The H3K27M mutation alters stem cell growth, epigenetic regulation, and differentiation potential.

BACKGROUND: Neurodevelopmental disorders increase brain tumor risk, suggesting that normal brain development may have protective properties. Mutations in epigenetic regulators are common in pediatric brain tumors, highlighting a potentially central role for disrupted epigenetic regulation of normal brain development in tumorigenesis. For example, lysine 27 to methionine mutation (H3K27M) in the H3F3A gene occurs frequently in Diffuse Intrinsic Pontine Gliomas (DIPGs), the most aggressive pediatric glioma. As H3K27M mutation is necessary but insufficient to cause DIPGs, it is accompanied by additional mutations in tumors. However, how H3K27M alone increases vulnerability to DIPG tumorigenesis remains unclear. RESULTS: Here, we used human embryonic stem cell models with this mutation, in the absence of other DIPG contributory mutations, to investigate how H3K27M alters cellular proliferation and differentiation. We found that H3K27M increased stem cell proliferation and stem cell properties. It interfered with differentiation, promoting anomalous mesodermal and ectodermal gene expression during both multi-lineage and germ layer-specific cell specification, and blocking normal differentiation into neuroectoderm. H3K27M mutant clones exhibited transcriptomic diversity relative to the more homogeneous wildtype population, suggesting reduced fidelity of gene regulation, with aberrant expression of genes involved in stem cell regulation, differentiation, and tumorigenesis. These phenomena were associated with global loss of H3K27me3 and concordant loss of DNA methylation at specific genes in H3K27M-expressing cells. CONCLUSIONS: Together, these data suggest that H3K27M mutation disrupts normal differentiation, maintaining a partially differentiated state with elevated clonogenicity during early development. This disrupted response to early developmental cues could promote tissue properties that enable acquisition of additional mutations that cooperate with H3K27M mutation in genesis of DMG/DIPG. Therefore, this work demonstrates for the first time that H3K27M mutation confers vulnerability to gliomagenesis through persistent clonogenicity and aberrant differentiation and defines associated alterations of histone and DNA methylation.

Parent-of-origin in individuals with familial neurofibromatosis type 1 and optic pathway gliomas.

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant cancer syndromes worldwide. Individuals with NF1 have a wide variety of clinical features including a strongly increased risk for pediatric brain tumors. The etiology of pediatric brain tumor development in NF1 is largely unknown. Recent studies have highlighted the contribution of parent-of-origin effects to tumorigenesis in sporadic cancers and cancer predisposition syndromes; however, there is limited data on this effect for cancers arising in NF1. To increase our understanding of brain tumor development in NF1, we conducted a multi-center retrospective chart review of 240 individuals with familial NF1 who were diagnosed with a pediatric brain tumor (optic pathway glioma; OPG) to determine whether a parent-of-origin effect exists overall or by the patient's sex. Overall, 50 % of individuals with familial NF1 and an OPG inherited the NF1 gene from their mother. Similarly, by sex, both males and females were as likely to inherit the NF1 gene from their mother as from their father, with 52 % and 48 % of females and males with OPGs inheriting the NF1 gene from their mother. In conclusion, in contrast to findings from other studies of sporadic cancers and cancer predisposition syndromes, our results indicate no parent-of-origin effect overall or by patient sex for OPGs in NF1.

The role of surgical biopsy in the diagnosis of glioma in individuals with neurofibromatosis-1.

Most gliomas in neurofibromatosis type 1 (NF1) are pilocytic astrocytomas (PAs) of the optic pathway occurring in young children. However, some individuals develop gliomas that lack the typical NF1-associated clinical features or radiographic appearance. We identified 17 atypical presentations from a review of 100 patients with NF1-associated gliomas. Biopsy showed that 9 were not classic PAs. These data highlight the value of biopsy in NF1-associated gliomas with unusual clinical or radiographic presentations.

SDF-1 alpha induces chemotaxis and enhances Sonic hedgehog-induced proliferation of cerebellar granule cells.

