Consensus statement from the international consensus meeting on post-traumatic cranioplasty.

BACKGROUND: Due to the lack of high-quality evidence which has hindered the development of evidence-based guidelines, there is a need to provide general guidance on cranioplasty (CP) following traumatic brain injury (TBI), as well as identify areas of ongoing uncertainty via a consensus-based approach. METHODS: The international consensus meeting on post-traumatic CP was held during the International Conference on Recent Advances in Neurotraumatology (ICRAN), in Naples, Italy, in June 2018. This meeting was endorsed by the Neurotrauma Committee of the World Federation of Neurosurgical Societies (WFNS), the NIHR Global Health Research Group on Neurotrauma, and several other neurotrauma organizations. Discussions and voting were organized around 5 pre-specified themes: (1) indications and technique, (2) materials, (3) timing, (4) hydrocephalus, and (5) paediatric CP. RESULTS: The participants discussed published evidence on each topic and proposed consensus statements, which were subject to ratification using anonymous real-time voting. Statements required an agreement threshold of more than 70% for inclusion in the final recommendations. CONCLUSIONS: This document is the first set of practical consensus-based clinical recommendations on post-traumatic CP, focusing on timing, materials, complications, and surgical procedures. Future research directions are also presented.

Vein of Galen aneurysmal malformation (VGAM) in the fetus: retrospective analysis of perinatal prognostic indicators in a two-center series of 49 cases.

OBJECTIVE: Vein of Galen aneurysmal malformation (VGAM) is a rare fetal anomaly, the neurological outcome of which can be good with appropriate perinatal management. However, most fetal series are too small to allow reliable statistical assessment of potential prognostic indicators. Our aim was to assess, in a two-center series of 49 cases, the prognostic value of several prenatal variables, in order to identify possible prenatal indicators of poor outcome, in terms of mortality and cerebral disability. METHODS: This was a retrospective study involving 49 cases of VGAM diagnosed prenatally and managed at two centers over a 17-year period (1999-2015). All cases had undergone detailed prenatal cerebral and cardiac assessment by grayscale ultrasound, color and pulsed-wave Doppler and magnetic resonance imaging (MRI). Ultrasound and MRI examination reports and images were reviewed and outcome information was obtained from medical reports. Volume of the VGAM (on ultrasound and MRI) was calculated and development of straight-sinus dilatation, ventriculomegaly and other major brain abnormalities was noted. Cardiothoracic ratio, tricuspid regurgitation and reversed blood flow across the aortic isthmus were evaluated on fetal echocardiography. Major brain lesions were considered by definition to be associated with poor outcome in all cases. Pregnancy and fetoneonatal outcome were known in all cases. Fetoneonatal outcome and brain damage were considered as dependent variables in the statistical evaluation. Poor outcome was defined as death, late termination of pregnancy due to association with related severe brain anomalies or severe neurological impairment. RESULTS: At a mean follow-up time of 20 (range, 0-72) months, 36.7% of the whole series and 52.9% of the cases which did not undergo late termination were alive and free of adverse sequelae. Five (10.2%) cases showed progression of the lesion between diagnosis and delivery. On univariate analysis, dilatation of the straight sinus, VGAM volume ≥ 20 000 mm3 and tricuspid regurgitation were all significantly related to poor outcome. However, on logistic regression analysis, the only variables associated significantly with poor outcome were tricuspid regurgitation and, to a lesser extent, VGAM volume ≥ 20 000 mm3 . The former was also the only variable associated with brain damage. CONCLUSIONS: Major brain lesions, tricuspid regurgitation and, to a lesser extent, VGAM volume ≥ 20 000 mm3 are the only prenatal variables associated with poor outcome in fetal VGAM. Prenatal multidisciplinary counseling should be based on these variables. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

Expanding the mutational spectrum associated to neural tube defects: literature revision and description of novel VANGL1 mutations.

