Goekhan Uyanik

High efficacy of clonal growth and expansion of adult neural stem cells.

Neural stem cells (NSCs) from the adult central nervous system are currently being investigated for their potential use in autologous cell replacement strategies. High expansion rates of NSCs in culture are crucial for the generation of a sufficient amount of cells needed for transplantation. Here, we describe efficient growth of adult NSCs in Neurobasal medium containing B27 supplement under clonal and low-density conditions in the absence of serum or conditioned medium. Expansion of up to 15-fold within 1 week was achieved on low-density NSC cultures derived from the lateral ventricle wall, the hippocampal formation, and the spinal cord of adult rats. A 27% single-cell cloning efficiency in Neurobasal/B27 combination further demonstrates its growth-promoting ability. Multipotency and nontumorgenicity of NSCs were retained despite the high rate of culture expansion. In addition, increased cell survival was obtained when Accutase, instead of trypsin, was used for enzymatic dissociation of NSC cultures. This work provides an important step toward the development of standardized protocols for highly efficient in vitro expansion of NSCs from the adult central nervous system to move more closely to the clinical use of NSCs.

Long‐term course and mutational spectrum of spatacsin‐linked spastic paraplegia

Hereditary spastic paraplegias (HSPs) comprise a heterogeneous group of neurodegenerative disorders resulting in progressive spasticity of the lower limbs. One form of autosomal recessive hereditary spastic paraplegia (ARHSP) with thin corpus callosum (TCC) was linked to chromosomal region 15q13‐21 (SPG11) and associated with mutations in the spatacsin gene. We assessed the long‐term course and the mutational spectrum of spatacsin‐associated ARHSP with TCC.

Novel POMGnT1 mutations define broader phenotypic spectrum of muscle–eye–brain disease

Muscle–eye–brain disease (MEB, OMIM 253280) is an autosomal recessive disorder characterized by a distinct triad of congenital muscular dystrophy, structural eye abnormalities, and cobblestone lissencephaly. Clinically, MEB patients present with early onset muscular hypotonia, severely compromised motor development, and mental retardation. Magnetic resonance imaging reveals a lissencephaly type II with hypoplasia of the brainstem and cerebellum. MEB is associated with mutations in the gene for protein O-mannose beta-1,2-N-acetylglucosaminyltransferase (POMGnT1, OMIM 606822). In this paper, we report the clinical findings of nine MEB patients from eight families. Eight of the nine patients presented typical features of MEB. However, a broad phenotypic variability was observed, ranging from two patients with severe autistic features to another patient with an unusually mild phenotype, initially diagnosed as congenital muscular dystrophy. Furthermore, severe hydrocephalus was reported in two families during a previous pregnancy, emphasizing the phenotypic overlap with Walker–Warburg syndrome. In addition to three previously reported mutations, we identified six novel POMGnT1 mutations (one missense, five truncating) in the present patient cohort. Our data suggest mutational hotspots within the minimal catalytic domain at arginine residue 442 (exon 16) and in intron 17. It is interesting to note that all mutations analyzed so far result in a complete loss of enzyme activity. Therefore, we conclude that the type and position of the POMGnT1 mutations are not of predictive value for the clinical severity. This supports the notion that additional environmental and/or genetic factors may contribute to the observed broad spectrum of POMGnT1-associated phenotypes.

A filamin A splice mutation resulting in a syndrome of facial dysmorphism, periventricular nodular heterotopia, and severe constipation reminiscent of cerebro-fronto-facial syndrome

Background: Mutations of the filamin A locus (FLNA) on Xq28 have been established in girls with periventricular nodular heterotopia and in patients with otopalatodigital and overlapping phenotypes, the pathogenesis of these phenotypes being thought to be quite distinct. To date only six male cases of periventricular nodular heterotopia (PVNH) have been reported and these almost invariably associated with severe neurological signs. Methods and Results: We report a new phenotype of male PVNH, with relatively normal development, no epilepsy or other neurological abnormality, severe constipation, and facial dysmorphism and without a discernible skeletal phenotype. This phenotype is associated with a splice site mutation in FLNA c.1923C>T, resulting in the generation of both normal and aberrant mRNA. Conclusions: We postulate that the patient retains enough FLNA function to avoid the usual lethality associated with loss of function mutations in males and suggest that the severe constipation may be a clue to the molecular aetiology of other X linked conditions associated with severe constipation.

