(1)H magnetic resonance spectroscopy in the diagnosis of paediatric low grade brain tumours.

INTRODUCTION: Low grade gliomas are the commonest brain tumours in children but present in a myriad of ways, each with its own treatment challenges. Conventional MRI scans play an important role in their management but have limited ability to identify likely clinical behaviour. The aim of this study is to investigate (1)H magnetic resonance spectroscopy (MRS) as a method for detecting differences between the various low grade gliomas and related tumours in children. PATIENTS AND METHODS: Short echo time single voxel (1)H MRS at 1.5 or 3.0 T was performed prior to treatment on children with low grade brain tumours at two centres and five MR scanners, 69 cases had data which passed quality control. MRS data was processed using LCModel to give mean spectra and metabolite concentrations which were compared using T-tests, ANOVA, Receiver Operator Characteristic curves and logistic regression in SPSS. RESULTS: Significant differences were found in concentrations of key metabolites between glioneuronal and glial tumours (T-test p<0.05) and between most of the individual histological subtypes of low grade gliomas. The discriminatory metabolites identified, such as choline and myoinositol, are known tumour biomarkers. In the set of pilocytic astrocytomas and unbiopsied optic pathway gliomas, significant differences (p<0.05, ANOVA) were found in metabolite profiles of tumours depending on location and patient neurofibromatosis type 1 status. Logistic regression analyses yielded equations which could be used to assess the probability of a tumour being of a specific type. CONCLUSIONS: MRS can detect subtle differences between low grade brain tumours in children and should form part of the clinical assessment of these tumours.

Direct effects of Bmi1 on p53 protein stability inactivates oncoprotein stress responses in embryonal cancer precursor cells at tumor initiation.

Embryonal cancer can arise from postnatally persistent embryonal remnant or rest cells, which are uniquely characterized by the absence of p53 mutations. Perinatal overexpression of the MycN oncoprotein in embryonal cancer precursor cells causes postnatal rests, and later tumor formation through unknown mechanisms. However, overexpression of Myc in adult tissues normally activates apoptosis and/or senescence signals as an organismal defense mechanism against cancer. Here, we show that perinatal neuroblastoma precursor cells exhibited a transiently diminished p53 response to MycN oncoprotein stress and resistance to trophic factor withdrawal, compared with their adult counterpart cells from the TH-MYCN(+/+) transgenic mouse model of neuroblastoma. The adult stem cell maintenance factor and Polycomb group protein, Bmi1 (B-cell-specific Moloney murine leukemia virus integration site), had a critical role at neuroblastoma initiation in the model, by repressing p53 responses in precursor cells. We further show in neuroblastoma tumor cells that Bmi1 could directly bind p53 in a complex with other Polycomb complex proteins, Ring1A or Ring1B, leading to increased p53 ubiquitination and degradation. Repressed p53 signal responses were also seen in precursor cells for other embryonal cancer types, medulloblastoma and acute lymphoblastic leukemia. Collectively, these date indicate a general mechanism for p53 inactivation in some embryonal cell types and consequent susceptibility to MycN oncogenesis at the point of embryonal tumor initiation.

Non-invasive detection of glycine as a biomarker of malignancy in childhood brain tumours using in-vivo 1H MRS at 1.5 tesla confirmed by ex-vivo high-resolution magic-angle spinning NMR.

