KCNN2 mutation in autosomal-dominant tremulous myoclonus-dystonia.

BACKGROUND AND PURPOSE: Despite recent advances in neurogenetics that have facilitated the identification of a number of dystonia genes, many familial dystonia syndromes remain without known cause. The aim of the study was to identify the cause of autosomal dominant tremulous myoclonus-dystonia in a UK kindred with affected individuals in three generations. METHODS: Known genetic causes of myoclonus-dystonia were excluded. We combined clinical and electrophysiological phenotyping with whole-exome sequencing and Sanger sequencing to identify candidate causal variants in a family with tremulous myoclonus-dystonia. RESULTS: The core phenotype consisted of childhood-onset dystonia predominantly affecting hands and neck, with a fast tremor with superimposed myoclonus and, in some individuals, subtle cerebellar signs. We identified a novel missense variant in potassium calcium-activated channel subfamily N member 2 (KCNN2) [NM_021614:c.1112G>A:p.(Gly371Glu)], which was the only variant that we were able to identify as segregating with the phenotype over three generations. This variant, which is absent from the most recent version of gnomAD, was predicted to be deleterious by SIFT and PolyPhen-2 and had an overall CADD score of 29.7. CONCLUSIONS: KCNN2, a member of the KCNN family of potassium channel genes, is highly conserved across species and in humans is highly expressed in the brain, particularly the cerebellum. KCNN2 mutations have never been described as pathological in human disease, but are recognized abnormalities in two rodent models of fast, jerky tremor. Segregation, absence of the variant in the normal population and in-silico prediction of a deleterious effect together with animal models compatible with the clinical phenotype are all in line with KCNN2 mutations being a plausible cause underlying myoclonus-dystonia.

Primary familial brain calcification linked to deletion of 5' noncoding region of SLC20A2.

OBJECTIVES: Primary familial brain calcification (PFBC) is a rare neurological disease often inherited as a dominant trait. Mutations in four genes (SLC20A2, PDGFB, PDGFRB, and XPR1) have been reported in patients with PFBC. Of these, point mutations or small deletions in SLC20A2 are most common. Thus far, only one large deletion covering entire SLC20A2 and several smaller, exonic deletions of SLC20A2 have been reported. The aim of this study was to identify the causative gene defect in a Finnish PFBC family with three affected patients. MATERIALS AND METHODS: A Finnish family with three PFBC patients and five unaffected subjects was studied. Sanger sequencing was used to exclude mutations in the coding and splice site regions of SLC20A2, PDGFRB, and PDGFB. Whole-exome (WES) and whole-genome sequencing (WGS) were performed to identify the causative mutation. A SNP array was used in segregation analysis. RESULTS: Copy number analysis of the WGS data revealed a heterozygous deletion of ~578 kb on chromosome 8. The deletion removes the 5' UTR region, the noncoding exon 1 and the putative promoter region of SLC20A2 as well as the coding regions of six other genes. CONCLUSIONS: Our results support haploinsufficiency of SLC20A2 as a pathogenetic mechanism in PFBC. Analysis of copy number variations (CNVs) is emerging as a crucial step in the molecular genetic diagnostics of PFBC, and it should not be limited to coding regions, as causative variants may reside in the noncoding parts of known disease-associated genes.

Rare variants analysis of cutaneous malignant melanoma genes in Parkinson's disease.

A shared genetic susceptibility between cutaneous malignant melanoma (CMM) and Parkinson's disease (PD) has been suggested. We investigated this by assessing the contribution of rare variants in genes involved in CMM to PD risk. We studied rare variation across 29 CMM risk genes using high-quality genotype data in 6875 PD cases and 6065 controls and sought to replicate findings using whole-exome sequencing data from a second independent cohort totaling 1255 PD cases and 473 controls. No statistically significant enrichment of rare variants across all genes, per gene, or for any individual variant was detected in either cohort. There were nonsignificant trends toward different carrier frequencies between PD cases and controls, under different inheritance models, in the following CMM risk genes: BAP1, DCC, ERBB4, KIT, MAPK2, MITF, PTEN, and TP53. The very rare TYR p.V275F variant, which is a pathogenic allele for recessive albinism, was more common in PD cases than controls in 3 independent cohorts. Tyrosinase, encoded by TYR, is the rate-limiting enzyme for the production of neuromelanin, and has a role in the production of dopamine. These results suggest a possible role for another gene in the dopamine-biosynthetic pathway in susceptibility to neurodegenerative Parkinsonism, but further studies in larger PD cohorts are needed to accurately determine the role of these genes/variants in disease pathogenesis.

