The Polygenic and Monogenic Basis of Paediatric Fractures.

PURPOSE OF REVIEW: Fractures are frequently encountered in paediatric practice. Although recurrent fractures in children usually unveil a monogenic syndrome, paediatric fracture risk could be shaped by the individual genetic background influencing the acquisition of bone mineral density, and therefore, the skeletal fragility as shown in adults. Here, we examine paediatric fractures from the perspective of monogenic and complex trait genetics. RECENT FINDINGS: Large-scale genome-wide studies in children have identified ~44 genetic loci associated with fracture or bone traits whereas ~35 monogenic diseases characterized by paediatric fractures have been described. Genetic variation can predispose to paediatric fractures through monogenic risk variants with a large effect and polygenic risk involving many variants of small effects. Studying genetic factors influencing peak bone attainment might help in identifying individuals at higher risk of developing early-onset osteoporosis and discovering drug targets to be used as bone restorative pharmacotherapies to prevent, or even reverse, bone loss later in life.

p38 MAP kinase mediates bax translocation in nitric oxide-induced apoptosis in neurons.

Nitric oxide is a chemical messenger implicated in neuronal damage associated with ischemia, neurodegenerative disease, and excitotoxicity. Excitotoxic injury leads to increased NO formation, as well as stimulation of the p38 mitogen-activated protein (MAP) kinase in neurons. In the present study, we determined if NO-induced cell death in neurons was dependent on p38 MAP kinase activity. Sodium nitroprusside (SNP), an NO donor, elevated caspase activity and induced death in human SH-SY5Y neuroblastoma cells and primary cultures of cortical neurons. Concomitant treatment with SB203580, a p38 MAP kinase inhibitor, diminished caspase induction and protected SH-SY5Y cells and primary cultures of cortical neurons from NO-induced cell death, whereas the caspase inhibitor zVAD-fmk did not provide significant protection. A role for p38 MAP kinase was further substantiated by the observation that SB203580 blocked translocation of the cell death activator, Bax, from the cytosol to the mitochondria after treatment with SNP. Moreover, expressing a constitutively active form of MKK3, a direct activator of p38 MAP kinase promoted Bax translocation and cell death in the absence of SNP. Bax-deficient cortical neurons were resistant to SNP, further demonstrating the necessity of Bax in this mode of cell death. These results demonstrate that p38 MAP kinase activity plays a critical role in NO-mediated cell death in neurons by stimulating Bax translocation to the mitochondria, thereby activating the cell death pathway.

Contribution of p53-dependent caspase activation to neuronal cell death declines with neuronal maturation.

Caspases play a pivotal role in neuronal cell death during development and after trophic factor withdrawal. However, the mechanisms regulating caspase activity and the role played by caspase activation in response to neuronal injury is poorly understood. The tumor suppressor gene p53 has been implicated in the loss of neuronal viability caused by excitotoxic and DNA damaging agents. In the present study we determined if p53-mediated neuronal cell death required caspase activation. DNA damage increased caspase activity in both cultured embryonic telencephalic and postnatal cortical neurons in a p53-dependent manner. Caspase inhibitors protected embryonic telencephalic neurons, but not postnatal cortical neurons, from DNA damage-induced cell death as measured by direct cell counting and annexin V staining. In marked contrast to the caspase inhibitors, an inhibitor of the DNA repair enzyme, poly(ADP-ribose) polymerase, conferred significant protection from genotoxic and excitotoxic cell death on postnatal cortical neurons but had no effect on embryonic neurons. Glutamate-mediated excitotoxicity in postnatal neurons was not associated with measurable changes in caspase activity, consistent with the failure of caspase inhibitors to prevent cell death under these conditions. Moreover, adenovirus-mediated overexpression of p53 killed embryonic and postnatal neurons without activating caspases. Thus, p53-mediated neuronal cell death may occur via both caspase-dependent and caspase-independent pathways. These results demonstrate that p53 is required for caspase activation in response to some forms of neuronal injury. However, the relative importance of caspase activation in neurons depends on the developmental status of the cell and the specific nature of the death stimulus.