Management of brain tumours in children would benefit from improved non-invasive diagnosis, characterisation and prognostic biomarkers. Metabolite profiles derived from in-vivo MRS have been shown to provide such information. Studies indicate that using optimum a priori information on metabolite contents in the construction of linear combination (LC) models of MR spectra leads to improved metabolite profile estimation. Glycine (Gly) is usually neglected in such models due to strong overlap with myo-inositol (mI) and a low concentration in normal brain. However, biological studies indicate that Gly is abundant in high-grade brain tumours. This study aimed to investigate the quantitation of Gly in paediatric brain tumours using MRS analysed by LCModel, and its potential as a non-invasive biomarker of malignancy. Single-voxel MRS was performed using PRESS (TR 1500 ms, TE 30 ms/135 ms) on a 1.5 T scanner. Forty-seven cases (18 high grade (HG), 17 low grade (LG), 12 ungraded) were retrospectively selected if both short-TE and long-TE MRS (n = 33) or short-TE MRS and high-resolution magic-angle spinning (HRMAS) of matched surgical samples (n = 15) were available. The inclusion of Gly in LCModel analyses led to significantly reduced fit residues for both short-TE and long-TE MRS (p < 0.05). The Gly concentrations estimated from short-TE MRS were significantly correlated with the long-TE values (R = 0.91, p < 0.001). The Gly concentration estimated by LCModel was significantly higher in HG versus LG tumours for both short-TE (p < 1e-6) and long-TE (p = 0.003) MRS. This was consistent with the HRMAS results, which showed a significantly higher normalised Gly concentration in HG tumours (p < 0.05) and a significant correlation with the normalised Gly concentration measured from short-TE in-vivo MRS (p < 0.05). This study suggests that glycine can be reliably detected in paediatric brain tumours using in-vivo MRS on standard clinical scanners and that it is a promising biomarker of tumour aggressiveness.

In-utero stenting: development of a low-cost high-fidelity task trainer.

OBJECTIVE: To develop an in-utero stent placement training model. METHODS: The in-utero stent task trainer was constructed using a formalin-preserved gravid pig uterus. Altering the size of the uterine segment, changing the fluid level in the uterus and addition of a large Ziploc freezer bag variably filled with differing amounts of ultrasound gel can vary the procedural skill required. RESULTS: Thoracoamniotic and vesicoamniotic shunts can be simulated using this life-like model. The cost of eight to 10 learning stations is approximately US $ 60. Fetal position, maternal size and amniotic fluid status can be altered rapidly to increase the complexity of the procedure. CONCLUSIONS: This low-cost and realistic task trainer can provide the opportunity to practice in-utero shunt procedures in a non-clinical environment. This model should enhance learning and reinforce acquired skills.

Identification and characterisation of childhood cerebellar tumours by in vivo proton MRS.

(1)H MRS has great potential for the clinical investigation of childhood brain tumours, but the low incidence in, and difficulties of performing trials on, children have hampered progress in this area. Most studies have used a long-TE, thus limiting the metabolite information obtained, and multivariate analysis has been largely unexplored. Thirty-five children with untreated cerebellar tumours (18 medulloblastomas, 12 pilocytic astrocytomas and five ependymomas) were investigated using a single-voxel short-TE PRESS sequence on a 1.5 T scanner. Spectra were analysed using LCModel to yield metabolite profiles, and key metabolite assignments were verified by comparison with high-resolution magic-angle-spinning NMR of representative tumour biopsy samples. In addition to univariate metabolite comparisons, the use of multivariate classifiers was investigated. Principal component analysis was used for dimension reduction, and linear discriminant analysis was used for variable selection and classification. A bootstrap cross-validation method suitable for estimating the true performance of classifiers in small datasets was used. The discriminant function coefficients were stable and showed that medulloblastomas were characterised by high taurine, phosphocholine and glutamate and low glutamine, astrocytomas were distinguished by low creatine and high N-acetylaspartate, and ependymomas were differentiated by high myo-inositol and glycerophosphocholine. The same metabolite features were seen in NMR spectra of ex vivo samples. Successful classification was achieved for glial-cell (astrocytoma + ependymoma) versus non-glial-cell (medulloblastoma) tumours, with a bootstrap 0.632 + error, e(B.632+), of 5.3%. For astrocytoma vs medulloblastoma and astrocytoma vs medulloblastoma vs ependymoma classification, the e(B.632+) was 6.9% and 7.1%, respectively. The study showed that (1)H MRS detects key differences in the metabolite profiles for the main types of childhood cerebellar tumours and that discriminant analysis of metabolite profiles is a promising tool for classification. The findings warrant confirmation by larger multi-centre studies.