Gastric adenocarcinoma in a 13-year-old boy: a diagnosis not often seen in this age group.

Gastric adenocarcinoma is not uncommon in the adult population, but in the pediatric population it is an extremely rare entity. A 13-year-old boy was referred to a pediatric oncology unit for evaluation of a tumor in the upper abdomen. Further investigation revealed an advanced stage gastric carcinoma with metastases suggestive for a hereditary cause. Awareness for uncommon diagnoses is a key issue in regard of accurate treatment and overall prognosis.

[Dissertations 25 years after the date 13. Sarcomas of the jaws]. / Proefschriften 25 jaar na dato 13. Sarcomen van de kaak.

On the basis of the dissertation 'Sarcomas of the jaws'from 1982, developments in the imaging and treatment of osteosarcoma in general and of jaws in particular are discussed. The majority of osteosarcomas is found outside the craniofacial skeleton (extracraniofacial). The most frequent primary sites are the distal femur and proximal tibia. Most of these patients are children and adolescents. Only 5% of all osteosarcomas is found within the craniofacial skeleton, especially the jaws. Usually these patients are older. During the past 30 years the treatment of extracraniofacial osteosarcoma has changed from being purely surgical to being surgical in combination with multidrug, multicycle chemotherapy within prospective trials; the 5-year survival rate has risen from 10-20% to 60-70%. For craniofacial osteosarcomas such studies are missing and there is no unanimous opinion about the benefits of chemotherapy. In this group the 5-year survival rate has risen from 25-40% to 60-80%. Factors associated with a good prognosis are tumour size < 4 cm and tumour free resection margins. Developments in imaging have made improvements in preoperative evaluations possible. A plea is made for a prospective study.

Different susceptibility of osteosarcoma cell lines and primary cells to treatment with oncolytic adenovirus and doxorubicin or cisplatin.

Despite improvements in treatment regimens for osteosarcoma (OS) patients, survival rate has not increased over the last two decades. New treatment modalities are therefore warranted. Preclinical results with conditionally replicative adenoviruses (CRAds) to treat OS are promising. One type of CRAd that was effective against OS cells is Ad5-Delta24RGD. In other types of cancer, CRAds have been shown to interact synergistically with chemotherapeutic agents. Chemotherapy for OS often includes doxorubicin and cisplatin. Therefore, we explored combination treatment of OS cell lines and primary OS cell cultures with Ad5-Delta24RGD and doxorubicin or cisplatin. On OS cell lines, combination treatment was additive to synergistic. Surprisingly, however, on seven of eight primary OS samples no such combination effects were observed. In contrast, in many cases chemotherapy even inhibited CRAd-mediated cell killing. The inhibitory effect of doxorubicin on Ad5-Delta24RGD in primary OS cells appeared to correlate with slow cell growth rate; reduced viral replication and absence of chemotherapy-induced G2 cell cycle arrest. Our results point to the possibility that, at least for OS, virotherapy and chemotherapy should best not be performed simultaneously. In general, our work underscores the importance of testing new genetic anticancer agents and treatment regimens on primary cancer specimens.

[Imported malaria in 2000 in 2 northern Paris hospitals]. / Le paludisme d'importation en 2000 dans deux hôpitaux du nord de la région parisienne.