BACKGROUND: Neural Tube Defects (NTD) are a common class of birth defects that occur in approximately 1 in 1000 live births. Both genetic and nongenetic factors are involved in the etiology of NTD. Planar cell polarity (PCP) genes plays a critical role in neural tube closure in model organisms. Studies in humans have identified nonsynonymous mutations in PCP pathway genes, including the VANGL genes, that may play a role as risk factors for NTD. METHODS: Here, we present the results of VANGL1 and VANGL2 mutational screening in a series of 53 NTD patients and 27 couples with a previous NTD affected pregnancy. RESULTS: We identified three heterozygous missense variants in VANGL1, p.Ala187Val, p.Asp389His, and p.Arg517His, that are absent in controls and predicted to be detrimental on the protein function and, thus, we expanded the mutational spectrum of VANGL1 in NTD cases. We did not identify any new variants having an evident pathogenic effect on protein function in VANGL2. Moreover, we reviewed all the rare nonsynonymous or synonymous variants of VANGL1 and VANGL2 found in patients and controls so far published and re-evaluated them for their pathogenic role by in silico prediction tools. Association tests were performed to demonstrate the enrichment of deleterious variants in reviewed cases versus controls from Exome Variant Server (EVS). CONCLUSION: We showed a significant (p = 7.0E-5) association between VANGL1 rare genetic variants, especially missense mutations, and NTDs risk.

Pineal germinoma in a child with interferon-γ receptor 1 deficiency. case report and literature review.

Interferon-γ receptor 1 (IFN-γR1) deficiency is one of the primary immunodeficiencies conferring Mendelian Susceptibility to Mycobacterial Disease (MSMD). Some cases of neoplasms have been recently reported in patients with MSMD, underlying the already known link between immunodeficiency and carcinogenesis. We report the first case of intracranial tumour, i.e. pineal germinoma, in a 11-year-old patient with complete IFN-γR1 deficiency. The first clinical presentation of the genetic immunodeficiency dates back to when the child was aged 2 y and 10 mo, when he presented a multi-focal osteomyelitis caused by Mycobacterium scrofulaceum. The diagnosis of IFN-γR1 deficiency (523delT/523delT in IFNGR1 gene) was subsequently made. The child responded to antibiotic therapy and remained in stable clinical condition until the age of 11 years, when he started complaining of frontal, chronic headache. MRI revealed a solid pineal region mass lesion measuring 20 × 29 × 36 mm. Histological findings revealed a diagnosis of pineal germinoma. The patient received chemotherapy followed by local whole ventricular irradiation with boost on pineal site, experiencing complete remission, and to date he is tumor-free at four years follow-up. Four other cases of tumors have been reported in patients affected by MSMD in our knowledge: a case of Kaposi sarcoma, a case of B-cell lymphoma, a case of cutaneous squamous cell carcinoma and a case of oesophageal squamous cell carcinoma. In conclusion, in patients with MSMD, not only the surveillance of infectious diseases, but also that of tumors is important.

IRS1 G972R polymorphism and type 2 diabetes: a paradigm for the difficult ascertainment of the contribution to disease susceptibility of 'low-frequency-low-risk' variants.

AIMS/HYPOTHESIS: The aim of the study was to determine the association between IRS1 G972R polymorphism and type 2 diabetes; published data concerning this association have been conflicting. To obtain further insight into this topic, we performed a meta-analysis of all available case-control studies. METHODS: We performed a meta-analysis of 32 studies (12,076 cases and 11,285 controls). RESULTS: The relatively infrequent R972 variant was not significantly associated with type 2 diabetes (OR 1.09, 95% CI 0.96-1.23, p = 0.184 under a dominant model). Some evidence of heterogeneity was observed across studies (p = 0.1). In the 14 studies (9,713 individuals) in which the mean age at type 2 diabetes diagnosis was available, this variable explained 52% of the heterogeneity (p = 0.03). When these studies were subdivided into tertiles of mean age at diagnosis, the OR for diabetes was 1.48 (95% CI 1.17-1.87), 1.22 (95% CI 0.97-1.53) and 0.88 (95% CI 0.68-1.13) in the youngest, intermediate and oldest tertile, respectively (p = 0.0022 for trend of ORs). CONCLUSIONS/INTERPRETATION: Our findings illustrate the difficulties of ascertaining the contribution of 'low-frequency-low-risk' variants to type 2 diabetes susceptibility. In the specific context of the R972 variant, approximately 200,000 study individuals would be needed to have 80% power to identify a 9% increase in diabetes risk at a genome-wide significance level. Under these circumstances, a strategy aimed at improving outcome definition and decreasing its heterogeneity may critically enhance our ability to detect genetic effects, thereby decreasing the required sample size. Our data suggest that focusing on early-onset diabetes, which is characterised by a stronger genetic background, may be part of such a strategy.

