Tarja Linnankivi

Mutations in GRIN2A cause idiopathic focal epilepsy with rolandic spikes

Idiopathic focal epilepsy (IFE) with rolandic spikes is the most common childhood epilepsy, comprising a phenotypic spectrum from rolandic epilepsy (also benign epilepsy with centrotemporal spikes, BECTS) to atypical benign partial epilepsy (ABPE), Landau-Kleffner syndrome (LKS) and epileptic encephalopathy with continuous spike and waves during slow-wave sleep (CSWS). The genetic basis is largely unknown. We detected new heterozygous mutations in GRIN2A in 27 of 359 affected individuals from 2 independent cohorts with IFE (7.5%; P = 4.83 × 10−18, Fishers exact test). Mutations occurred significantly more frequently in the more severe phenotypes, with mutation detection rates ranging from 12/245 (4.9%) in individuals with BECTS to 9/51 (17.6%) in individuals with CSWS (P = 0.009, Cochran-Armitage test for trend). In addition, exon-disrupting microdeletions were found in 3 of 286 individuals (1.0%; P = 0.004, Fishers exact test). These results establish alterations of the gene encoding the NMDA receptor NR2A subunit as a major genetic risk factor for IFE.

De novo loss- or gain-of-function mutations in KCNA2 cause epileptic encephalopathy.

Epileptic encephalopathies are a phenotypically and genetically heterogeneous group of severe epilepsies accompanied by intellectual disability and other neurodevelopmental features. Using next-generation sequencing, we identified four different de novo mutations in KCNA2, encoding the potassium channel KV1.2, in six isolated patients with epileptic encephalopathy (one mutation recurred three times independently). Four individuals presented with febrile and multiple afebrile, often focal seizure types, multifocal epileptiform discharges strongly activated by sleep, mild to moderate intellectual disability, delayed speech development and sometimes ataxia. Functional studies of the two mutations associated with this phenotype showed almost complete loss of function with a dominant-negative effect. Two further individuals presented with a different and more severe epileptic encephalopathy phenotype. They carried mutations inducing a drastic gain-of-function effect leading to permanently open channels. These results establish KCNA2 as a new gene involved in human neurodevelopmental disorders through two different mechanisms, predicting either hyperexcitability or electrical silencing of KV1.2-expressing neurons.

Mutations in CTC1, Encoding the CTS Telomere Maintenance Complex Component 1, Cause Cerebroretinal Microangiopathy with Calcifications and Cysts

Cerebroretinal microangiopathy with calcifications and cysts (CRMCC) is a rare multisystem disorder characterized by extensive intracranial calcifications and cysts, leukoencephalopathy, and retinal vascular abnormalities. Additional features include poor growth, skeletal and hematological abnormalities, and recurrent gastrointestinal bleedings. Autosomal-recessive inheritance has been postulated. The pathogenesis of CRMCC is unknown, but its phenotype has key similarities with Revesz syndrome, which is caused by mutations in TINF2, a gene encoding a member of the telomere protecting shelterin complex. After a whole-exome sequencing approach in four unrelated individuals with CRMCC, we observed four recessively inherited compound heterozygous mutations in CTC1, which encodes the CTS telomere maintenance complex component 1. Sanger sequencing revealed seven more compound heterozygous mutations in eight more unrelated affected individuals. Two individuals who displayed late-onset cerebral findings, a normal fundus appearance, and no systemic findings did not have CTC1 mutations, implying that systemic findings are an important indication for CTC1 sequencing. Of the 11 mutations identified, four were missense, one was nonsense, two resulted in in-frame amino acid deletions, and four were short frameshift-creating deletions. All but two affected individuals were compound heterozygous for a missense mutation and a frameshift or nonsense mutation. No individuals with two frameshift or nonsense mutations were identified, which implies that severe disturbance of CTC1 function from both alleles might not be compatible with survival. Our preliminary functional experiments did not show evidence of severely affected telomere integrity in the affected individuals. Therefore, determining the underlying pathomechanisms associated with deficient CTC1 function will require further studies.

