Background

Barth syndrome (BS) is an X-linked infantile-onset cardioskeletal disease characterized by cardiomyopathy, hypotonia, growth delay, neutropenia and 3-methylglutaconic aciduria. It is caused by mutations in the TAZ gene encoding tafazzin, a protein involved in the metabolism of cardiolipin, a mitochondrial-specific phospholipid involved in mitochondrial energy production.

Methods

Clinical, biochemical and molecular characterization of a group of six male patients suspected of having BS. Three patients presented early with severe metabolic decompensation including respiratory distress, oxygen desaturation and cardiomyopathy and died within the first year of life. The remaining three patients had cardiomyopathy, hypotonia and growth delay and are still alive. Cardiomyopathy was detected during pregnancy through a routine check-up in one patient. All patients exhibited 3-methylglutaconic aciduria and neutropenia, when tested and five of them also had lactic acidosis.

Results

We confirmed the diagnosis of BS with sequence analysis of the TAZ gene, and found five new mutations, c.641A>G p.His214Arg, c.284dupG (p.Thr96Aspfs*37), c.678_691del14 (p.Tyr227Trpfs*79), g.8009_16445del8437 and g.[9777_9814del38; 9911-?_14402del] and the known nonsense mutation c.367C>T (p.Arg123Term). The two gross rearrangements ablated TAZ exons 6 to 11 and probably originated by non-allelic homologous recombination and by Serial Replication Slippage (SRS), respectively. The identification of the breakpoints boundaries of the gross deletions allowed the direct detection of heterozygosity in carrier females.

Conclusions

Lactic acidosis associated with 3-methylglutaconic aciduria is highly suggestive of BS, whilst the severity of the metabolic decompensation at disease onset should be considered for prognostic purposes. Mutation analysis of the TAZ gene is necessary for confirming the clinical and biochemical diagnosis in probands in order to identify heterozygous carriers and supporting prenatal diagnosis and genetic counseling.

Infantile spasms (ISS) are an epilepsy disorder frequently associated with severe developmental outcome and have diverse genetic etiologies. We ascertained 11 subjects with ISS and novel copy number variants (CNVs) and combined these with a new cohort with deletion 1p36 and ISS, and additional published patients with ISS and other chromosomal abnormalities. Using bioinformatics tools, we analyzed the gene content of these CNVs for enrichment in pathways of pathogenesis. Several important findings emerged. First, the gene content was enriched for the gene regulatory network involved in ventral forebrain development. Second, genes in pathways of synaptic function were overrepresented, significantly those involved in synaptic vesicle transport. Evidence also suggested roles for GABAergic synapses and the postsynaptic density. Third, we confirm the association of ISS with duplication of 14q12 and maternally inherited duplication of 15q11q13, and report the association with duplication of 21q21. We also present a patient with ISS and deletion 7q11.3 not involving MAGI2. Finally, we provide evidence that ISS in deletion 1p36 may be associated with deletion of KLHL17 and expand the epilepsy phenotype in that syndrome to include early infantile epileptic encephalopathy. Several of the identified pathways share functional links, and abnormalities of forebrain synaptic growth and function may form a common biologic mechanism underlying both ISS and autism. This study demonstrates a novel approach to the study of gene content in subjects with ISS and copy number variation, and contributes further evidence to support specific pathways of pathogenesis.

Many pathogenic structural variants of the human genome are known to cause facial dysmorphism. During the past decade, pathogenic structural variants have also been found to be an important class of genetic risk factor for epilepsy. In other fields, face shape has been assessed objectively using 3D stereophotogrammetry and dense surface models. We hypothesized that computer-based analysis of 3D face images would detect subtle facial abnormality in people with epilepsy who carry pathogenic structural variants as determined by chromosome microarray. In 118 children and adults attending three European epilepsy clinics, we used an objective measure called Face Shape Difference to show that those with pathogenic structural variants have a significantly more atypical face shape than those without such variants. This is true when analysing the whole face, or the periorbital region or the perinasal region alone. We then tested the predictive accuracy of our measure in a second group of 63 patients. Using a minimum threshold to detect face shape abnormalities with pathogenic structural variants, we found high sensitivity (4/5, 80% for whole face; 3/5, 60% for periorbital and perinasal regions) and specificity (45/58, 78% for whole face and perinasal regions; 40/58, 69% for periorbital region). We show that the results do not seem to be affected by facial injury, facial expression, intellectual disability, drug history or demographic differences. Finally, we use bioinformatics tools to explore relationships between facial shape and gene expression within the developing forebrain. Stereophotogrammetry and dense surface models are powerful, objective, non-contact methods of detecting relevant face shape abnormalities. We demonstrate that they are useful in identifying atypical face shape in adults or children with structural variants, and they may give insights into the molecular genetics of facial development.