Two mutant alleles of the insulin receptor gene in a patient with extreme insulin resistance.

Insulin receptor complementary DNA has been cloned from an insulin-resistant patient with leprechaunism whose receptors exhibited multiple abnormalities in insulin binding. The patient is a compound heterozygote, having inherited two different mutant alleles of the insulin receptor gene. One allele contains a missense mutation encoding the substitution of glutamic acid for lysine at position 460 in the alpha subunit of the receptor. The second allele has a nonsense mutation causing premature chain termination after amino acid 671 in the alpha subunit, thereby deleting both the transmembrane and tyrosine kinase domains of the receptor. Interestingly, the father is heterozygous for this nonsense mutation and exhibits a moderate degree of insulin resistance. This raises the possibility that mutations in the insulin receptor gene may account for the insulin resistance in some patients with non-insulin-dependent diabetes mellitus.

Successful isolation and long-term establishment of a cell line with stem cell-like features from an anaplastic medulloblastoma.

AIMS: Herein we report on the successful isolation and establishment of a novel, long-term, primary, neurosphere-like cell line called 1603-MED from a 5-year-old boy affected by a highly aggressive anaplastic medulloblastoma. METHODS: Elaboration of the new protocol for neurosphere assay is extensively discussed, together with a complete immuno-histochemical and cytogenetic characterization of 1603-MED. RESULTS: Clinical course and histopathology are briefly discussed. The 1603-MED possesses a high capacity for proliferation, CD133 expression, self-renewal and differentiation, thus indicating that anaplastic medulloblastoma contains a subpopulation of cancer stem cells as observed in classic medulloblastoma. CONCLUSIONS: 1603-MED provides us with the first in vitro model of anaplastic medulloblastoma that may be suitable for studying both tumour progression and the genetic mechanisms related to therapy resistance, and may lead to the development and testing of chemosensitivity and new therapeutic targets.

Noninvasive Assessment of Hemodynamic Stress Distribution after Indirect Revascularization for Pediatric Moyamoya Vasculopathy.

BACKGROUND AND PURPOSE: Indirect revascularization surgery is an effective treatment in children with Moyamoya vasculopathy. In the present study, we hypothesized that DSC-PWI may reliably assess the evolution of CBF-related parameters after revascularization surgery, monitoring the outcome of surgical pediatric patients with Moyamoya vasculopathy. Thus, we aimed to evaluate differences in DSC-PWI parameters, including the hemodynamic stress distribution, in surgical and nonsurgical children with Moyamoya vasculopathy and to correlate them with long-term postoperative outcome. MATERIALS AND METHODS: Pre- and postoperative DSC parameters of 28 patients (16 females; mean age, 5.5 ± 4.8 years) treated with indirect revascularization were compared with those obtained at 2 time points in 10 nonsurgical patients (6 females; mean age, 6.9 ± 4.7 years). We calculated 4 normalized CBF-related parameters and their percentage variance: mean normalized CBF of the MCA territory, mean normalized CBF of the proximal MCA territory, mean normalized CBF of cortical the MCA territory, and hemodynamic stress distribution. The relationship between perfusion parameters and postoperative outcomes (poor, fair, good, excellent) was explored using 1-way analysis of covariance (P < .05). RESULTS: A significant decrease of the mean normalized CBF of the proximal MCA territory and hemodynamic stress distribution and an increase of the mean normalized CBF of the cortical MCA territory were observed after revascularization surgery (P < .001). No variations were observed in nonsurgical children. Postoperative hemodynamic stress distribution and its percentage change were significantly different in outcome groups (P < .001). CONCLUSIONS: DSC-PWI indices show postoperative hemodynamic changes that correlate with clinical outcome after revascularization surgery in children with Moyamoya disease.