The chemokine SDF-1 alpha (CXC12) and its receptor CXCR4 have been shown to play a role in the development of normal cerebellar cytoarchitecture. We report here that SDF-1 alpha both induces chemotactic responses in granule precursor cells and enhances granule cell proliferative responses to Sonic hedgehog. Chemotactic and proliferative responses to SDF-1 alpha are greater in granule cells obtained from cerebella of animals in the first postnatal week, coinciding with the observed in vivo peak in cerebellar CXCR4 expression. SDF-1 alpha activation of neuronal CXCR4 differs from activation of CXCR4 in leukocytes in that SDF-1 alpha-induced calcium flux is activity dependent, requiring predepolarization with KCl or pretreatment with glutamate. However, as is the case in leukocytes, neuronal responses to SDF-1 alpha are all abolished by pretreatment of granule cells with pertussis toxin, suggesting they occur through G(alpha i) activation. In conclusion, SDF-1 alpha plays a role in two important processes of granule cell maturation - proliferation and migration - assisting in the achievement of appropriate cell number and position in the cerebellar cortex.

Molecular determinants of electrical rectification of single channel conductance in gap junctions formed by connexins 26 and 32.

The fully open state of heterotypic gap junction channels formed by pairing cells expressing connexin 32 (Cx32) with those expressing connexin 26 (Cx26) rectifies in a way that cannot be predicted from the current-voltage (I-V) relation of either homotypic channel. Using a molecular genetic analysis, we demonstrate that charged amino acids positioned in the amino terminus (M1 and D2) and first extracellular loop (E42) are major determinants of the current-voltage relation of the fully open state of homotypic and heterotypic channels formed by Cx26 and Cx32. The observed I-V relations of wild-type and mutant channels were closely approximated by those obtained with the electrodiffusive model of Chen and Eisenberg (Chen, D., and R. Eisenberg. 1993. Biophys. J. 64:1405-1421), which solves the Poisson-Nernst-Plank equations in one dimension using charge distribution models inferred from the molecular analyses. The rectification of the Cx32/Cx26 heterotypic channel results from the asymmetry in the number and position of charged residues. The model required the incorporation of a partial charge located near the channel surface to approximate the linear I-V relation observed for the Cx32*Cx26E1 homotypic channel. The best candidate amino acid providing this partial charge is the conserved tryptophan residue (W3). Incorporation of the partial charge of residue W3 and the negative charge of the Cx32E41 residue into the charge profile used in the Poisson-Nernst-Plank model of homotypic Cx32 and heterotypic Cx26/Cx32 channels resulted in I-V relations that closely resembled the observed I-V relations of these channels. We further demonstrate that some channel substates rectify. We suggest that the conformational changes associated with transjunctional voltage (V(j))-dependent gating to these substates involves a narrowing of the cytoplasmic entry of the channel that increases the electrostatic effect of charges in the amino terminus. The rectification that is observed in the Cx32/Cx26 heterotypic channel is similar although less steep than that reported for some rectifying electrical synapses. We propose that a similar electrostatic mechanism, which results in rectification through the open and substates of heterotypic channels, is sufficient to explain the properties of steeply rectifying electrical synapses.

Innovative therapies for pediatric brain tumors.

Success in the treatment of pediatric brain tumors has lagged behind that of other pediatric cancers. This paper highlights many of the advances that have taken place over the past few years in the surgical, radiotherapeutic, and chemotherapeutic approaches to central nervous system lesions that we hope will lead to a dramatic improvement in outcome. Innovations in neurosurgical and radiotherapeutic techniques have resulted in decreasing toxicity although substantial improvement in cure rates has not been observed. Many new techniques such as gene therapy, angiogenesis inhibitors, immunotherapy, and others that have not been part of the classic approach to these lesions are now in clinical trials in the hope that they will impact on the survival of these patients. The scientific basis for these new treatment modalities and preliminary clinical results are discussed.

A domain substitution procedure and its use to analyze voltage dependence of homotypic gap junctions formed by connexins 26 and 32.

We have developed a procedure for the replacement of defined domains with specified domains from other proteins that we used to examine the molecular basis for the differences in voltage-dependent gating between connexins 26 (Cx26) and 32 (Cx32). This technique does not depend on sequence homology between the domains to be exchanged or the presence of restriction endonuclease sites. Rather, it makes use of a PCR strategy to create an adhesive "band-aid" that directs the annealing of the amplified sequence to the correct location in the recipient clone. With this technique we created a series of chimeras involving the replacement of topologically defined protein domains of Cx32 with the corresponding sequences of Cx26. We focused on domains that are predicted to line the gap junction channel as we expect that a component of the voltage-sensing mechanism resides there. Differences between Cx26 and Cx32 in the sequences of their first and second extracellular loops, the cytoplasmic loop, and the third transmembrane domain did not account for the difference in their calculated gating charges. Shifts along the voltage axis in the steady-state conductance-voltage relations of the chimeric connexins were produced by replacement of the first extracellular loop or the cytoplasmic loop and the amino-terminal half of the third transmembrane domain. These data suggest that the voltage-sensing mechanism arises from the interaction of domains lining the aqueous channel and domains deeper in the channel wall.