Location and type of mutation in the LIS1 gene do not predict phenotypic severity

Background: Lissencephaly is a neuronal migration disorder leading to absent or reduced gyration and a broadened but poorly organized cortex. The most common form of lissencephaly is isolated, referred as classic or type 1 lissencephaly. Type 1 lissencephaly is mostly associated with a heterozygous deletion of the entire LIS1 gene, whereas intragenic heterozygous LIS1 mutations or hemizygous DCX mutations in males are less common. Methods: Eighteen unrelated patients with type 1 lissencephaly were clinically and genetically assessed. In addition, patients with subcortical band heterotopia (n = 1) or lissencephaly with cerebellar hypoplasia (n = 2) were included. Results: Fourteen new and seven previously described LIS1 mutations were identified. We observed nine truncating mutations (nonsense, n = 2; frameshift, n = 7), six splice site mutations, five missense mutations, and one in-frame deletion. Somatic mosaicism was assumed in three patients with partial subcortical band heterotopia in the occipital-parietal lobes or mild pachygyria. We report three mutations in exon 11, including a frameshift which extends the LIS1 protein, leading to type 1 lissencephaly and illustrating the functional importance of the WD domains at the C terminus. Furthermore, we present two patients with novel LIS1 mutations in exon 10 associated with lissencephaly with cerebellar hypoplasia type a. Conclusion: In contrast to previous reports, our data suggest that neither type nor position of intragenic mutations in the LIS1 gene allows an unambiguous prediction of the phenotypic severity. Furthermore, patients presenting with mild cerebral malformations such as subcortical band heterotopia or cerebellar hypoplasia should be considered for genetic analysis of the LIS1 gene.

Novel truncating and missense mutations of the KCC3 gene associated with Andermann syndrome

Background: Andermann syndrome (OMIM 218000) is an autosomal recessive motor-sensory neuropathy associated with developmental and neurodegenerative defects. The cerebral MRI reveals a variable degree of agenesis of the corpus callosum. Recently, truncating mutations of the KCC3 gene (also known as SLC12A6) have been associated with Andermann syndrome. Methods: The authors assessed clinically and genetically three isolated cases from Germany and Turkey with symptoms consistent with Andermann syndrome. Results: The authors detected four novel mutations within the KCC3 gene in their patients: two different truncating mutations in the first patient, a homozygous truncating mutation in the second, and a homozygous missense mutation in the third patient. In contrast to the classic phenotype of the Andermann syndrome linked to truncating KCC3 mutations the phenotype and the course of the disease linked to the missense mutation appeared to be different (i.e., showing additional features like diffuse and widespread white matter abnormalities). Conclusions: Not only truncating but also missense mutations of the KCC3 gene are associated with Andermann syndrome. Different types of KCC3 mutations may determine different clinical phenotypes.

Somatic mosaicism and variable penetrance in doublecortin-associated migration disorders

X-linked isolated lissencephaly sequence (XLIS) and subcortical band heterotopia (SBH) are allelic disorders caused by mutations in the doublecortin (DCX) gene. This genetic analysis of seven families revealed four novel mutations in the DCX gene. The authors detected a high rate of somatic mosaicism in male and female patients with variable penetrance of bilateral SBH including nonpenetrance in a heterozygous woman. In addition, the authors implemented prenatal diagnosis in a family with SBH/XLIS.

Heterozygous mutations in SIX3 and SHH are associated with schizencephaly and further expand the clinical spectrum of holoprosencephaly.

Schizencephaly (SCH) is a clinically and etiologically heterogeneous cerebral malformation presenting as unilateral or bilateral hemispheric cleft with direct connection between the inner and outer liquor spaces. The SCH cleft is usually lined by gray matter, which appears polymicrogyric implying an associated impairment of neuronal migration. The majority of SCH patients are sporadic, but familial SCH has been described. An initial report of heterozygous mutations in the homeobox gene EMX2 could not be confirmed in 52 patients investigated in this study in agreement with two independent SCH patient cohorts published previously. SCH frequently occurs with additional cerebral malformations like hypoplasia or aplasia of the septum pellucidum or optic nerve, suggesting the involvement of genes important for the establishment of midline forebrain structures. We therefore considered holoprosencephaly (HPE)-associated genes as potential SCH candidates and report for the first time heterozygous mutations in SIX3 and SHH in a total of three unrelated patients and one fetus with SCH; one of them without obvious associated malformations of midline forebrain structures. Three of these mutations have previously been reported in independent patients with HPE. SIX3 acts directly upstream of SHH, and the SHH pathway is a key regulator of ventral forebrain patterning. Our data indicate that in a subset of patients SCH may develop as one aspect of a more complex malformation of the ventral forebrain, directly result from mutations in the SHH pathway and hence be considered as yet another feature of the broad phenotypic spectrum of holoprosencephaly.