Comparative study between biodegradable nasopore (BNP) and Merocel hemox 10 cm after septo-turbinoplasty procedure.

OBJECTIVE: The purpose of our study was to compare Merocel (Merocel Hemox 10 cm) and BNP (biodegradable nasopore) during a septoturbinoplasty procedure in terms of efficiency and patient comfort. PATIENTS AND METHODS: We carried out a retrospective review of 72 patients who had undergone septoturbinoplasty between January 2015 and January 2016. Each group, packed with BNP or Merocel Hemox 10 cm was composed of 36 patients. A standard visual analogue scale ranging from zero (no symptoms) to 10 (the most severe symptoms) was used to assess subjective symptoms. To compare the usefulness of materials we evaluated the postoperative bleeding, infection and adhesion after the removal of packing with and endoscopic examination using a 5-point scale (zero, absent; 1, mild; 2, moderate; 3, severe; and 4, very severe). Secretions and crusts were evaluated 1 week and 4 weeks after surgery in both groups using a 5-point scale (zero, absent; 1, mild; 2, moderate; 3, severe; and 4, very severe). RESULTS: A total of 72 patients were enrolled in the study, 45 women and 27 men; age range 15-78 with a mean age of 47 years. In the group A (Merocel group), 21 cases showed grade 1 bleeding (58%), 11 cases grade 2 (30%) and 4 patients grade 0 (11.1%). In the group B (BNP group), 29 cases showed grade 0 bleeding (80.56%) and 7 cases showed grade 1 bleeding (19.44%). There was a statistical significant difference between the Merocel group and the BNP group in terms of bleeding after removal of packing material (p < 0.05). In the group A, 16 patients developed mild adhesion (44%), 8 patients moderate adhesion (22.2%), 3 patients severe adhesion (8.33%) and 1 patient very severe adhesion (2.77%). BNP nasal packing didn't cause any adhesion in 25 patients (69.4%), 11 patients developed mild adhesion (30.5%). So there was a statistical significant difference between group A and group B regarding the adhesion (p < 0.05). There was a statistically significance reduction of nasal secretions and crusts at a week after surgery in the BNP group vs. Merocel group. The difference is not statistically significant 4 weeks after surgery. About the severity of symptoms related to nasal packing, we found a statistically significant difference (p < 0.05) between Merocel and BNP group regarding the pain during packing removal, the general satisfaction and the pressure. CONCLUSIONS: Biodegradable nasopore reduced pain and patient discomfort during packing removal and causes less bleeding compared to Merocel hemox 10 cm. This type of material can be used after septoturbinolplasty.

Patterns of K-ras mutation in colorectal carcinomas from Iran and Italy (a Gruppo Oncologico dell'Italia Meridionale study): influence of microsatellite instability status and country of origin.

BACKGROUND: K-ras mutations are a key step in colorectal cancer progression. Such mutations have been widely studied in case series from Western countries but there are few data on the rate and spectrum of mutations in tumors from countries where the epidemiological features of the disease are different. PATIENTS AND METHODS: Tumor samples from 182 Iranian colorectal cancer patients (170 sporadic cases and 12 HNPCC cases) were screened for K-ras mutations at codons 12, 13 and 61 by sequencing analysis. The cases were also characterized for microsatellite instability at mononucleotide repeats by PCR and fragment analysis, and classified according to microsatellite instability status. The frequency and the spectrum of K-ras mutations were compared with those observed in a series of colorectal cancer patients from Italy. RESULTS: K-ras mutations were observed in 68/182 (37.4%) cases. Mutation frequencies were similar in HNPCC-associated, sporadic MSI-H and sporadic microsatellite-stable (MSS) tumors. However, the G13D substitution was more frequent in HNPCC (3/4, 75%) and sporadic MSI-H (7/11, 63.6%) tumors compared to sporadic MSS tumors (11/53, 20.4%) (P <0.01). Comparison of mutations in the two series from Iran and Italy showed a significantly higher frequency of G13D among Italian patients. CONCLUSIONS: While the frequency of K-ras mutations could be similar, the mutational spectrum could be differentially influenced by genetic and environmental factors.