Background

Despite progresses in neonatal care, the mortality and the incidence of neuro-motor disability after perinatal asphyxia have failed to show substantial improvements. In countries with a high level of perinatal care, the incidence of asphyxia responsible for moderate or severe encephalopathy is still 2–3 per 1000 term newborns. Recent trials have demonstrated that moderate hypothermia, started within 6 hours after birth and protracted for 72 hours, can significantly improve survival and reduce neurologic impairment in neonates with hypoxic-ischemic encephalopathy. It is not currently known whether neuroprotective drugs can further improve the beneficial effects of hypothermia. Topiramate has been proven to reduce brain injury in animal models of neonatal hypoxic ischemic encephalopathy. However, the association of mild hypothermia and topiramate treatment has never been studied in human newborns. The objective of this research project is to evaluate, through a multicenter randomized controlled trial, whether the efficacy of moderate hypothermia can be increased by concomitant topiramate treatment.

Methods/Design

Term newborns (gestational age ≥ 36 weeks and birth weight ≥ 1800 g) with precocious metabolic, clinical and electroencephalographic (EEG) signs of hypoxic-ischemic encephalopathy will be randomized, according to their EEG pattern, to receive topiramate added to standard treatment with moderate hypothermia or standard treatment alone. Topiramate will be administered at 10 mg/kg once a day for the first 3 days of life. Topiramate concentrations will be measured on serial dried blood spots. 64 participants will be recruited in the study. To evaluate the safety of topiramate administration, cardiac and respiratory parameters will be continuously monitored. Blood samplings will be performed to check renal, liver and metabolic balance. To evaluate the efficacy of topiramate, the neurologic outcome of enrolled newborns will be evaluated by serial neurologic and neuroradiologic examinations. Visual function will be evaluated by means of behavioural standardized tests.

Discussion

This pilot study will explore the possible therapeutic role of topiramate in combination with moderate hypothermia. Any favourable results of this research might open new perspectives about the reduction of cerebral damage in asphyxiated newborns.

Trial registration

Current Controlled Trials ISRCTN62175998; ClinicalTrials.gov Identifier NCT01241019; EudraCT Number 2010-018627-25

Malformations of cerebral cortical development include a wide range of developmental disorders that are common causes of neurodevelopmental delay and epilepsy. In addition, study of these disorders contributes greatly to the understanding of normal brain development and its perturbations. The rapid recent evolution of molecular biology, genetics and imaging has resulted in an explosive increase in our knowledge of cerebral cortex development and in the number and types of malformations of cortical development that have been reported. These advances continue to modify our perception of these malformations. This review addresses recent changes in our perception of these disorders and proposes a modified classification based upon updates in our knowledge of cerebral cortical development.

Mutations in the ALDOB gene impair the activity of the hepatic aldolase B enzyme, causing hereditary fructose intolerance (HFI), an inherited autosomic recessive disease of carbohydrate metabolism, that can result in hypoglycemia, liver and kidney failure, coma, and death. Noninvasive diagnosis is possible by identifying mutant ALDOB alleles in suspected patients. We report the genetic characterization of a cohort of 18 HFI Caucasian patients, based on PCR-sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA), with the identification of two novel genetic lesions: a small duplication c.940_941dupT (p.Trp314fsX22) and a large deletion encompassing the promoter region and exon 1. MLPA and long range-PCR (LR-PCR) also identified the recently reported g.7840_14288del6448 allele with a surprisingly high frequency (11%) within our patients’ cohort. The most common p.Ala150Pro (44%), p.Ala175Asp (19%), p.Asn335Lys (8%), and/or the known c.360-363del4 (5%), p.Tyr204X (2.8%), IVS6 −2A>G (2.8%) mutant alleles were identified in 14 patients at a homozygous or compound-heterozygous level. The integration of PCR-sequencing analysis with exon-dosage tools [MLPA and quantitative fluorescent multiplex-PCR (QFM-PCR)] led to the full genotyping of patients within our cohort and to the identification of the new deletion encompassing the promoter region and exon 1.