Characterization and purification of glycosaminoglycans from crude biological samples.

Chondroitin sulfate (CS) is a glycosaminoglycan derived from cartilage and commonly used to treat osteoarthritis, psoriasis, and other conditions. The dimethylmethylene blue (DMMB) assay has been used often to measure glycosaminoglycan levels in relatively pure samples. In this study, we verified the accuracy of the DMMB assay in measuring CS levels in unpurified extract from bovine trachea and shark cartilage, despite potential interference from salts, proteins, and DNA. We found that the glycosaminoglycan signal obtained was due to CS and not to other glycosaminoglycan species. This was confirmed using fluorophore-assisted carbohydrate electrophoresis, which also revealed that the majority of the CS was monosulfated at the C4 or C6 position. Finally, we used anion-exchange chromatography to purify the bovine extract and obtained complete recovery of the glycosaminoglycans, with no contaminating protein. The results of this study should be very useful for future purification and analysis of this common supplement.

Chloride channel myotonia: exon 8 hot-spot for dominant-negative interactions.

Myotonia congenita (MC) is the commonest genetic skeletal muscle ion channelopathy. It is caused by mutations in CLCN1 on chromosome 7q35, which alter the function of the major skeletal muscle voltage-gated chloride channel. Dominant and recessive forms of the disease exist. We have undertaken a clinical, genetic and molecular expression study based upon a large cohort of over 300 UK patients. In an initial cohort of 22 families, we sequenced the DNA of the entire coding region of CLCN1 and identified 11 novel and 11 known mutations allowing us to undertake a detailed genotype-phenotype correlation study. Generalized muscle hypertrophy, transient weakness and depressed tendon reflexes occurred more frequently in recessive than dominant MC. Mild cold exacerbation and significant muscle pain were equally common features in dominant and recessive cases. Dominant MC occurred in eight families. We noted that four newly identified dominant mutations clustered in exon 8, which codes for a highly conserved region of predicted interaction between the CLC-1 monomers. Expressed in Xenopus oocytes these mutations showed clear evidence of a dominant-negative effect. Based upon the analysis of mutations in this initial cohort as well as a review of published CLCN1 mutations, we devised an exon hierarchy analysis strategy for genetic screening. We applied this strategy to a second cohort of 303 UK cases with a suspected diagnosis of MC. In 23 individuals, we found two mutations and in 86 individuals we identified a single mutation. Interestingly, 40 of the cases with a single mutation had dominant exon 8 mutations. In total 48 individuals (from 34 families) in cohort 1 and 2 were found to harbour dominant mutations (37% of mutation positive individuals, 30% of mutation positive families). In total, we have identified 23 new disease causing mutations in MC, confirming the high degree of genetic heterogeneity associated with this disease. The DNA-based strategy we have devised achieved a genetic diagnosis in 36% of individuals referred to our centre. Based on these results, we propose that exon 8 of CLCN1 is a hot-spot for dominant mutations. Our molecular expression studies of the new exon 8 mutations indicate that this region of the chloride channel has an important role in dominant negative interactions between the two chloride channel monomers. Accurate genetic counselling in MC should be based not only upon clinical features and the inheritance pattern but also on molecular genetic analysis and ideally functional expression data.

Age-related changes in the normal sagittal relationship between globe and orbit.