Microsatellite instability in gastric cancer is associated with tumor location and family history in a high-risk population from Tuscany.

We studied the presence of microsatellite instability (MSI) in a series of 108 gastric cancers (GCs) previously identified in an epidemiological study carried out in a high-risk area around Florence. To investigate associations between MSI and GC family history, 34 cases (31.5%) who had a GC-affected first-degree relative were included in the series. A family history positive for colorectal cancer was reported quite rarely (5.6%). The analysis of 6 microsatellite loci in DNA from paired normal tissue and tumor samples microdissected from paraffin-embedded specimens revealed varying degrees of instability: 56 cases (51.8%) did not show instability at any of the 6 loci; 19 (17.6%) showed instability at 1 locus; 16 (14.8%) showed instability at 2 loci; 11 (10.2%) showed instability at 3 loci; 4 (3.7%) showed instability at 4 loci; and 2 (1.9%) showed instability at 5 loci. The replication error-positive (RER+) phenotype, defined as the presence of MSI at 2 or more loci, had a frequency of 30.6% (33 of 108) and tended to be positively associated with female sex, intestinal histological type, advanced tumor stage, vascular invasion, positive GC family history, and blood group of A type. No correlation emerged between age at diagnosis and RER+ phenotype, whereas a significant association with the RER+ phenotype was shown by the antral location. A multivariate analysis adjusting for a selected group of potential confounding factors confirmed the strong association of the RER+ phenotype with the antral location (P = 0.001) and with a positive GC family history (P < 0.05). Survival analyses at 5 and 8 years showed no difference between RER+ and RER- patients, even when corrected for stage distribution. By the microdissection technique, we also used microsatellite allele patterns to investigate intratumoral heterogeneity and genetic relationships between tumors and adjacent dysplasia and/or intestinal metaplasia. Areas of metaplasia and dysplasia demonstrated MSI only in cases with MSI-positive tumors. In MSI-positive tumors, there was consistent evidence of intratumoral microsatellite allele heterogeneity, indicating the presence of genetically divergent tumor cell clones within the same neoplasm.

Unbalanced germ-line expression of hMLH1 and hMSH2 alleles in hereditary nonpolyposis colorectal cancer.

We analyzed the hMLH1 and hMSH2 genes in 30 unrelated hereditary nonpolyposis colorectal cancer (HNPCC) patients using mutational and immunohistochemical analyses combined whenever possible with primer extension assays, designed to estimate hMLH1 and hMSH2 transcript expression in peripheral blood lymphocytes. Single-strand conformational polymorphism screening and PCR-direct sequencing revealed seven hMLH1 and five hMSH2 sequence variants in 14 unrelated HNPCC patients, including three definite pathogenic mutations, four amino acid substitutions of uncertain pathogenic significance, and five polymorphisms. Immunohistochemistry indicated the lack of either hMLH1 or hMSH2 protein expression in tumors from 13 patients, and the absence of both hMLH1 and hMSH2 immunostaining was observed in the tumor from one additional case. The lack of hMLH1 or hMSH2 immunostaining was associated with the presence of microsatellite instability in the corresponding tumor and was also observed in tumors from patients negative for pathogenic mutations by mutational screening. There was a marked unbalance in the allelic expression of either hMLH1 or hMSH2 transcripts in three of eight unrelated HNPCC patients that could be analyzed, although a less marked unbalance was detected in two additional patients. Tumors from patients with germ-line unbalance in hMLH1 or hMSH2 transcript expression did not express the corresponding mismatch repair protein and displayed microsatellite instability. Our results indicate that constitutional alterations in hMLH1 and hMSH2 transcript expression may represent genetic markers for HNPCC carrier status also in cases in which mutational analysis did not detect a definite pathogenic variant. This suggests that transcript deregulation may represent a relevant mode of germ-line inactivation for mismatch repair genes.