Focal cortical dysplasias (FCDs) are localized regions of malformed cerebral cortex and are very frequently associated with epilepsy in both children and adults. A broad spectrum of histopathology has been included in the diagnosis of FCD. Characteristic findings include aberrant radial or tangential lamination of the neocortex (FCD Type I) and cytological abnormalities (FCD Type II). An ILAE task force has re-evaluated available data and proposes a clinico-pathologic classification system of FCDs. The major change since a prior classification represents the introduction of FCD Type III, which occurs in combination with Hippocampal Sclerosis (FCD Type IIIa), or with epilepsy-associated tumors (FCD Type IIIb). FCD Type IIIc is found adjacent to vascular malformations, whereas FCD Type IIId can be diagnosed in association with epileptogenic lesions acquired in early life (i.e., traumatic injury, ischemic injury or encephalitis). Hence, FCD Type I will now refer to isolated lesions, which present either as radial (FCD Type Ia) or tangential (FCD Type Ib) dyslamination of the neocortex, microscopically identified in one or multiple lobes. FCD Type II is an isolated lesion characterized by cortical dyslamination and dysmorphic neurons without (Type IIa) or with balloon cells (Type IIb). Architectural abnormalities adjacent to or within gross malformations of cortical development are frequently observed and not distinguished as a specific FCD variant. This three-tiered classification system will help to better characterize specific clinico-pathological entities and is an important basis to further explore imaging, electro-clinical features, and postsurgical seizure control as well as underlying molecular pathomechanisms.

GM1 gangliosidosis and Morquio B syndrome, both arising from beta-galactosidase (GLB1) deficiency, are very rare lysosomal storage diseases with an incidence of about 1:100,000– 1:200,000 live births worldwide. Here we report the beta-galactosidase gene (GLB1) mutation analysis of 21 unrelated GM1 gangliosidosis patients, and of 4 Morquio B patients, of whom two are brothers. Clinical features of the patients were collected and compared with those in literature. In silico analyses were performed by standard alignments tools and by an improved version of GLB1 three-dimensional models. The analysed cohort includes remarkable cases. One patient with GM1 gangliosidosis had a triple X syndrome. One patient with juvenile GM1 gangliosidosis was homozygous for a mutation previously identified in Morquio type B. A patient with infantile GM1 gangliosidosis carried a complex GLB1 allele harbouring two genetic variants leading to p.R68W and p.R109W amino acid changes, in trans with the known p.R148C mutation.

Molecular analysis showed 27 mutations, 9 of which are new: 5 missense, 3 microdeletions and a nonsense mutation. We also identified four new genetic variants with a predicted polymorphic nature that was further investigated by in silico analyses.

Three-dimensional structural analysis of GLB1 homology models including the new missense mutations and the p.R68W and p.R109W amino acid changes, showed that all the amino acids replacements affected the resulting protein structures in different ways, from changes in polarity to folding alterations. Genetic and clinical associations led us to undertake a critical review of the classifications of late-onset GM1 gangliosidosis and Morquio B disease.

In this study, we used deletions at 22q13, which represent a substantial source of human pathology (Phelan/McDermid syndrome), as a model for investigating the molecular mechanisms of terminal deletions that are currently poorly understood. We characterized at the molecular level the genomic rearrangement in 44 unrelated patients with 22q13 monosomy resulting from simple terminal deletions (72%), ring chromosomes (14%), and unbalanced translocations (7%). We also discovered interstitial deletions between 17–74 kb in 9% of the patients. Haploinsufficiency of the SHANK3 gene, confirmed in all rearrangements, is very likely the cause of the major neurological features associated with PMS. SHANK3 mutations can also result in language and/or social interaction disabilities. We determined the breakpoint junctions in 29 cases, providing a realistic snapshot of the variety of mechanisms driving non-recurrent deletion and repair at chromosome ends. De novo telomere synthesis and telomere capture are used to repair terminal deletions; non-homologous end-joining or microhomology-mediated break-induced replication is probably involved in ring 22 formation and translocations; non-homologous end-joining and fork stalling and template switching prevail in cases with interstitial 22q13.3. For the first time, we also demonstrated that distinct stabilizing events of the same terminal deletion can occur in different early embryonic cells, proving that terminal deletions can be repaired by multistep healing events and supporting the recent hypothesis that rare pathogenic germline rearrangements may have mitotic origin. Finally, the progressive clinical deterioration observed throughout the longitudinal medical history of three subjects over forty years supports the hypothesis of a role for SHANK3 haploinsufficiency in neurological deterioration, in addition to its involvement in the neurobehavioral phenotype of PMS.