18q deletions : Clinical, molecular, and brain MRI findings of 14 individuals

We studied 14 individuals with partial deletions of the long arm of chromosome 18, including terminal and interstitial de novo and inherited deletions. Study participants were examined clinically and by brain MRI. The size of the deletion was determined by segregation analysis using microsatellite markers. We observed that the phenotype was highly variable, even in two families with three 1st degree relatives. Among the 14 individuals, general intelligence varied from normal to severe mental retardation. The more common features of 18q‐deletions (e.g., foot deformities, aural atresia, palatal abnormalities, dysmyelination, and nystagmus) were present in individuals lacking only the distal portion 18q22.3‐qtel. Interstitial deletions exerted very heterogeneous effects on phenotype. In individuals with distal 18q22.3‐q23 deletions, brain MRI was very distinctive with poor differentiation of gray and white matter on T2‐weighted images.

Molecular characterization of folate receptor 1 mutations delineates cerebral folate transport deficiency.

Cerebral folate transport deficiency is an inherited brain-specific folate transport defect that is caused by mutations in the folate receptor 1 gene coding for folate receptor alpha (FRα). This genetic defect gives rise to a progressive neurological disorder with late infantile onset. We screened 72 children with low 5-methyltetrahydrofolate concentrations in the cerebrospinal fluid and neurological symptoms that developed after infancy. We identified nucleotide alterations in the folate receptor 1 gene in 10 individuals who shared developmental regression, ataxia, profound cerebral hypomyelination and cerebellar atrophy. We found four novel pathogenic alleles, one splice mutation and three missense mutations. Heterologous expression of the missense mutations, including previously described mutants, revealed minor decrease in protein expression but loss of cell surface localization, mistargeting to intracellular compartments and thus absence of cellular binding of folic acid. These results explain the functional loss of folate receptor alpha for all detected folate receptor 1 mutations. Three individuals presenting a milder clinical phenotype revealed very similar biochemical and brain imaging data but partially shared pathogenic alleles with more severely affected patients. Thus, our studies suggest that different clinical severities do not necessarily correlate with residual function of folate receptor alpha mutants and indicate that additional factors contribute to the clinical phenotype in cerebral folate transport deficiency.

Five new cases of a recently described leukoencephalopathy with high brain lactate

Background: A new leukoencephalopathy with brainstem and spinal cord involvement and high brain lactate was recently defined. The authors describe five new patients with this entity. Methods: Brain MRI was performed in all patients and spinal MRI and proton magnetic resonance spectroscopy (1H-MRS) in four patients. Laboratory examinations ruled out classic leukodystrophies. Results: MRI showed signal abnormalities in the periventricular and deep white matter, in the pyramidal tracts, mesencephalic trigeminal tracts, in the cerebellar connections, and in dorsal columns of the spinal cord. MRS showed decreased N-acetylaspartate and increased lactate in the white matter of all patients. In one patient choline-containing compounds were elevated. A slowly progressive sensory ataxia and tremor manifested at the age of 3 to 16 years and distal spasticity in adolescence. One 13-year-old patient was asymptomatic. Conclusions: A slowly progressive sensory ataxia is a typical feature in this new leukodystrophy. MRS favors a primary axonal degeneration.

Cerebroretinal microangiopathy with calcifications and cysts

Background: Extensive cerebral calcifications and leukoencephalopathy have been reported in two rare disorders Coats plus and leukoencephalopathy with calcifications and cysts. In the latter, a progressive formation of parenchymal brain cysts is a special feature, whereas Coats plus is characterized by intrauterine growth retardation, bilateral retinal telangiectasias and exudations (Coats disease), sparse hair, and dysplastic nails without cyst formation. Methods: We identified 13 patients, including two pairs of siblings, with extensive cerebral calcifications and leukoencephalopathy. We reviewed clinical, ophthalmologic, radiologic and neuropathologic data of seven deceased patients and studied five patients prospectively. Results: Eleven patients were small for gestational age; the other symptoms emerged from infancy to adolescence. All patients had neurologic symptoms including seizures, spasticity, dystonia, ataxia, and cognitive decline. Progressive intracerebral calcifications involved deep gray nuclei, brainstem, cerebral and cerebellar white matter, and dentate nuclei and were accompanied by diffuse white matter signal changes and, in five patients, cerebral cysts. Eleven patients had retinal telangiectasias or angiomas. Additional features were skeletal and hematologic abnormalities, intestinal bleeding, and poor growth. Neuropathologic examination showed extensive calcinosis and abnormal small vessels with thickened, hyalinized wall and reduced lumen. Conclusions: Our data suggest that Coats plus syndrome and leukoencephalopathy with calcifications and cysts belong to the same spectrum. The primary abnormality seems to be an obliterative cerebral angiopathy involving small vessels, leading to dystrophic calcifications via slow necrosis and finally to formation of cysts and secondary white matter abnormalities.