Familial West syndrome and dystonia caused by an Aristaless related homeobox gene mutation

Boys with unexplained West syndrome should be examined for a mutation in the Aristaless related homeobox gene, especially, when the family history is positive for mental retardation and epilepsy. X-linked West syndrome is very rare. We report on two brothers with West syndrome and dystonia with polyalanine expansion of the Aristaless related homeobox gene (ARX). The index patient (Fig. 1a; III-2) was the second child of non-consanguineous parents, born in 2001 at term, after an uneventful pregnancy and delivery (birth weight 3659 g, 75th percentile; length 51 cm, 75th percentile; head circumference 35 cm, 50th percentile). At the age of 3 months he developed infantile spasms and a hypsarrhythmic EEG pattern. He promptly responded to vigabatrin therapy. At 5 months a generalised dystonia, i.e. increased muscle tone with dystonic posturing of limbs, was evident. At the age of 3 years, he is able to walk a few steps with help and grasping objects is very difficult. He has no expressive speech. The elder brother of the index patient (Fig. 1a; III-1) was born in 1996 by caesarean section because of neonatal macrosomia (birth weight 4200 g, 90th percentile; length 53 cm, 90th percentile; head circumference 38 cm, >90th percentile). At the age of 3 months he showed dystonic movements and marked truncal hypotonia. One month later he developed infantile spasms and a hypsarrhythmic EEG pattern. He did not respond to vigabatrin, but to ACTH. At the age of 8 years he is wheel-chair bound, not talking, grasping objects is not possible. In both children metabolic tests and neuroimaging (MRI) were normal. Actually, both brothers suffer from a severe dystonia, mental impairment and rare generalised tonic-clonic seizures (the older brother), treated with valproic acid. The family history was remarkable. In the maternal uncle (Fig. 1a; II-3, now 37 years old), spastic tetraplegia, mental retardation and epilepsy have been present since early infancy. In retrospect, the epilepsy syndrome could not be classified. MRI was not performed in this uncle, the mother or the grandmother. From the pedigree and the clinical findings we suspected a mutation in the ARX gene. Following informed consent, a sequence analysis of the coding region and flanking intronic sequences of the ARX gene was performed. The male proband (index patient; Fig. 1a; III-2) as well as his brother and uncle were found to be hemizygous for a 21 bp GCG repeat expansion in exon 2 of the ARX gene c.333_334ins(GCG)7, which expands the first of four alanine tracts from normally 16 to 23 alanine residues (Fig. 1b,c). Both the mother (Fig. 11; II-2) and the maternal grandmother (Fig. 1a; I-2) were identified as heterozygous mutation carriers using an optimised fluorescence-based PCR assay. Mutations in the ARX gene have been found in a broad spectrum of disorders including X-linked infantile spasms (ISSX)/West syndrome, mental retardation [2], ataxia and dystonia (Partington syndrome), syndromic and non-syndromic forms of mental retardation, myoclonic epilepsy and X-linked lissencephaly with abnormal genitalia (XLAG) [5, 6,8]. The mutation found in our Swiss family, which is not related to previously reported families, has been described before in boys with infantile spasms and normal MRI, severe mental and motor retardation [7]. In addition to Partington syndrome, dystonia was described in a few unpublished Australian and Norwegian cases [6], but G. Wohlrab (&) AE B. Schmitt AE E. Boltshauser Department of Neuropaediatrics and Neurophysiology, University Children’s Hospital, Steinwiesstrasse 75, 8032 Zurich, Switzerland E-mail: gabriele.wohlrab@kispi.unizh.ch Tel.: +41-1-2667701 Fax: +41-1-2667165

Relation between laterality and immune response after acute cerebral ischemia

Objective: During the last 2 decades, right/left hemisphere dominance was supposed to affect the immune system differently. Experimental and clinical observations indicate that the left hemisphere plays a crucial role in the development of the immune system. The true relationship between immune response and acute ischemic stroke laterality remains to be elucidated. Methods: We studied acute right-handed stroke patients admitted to a single acute neurology department with a specialized stroke unit. Being part of our clinical protocol, blood samples were taken within the first 24 h after the onset of stroke symptoms. The medical record of each patient was reviewed, and demographic, clinical laboratory (key criteria: C-reactive protein, CRP, and white blood cell count, WBC) and neuroimaging information was retrieved. All data were presented descriptively, and bivariate test statistics, ANOVA (log-transformed data) or linear correlations were calculated. Results: Fifty-six of the 187 patients admitted to our Stroke Unit between October 2003 and March 2004 with different stroke subtypes according to the TOAST criteria were retrospectively evaluated in order to characterize the impact of stroke laterality on immunoregulatory response measured by CRP levels and WBC. Correlation analysis revealed that left-sided ischemic stroke yielded a significantly higher correlation between CRP levels and WBC. Following left-sided stroke, a more marked variability in CRP and WBC was found compared to patients with right-sided ischemic stroke, although ANOVA did not show significant differences between immune response values as a function of stroke subtypes. Conclusions: We identified an association between stroke laterality and immunoregulatory response in patients with acute ischemic stroke. Left-sided stroke may be considered as a direct risk factor for infectious disease or immune deficits and should attract special attention. However, these preliminary results need be confirmed by controlled studies.