Author Summary

Terminal chromosome deletions are among the most commonly observed rearrangements detected by cytogenetics and may result in several well-known genetic syndromes. We used 22q13 deletions to study how these types of chromosome abnormalities arise. Children with Phelan/McDermid syndrome, caused by deletion of the terminal portion of chromosome 22, experience developmental delay, absent or severely delayed speech, and frequent behavioral problems. Lack of one copy of SHANK3, a key gene for the correct development and organization of brain synapses, is very likely the basis of the syndrome's major neurological features. Deletion of additional genes probably causes more complex phenotypes in subjects with larger deletions. We also studied patients who only lack a portion of SHANK3 and demonstrated that small, hard-to-detect deletions of this gene may cause substantial clinical problems. Until now, the 22q distal deletion had been only diagnosed in very young people. We studied a large group of patients of different ages and discovered that all adult patients face progressive cognitive decline. Our study demonstrates that deletion of the terminal portion of chromosome 22, a prototype for terminal deletions in human chromosomes, can occur in several ways. Mosaic deletions of different size can also form in early embryogenesis.

Tyrosinemia type I is a genetic disorder characterized by accumulation in the blood and urine of the toxic metabolite succinylacetone (SUAC), not detectable in healthy samples. In many countries, newborns are screened for tyrosinemia type I using tyrosine as a primary marker. Unfortunately, tyrosine accumulation may take longer to occur and it may be not obvious when specimens are collected, in the first few days of life, as for newborn screening. In 2008, we reported changes to simultaneously measure acylcarnitines, amino acids, and SUAC during expanded newborn screening. We established the usefulness of this method after identifying a first asymptomatic newborn affected by tyrosinemia type I. Now we report a second infant with positive SUAC screening result (14.1 μmol/L, n.v. < 2) and normal tyrosine concentration (74 μmol/L; n.v. < 250). We also performed molecular analysis of FAH gene in both patients after diagnosis at newborn screening. They had consanguineous parents and were both homozygous for two known disease-causing mutations of the FAH gene. The outcome of patients detected in the MS/MS screening is significantly favorable. We also report our results of newborn screening for tyrosinemia type I before and after inclusion of SUAC as a primary marker for this disease.

Polymicrogyria is one of the most common malformations of cortical development and is associated with a variety of clinical sequelae including epilepsy, intellectual disability, motor dysfunction and speech disturbance. It has heterogeneous clinical manifestations and imaging patterns, yet large cohort data defining the clinical and imaging spectrum and the relative frequencies of each subtype are lacking. The aims of this study were to determine the types and relative frequencies of different polymicrogyria patterns, define the spectrum of their clinical and imaging features and assess for clinical/imaging correlations. We studied the imaging features of 328 patients referred from six centres, with detailed clinical data available for 183 patients. The ascertainment base was wide, including referral from paediatricians, geneticists and neurologists. The main patterns of polymicrogyria were perisylvian (61%), generalized (13%), frontal (5%) and parasagittal parieto-occipital (3%), and in 11% there was associated periventricular grey matter heterotopia. Each of the above patterns was further divided into subtypes based on distinguishing imaging characteristics. The remaining 7% were comprised of a number of rare patterns, many not described previously. The most common clinical sequelae were epileptic seizures (78%), global developmental delay (70%), spasticity (51%) and microcephaly (50%). Many patients presented with neurological or developmental abnormalities prior to the onset of epilepsy. Patients with more extensive patterns of polymicrogyria presented at an earlier age and with more severe sequelae than those with restricted or unilateral forms. The median age at presentation for the entire cohort was 4 months with 38% presenting in either the antenatal or neonatal periods. There were no significant differences between the prevalence of epilepsy for each polymicrogyria pattern, however patients with generalized and bilateral forms had a lower age at seizure onset. There was significant skewing towards males with a ratio of 3:2. This study expands our understanding of the spectrum of clinical and imaging features of polymicrogyria. Progression from describing imaging patterns to defining anatomoclinical syndromes will improve the accuracy of prognostic counselling and will aid identification of the aetiologies of polymicrogyria, including genetic causes.

Periventricular heterotopia (PH) is a disorder characterized by neuronal nodules, ectopically positioned along the lateral ventricles of the cerebral cortex. Mutations in either of two human genes, Filamin A (FLNA) or ADP-ribosylation factor guanine exchange factor 2 (ARFGEF2), cause PH (Fox et al. in ‘Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia'. Neuron, 21, 1315–1325, 1998; Sheen et al. in ‘Mutations in ARFGEF2 implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex'. Nat. Genet., 36, 69–76, 2004). Recent studies have shown that mutations in mitogen-activated protein kinase kinase kinase-4 (Mekk4), an indirect interactor with FlnA, also lead to periventricular nodule formation in mice (Sarkisian et al. in ‘MEKK4 signaling regulates filamin expression and neuronal migration'. Neuron, 52, 789–801, 2006). Here we show that neurons in post-mortem human PH brains migrated appropriately into the cortex, that periventricular nodules were primarily composed of later-born neurons, and that the neuroependyma was disrupted in all PH cases. As studied in the mouse, loss of FlnA or Big2 function in neural precursors impaired neuronal migration from the germinal zone, disrupted cell adhesion and compromised neuroepithelial integrity. Finally, the hydrocephalus with hop gait (hyh) mouse, which harbors a mutation in Napa [encoding N-ethylmaleimide-sensitive factor attachment protein alpha (α-SNAP)], also develops a progressive denudation of the neuroepithelium, leading to periventicular nodule formation. Previous studies have shown that Arfgef2 and Napa direct vesicle trafficking and fusion, whereas FlnA associates dynamically with the Golgi membranes during budding and trafficking of transport vesicles. Our current findings suggest that PH formation arises from a final common pathway involving disruption of vesicle trafficking, leading to impaired cell adhesion and loss of neuroependymal integrity.

