Cengiz Yalcinkaya

Whole-exome sequencing identifies recessive WDR62 mutations in severe brain malformations

The development of the human cerebral cortex is an orchestrated process involving the generation of neural progenitors in the periventricular germinal zones, cell proliferation characterized by symmetric and asymmetric mitoses, followed by migration of post-mitotic neurons to their final destinations in six highly ordered, functionally specialized layers. An understanding of the molecular mechanisms guiding these intricate processes is in its infancy, substantially driven by the discovery of rare mutations that cause malformations of cortical development. Mapping of disease loci in putative Mendelian forms of malformations of cortical development has been hindered by marked locus heterogeneity, small kindred sizes and diagnostic classifications that may not reflect molecular pathogenesis. Here we demonstrate the use of whole-exome sequencing to overcome these obstacles by identifying recessive mutations in WD repeat domain 62 (WDR62) as the cause of a wide spectrum of severe cerebral cortical malformations including microcephaly, pachygyria with cortical thickening as well as hypoplasia of the corpus callosum. Some patients with mutations in WDR62 had evidence of additional abnormalities including lissencephaly, schizencephaly, polymicrogyria and, in one instance, cerebellar hypoplasia, all traits traditionally regarded as distinct entities. In mice and humans, WDR62 transcripts and protein are enriched in neural progenitors within the ventricular and subventricular zones. Expression of WDR62 in the neocortex is transient, spanning the period of embryonic neurogenesis. Unlike other known microcephaly genes, WDR62 does not apparently associate with centrosomes and is predominantly nuclear in localization. These findings unify previously disparate aspects of cerebral cortical development and highlight the use of whole-exome sequencing to identify disease loci in settings in which traditional methods have proved challenging.

CLP1 founder mutation links tRNA splicing and maturation to cerebellar development and neurodegeneration.

Neurodegenerative diseases can occur so early as to affect neurodevelopment. From a cohort of more than 2,000 consanguineous families with childhood neurological disease, we identified a founder mutation in four independent pedigrees in cleavage and polyadenylation factor I subunit 1 (CLP1). CLP1 is a multifunctional kinase implicated in tRNA, mRNA, and siRNA maturation. Kinase activity of the CLP1 mutant protein was defective, and the tRNA endonuclease complex (TSEN) was destabilized, resulting in impaired pre-tRNA cleavage. Germline clp1 null zebrafish showed cerebellar neurodegeneration that was rescued by wild-type, but not mutant, human CLP1 expression. Patient-derived induced neurons displayed both depletion of mature tRNAs and accumulation of unspliced pre-tRNAs. Transfection of partially processed tRNA fragments into patient cells exacerbated an oxidative stress-induced reduction in cell survival. Our data link tRNA maturation to neuronal development and neurodegeneration through defective CLP1 function in humans.

Brain white matter oedema due to ClC-2 chloride channel deficiency: an observational analytical study.

BACKGROUND Mutant mouse models suggest that the chloride channel ClC-2 has functions in ion and water homoeostasis, but this has not been confirmed in human beings. We aimed to define novel disorders characterised by distinct patterns of MRI abnormalities in patients with leukoencephalopathies of unknown origin, and to identify the genes mutated in these disorders. We were specifically interested in leukoencephalopathies characterised by white matter oedema, suggesting a defect in ion and water homoeostasis. METHODS In this observational analytical study, we recruited patients with leukoencephalopathies characterised by MRI signal abnormalities in the posterior limbs of the internal capsules, midbrain cerebral peduncles, and middle cerebellar peduncles from our databases of patients with leukoencephalopathies of unknown origin. We used exome sequencing to identify the gene involved. We screened the candidate gene in additional patients by Sanger sequencing and mRNA analysis, and investigated the functional effects of the mutations. We assessed the localisation of ClC-2 with immunohistochemistry and electron microscopy in post-mortem human brains of individuals without neurological disorders. FINDINGS Seven patients met our inclusion criteria, three with adult-onset disease and four with childhood-onset disease. We identified homozygous or compound-heterozygous mutations in CLCN2 in three adult and three paediatric patients. We found evidence that the CLCN2 mutations result in loss of function of ClC-2. The remaining paediatric patient had an X-linked family history and a mutation in GJB1, encoding connexin 32. Clinical features were variable and included cerebellar ataxia, spasticity, chorioretinopathy with visual field defects, optic neuropathy, cognitive defects, and headaches. MRI showed restricted diffusion suggesting myelin vacuolation that was confined to the specified white matter structures in adult patients, and more diffusely involved the brain white matter in paediatric patients. We detected ClC-2 in all components of the panglial syncytium, enriched in astrocytic endfeet at the perivascular basal lamina, in the glia limitans, and in ependymal cells. INTERPRETATION Our observations substantiate the concept that ClC-2 is involved in brain ion and water homoeostasis. Autosomal-recessive CLCN2 mutations cause a leukoencephalopathy that belongs to an emerging group of disorders affecting brain ion and water homoeostasis and characterised by intramyelinic oedema. FUNDING European Leukodystrophies Association, INSERM and Assistance Publique-Hôpitaux de Paris, Dutch Organisation for Scientific Research (ZonMw), E-Rare, Hersenstichting, Optimix Foundation for Scientific Research, Myelin Disorders Bioregistry Project, National Institute of Neurological Disorders and Stroke, and Genetic and Epigenetic Networks in Cognitive Dysfunction (GENCODYS) Project (funded by the European Union Framework Programme 7).