Molecular analysis of voltage dependence of heterotypic gap junctions formed by connexins 26 and 32.

Heterotypic gap junctions formed by pairing Xenopus oocytes expressing hemichannels formed of Cx32 with those expressing hemichannels formed of Cx26 displayed novel transjunctional voltage (Vj) dependence not predicted by the behavior of these connexins in homotypic configurations. Rectification of initial and steady-state currents was observed. Relative positivity and negativity on the Cx26 side of the junction resulted in increased and decreased initial conductance (gj0), respectively. Only relative positivity on the Cx26 decreased steady-state conductance (gj infinity). This behavior suggested that interactions between hemichannels influences gap junction gating. The role of the first extracellular loop (E1) in these interactions was examined by pairing Cx32 and Cx26 with a chimeric connexin in which Cx32 E1 was replaced with Cx26 E1 (Cx32*26E1). Both junctions rectified with gj0/Vj relations that were less steep than that observed for Cx32/Cx26. Decreases in gj infinity occurred for either polarity Vj in the Cx32/Cx32*26E1 junction. Mutation of two amino acids in Cx26 E1 increased the steepness of both the gj0/Vj and gj infinity/Vj relations. These data demonstrate that fast rectification can arise from mismatched E1 domains and that E1 may contribute to the voltage sensing mechanisms underlying both fast and slow Vj-dependent processes.

Short TR, variable flip angle, gradient echo scans of the cervical spine: comparison of 2DFT and 3DFT techniques.

A prospective study of 16 patients was performed to compare quantitatively a contiguous single slice 2DFT version with a 3DFT version of a short TR, variable flip angle, gradient echo (GRASS) pulse sequence. The 3DFT GRASS scans had higher signal-to-noise ratios (SNR) of cord and CSF compared to the single slice 2DFT GRASS scans. The 3DFT GRASS scans, however, had lower CSF-cord and CSF-disc contrast than the single slice 2DFT version. The 3DFT GRASS sequence demonstrated comparable contrast only on the end slices of an imaging volume suggesting influence of an entry phenomenon. The lower CSF-cord and CSF-disc contrast of the 3DFT GRASS technique diminished its usefulness in the diagnosis of cervical disc disease compared to the single slice 2DFT GRASS technique. Two different slice thicknesses (3 mm and 5 mm) were investigated with the 2DFT GRASS technique and found to be comparable although the 3 mm scans had sharper disc and dural margins because of less partial volume artifact.

Specific trophic factor-receptor interactions. Key selective elements in brain development and "regeneration".

An hypothesis is presented which emphasizes the key role of specific trophic factor-receptor interactions in the development of the brain. We postulate that very early in development neurons become dependent on external factors (mainly neuropeptides) for guidance and survival. These requirements are the key to the selection process which results in the creation of a functional nervous system. These specific localized trophic factor requirements are postulated to persist throughout life. Disruptions in specific trophic factor-receptor systems are postulated to be responsible for a variety of age-related neurodegenerative diseases. The implications of recent animal and human transplant experiments in the context of the theoretical framework discussed above are profound. It would appear that the mature mammalian brain possesses an exquisite ability to regenerate specific connections to replace those lost due to death or injury to nerve cells. Unfortunately, it does not contain a population of undifferentiated stem cells to supply the necessary healthy neurons. The reason for this appears obvious based on the theoretical considerations given above, that the specific trophic factor-receptor interactions needed to produce a functional brain circuitry are necessarily stringently selective. Therefore, a significant stem cell population does not survive. However, if an appropriate stem cell population, ie, a fetal transplant, is provided, the brain will "heal itself" according to the program outlined above. In the future it may be technically feasible to perform genetic testing of newborns to determine to which genetic neurological diseases they are susceptible and at an appropriate time provide the appropriate fetal transplant. Obviously, society will have to deal with the profound ethical questions this technology will raise.

Lumbar spine: motion compensation for cerebrospinal fluid on MR imaging.

