Epigenetic mutations of the imprinted IGF2-H19 domain in Silver-Russell syndrome (SRS): results from a large cohort of patients with SRS and SRS-like phenotypes.

BACKGROUND: Silver-Russell syndrome (SRS) is a clinically and genetically heterogeneous condition characterised by severe intrauterine and postnatal growth retardation. Loss of DNA methylation at the telomeric imprinting control region 1 (ICR1) on 11p15 is an important cause of SRS. METHODS: We studied the methylation pattern at the H19-IGF2 locus in 201 patients with suspected SRS. In an attempt to categorise the patients into different subgroups, we developed a simple clinical scoring system with respect to readily and unambiguously assessable clinical features. In a second step, the relationship between clinical score and epigenetic status was analysed. RESULTS AND CONCLUSIONS: The scoring system emerged as a powerful tool for identifying those patients with both a definite SRS phenotype and carrying an epimutation at 11p15. 53% of the 201 patients initially enrolled fulfilled the criteria for SRS and about 40% of them exhibited an epimutation at the H19-IGF2 locus. Methylation defects were restricted to patients who fulfilled the diagnostic criteria for SRS. Patients carrying epimutations had a more severe phenotype than either the SRS patients with mUPD7 or the idiopathic SRS patients. The majority of patients with methylation abnormalities showed hypomethylation at both the H19 and IGF2 genes. However, we also identified SRS patients where hypomethylation was restricted to either the H19 or the IGF2 gene. Interestingly, we detected epimutations in siblings of normal parents, most likely reflecting germ cell mosaicism in the fathers. In one family, we identified an epimutation in an affected father and his likewise affected daughter.

Clinical profiles of four patients with Rett syndrome carrying a novel exon 1 mutation or genomic rearrangement in the MECP2 gene.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked MECP2 gene encoding methyl CpG binding protein 2 (MeCP2). Recently, a new isoform of MeCP2 including exon 1 was identified. This new isoform is more abundantly expressed in brain than the isoform including exons 2-4. Very little is known about the phenotypes associated with mutations in exon 1 of MECP2 since only a limited number of RTT patients carrying such mutations have been identified so far. In this study, we screened a cohort of 20 girls with RTT for exon 1 mutations by sequencing and multiplex ligation-dependent probe amplification (MLPA). We identified one girl with a novel exon 1 mutation (c.30delCinsGA) by sequencing and three with genomic rearrangements by MLPA. Comparison of the phenotypes showed that the girls carrying a mutation or rearrangement encompassing exon 1 were more severely affected than the girls with rearrangements not affecting exon 1.

The promoters of the survival motor neuron gene (SMN) and its copy (SMNc) share common regulatory elements.

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration of motor neurons of the spinal cord. The survival motor neuron gene (SMN) has been recognized as the disease-causing gene. SMN is duplicated, and the almost identical copy gene (SMNc) remains functional in patients with SMA. The expression level of SMNc is tightly correlated with the clinical severity of the disease. Here, we define the transcription initiation site, delineate the region containing promoter activity, and analyze the sequence of the promoter region of both SMN and SMNc. We show that the promoter sequence and activity of the two genes are quasi identical, providing strong evidence for similar transcription regulation of the two genes. Therefore, the difference in the level of protein encoded by SMN and SMNc is the result of either different regulatory region(s) further apart or different posttranscriptional regulation. Interestingly, sequence analysis of the promoter region revealed several consensus binding sites for transcription factors. Therefore, the identification of transcription factors involved in the regulation of SMNc gene expression may lead to attractive strategies for therapy in SMA.

Oligodendroglial reaction following spinal cord injury in rat: transient upregulation of MBP mRNA.