Recessive loss of function of the neuronal ubiquitin hydrolase UCHL1 leads to early-onset progressive neurodegeneration

Ubiquitin C-terminal hydrolase-L1 (UCHL1), a neuron-specific de-ubiquitinating enzyme, is one of the most abundant proteins in the brain. We describe three siblings from a consanguineous union with a previously unreported early-onset progressive neurodegenerative syndrome featuring childhood onset blindness, cerebellar ataxia, nystagmus, dorsal column dysfuction, and spasticity with upper motor neuron dysfunction. Through homozygosity mapping of the affected individuals followed by whole-exome sequencing of the index case, we identified a previously undescribed homozygous missense mutation within the ubiquitin binding domain of UCHL1 (UCHL1GLU7ALA), shared by all affected subjects. As demonstrated by isothermal titration calorimetry, purified UCHL1GLU7ALA, compared with WT, exhibited at least sevenfold reduced affinity for ubiquitin. In vitro, the mutation led to a near complete loss of UCHL1 hydrolase activity. The GLU7ALA variant is predicted to interfere with the substrate binding by restricting the proper positioning of the substrate for tunneling underneath the cross-over loop spanning the catalytic cleft of UCHL1. This interference with substrate binding, combined with near complete loss of hydrolase activity, resulted in a >100-fold reduction in the efficiency of UCHL1GLU7ALA relative to WT. These findings demonstrate a broad requirement of UCHL1 in the maintenance of the nervous system.

Recessive LAMC3 mutations cause malformations of occipital cortical development

The biological basis for regional and inter-species differences in cerebral cortical morphology is poorly understood. We focused on consanguineous Turkish families with a single affected member with complex bilateral occipital cortical gyration abnormalities. By using whole-exome sequencing, we initially identified a homozygous 2-bp deletion in LAMC3, the laminin γ3 gene, leading to an immediate premature termination codon. In two other affected individuals with nearly identical phenotypes, we identified a homozygous nonsense mutation and a compound heterozygous mutation. In human but not mouse fetal brain, LAMC3 is enriched in postmitotic cortical plate neurons, localizing primarily to the somatodendritic compartment. LAMC3 expression peaks between late gestation and late infancy, paralleling the expression of molecules that are important in dendritogenesis and synapse formation. The discovery of the molecular basis of this unusual occipital malformation furthers our understanding of the complex biology underlying the formation of cortical gyrations.

Functional deficiencies of sulfite oxidase: Differential diagnoses in neonates presenting with intractable seizures and cystic encephalomalacia

Sulfite oxidase is a mitochondrial enzyme encoded by the SUOX gene and essential for the detoxification of sulfite which results mainly from the catabolism of sulfur-containing amino acids. Decreased activity of this enzyme can either be due to mutations in the SUOX gene or secondary to defects in the synthesis of its cofactor, the molybdenum cofactor. Defects in the synthesis of the molybdenum cofactor are caused by mutations in one of the genes MOCS1, MOCS2, MOCS3 and GEPH and result in combined deficiencies of the enzymes sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase. Although present in many ethnic groups, isolated sulfite oxidase deficiency and molybdenum cofactor deficiency are rare inborn errors of metabolism, which makes awareness of key clinical and laboratory features of affected individuals crucial for early diagnosis. We report clinical, radiologic, biochemical and genetic data on a Brazilian and on a Turkish child with sulfite oxidase deficiency due to the isolated defect and impaired synthesis of the molybdenum cofactor, respectively. Both patients presented with early onset seizures and neurological deterioration. They showed no sulfite oxidase activity in fibroblasts and were homozygous for the mutations c.1136A>G in the SUOX gene and c.667insCGA in the MOCS1 gene, respectively. Widely available routine laboratory tests such as assessment of total homocysteine and uric acid are indicated in children with a clinical presentation resembling that of hypoxic ischemic encephalopathy and may help in obtaining a tentative diagnosis locally, which requires confirmation by specialized laboratories.