PURPOSE: To establish the pattern of change in globe protrusion with advancing age. The findings contribute to our understanding of orbital ageing, and are useful in the longitudinal assessment of patients with orbital disease, craniofacial abnormalities and trauma. METHODS: Ocular protrusion from the lateral orbital rim to the corneal apex was measured in 653 Caucasians aged 21-80 years. Healthy subjects only were included in the study excluding those with ocular or orbital diseases. Measurements were taken using a single instrument and observer. Data were analysed for both sexes and each eye separately. RESULTS: The mean exophthalmometry reading in both sexes (318 female and 335 male) was 19+/-2mm. Ninety-eight percent of readings between the two eyes were within 1mm of each other and no subject had greater than 2mm of asymmetry. In all groups there was a negative linear correlation between ocular protrusion and age. This correlation was found to be highly statistically significant in all groups (r=0.56-0.65, p<0.0001). There was no statistically significant difference between change in ocular protrusion with age between the left and right eye for females or males. This study demonstrates a strong association between ocular protrusion and age in a Caucasian population. This association is an almost linear reduction in ocular protrusion with increasing age between the ages of 31 and 80. Asymmetry in ocular protrusion between the two eyes does not develop with increasing age.

Andersen-Tawil syndrome: new potassium channel mutations and possible phenotypic variation.

OBJECTIVE: To evaluate clinical, genetic, and electrophysiologic features of patients with Andersen-Tawil syndrome (ATS) in the United Kingdom. METHODS: Clinical and neurophysiologic evaluation was conducted of 11 families suspected to have ATS. Molecular genetic analysis of each proband was performed by direct DNA sequencing of the entire coding region of KCNJ2. Control samples were screened by direct DNA sequencing. The electrophysiologic consequences of several new mutations were studied in an oocyte expression system. RESULTS: All 11 ATS families harbored pathogenic mutations in KCNJ2 with six mutations not previously reported. Some unusual clinical features including renal tubular defect, CNS involvement, and dental and phonation abnormalities were observed. Five mutations (T75M, D78G, R82Q, L217P, and G300D) were expressed, all of which resulted in nonfunctional channels when expressed alone, and co-expression with wild-type (WT) KCNJ2 demonstrated a dominant negative effect. CONCLUSION: Six new disease-causing mutations in KCNJ2 were identified, one of which was in a PIP2 binding site. Molecular expression studies indicated that five of the mutations exerted a dominant negative effect on the wild-type allele. KCNJ2 mutations are an important cause of ATS in the UK.

Corroboration of in utero MRI using post-mortem MRI and autopsy in foetuses with CNS abnormalities.

AIMS: To corroborate the findings of in utero magnetic resonance imaging (MRI) with autopsy and post-mortem MRI in cases of known or suspected central nervous system (CNS) abnormalities on ultrasound and to compare the diagnostic accuracy of ante-natal ultrasound and in utero MRI. METHODS: Twelve pregnant women, whose foetuses had suspected central nervous system abnormalities underwent in utero MRI. The foetuses were imaged using MRi before autopsy. The data were used to evaluate the diagnostic accuracy of in utero MRI when compared with a reference standard of autopsy and post-mortem MRI in 10 cases and post-mortem MRI alone in two cases. RESULTS: The diagnostic accuracy of antenatal ultrasound and in utero MRI in correctly characterizing brain and spine abnormalities were 42 and 100%, respectively. CONCLUSION: In utero MRI provides a useful adjuvant to antenatal ultrasound when assessing CNS abnormalities by providing more accurate anatomical information. Post-mortem MRI assists the diagnosis of macroscopic structural abnormalities.

Comparison of ultrasound and magnetic resonance imaging in 100 singleton pregnancies with suspected brain abnormalities.

OBJECTIVE: To compare the diagnostic accuracy of the current reference standard-ultrasound with in utero magnetic resonance imaging, in a selected group of patients. DESIGN: Prospective study. SETTING: Five fetal maternal tertiary referral centres and an academic radiology unit. SAMPLE: One hundred cases of fetuses with central nervous system abnormalities where there has been diagnostic difficulties on ultrasound. In 48 cases the women were less than 24 weeks of gestation and in 52 cases later in pregnancy. METHODS: All women were imaged on a 1.5 T clinical system using a single shot fast spin echo technique. The results of antenatal ultrasound and in utero magnetic resonance were compared. MAIN OUTCOME MEASURES: The definitive diagnosis was made either at autopsy or by postmortem magnetic resonance imaging, in cases that went to termination of pregnancy, or a combination of postnatal imaging and clinical follow up in the others. RESULTS: In 52 of cases, ultrasound and magnetic resonance gave identical results and in a further 12, magnetic resonance provided extra information that was judged not to have had direct effects on management. In 35 of cases, magnetic resonance either changed the diagnosis (29) or gave extra information that could have altered management (6). In 11 of the 30 cases where magnetic resonance changed the diagnosis, the brain was described as normal on magnetic resonance. CONCLUSIONS: In utero magnetic resonance imaging is a powerful tool in investigating fetal brain abnormalities. Our results suggest that in selected cases of brain abnormalities, detected by ultrasound, antenatal magnetic resonance may provide additional, clinically useful information that may alter management.