The reaction of oligodendrocytes in response to traumatic injury of the CNS are poorly understood. In the present report we studied changes in the expression of a major constituent of CNS myelin, myelin basic protein (MBP), by immunohistochemistry and in situ hybridization from 6 h up to 2 weeks following partial transection of the spinal cord in adult rats. MBP immunohistochemistry showed degeneration of myelin at the lesion center and signs of myelin breakdown in necrotic foci in the dorsal and ventral funiculi proximal and distal to the lesion. In situ hybridization revealed that mRNA for MBP was downregulated at the local lesion site within the first day following injury, probably reflecting oligodendrocytes to undergo cell death. From 2 days on, however, MBP mRNA was conspicuously upregulated at the border of the lesion area. This "reactive" response of surviving oligodendrocytes, as indicated by increased levels of MBP mRNA, peaked around 8 days. At this time, oligodendrocytes displaying strong MBP in situ signal formed stripe-like structures which were oriented radially toward the lesion center and arranged in parallel to neurofilament-positive axons. At around 2 weeks post-injury, MBP mRNA at the border of the lesion area was again downregulated to levels comparable to uninjured controls. These results show that traumatic injury of the spinal cord induces a "reactive" response of surviving oligodendrocytes adjacent to lesion sites. This response might represent an important component of local repair mechanisms.

Expression of pro-inflammatory cytokine and chemokine mRNA upon experimental spinal cord injury in mouse: an in situ hybridization study.

Injury to the spinal cord induces a complex cascade of cellular reactions at the local lesion area: secondary cell death and inflammatory reactions as well as scar and cavity formation take place. In order to investigate the molecular features underlying this local wounding response and to determine their pathophysiological implications, we studied the expression pattern of pro-inflammatory and chemoattractant cytokines in an experimental spinal cord injury model in mouse. We show by in situ hybridization that transcripts for the pro-inflammatory cytokines TNF alpha and IL-1 as well as the chemokines MIP-1alpha and MIP-1beta are upregulated within the first hour following injury. In this early phase, the expression of the pro-inflammatory cytokines is restricted to cells in the surroundings of the lesion area probably resident CNS cells. While TNF alpha is expressed in a very narrow time window, IL-1 can be detected in a second phase in a subset of polymorphonuclear granulocytes which immigrate into the spinal cord around 6 h. Message for the chemokines MIP-1alpha and beta is expressed in a generalized way in the grey matter of the entire spinal cord around 24 h and gets again restricted to the cellular infiltrate at the lesion site at 4 days following injury. Interestingly, our data suggest that resident CNS cells, most probably microglial cells, and not peripheral inflammatory cells, are the main source for cytokine and chemokine mRNAs. The defined cytokine pattern observed indicates that the inflammatory events upon lesioning the CNS are tightly controlled. The very early expression of pro-inflammatory cytokine and chemokine messages may represent an important element of the recruitment of inflammatory cells. Additional pathophysiological consequences of the specific cytokine pattern observed remain to be determined.

VEGF mRNA induction correlates with changes in the vascular architecture upon spinal cord damage in the rat.

The multiple cellular and molecular processes induced by injury to the central nervous system (CNS) are still poorly understood. In the present study, we investigated the response of the vasculature and the expression of mRNA for the angiogenic vascular endothelial growth factor (VEGF) following X-irradiation of the spinal cord in the newborn and following traumatic spinal cord injury in the adult rat. Both lesion models induced changes in the density and the distribution pattern of blood vessels: while X-irradiation led to a permanent local increase in vascular density in the fibre tracts of the exposed segments, a transient local sprouting of vessels was induced upon traumatic spinal cord injury. In situ hybridization showed that an increase of VEGF mRNA anticipated and overlapped with the vascular responses in both lesion models. In addition to the temporal correlation of VEGF expression and vascular sprouting, there was a clear correlation in the spatial distribution patterns. Following X-irradiation, the expression of VEGF mRNA was restricted to the fibre tracts, precisely the areas where the changes in the vasculature were observed later on. Upon transection in the adult animal, VEGF was mainly detectable at the border of the lesion area, where the transient increase in vascular density could be observed. Interestingly, according to the type of lesion applied, astrocytes (X-irradiation) or inflammatory cells (presumably microglial cells or macrophages; traumatic lesion) are the cellular sources of VEGF mRNA. Our results strongly indicate that VEGF is crucially involved in mediating vascular changes following different types of injury in the CNS.

