Grace Vassallo

Mutations in ADAR1 cause Aicardi-Goutières syndrome associated with a type I interferon signature

Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosine to inosine in double-stranded RNA (dsRNA) and thereby potentially alter the information content and structure of cellular RNAs. Notably, although the overwhelming majority of such editing events occur in transcripts derived from Alu repeat elements, the biological function of non-coding RNA editing remains uncertain. Here, we show that mutations in ADAR1 (also known as ADAR) cause the autoimmune disorder Aicardi-Goutières syndrome (AGS). As in Adar1-null mice, the human disease state is associated with upregulation of interferon-stimulated genes, indicating a possible role for ADAR1 as a suppressor of type I interferon signaling. Considering recent insights derived from the study of other AGS-related proteins, we speculate that ADAR1 may limit the cytoplasmic accumulation of the dsRNA generated from genomic repetitive elements.

Use of Whole-Exome Sequencing to Determine the Genetic Basis of Multiple Mitochondrial Respiratory Chain Complex Deficiencies

IMPORTANCE Mitochondrial disorders have emerged as a common cause of inherited disease, but their diagnosis remains challenging. Multiple respiratory chain complex defects are particularly difficult to diagnose at the molecular level because of the massive number of nuclear genes potentially involved in intramitochondrial protein synthesis, with many not yet linked to human disease. OBJECTIVE To determine the molecular basis of multiple respiratory chain complex deficiencies. DESIGN, SETTING, AND PARTICIPANTS We studied 53 patients referred to 2 national centers in the United Kingdom and Germany between 2005 and 2012. All had biochemical evidence of multiple respiratory chain complex defects but no primary pathogenic mitochondrial DNA mutation. Whole-exome sequencing was performed using 62-Mb exome enrichment, followed by variant prioritization using bioinformatic prediction tools, variant validation by Sanger sequencing, and segregation of the variant with the disease phenotype in the family. RESULTS Presumptive causal variants were identified in 28 patients (53%; 95% CI, 39%-67%) and possible causal variants were identified in 4 (8%; 95% CI, 2%-18%). Together these accounted for 32 patients (60% 95% CI, 46%-74%) and involved 18 different genes. These included recurrent mutations in RMND1, AARS2, and MTO1, each on a haplotype background consistent with a shared founder allele, and potential novel mutations in 4 possible mitochondrial disease genes (VARS2, GARS, FLAD1, and PTCD1). Distinguishing clinical features included deafness and renal involvement associated with RMND1 and cardiomyopathy with AARS2 and MTO1. However, atypical clinical features were present in some patients, including normal liver function and Leigh syndrome (subacute necrotizing encephalomyelopathy) seen in association with TRMU mutations and no cardiomyopathy with founder SCO2 mutations. It was not possible to confidently identify the underlying genetic basis in 21 patients (40%; 95% CI, 26%-54%). CONCLUSIONS AND RELEVANCE Exome sequencing enhances the ability to identify potential nuclear gene mutations in patients with biochemically defined defects affecting multiple mitochondrial respiratory chain complexes. Additional study is required in independent patient populations to determine the utility of this approach in comparison with traditional diagnostic methods.

Assessment of interferon-related biomarkers in Aicardi-Goutières syndrome associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR: A case-control study