MRI and MRS in HMG-CoA lyase deficiency

3-Hydroxy-3-Methylglutaryl coenzyme A lyase (HMG-CoA) deficiency is a rare inborn error of leucine catabolism. The disease is characterized by recurrent episodes of metabolic acidosis, hyperammonemia without ketosis, hypoglycemia, lethargy, hepatomegaly, and seizures. This study has evaluated the magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) findings of three patients with HMG-CoA deficiency. The common findings on all of the MRI scans were multiple, coalescent, marked lesions in periventricular white matter and arcuate fibers, most prominently in frontal or periatrial regions that were superimposed on diffuse, slightly hyperintense subcortical white matter signal. Involvement of the caudate nucleus and the dentate nucleus were observed in the reported patients. MRS studies by both STEAM and PRESS spectra of all patients revealed a decrease in N-acetylaspartate and elevation in both myoinositol and choline. A pathologic peak at 1.33 ppm, which is compatible with lactate, and a particular peak at 2.42 ppm in all patients were also found. The combination of both MRI and MRS findings could be considered as being specific in patients with HMG-CoA lyase deficiency.

Ictal and interictal SPECT findings in childhood absence epilepsy

The purpose of this study was to investigate the informative value of single photon emission tomography (SPECT) in relation to the pathophysiological functioning of the brain during absence seizures and the origin of ictal discharges in idiopathic generalized epilepsies (IGEs). Six patients with childhood absence epilepsy (CAE) were selected for the study and two consecutive SPECT sessions were performed concomitant with EEG recordings revealing normal results and during hyperventilation (HV) studies where the ictal discharges were induced either alone or accompanied by clinical absence seizures. All six patients had ictal discharges in their EEGs during HV and two of them also had clinical absences. SPECT findings during HV revealed an overall increase in the cerebral blood flow (CBF) with significantly higher values as compared to the baseline data. There was no indication for any focal origin in either the interictal or the ictal SPECT findings. Results of the study were supportive for the concept of subcortical origin for the absence seizures and they were also promising for the diagnostic value of ictal SPECT in epileptic cases with undetermined origin as to whether they were localization-related or generalized.

Neuroimaging findings of four patients with Sandhoff disease.

Sandhoff disease is a severe form of GM2 gangliosidosis that is caused by the deficiency of both hexosaminidase A and B. Startle reaction, hypotonia, psychomotor retardation, and blindness are the main clinical features. Presented are computed tomography and magnetic resonance imaging findings of four patients with Sandhoff disease diagnosed by enzymatic analyses. Bilateral homogeneous thalamic hyperdensity was evident on computed tomography. Magnetic resonance imaging scans revealed mild cortical atrophy, a thin corpus callosum, and abnormal signal intensities in the caudate nucleus, globus pallidum, putamen, cerebellum, and brainstem. No correlation was evident between the severity of the central nervous system imaging findings and the clinical pictures. In this article the neuroimaging findings of four patients with Sandhoff disease are discussed.

Novel NDE1 homozygous mutation resulting in microhydranencephaly and not microlyssencephaly.

Lissencephaly is characterized by deficient cortical lamination. Recently homozygous NDE1 mutations were reported in three kindred afflicted with extreme microcephaly with lissencephaly or microlissencephaly. Another severe developmental defect that involves the brain is microhydranencephaly which manifests with microcephaly, motor and mental retardation and brain malformations that include gross dilation of the ventricles with complete absence of the cerebral hemispheres or severe delay in their development. In the three related patients with microhydranencephaly that we had reported previously, we identified a homozygous deletion that encompasses NDE1 exon 2 containing the initiation codon. The mutation is predicted to result in a null allele. Herein we compare the clinical phenotypes of our research patients to those reported as microlissencephaly. The clinical findings in our patients having the fourth NDE1 mutation reported so far widen the spectrum of brain malformations resulting from mutations in NDE1.