The number of travellers in malaria striken areas increases each year (2). The risk of infection is high in Sub-Saharan Africa, but appropriate chemoprophylaxis can reduce the morbidity and mortality rate of malaria. Half of the samples of malaria cases received by the National reference centre of malaria chemosensibility (CNRCP) for chemosensibility analysis came from two hospitals in the north of Paris: Bichat Claude Bernard in Paris and Delafontaine in Saint-Denis. In 2000, quite all the malaria cases (n = 387) observed at the Bichat and Delafontaine Hospitals came from Africa (99%). Plasmodium falciparum remains the most represented (87.6%) species, with an average parasitic density of 0.3%. Patients with P falciparum came for medical advice on the tenth day after return (median, extremes 0-174 days). More than half of the patients (58%) did not take any medication for chemoprophylaxis and even if they took some, it was irregular or inappropriate. The most used drug chemoprophylaxis is the association of chloroquine and proguanil or Savarine. In 15% of the cases, the travellers took chloroquine as a prophylaxis and 4% other medicine not recommended by the French authorities. An average of 43.7% of these travellers took inappropriate chemoprophylaxis. In total, 27 chemoprophylaxis failures are reported. Some patients (22%) have already taken self treatment which was readjusted during admission at hospital. The first treatment of malaria in 2000 was monotherapy with quinine (P. falciparum) and chloroquine (P. ovale, malariae, vivax). The treatment associations in case of suspicious resistance were quinine + doxycycline and atovaquone + proguanil. Treatment failure was infrequent and resulted above all from a bad observance. More information should be given to travellers as well as doctors about recommendations and treatments.

Epidermal growth factor receptor targeting enhances adenoviral vector based suicide gene therapy of osteosarcoma.

BACKGROUND: Despite improvements in the treatment of osteosarcoma (OS) there are still too many patients who cannot benefit from current treatment modalities. Therefore, new therapeutic approaches are warranted. Here we explore the efficacy of targeted adenoviral based suicide gene therapy. METHODS AND RESULTS: Immunohistochemistry and FACS analysis detected low or absent expression levels of the primary adenovirus receptor CAR on human primary OS and human OS cell lines. These results predict a low infection efficiency and thus a reduced therapeutic effect. Targeting the adenoviruses to another receptor highly expressed on OS could overcome this limitation. We found epidermal growth factor receptor (EGFR) to be widely expressed on primary OS. Immunohistochemistry on primary tumor samples and FACS analysis on primary short-term cultures and four OS cell lines showed that EGFR was consistently expressed. The recombinant bispecific single-chain antibody 425-s11 redirects adenoviral vectors towards the EGFR. Adenovirus transduction experiments in the presence or absence of 425-s11 showed significantly enhanced gene transfer with the targeted adenoviral vector compared with the native vector (OS cell lines 2.5 to 7.2 times enhanced gene transfer and OS primary short term cultures 1.7 to 10 times enhanced gene transfer). On this basis, targeted suicide gene therapy experiments with AdCMVHSV-TK in combination with ganciclovir were performed. These experiments demonstrated up to 3.5-fold enhanced kill of OS cell lines and primary short-term cultures by the EGFR targeted vector. CONCLUSIONS: Suicide gene therapy with adenovirus targeted towards EGFR may have favorable therapeutic characteristics for future gene therapy applications in OS.

Iron chelators as antimalarial agents: in vitro activity of dicatecholate against Plasmodium falciparum.

The present study was undertaken to explore the antimalarial effect of a series of dicatecholate iron chelators. They may be made more or less lipophilic by increasing or reducing the length of the R substituent on the nitrogen. In vitro activity against the W2 and 3D7 clones of Plasmodium falciparum, toxicity on Vero cells and toxicity on uninfected erythrocytes by measure of the released haemoglobin were assessed for each compound. These findings were compared with the ability of iron(III), iron(II) and ferritin to reverse the inhibitory effect of catecholates. This study shows that increased lipid solubility of catecholate iron chelators does not lead to improved antimalarial activity. However, their activity is well correlated with their interaction with iron and with their toxicity against Vero cells. This study demonstrates a potent antimalarial effect of FR160 (R = C9H19) on five different strains of P. falciparum in vitro. FR160 inhibited parasite growth with an IC50 between 0.8 and 1.5 micro M. The effects of FR160 on mammalian cells were minimal compared with those obtained with malaria parasites. FR160 acted on parasites at considerably higher rates than desferrioxamine, and at all stages of parasite growth. The drug was more effective at the late trophozoite and young schizont stages, although FR160 affected rings and schizonts as well. Ascorbic acid, a free radical scavenger, reduced the activities of FR160 and artesunate. FR160 might induce formation of free radicals, which could explain why FR160 antagonized the effects of artesunate and dihydroartemisinin.

