize a subgroup of pediatric patients with recurrent demyelinating central nervous system disorders. METHODS: This study includes a case series of pediatric patients with monophasic or recurrent acute disseminated encephalomyelitis (ADEM) who later presented with either monophasic or recurrent optic neuritis (ON). RESULTS: We describe seven patients with a median follow-up of six years (five females, two males) who presented at a median age of 6 years (range 4-8 years) with monophasic (n = 4) or recurrent ADEM (two to four attacks) followed by monophasic (n = 3) or recurrent ON (two to nine attacks). Cranial magnetic resonance imaging (MRI) was typical for ADEM (n = 6) with complete or almost complete resolution of lesions on follow-up. Cerebrospinal (CSF) studies at the time of ADEM showed a pleocytosis in six patients and were negative for oligoclonal bands (OCBs) in all. In all patients high titers for serum anti-MOG antibodies were detected. CONCLUSION: ADEM followed by ON is a rare but distinct clinical phenotype among pediatric patients. Further studies are needed to allow recommendations on treatment or prognosis.

Therapeutic apheresis in pediatric patients with acute CNS inflammatory demyelinating disease.

BACKGROUND/AIMS: In adults, plasma exchange (PE) has been shown to be an efficient treatment for severe relapses of acute inflammatory CNS demyelinating diseases. The aim of this study was to evaluate the safety and efficacy of this treatment in pediatric patients. METHODS: We retrospectively analyzed a single-center cohort of pediatric patients with inflammatory CNS demyelinating disorders who underwent apheresis between 2007 and 2011. RESULTS: Ten patients (mean age: 11.6 ± 3.4 years) with an acute relapse of multiple sclerosis (n = 5), neuromyelitis optica (n = 2) or acute disseminated encephalomyelitis were included. All received methylprednisolone prior to treatment with either PE (n = 5) or immunoadsorption (n = 5). Apheresis-related side effects were either self-limiting or easily managed. EDSS (Expanded Disability Status Scale) improved in 7 of 8 patients during apheresis and in all patients within 30 days from a median of 7.5 to 1 (p < 0.01). The visual acuity initially worsened during the procedure in 3 of 7 affected eyes (mean 0.09), but improved in all at follow-up (mean: 0.5; p = 0.008). CONCLUSIONS: Apheresis was well tolerated and associated with a favorable outcome in all pediatric patients similar to reports in adults.

Pretreatment Neurofilament Light Chain Serum Levels, Early Disease Severity, and Treatment Response in Pediatric Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: High disease activity and frequent therapy failure in pediatric multiple sclerosis (MS) make prognostic biomarkers urgently needed. We investigated whether serum neurofilament light chain (sNfL) levels in treatment-naive pediatric patients with MS are associated with early disease severity and indicate treatment outcomes. METHODS: A retrospective cohort study of patients seen in the Göttingen Center for MS in Childhood and Adolescence, Germany. Inclusion criteria were MS diagnosis according to the McDonald criteria, MS onset <18 years, and available pretreatment serum sample. sNfL levels were analyzed using a single-molecule array assay. Associations with clinical and MRI evidence of disease severity at sampling were evaluated using the Spearman correlations and nonparametric tests for group comparisons. Correlations between pretreatment sNfL and annualized relapse and new T2 lesion rate on first-line therapy, and odd ratios for switch to high-efficacy therapy were assessed. RESULTS: A total of 178 patients (116 women [65%]) with a mean sampling age of 14.3 years were included in the study. Pretreatment sNfL levels were above the ≥90th percentile reported for healthy controls in 80% of patients (median 21.1 pg/mL) and correlated negatively with age, but no correlation was seen with sex, oligoclonal band status, or body mass index. High pretreatment sNfL levels correlated significantly with a high number of preceding relapses, a shorter first interattack interval, a high T2 lesion count, and recent gadolinium-enhancing lesions. Of interest, sNfL levels reflected more strongly MRI activity rather than clinical activity. Pretreatment sNfL levels also correlated significantly with the relapse rate and occurrence of new/enlarging T2 lesions while on first-line injectable therapy. Odds of future therapy escalation increased from 0.14 for sNfL below 7.5 pg/mL to 6.38 for sNfL above 15 pg/mL. In patients with a recent relapse, higher sNfL levels were associated with poorer recovery 3 months after attack. DISCUSSION: The results of this study have 3 important implications: First, pretreatment sNfL levels are a valuable biomarker for underlying disease activity in pediatric patients with MS. Second, pretreatment sNfL levels in pediatric patients with MS have a predictive value for the response to first-line therapy and the necessity of future therapy escalation. Third, high sNfL levels during a relapse are associated with poor recovery in this age group.

Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase.

Copper (Cu) is a trace metal that readily gains and donates electrons, a property that renders it desirable as an enzyme cofactor but dangerous as a source of free radicals. To regulate cellular Cu metabolism, an elaborate system of chaperones and transporters has evolved, although no human Cu chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the Cu chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution of a highly conserved arginine residue at position 163, with tryptophan in domain II of CCS, which interacts directly with superoxide dismutase 1 (SOD1). Biochemical analyses of the patient's fibroblasts, mammalian cell transfections, immunoprecipitation assays, and Lys7Δ (CCS homolog) yeast complementation support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result, this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal Cu homeostasis. CCS-Arg163Trp represents the primary example of a human mutation in a gene coding for a Cu chaperone.