O6-methylguanine-DNA methyltransferase-deficient phenotype in human gliomas: frequency and time to tumor progression after alkylating agent-based chemotherapy.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) contributes to the resistance of human brain tumor cell lines and xenografts to methylating and chloroethylating agents. We assayed MGMT in 174 newly diagnosed or recurrent gliomas to (a) quantitate changes in MGMT activity associated with alkylating agent-based chemotherapy; and (b) assess the contribution of MGMT to clinical outcome. Glioma MGMT activity ranged 300-fold, averaging 3,800+/-7,200 molecules/cell. Twenty-four percent of tumors lacked detectable activity [Methyl repair-deficient (Mer-) phenotype, defined here as <151 molecules/cell or <0.25 fmol/10(6) cells]. Tumors treated with surgery alone and tumors recurring after surgery and radiotherapy did not differ significantly in frequency of the Mer- phenotype (29% versus 24%). However, the frequency of the Mer- phenotype among tumors recurring after surgery, radiation, and alkylating agent-based chemotherapy was 7-fold lower than in tumors treated with surgery alone (4.3% versus 29%; P < or = 0.02) and 6-fold lower than in tumors recurring after surgery and radiation (4.3% versus 24%; P < or = 0.05). In contrast to gliomas, there was no relationship of alkylating agent-based therapy with the frequency of the Mer- phenotype in paired histologically normal brain. These data suggest that alkylating agents, either alone or synergistically with radiotherapy, selectively kill Mer- glioma cells in situ. Importantly, Mer- and Mer+ tumors did not differ in time to tumor progression following treatment with alkylating agents, indicating that although Mer- glioma cells may be differentially killed by alkylators, factors other than Mer phenotype were the principal determinants of time to clinical progression. Nonetheless, our results support the possibility that complete ablation of glioma MGMT with substrate analogue inhibitors could improve the efficacy of alkylating agent-based chemotherapy.

O6-methylguanine-DNA methyltransferase activity in adult gliomas: relation to patient and tumor characteristics.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) confers resistance to therapeutic methylating and chloroethylating agents in human brain tumor-derived cell lines. In this work, we assayed MGMT activity in 152 adult gliomas to establish correlates with patient and tumor characteristics. We also assayed MGMT in histologically normal brain adjacent to 87 tumors to characterize changes in activity accompanying neurocarcinogenesis. MGMT activity was detectable in 76% (115 of 152) of tumors, ranging approximately 300-fold from 0.30 to 89 fmol/10(6) cells (180-57,000 molecules/cell). Mean activity was 6.6 +/- 13 fmol/10(6) cells and varied 4-fold among diagnostic groups. The mean for oligodendrogliomas was 2-fold lower (P < 0.03), and for mixed oligodendroglioma-astrocytomas, the mean was 4-fold lower (P < 0.006) than for astroglial tumors. Twenty-five % of gliomas had no detectable MGMT activity (Mer- phenotype; < 0.25 fmol/10(6) cells or 150 molecules/cell). Glioma MGMT was inversely correlated with age (P < 0.01), consistent with the observed age dependence in the progenitor tissue of brain tumors (J. R. Silber et al., Proc. Natl. Acad. Sci. USA, 93: 6941-6946, 1996). Neither MGMT activity nor proportion of Mer- tumors differed by sex. Glioma MGMT was correlated with degree of aneuploidy (P < 0.006) but not with fraction of S-phase cells. Mean activity in tumors was 5-fold higher than in adjacent histologically normal brain (5.0 +/- 7.6 versus 1.1 +/- 1.9 fmol/10(6) cells; P < 0.001). Notably, elevation of tumor activity was observed in 62% of tissue pairs, ranging from 2-fold to > 105-fold. Moreover, 64% of Mer- normal tissue was accompanied by Mer+ tumor. These observations indicate that expression of MGMT activity is frequently activated and/or increased during human neurocarcinogenesis, and that the enhancement is not related to proliferation per se. Significantly, enhanced MGMT activity may heighten the resistance of brain tumors to therapeutic alkylating agents.

Cerebral autoregulation following minor head injury.

The purpose of this study was to determine whether patients with minor head injury experience impairments in cerebral autoregulation. Twenty-nine patients with minor head injuries defined by Glasgow Coma Scale (GCS) scores of 13 to 15 underwent testing of dynamic cerebral autoregulation within 48 hours of their injury using continuous transcranial Doppler velocity recordings and blood pressure recordings. Twenty-nine age-matched normal volunteers underwent autoregulation testing in the same manner to establish comparison values. The function of the autoregulatory response was assessed by the cerebral blood flow velocity response to induced rapid brief changes in arterial blood pressure and measured as the autoregulation index (ARI). Eight (28%) of the 29 patients with minor head injury demonstrated poorly functioning or absent cerebral autoregulation versus none of the controls, and this difference was highly significant (p = 0.008). A significant correlation between lower blood pressure and worse autoregulation was found by regression analysis in head-injured patients (r = 0.6, p < 0.001); however, lower blood pressure did not account for the autoregulatory impairment in all patients. Within this group of head-injured patients there was no correlation between ARI and initial GCS or 1-month Glasgow Outcome Scale scores. This study indicates that a significant number of patients with minor head injury may have impaired cerebral autoregulation and may be at increased risk for secondary ischemic neuronal damage.