Red meat, family history, and increased risk of gastric cancer with microsatellite instability.

Microsatellite instability (MSI) occurs frequently in sporadic gastric cancer (GC) and may define a distinctive molecular pathway of carcinogenesis. We evaluated the role of dietary risk factors in GC according to MSI status. A large series of 382 GC cases and 561 controls were originally identified in a population-based case-control study carried out in the high-risk area around Florence, Italy; 126 GC patients were typed for MSI status. A MSI+ phenotype was detected in 43 of 126 cases (34.1%), whereas 83 cases were classified as MSI-. A multinomial logistic regression model was used to compare the two subgroups of GC classified according to MSI status in the same analysis, with all of the available population controls. A case-case approach was also used. The risk of MSI+ tumors was positively associated with high consumption of red meat and meat sauce and negatively associated with consumption of white meat. A positive association was also seen with total protein and nitrite intake, whereas no relation was found with micronutrient intake. Risk was especially high among subjects reporting both a positive GC family history and a high consumption of red meat (odds ratio, 25.7; 95% confidence interval, 6.4-102.8). For MSI- tumors, a significant protective effect was associated with frequent consumption of citrus and other fresh fruit, garlic, legumes, vegetables, and olive oil and with high intake of beta-carotene and other antioxidants and sugar, whereas positive associations were seen with protein and sodium intake. In summary, a specific dietary pattern emerged for MSI+ gastric tumors, suggesting that factors related to red meat consumption are involved in this pathway, particularly among individuals with a positive family history. In contrast, the risk of MSI- tumors was strongly reduced by the frequent consumption of fresh fruit and vegetables.

Mutations at coding mononucleotide repeats in gastric cancer with the microsatellite mutator phenotype.

We analysed 50 gastric carcinomas (GCs) to verify whether mutations at coding repeats were associated with microsatellite instability (MSI). The tumors included: ten cases with no MSI, 14 cases with MSI = 1 locus, 13 cases with MSI = two loci and 13 cases with MSI > or = 3 loci. We investigated coding repeats within the TGF-beta RII, IGFIIR, BAX, hMSH6, hMSH3 and BRCA2 genes. The TGF-beta RII, IGFIIR, BAX, hMSH6 and hMSH3 repeats were altered in 11 (22%), five (10%), four (8%), 16 (32%) and five (10%) cases respectively. Mutations occurred only in MSI-positive (MSI+) tumors and correlated with increasing MSI levels. No alterations of the BRCA2 repeat were found. Mutations in genes other than hMSH6 were strongly associated to hMSH6 mutations, suggesting a key role of this gene. The non-coding BAT-26 and E-Cadherin 3' UTR poly(A)8/(T)15 repeats were analysed in 44 of the 50 cases. Novel tumor-associated alleles were observed only in MSI-positive GCs and were in most cases associated with mutations at coding repeats. Further investigations with BAT-40 confirmed that four cases manifested mononucleotide repeat alterations restricted to hMSH6 and one case to TGF-beta RII. A subset of tumors with MSI at two or more dinucleotide loci resulted negative for mutations at coding and non-coding mononucleotide repeats.

Immunological abnormalities in insulin receptors on cultured EBV-transformed lymphocytes from insulin-resistant patient with leprechaunism.