Frequency and natural history of subdural haemorrhages in babies and relation to obstetric factors.

BACKGROUND: Subdural haematomas are thought to be uncommon in babies born at term. This view is mainly based on findings in symptomatic neonates and babies in whom subdural haemorrhages are detected fortuitously. We aimed to establish the frequency of subdural haemorrhages in asymptomatic term neonates; to study the natural history of such subdural haematomas; and to ascertain which obstetric factors, if any, are associated with presence of subdural haematoma. METHODS: We did a prospective study in babies who were born in the Jessop wing of the Central Sheffield University Hospitals between March, 2001, and November, 2002. We scanned neonates with a 0.2 T magnetic resonance machine. FINDINGS: 111 babies underwent MRI in this study. 49 were born by normal vertex delivery without instrumentation, 25 by caesarean section, four with forceps, 13 ventouse, 18 failed ventouse leading to forceps, one failed ventouse leading to caesarean section, and one failed forceps leading to caesarean section. Nine babies had subdural haemorrhages: three were normal vaginal deliveries (risk 6.1%), five were delivered by forceps after an attempted ventouse delivery (27.8%), and one had a traumatic ventouse delivery (7.7%). All babies with subdural haemorrhage were assessed clinically but no intervention was needed. All were rescanned at 4 weeks and haematomas had completely resolved. INTERPRETATION: Presence of unilateral and bilateral subdural haemorrhage is not necessarily indicative of excessive birth trauma.

Severe infantile hyperkalaemic periodic paralysis and paramyotonia congenita: broadening the clinical spectrum associated with the T704M mutation in SCN4A.

The authors describe an Italian kindred with nine individuals affected by hyperkalaemic periodic paralysis associated with paramyotonia congenita (hyperPP/PMC). Periodic paralysis was particularly severe, with several episodes a day lasting for hours. The onset of episodes was unusually early, beginning in the first year of life and persisting into adult life. The paralytic episodes were refractory to treatment. Patients described minimal paramyotonia, mainly of the eyelids and hands. All affected family members carried the threonine to methionine substitution at codon 704 (T704M) in exon 13 of the skeletal muscle voltage gated sodium channel gene (SCN4A). The association between T704M and the hyperPP/PMC phenotype has been only recently revealed. Nevertheless, such a severe phenotype has never been reported so far in families with either hyperPP or hyperPP/PMC. These data further broaden the clinical spectrum of T704M and support the evidence that this mutation is a common cause of hyperPP/PMC.

Sodium channel gene mutations in hypokalemic periodic paralysis: an uncommon cause in the UK.

Eleven of 36 families with hypokalemic periodic paralysis (hypoPP) harbored mutations in the skeletal muscle calcium channel gene (CACNA1S). The authors screened the skeletal muscle sodium channel gene (SCN4A) in the remainder. One family harbored a new heterozygous point mutation C2014A in exon 12 (R672S) of SCN4A. The authors identified the genetic defect underlying hypoPP in 33% of individuals tested. The authors conclude that SCN4A mutations are an uncommon cause of hypoPP in this UK population.

The skeletal muscle channelopathies: basic science, clinical genetics and treatment.