To determine whether the motion of cerebrospinal fluid (CSF) in the lumbar spine degrades T2-weighted magnetic resonance (MR) images, a spine phantom, three healthy volunteers, and a prospective series of 20 patients suspected of having lumbar spine disease underwent MR imaging with and without motion-compensation techniques. In the phantom, pulsation amplitudes as low as 3 mm (within the physiologic range of human lumbar CSF motion) reduced image quality on conventional images but not on motion-compensated images. Similar findings were observed in two volunteers and 11 patients. The magnitude of the artifacts was variable; they could impair visualization of the conus, decrease contrast or reduce the sharpness of the CSF-thecal sac interface, and cause focal regions of reduced CSF signal intensity adjacent to bulging disks. Image quality was most improved when peripheral gating was combined with even-echo rephasing. In the patient group, the use of motion-compensation techniques increased the CSF signal-to-noise ratio by an average of 29% (P less than .01); this resulted in improved contrast between the conus and extradural structures. The data suggest that CSF motion compensation is clinically useful during T2-weighted MR imaging of the lumbar spine.

Cervical spine: MR imaging with a partial flip angle, gradient-refocused pulse sequence. Part I. General considerations and disk disease.

A magnetic resonance imaging pulse sequence with a short repetition time (TR), short echo time (TE), partial flip angle, and gradient refocused echo was evaluated for the detection of cervical disk disease in a prospective study of 90 patients. These parameters were manipulated to adjust signal-to-noise ratio (S/N) and contrast: flip angle (3 degrees-18 degrees), TR (22-60 msec), and TE (12.5-25 msec). Flip angle had the greatest effect on S/N and contrast; its effect differed between axial and sagittal imaging. Cerebrospinal fluid S/N reached a peak at a smaller flip angle in sagittal imaging than in axial imaging. The useful range of flip angles depended on TR. Increasing TR had minimal direct effect on S/N or contrast, but because a longer TR allowed the use of larger flip angles for both axial and sagittal imaging, higher S/N could be achieved with similar contrast. This effect of increasing TR had to be balanced against increased imaging time and increased probability of motion artifact. Increasing TE decreased S/N, increased contrast, and increased magnetic susceptibility artifacts. For the diagnosis of cervical disk disease, the best sequence appears to be one with a very short TR, short TE, and small flip angles within a narrow range.

Cervical spine: MR imaging with a partial flip angle, gradient-refocused pulse sequence. Part II. Spinal cord disease.

A magnetic resonance imaging pulse sequence (GRASS) with a short repetition time (TR), short echo time (TE), partial flip angle, and gradient refocused echo was prospectively evaluated for the detection of cervical cord disease that caused minimal or no cord enlargement in eight patients. Sagittal T2-weighted, cerebrospinal fluid (CSF)-gated images and sagittal and axial GRASS images were obtained in all patients. The following GRASS parameters were manipulated to determine their effect on signal-to-noise ratio (S/N) and contrast: flip angle (4 degrees-18 degrees), TR (22-50 msec), and TE (12.5-25 msec). Flip angle had the greatest effect on S/N and contrast. There were no differences between axial and sagittal imaging for the spinal cord or lesion. However, because the signal intensity of CSF did differ on sagittal and axial images and because this influenced the conspicuity of lesions, there was a difference in the useful flip angle range for axial and sagittal imaging. No one set of imaging parameters was clearly superior, and in all patients, the gated image was superior to the sagittal GRASS image in lesion detection. GRASS images should be used in the axial plane primarily to confirm spinal cord disease detected on sagittal CSF-gated images. For this, a balanced approach is suggested (TR = 40 msec, TE = 20 msec, with flip angles of 4 degrees-6 degrees for sagittal and 6 degrees-8 degrees for axial imaging).

Potential false-negative MR images of the thoracic spine in disk disease with switching of phase- and frequency-encoding gradients.

Two patients with thoracic disk protrusion were evaluated with magnetic resonance imaging. A T1-weighted spin-echo sequence was used, with and without switching of the phase- and frequency-encoding gradients. Both disks were well delineated when the frequency-encoding gradient was parallel to the spinal axis. When the gradients were switched (with the phase-encoding gradient parallel to the spinal axis), both herniated disks were partially obscured by a posteriorly displaced fat signal from marrow, caused by a chemical shift artifact. In addition, the anterior subarachnoid space appeared falsely narrowed, and the cerebrospinal fluid (CSF) signal intensity was increased, which reduced the CSF-cord contrast. These findings suggest that switching the orientation of the frequency- and phase-encoding gradients may result in false-negative T1-weighted sagittal images of the thoracic spine.