Defects in insulin-receptor structure can impair insulin-receptor function. We have previously identified qualitative abnormalities in insulin binding to insulin receptors from an insulin-resistant patient (Lep/Ark-1). The defects in insulin binding are probably caused by a defect in receptor structure. In this study, we used immunological probes to investigate the structural defect(s) responsible for the abnormal function. Several anti-receptor antibodies were impaired in their abilities to bind to the insulin receptor of Lep/Ark-1. For example, monoclonal antibody MoAb-51 was much less effective in inhibiting binding to insulin receptors from Lep/Ark-1 (ID50 70 nM) than to those of normal subjects (ID50 8 nM). In addition, there was a 10-fold reduction of the avidity with which human polyclonal antibody B-d immunoprecipitated the patient's insulin receptors. The avidity of antibody B-10 was also reduced, although to a lesser extent. In contrast, several site-specific antibodies against epitopes on the beta-subunit of the receptor bound to receptors from Lep/Ark-1 with normal avidity. The data with monoclonal and polyclonal antibodies are consistent with the hypothesis that the structural defect resides in the extracellular domain of this patient's insulin receptor. The normal immunoreactivity of two putative phosphorylation sites on the beta-subunit with site-specific antibodies gives further support to the conclusion that this patient's receptors have normal tyrosine kinase activity.

Normal coding sequence of insulin gene in Pima Indians and Nauruans, two groups with highest prevalence of type II diabetes.

The nucleotide sequence of the insulin gene was determined in American Pima Indians and Micronesian Nauruans, two populations in whom the prevalence of non-insulin-dependent (type II) diabetes mellitus is the highest in the world. The insulin gene was amplified by the polymerase chain reaction to generate single-stranded DNA suitable for direct sequencing. The nucleotide sequences of the coding and adjacent regions of the insulin gene in six Pima Indians and two Nauruans with type II diabetes were identical to previously published insulin gene sequences of nondiabetic subjects.

Detection of mutations in insulin receptor gene by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) has been used to screen for mutations in the insulin receptor gene. Each of the 22 exons was amplified by the polymerase chain reaction (PCR). For each exon, one of the two PCR primers contained a guanine-cytosine (GC) clamp at its 5' end. The DNA was analyzed by electrophoresis through a polyacrylamide gel containing a gradient of denaturants. Two geometries for the gels were compared; the gradient of denaturants was oriented either parallel or perpendicular to the electric field. The sensitivity of the technique was evaluated by determining whether DGGE succeeded in detecting known mutations and polymorphisms in the insulin receptor gene. With parallel gels, 12 of 16 sequence variants were detected. The use of perpendicular gels increased the sensitivity of detection so that all 16 sequence variants were successfully detected when DNA was analyzed by a combination of perpendicular and parallel gels. Furthermore, DGGE was used to investigate a patient with leprechaunism whose insulin receptor genes had not previously been studied. Two mutant alleles were identified in this patient. The allele inherited from the father had a mutation substituting alanine for Val-28; in the allele inherited from the mother, arginine was substituted for Gly-366.

Evolving role of myeloablative chemotherapy in the treatment of childhood brain tumours.

Primary brain tumours, a heterogeneous group of cancer that constitute the second most common cancer in childhood, were historically treated with neurosurgical resection and radiation therapy. Chemotherapy has proven to be beneficial for some histological types, which has since led to exploration of the role of high-dose chemotherapy and haematopoietic stem cell rescue. Patients with high-grade glial tumours, primitive neuroectodermal tumours and high-risk medulloblastoma usually fare poorly. The indicators of bad prognosis are metastatic status, extent of resection and age. Children <3 years at diagnosis carry worse prognosis. Rare cancers such as ependymoblastoma, atypical teratoid rhabdoid tumour and choroid plexus carcinoma have a dismal prognosis regardless of the above-mentioned indicators. The use of myeloablative therapy (MAT) has been investigated to improve the rate of long-term DFS, as well as to reduce and delay in the youngest children the use of the craniospinal irradiation associated with unacceptable late effects. We will overview the literature regarding patients with 'good and uncertain indications' to MAT. Ependymoma and brain stem tumours, for which the available data discourage the use of MAT, are excluded. Finally, we will summarize a single Institution experience (Giannina Gaslini Children's Hospital, Genoa) with MAT in the period 1997-2003.