BACKGROUND Aicardi-Goutières syndrome (AGS) is an inflammatory disorder caused by mutations in any of six genes (TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR). The disease is severe and effective treatments are urgently needed. We investigated the status of interferon-related biomarkers in patients with AGS with a view to future use in diagnosis and clinical trials. METHODS In this case-control study, samples were collected prospectively from patients with mutation-proven AGS. The expression of six interferon-stimulated genes (ISGs) was measured by quantitative PCR, and the median fold change, when compared with the median of healthy controls, was used to create an interferon score for each patient. Scores higher than the mean of controls plus two SD (>2·466) were designated as positive. Additionally, we collated historical data for interferon activity, measured with a viral cytopathic assay, in CSF and serum from mutation-positive patients with AGS. We also undertook neutralisation assays of interferon activity in serum, and looked for the presence of autoantibodies against a panel of interferon proteins. FINDINGS 74 (90%) of 82 patients had a positive interferon score (median 12·90, IQR 6·14-20·41) compared with two (7%) of 29 controls (median 0·93, IQR 0·57-1·30). Of the eight patients with a negative interferon score, seven had mutations in RNASEH2B (seven [27%] of all 26 patients with mutations in this gene). Repeat sampling in 16 patients was consistent for the presence or absence of an interferon signature on 39 of 41 occasions. Interferon activity (tested in 147 patients) was negatively correlated with age (CSF, r=-0·604; serum, r=-0·289), and was higher in CSF than in serum in 104 of 136 paired samples. Neutralisation assays suggested that measurable antiviral activity was related to interferon α production. We did not record significantly increased concentrations of autoantibodies to interferon subtypes in patients with AGS, or an association between the presence of autoantibodies and interferon score or serum interferon activity. INTERPRETATION AGS is consistently associated with an interferon signature, which is apparently sustained over time and can thus be used to differentiate patients with AGS from controls. If future studies show that interferon status is a reactive biomarker, the measurement of an interferon score might prove useful in the assessment of treatment efficacy in clinical trials. FUNDING European Unions Seventh Framework Programme; European Research Council.

Phospholipase C beta 1 deficiency is associated with early-onset epileptic encephalopathy

The epileptic encephalopathies of infancy and childhood are a collection of epilepsy disorders characterized by refractory, severe seizures and poor neurological outcome, in which the mechanism of disease is poorly understood. We report the clinical presentation and evolution of epileptic encephalopathy in a patient, associated with a loss-of-function mutation in the phospholipase C-β 1 gene. We ascertained a consanguineous family containing a male infant who presented with early-onset epileptic encephalopathy for detailed clinical phenotyping and molecular genetic investigation. In addition, a cohort of 12 consanguineous families of children with infantile spasms were analysed for linkage to the phospholipase C-β 1 gene locus. The male infant presented with tonic seizures in early infancy and subsequently developed infantile spasms. Over time, he developed drug-resistant epilepsy associated with severe neurological regression and failure to thrive. Molecular genetic investigation revealed a homozygous loss-of-function 0.5-Mb deletion, encompassing the promoter element and exons 1, 2 and 3 of phospholipase C-β 1 in the index case. Linkage to the phospholipase C-β 1 locus was excluded in the 12 other consanguineous families, consistent with genetic heterogeneity in this disorder. Although phospholipase C-β 1 deficiency has not previously been reported in humans, the Plcb1 homozygote knockout mouse displays early-onset severe tonic seizures and growth retardation, thus recapitulating the human phenotype. Phospholipase C-β 1 has important functions in both hippocampal muscarinic acetylcholine receptor signalling and in cortical development. Thus, the discovery of a phospholipase C-β 1 mutation allows us to propose a novel potential underlying mechanism in early-onset epileptic encephalopathy.

Identification and Characterization of an Inborn Error of Metabolism Caused by Dihydrofolate Reductase Deficiency

Dihydrofolate reductase (DHFR) is a critical enzyme in folate metabolism and an important target of antineoplastic, antimicrobial, and antiinflammatory drugs. We describe three individuals from two families with a recessive inborn error of metabolism, characterized by megaloblastic anemia and/or pancytopenia, severe cerebral folate deficiency, and cerebral tetrahydrobiopterin deficiency due to a germline missense mutation in DHFR, resulting in profound enzyme deficiency. We show that cerebral folate levels, anemia, and pancytopenia of DHFR deficiency can be corrected by treatment with folinic acid. The characterization of this disorder provides evidence for the link between DHFR and metabolism of cerebral tetrahydrobiopterin, which is required for the formation of dopamine, serotonin, and norepinephrine and for the hydroxylation of aromatic amino acids. Moreover, this relationship provides insight into the role of folates in neurological conditions, including depression, Alzheimer disease, and Parkinson disease.

Mutations in SLC12A5 in epilepsy of infancy with migrating focal seizures.