Degeneration and regeneration of axons in the lesioned spinal cord.

For many decades, the inability of lesioned central neurons to regrow was accepted almost as a "law of nature", and on the clinical level, spinal cord and brain lesions were seen as being irreversible. Today we are starting to understand the mechanisms of neuronal regeneration in the central nervous system and its presence in the periphery. There is now a rapid expansion in this field of neuroscience. Developmental neurobiology has produced tools and concepts that start to show their impact on regeneration research. This is particularly true for the availability of antibodies and factors and for the rapidly growing cellular and molecular understanding of crucial aspects of neurite growth, guidance, target finding, and synapse stabilization. New cell biological concepts on the mechanisms of neuron survival and death and on the interaction of inflammatory cells with the central nervous system also find their way into the field of spinal cord and brain lesions and have, indeed, led already to new therapeutic approaches. This review briefly summarizes the current knowledge on the mechanisms involved in degeneration and tissue loss and in axonal regeneration subsequent to spinal cord lesions, particularly in mammals and humans.

Experimental Listeria meningoencephalitis. Macrophage inflammatory protein-1 alpha and -2 are produced intrathecally and mediate chemotactic activity in cerebrospinal fluid of infected mice.

In bacterial meningitis, the recruitment of leukocytes across the blood-brain barrier into the central nervous system may be crucial for both elimination of pathogens and tissue injury. In addition to bacterial cell wall products, host factors including chemokines may lead to accumulation of phagocytes within the central nervous system. As shown by Northern analysis, brains of mice infected intracerebrally with Listeria monocytogenes (LM) express mRNA for three chemokines, the macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and MIP-2. The cellular sources of these chemokines comprise both the blood-derived polymorphonuclear leukocytes (PMNs) and monocytes infiltrating the meninges, the ventricular system and the periventricular area. In the course of meningitis a time-dependent increase of MIP-1 alpha and MIP-2 was found in the cerebrospinal fluid (CSF) by ELISA. CSF taken 24 h after infection (CSF-LM24) induced migration of human leukocytes when treated in chemotactic chambers in vitro. Neutralizing Abs to chemokines identified MIP-1 alpha and MIP-2 to be responsible for CSF-LM24 mediated chemotaxis of monocytes and PMNs, respectively. CSF obtained from mock-infected animals contained no MIP-1 alpha or MIP-2 and did not lead to migration of leukocytes. When testing CSF-LM24 on mouse spleen cells, the chemotactic activity detected for mononuclear cells was only partly inhibited by Abs to MIP-1 alpha and -1 beta. Thus, in addition to MIP-1 and -2 other not yet defined chemotactic factors are of importance for recruitment of leukocytes in bacterial meningitis.

Methylprednisolone inhibits early inflammatory processes but not ischemic cell death after experimental spinal cord lesion in the rat.

Experimental studies and clinical observations show that spinal cord lesions are greatly enlarged by a process called secondary cell death. A detailed understanding of the molecular and cellular processes underlying these events is still lacking. In clinical studies using methylprednisolone in spinal cord injured patients a mega-dose of methylprednisolone applied during the first few hours after injury was found to improve the neurological outcome. In the present study the possible neuroprotective mechanism of methylprednisolone was assessed by histologically studying its effect on the extent of secondary cell death and on early inflammatory reactions following partial transection of the spinal cord in the rat. Our results show that a single high dose of 30 or 60 mg/kg methylprednisolone affects neither the time course nor the extent of secondary cell death. In contrast, methylprednisolone markedly suppressed the invasion of the injured spinal cord tissue by polymorphonuclear granulocytes and macrophages. The role of these inflammatory cells in traumatic CNS lesions is very unclear at present. It is possible that they lead to further damage of the injured spinal cord tissue and that the beneficial effect of methylprednisolone is at least partially due to its anti-inflammatory effect, thereby inhibiting bystander damage of invading inflammatory cells.