Low-grade gliomas associated with intractable epilepsy: seizure outcome utilizing electrocorticography during tumor resection.

Adults and children with low-grade gliomas often present with medically refractory epilepsy. Currently, controversy exists regarding the need for intraoperative electrocorticography (ECoG) to identify and, separately, resect seizure foci versus tumor removal alone to yield maximum seizure control in this patient population. Forty-five patients with low-grade gliomas and intractable epilepsy were retrospectively analyzed with respect to preoperative seizure frequency and duration, number of antiepileptic drugs, intraoperative ECoG data (single versus multiple foci), histology of resected seizure foci, and postoperative control of seizures with or without antiepileptic drugs. Multiple versus single seizure foci were more likely to be associated with a longer preoperative duration of epilepsy. Of the 45 patients studied, 24 were no longer taking antiepileptic drugs and were seizure-free (mean follow-up interval 54 months). Seventeen patients, who all had complete control of their seizures, remained on antiepileptic drugs at lower doses (mean follow-up interval 44 months); seven of these patients were seizure-free postoperatively, yet the referring physician was reluctant to taper the antiepileptic drugs. Four patients continued to have seizures while receiving antiepileptic drugs, although at a reduced frequency and severity. In this series 41% of the adults versus 85% of the children were seizure-free while no longer receiving antiepileptic drugs, with mean postoperative follow-up periods of 50 and 56 months, respectively. This difference was statistically significant (p = 0.016). Therefore, based on this experience and in comparison with numerous retrospective studies involving similar patients, ECoG is advocated, especially in children and in any patient with a long-standing seizure disorder, to maximize seizure control while minimizing or abolishing the need for postoperative antiepileptic drugs.

Changes in gamma-aminobutyric acid and somatostatin in epileptic cortex associated with low-grade gliomas.

The role of specific neuronal populations in epileptic foci was studied by comparing epileptic and non-epileptic cortex removed from patients with low-grade gliomas. Epileptic and nearby (within 1 to 2 cm) non-epileptic temporal lobe neocortex was identified using electrocorticography. Cortical specimens taken from four patients identified as epileptic and nonepileptic were all void of tumor infiltration. Somatostatin- and gamma-aminobutyric acid (GABAergic)-immunoreactive neurons were identified and counted. Although there was no significant difference in the overall cell count, the authors found a significant decrease in both somatostatin- and GABAergic-immunoreactive neurons (74% and 51%, respectively) in the epileptic cortex compared to that in nonepileptic cortex from the same patient. It is suggested that these findings demonstrate changes in neuronal subpopulations that may account for the onset and propagation of epileptiform activity in patients with low-grade gliomas.

Seizure outcome in children with hemispheric tumors and associated intractable epilepsy: the role of tumor removal combined with seizure foci resection.

Children harboring hemispheric tumors associated with intractable epilepsy were retrospectively reviewed to assess seizure outcome following tumor resection and electrocorticography-guided seizure foci removal. Thirteen (93%) of our patients have remained seizure-free, off anticonvulsants or on tapering doses, following surgery with a mean follow-up of 33 months. Fifteen of 16 (93%) seizure foci examined histologically were void of tumor infiltration. A review of the literature is provided regarding the controversy of tumor removal versus additional seizure foci removal at the time of tumor removal in providing optimal seizure control.

Polyamine metabolism in isoproterenol-stimulated mouse salivary glands.

Isoproterenol (0.3 micronmole/gm body weight) was injected intraperitoneally every 24 h for three days. The synthesis of deoxyribonucleic acid, the concentration of putrescine, spermidine and spermine and the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were measured in parotid and submandibular glands at 4 to 8 h after each injection. The parotid glands responded with peaks of DNA synthesis at 24 and 72 h and peaks of putrescine content and decarboxylase activities 8 to 12 h after each injection. Spermidine increased steadily in the parotid, whereas there was little change in the spermine concentration throughout the 72 h. Polyamine metabolism showed much less response in the submandibular gland, and little or no increase in spermidine or spermine levels or in DNA synthesis was observed.