The potassium-chloride co-transporter KCC2, encoded by SLC12A5, plays a fundamental role in fast synaptic inhibition by maintaining a hyperpolarizing gradient for chloride ions. KCC2 dysfunction has been implicated in human epilepsy, but to date, no monogenic KCC2-related epilepsy disorders have been described. Here we show recessive loss-of-function SLC12A5 mutations in patients with a severe infantile-onset pharmacoresistant epilepsy syndrome, epilepsy of infancy with migrating focal seizures (EIMFS). Decreased KCC2 surface expression, reduced protein glycosylation and impaired chloride extrusion contribute to loss of KCC2 activity, thereby impairing normal synaptic inhibition and promoting neuronal excitability in this early-onset epileptic encephalopathy.

Clinical features of childhood‐onset paroxysmal kinesigenic dyskinesia with PRRT2 gene mutations

To define better the phenotype and genotype of familial and sporadic cases of paroxysmal kinesigenic dyskinesia (PKD) caused by mutations in the PRRT2 gene presenting in the paediatric age group.

An investigation into the relationship between vigabatrin, movement disorders, and brain magnetic resonance imaging abnormalities in children with infantile spasms

We aimed to investigate the relationship between movement disorders, changes on brain magnetic resonance imaging (MRI), and vigabatrin therapy in children with infantile spasms.

Cerebral palsy: An overview and the role of a paediatric neurologist in the evaluation of children with complex needs for cochlear implant

Abstract Cerebral palsy (CP) is primarily a non-progressive disorder of movement and posture affecting the developing brain, but many children with CP have other impairments which may affect their quality of life and life expectancy. It is the most common childhood physical disability and affects 2 to 2.5 children per 1,000 born in the developing world. The differential diagnosis of cerebral palsy includes metabolic and genetic disorders. The diagnostic strategy is based on the clinical picture, pattern of development of symptoms, family history, and other factors influencing the probability of specific diagnoses. Targeted laboratory tests and cerebral imaging using magnetic resonance imaging are useful diagnostic tools. Surveillance for associated disabilities such as hearing impairment can help determine the goals of treatment and to improve functionality and quality of life. Children with CP being evaluated for cochlear implant should have assessment by paediatric neurologists to establish the correct diagnosis and identify co-morbidities that can affect the childs use of cochlear implant. Multi-specialty teams should be developed around the needs of each patient to provide goal oriented care plans.

The clinical, biochemical and genetic features associated with RMND1-related mitochondrial disease

Background Mutations in the RMND1 (Required for Meiotic Nuclear Division protein 1) gene have recently been linked to infantile onset mitochondrial disease characterised by multiple mitochondrial respiratory chain defects. Methods We summarised the clinical, biochemical and molecular genetic investigation of an international cohort of affected individuals with RMND1 mutations. In addition, we reviewed all the previously published cases to determine the genotype–phenotype correlates and performed survival analysis to identify prognostic factors. Results We identified 14 new cases from 11 pedigrees that harbour recessive RMND1 mutations, including 6 novel variants: c.533C>A, p.(Thr178Lys); c.565C>T, p.(Gln189*); c.631G>A, p.(Val211Met); c.1303C>T, p.(Leu435Phe); c.830+1G>A and c.1317+1G>T. Together with all previously published cases (n=32), we show that congenital sensorineural deafness, hypotonia, developmental delay and lactic acidaemia are common clinical manifestations with disease onset under 2 years. Renal involvement is more prevalent than seizures (66% vs 44%). In addition, median survival time was longer in patients with renal involvement compared with those without renal disease (6 years vs 8 months, p=0.009). The neurological phenotype also appears milder in patients with renal involvement. Conclusions The clinical phenotypes and prognosis associated with RMND1 mutations are more heterogeneous than that were initially described. Regular monitoring of kidney function is imperative in the clinical practice in light of nephropathy being present in over 60% of cases. Furthermore, renal replacement therapy should be considered particularly in those patients with mild neurological manifestation as shown in our study that four recipients of kidney transplant demonstrate good clinical outcome to date.