The human neurological channelopathies are a rapidly expanding group of mainly genetic conditions that are characterized by dysfunction of membrane-bound glycoproteins (ion channels). The skeletal muscle channelopathies were the first to be characterized in this group. In recent years significant progress has been made in our understanding of the molecular genetic and cellular electrophysiological bases of these disorders. DNA-based diagnosis is now a reality for many of the channelopathies. The advances made have implications for both genetic counselling and for tailoring treatment to specific channelopathies.

Inherited muscle and brain channelopathies.

In the past 5 years, advances in the complementary fields of neurogenetics and cellular electrophysiology have resulted in an explosion of knowledge about a group of disorders now known as the neurological channelopathies. These advances have resulted in more accurate DNA-based diagnosis and have increased our understanding of cellular pathophysiology. This is leading to more tailored therapies for patients with these disorders.

The effect of chromatin structure on cisplatin damage in intact human cells.

The influence of chromatin structure on cisplatin DNA damage was investigated in intact human cells. The epsilon-globin gene promoter was utilised as the target DNA sequence and the terminal transferase-dependent PCR technique was employed to examine adduct formation at base pair resolution. It was found that cisplatin preferentially damaged at runs of consecutive guanine bases in intact cells. By comparing the relative intensity of adduct formation in intact cells and in purified genomic DNA, it was possible to assess the influence of chromatin proteins on the extent of cisplatin DNA damage. Enhanced damage in intact cells was found at the CACC site where a member of the Sp1 family of proteins is thought to bind. It is postulated that protein binding at this site bends the DNA double-helix so that enhanced cisplatin binding occurs. The altered DNA binding of cisplatin in the presence of chromatin proteins could be important in the properties of cisplatin as an anti-tumour drug.

Exposure of primary orbital implants in postenucleation retinoblastoma patients.

PURPOSE: To determine significant factors influencing the exposure of primary orbital implants in patients with retinoblastoma. DESIGN: Retrospective noncomparative case series. PARTICIPANTS: One hundred nine consecutive patients (110 sockets) who had undergone enucleation for retinoblastoma from January 1993 to December 1997. METHODS: Two patients with recurrence of orbital retinoblastoma were excluded from further analysis, leaving 107 patients (108 sockets). The parameters analyzed included the patient's age; gender; ocular diagnosis; surgeon; type, covering, and size of the implant; the use of chemotherapy or radiotherapy; and the timing of these treatments in relation to enucleation. Study patients were divided into two main groups: the "treated group"-patients who had undergone adjuvant external beam radiotherapy or chemotherapy, and the "untreated group"-patients had undergone enucleation with or without cryotherapy, laser thermotherapy, or brachytherapy to the index or fellow eye. The following additional parameters were noted in the patients with exposed implants: time to exposure from date of enucleation and treatment of exposure. MAIN OUTCOME MEASURE: Exposure of orbital implants. RESULTS: There were two exposures caused by orbital recurrence of retinoblastoma. The rate of nontumor recurrence exposure was 28% (30 of 108). The median time to exposure was 136 days (range, 1-630 days). There were 18 exposures (35%,18 of 51) in the treated group, with a 34% exposure rate (13 of 38) in the chemotherapy group. The exposure rate was 21% (12 of 57) in the untreated group. The rates of exposure according to implant were: Vicryl mesh-wrapped hydroxyapatite (2 of 18, 11%), Medpor (8 of 13, 53%), plain polymethylmethacrylate (PMMA) (4 of 50, 8%), Mersilene-wrapped PMMA (9 of 17, 53%) and Castroviejo (7 of 10, 70%). Eight of the exposures (27%) were managed conservatively; the remainder required surgical repair. CONCLUSIONS: Results suggested that implant type and covering (P = 0.000) had a highly significant effect on the rate of exposure in postenucleation retinoblastoma patients. There was no statistical evidence that age, gender, ocular diagnosis, surgeon, size of the implant, or radiotherapy had an effect on implant exposure. There was an increased rate of exposure in the chemotherapy group, although this did not achieve statistical significance (P = 0.058), but a detrimental effect could not be excluded.