DARS2 mutations in mitochondrial leucoencephalopathy and multiple sclerosis

Background Leucoencephalopathy with brain stem and spinal cord involvement and high brain lactate (LBSL) was first defined by characteristic magnetic resonance imaging and spectroscopic findings. The clinical features include childhood or juvenile onset slowly progressive ataxia, spasticity, and dorsal column dysfunction, occasionally accompanied by learning difficulties. Mutations in DARS2, encoding mitochondrial aspartyl-tRNA synthetase, were recently shown to cause LBSL. The signs and symptoms show some overlap with the most common leucoencephalopathy of young adults, multiple sclerosis (MS). Objective To clarify the molecular background of LBSL patients in Finland, and to look for DARS2 mutations in a group of MS patients. Methods Clinical evaluation of LBSL patients, DARS2 sequencing and haplotype analysis, and carrier frequency determination in Finland. Results All eight LBSL patients were compound heterozygotes for DARS2 mutations: all carried R76SfsX5 change, seven had M134_K165del, and one had C152F change. Axonal neuropathy was found in five of the eight patients. The carrier frequencies of the R76SfsX5 and M134_K165del mutations were 1:95 and 1:380, respectively. All patients shared common European haplotypes, suggestive of common European LBSL ancestors. No enrichment of the two common DARS2 mutations was found in 321 MS patients. Conclusion All LBSL patients were compound heterozygotes, which suggests that DARS2 mutation homozygosity may be lethal or manifest as a different phenotype. The authors show here that despite identical mutations the clinical picture was quite variable in the patients. Axonal neuropathy was an important feature of LBSL. DARS2 mutations cause childhood-to-adolescence onset leucoencephalopathy, but they do not seem to be associated with MS.

GRIN2B encephalopathy: novel findings on phenotype, variant clustering, functional consequences and treatment aspects

Background We aimed for a comprehensive delineation of genetic, functional and phenotypic aspects of GRIN2B encephalopathy and explored potential prospects of personalised medicine. Methods Data of 48 individuals with de novo GRIN2B variants were collected from several diagnostic and research cohorts, as well as from 43 patients from the literature. Functional consequences and response to memantine treatment were investigated in vitro and eventually translated into patient care. Results Overall, de novo variants in 86 patients were classified as pathogenic/likely pathogenic. Patients presented with neurodevelopmental disorders and a spectrum of hypotonia, movement disorder, cortical visual impairment, cerebral volume loss and epilepsy. Six patients presented with a consistent malformation of cortical development (MCD) intermediate between tubulinopathies and polymicrogyria. Missense variants cluster in transmembrane segments and ligand-binding sites. Functional consequences of variants were diverse, revealing various potential gain-of-function and loss-of-function mechanisms and a retained sensitivity to the use-dependent blocker memantine. However, an objectifiable beneficial treatment response in the respective patients still remains to be demonstrated. Conclusions In addition to previously known features of intellectual disability, epilepsy and autism, we found evidence that GRIN2B encephalopathy is also frequently associated with movement disorder, cortical visual impairment and MCD revealing novel phenotypic consequences of channelopathies.

Selenoprotein biosynthesis defect causes progressive encephalopathy with elevated lactate

Objective: We aimed to decipher the molecular genetic basis of disease in a cohort of children with a uniform clinical presentation of neonatal irritability, spastic or dystonic quadriplegia, virtually absent psychomotor development, axonal neuropathy, and elevated blood/CSF lactate. Methods: We performed whole-exome sequencing of blood DNA from the index patients. Detected compound heterozygous mutations were confirmed by Sanger sequencing. Structural predictions and a bacterial activity assay were performed to evaluate the functional consequences of the mutations. Mass spectrometry, Western blotting, and protein oxidation detection were used to analyze the effects of selenoprotein deficiency. Results: Neuropathology indicated laminar necrosis and severe loss of myelin, with neuron loss and astrogliosis. In 3 families, we identified a missense (p.Thr325Ser) and a nonsense (p.Tyr429*) mutation in SEPSECS, encoding the O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase, which was previously associated with progressive cerebellocerebral atrophy. We show that the mutations do not completely abolish the activity of SEPSECS, but lead to decreased selenoprotein levels, with demonstrated increase in oxidative protein damage in the patient brain. Conclusions: These results extend the phenotypes caused by defective selenocysteine biosynthesis, and suggest SEPSECS as a candidate gene for progressive encephalopathies with lactate elevation.