Summary

Childhood absence epilepsy (CAE) is an idiopathic generalised epilepsy (IGE) characterised by typical absence seizures manifested by transitory loss of awareness with 2.5–4 Hz spike-wave complexes on ictal EEG. A genetic component to the aetiology is well recognised but the mechanism of inheritance and the genes involved are yet to be fully established.

A genome wide single nucleotide polymorphism (SNP)-based high density linkage scan was carried out using 41 nuclear pedigrees with at least two affected members. Multipoint parametric and non-parametric linkage analyses were performed using MERLIN 1.1.1 and a susceptibility locus was identified on chromosome 3p23-p14 (Zmean = 3.9, p < 0.0001; HLOD = 3.3, α = 0.7). The linked region harbours the functional candidate genes TRAK1 and CACNA2D2. Fine-mapping using a tagSNP approach demonstrated disease association with variants in TRAK1.

Childhood absence epilepsy (CAE) is an idiopathic generalised epilepsy characterised by absence seizures manifested by transitory loss of awareness with 2.5-4Hz spike-wave complexes on ictal EEG. A genetic component to aetiology is established but the mechanism of inheritance and the genes involved are not fully defined. Available evidence suggests that genes encoding brain expressed voltage-gated calcium channels, including CACNG3 on chromosome 16p12-p13.1, may represent susceptibility loci for CAE. The aim of this work was to further evaluate CACNG3 as a susceptibility locus by linkage and association analysis. Assuming locus heterogeneity, a significant HLOD score (HLOD=3.54, α=0.62) was obtained for markers encompassing CACNG3 in 65 nuclear families with a proband with CAE. The maximum NPL score was 2.87 (p<0.002). Re-sequencing of the coding exons in 59 patients did not identify any putative causal variants. A linkage disequilibrium (LD) map of CACNG3 was constructed using 23 single nucleotide polymorphisms (SNPs). Transmission disequilibrium was sought using individual SNPs and SNP-based haplotypes with the pedigree disequilibrium test in 217 CAE trios and the 65 nuclear pedigrees.

Evidence for transmission disequilibrium (p≤0.01) was found for SNPs within a ∼35kb region of high LD encompassing the 5′UTR, exon 1 and part of intron 1 of CACNG3. Re-sequencing of this interval was undertaken in 24 affected individuals. Seventy-two variants were identified: 45 upstream; two 5′UTR; and 25 intronic SNPs. No coding sequence variants were identified, although four variants are predicted to affect exonic splicing.

This evidence supports CACNG3 as a susceptibility locus in a subset of CAE patients.

Malformations of cortical development (MCDs) are macroscopic or microscopic abnormalities of the cerebral cortex that arise as a consequence of an interruption to the normal steps of formation of the cortical plate. The human cortex develops its basic structure during the first two trimesters of pregnancy as a series of overlapping steps, beginning with proliferation and differentiation of neurons, which then migrate before finally organizing themselves in the developing cortex. Abnormalities at any of these stages, be they environmental or genetic in origin, may cause disruption of neuronal circuitry and predispose to a variety of clinical consequences, the most common of which is epileptic seizures, A large number of MCDs have now been described, each with characteristic pathological, clinical, and imaging features. The causes of many of these MCDs have been determined through the study of affected individuals, with many MCDs now established as being secondary to mutations in cortical development genes. This review will highlight the best-known of the human cortical malformations associated with epilepsy. The pathological, clinical, imaging, and etioiogic features of each MCD will be summarized, with representative magnetic resonance imaging (MRI) images shown for each MCD, The malformations tuberous sclerosis, focal cortical dysplasia, hemimegalencephaiy, classical iissencephaly, subcortical band heterotopia, periventricular nodular heterotopia, polymicrogyria, and schizencephaly will be presented.