The role of fine-needle aspiration cytology in children with persistent or suspicious lymphadenopathy.

PURPOSE: The aim of this study was (1) To determine the value of fine-needle aspiration cytology (FNAC) in children with persistent or suspicious peripheral lymphadenopathy and (2) to analyze whether a history of previous malignancy influenced the accuracy of FNAC. METHODS: A retrospective study in an Academic Children's Hospital of 73 FNACs of peripheral lymph nodes in 64 patients (35 boys and 29 girls; median age, 9 years; range, 15 months to 20 years) was performed between 1992 and 1997. Eleven patients were excluded because aspirated material appeared inadequate. Outcome was compared with results of subsequent surgical biopsies, clinical follow-up, or both. Patients were divided into group A "de novo" lymphadenopathy (n = 39) and group B lymphadenopathy and a history of previous malignancy (n = 23). RESULTS: Group A: FNAC showed a benign diagnosis in 25 cases, a malignancy in 13, and was inconclusive in 1. Outcome was false-negative in 2 and false-positive in 1. Sensitivity and specificity were 86% and 96%, respectively. FNAC helped avoid additional surgical procedures in 27 cases (61%). However, if FNAC showed malignant lymphoma, open biopsy was inevitable (8 of 13) to establish proper classification. Group B: FNAC showed a benign diagnosis in 10 cases and a malignancy in 13. Outcome was false-negative in 1, and false-positive in 1. Sensitivity and specificity were 92% and 90%, respectively. FNAC helped avoid additional surgical diagnostic procedures in 25 cases (86%). CONCLUSIONS: (1) Fine-needle aspiration cytology is a rapid, simple and accurate diagnostic procedure to differentiate between benign and malignant peripheral lymphadenopathy in children. FNAC can avoid open biopsy in at least 60% of cases. (2) A history of previous malignant disease does not influence the accuracy of the test.

[Considering disparities in resistance of Plasmodium falciparum in Africa in chemoprevention decisions]. / Prise en compte des disparités de résistance de Plasmodium falciparum en Afrique dans la décision chimioprophylactique.

OBJECTIVES: Assess the efficacy of preventive and curative treatments of imported malaria. METHODS: The in vitro drug susceptibility of mefloquine, chloroquine and cycloguanil was determined against African isolates of Plasmodium falciparum from imported malaria cases by an isotopic in vitro test or a genomic approach. RESULTS: Plasmodium falciparum resistance to mefloquine, chloroquine or to the dihydrofolate reductase inhibitor was present in 5.2%, 46% and 42% of isolates respectively. Plasmodium falciparum drug resistance to chloroquine or antifolinics was more frequent in permanent than in seasonal malarial transmission areas. Simultaneous resistance to chloroquine and antifolinics was observed in 17% of isolates between 1991 and 1994 and in 28% between 1995 and 1997.

Carney's triad.

The purpose of this study is to provide an overview of cases of Carney's triad and to discuss the clinical implications of this diagnosis. A search was made of the English-language literature for original articles, reviews and abstracts addressing Carney's triad. A new patient was described and added to the number of known patients. In literature 40 patients were found with a complete or incomplete triad. There exists no agreement on the pathology and possible common origin of these tumours. Directions are given for diagnostics and therapy. The diagnosis of Carney's triad has specific clinical implications that make a good knowledge of this syndrome mandatory.

Herpes simplex virus 1-mediated transfer of preproenkephalin A in rat dorsal root ganglia.

Recombinant herpes simplex virus-1 encoding the rat preproenkephalin A (HSVLatEnk1) was generated for driving the expression of preproenkephalin A-derived peptides in dorsal root ganglia of rats in vivo. Three weeks after infection via the hind footpads, quantitative RT-PCR and in situ hybridization experiments showed a strong expression of preproenkephalin A mRNA in lumbar dorsal root ganglia. In addition, a 40-160% increase in radioimmunoassayable Met-enkephalin-like material concentrations was found in the dorsal spinal cord and dorsal root ganglia, respectively, at the lumbar level in HSVLatEnk1-infected rats as compared with animals infected with beta-galactosidase-encoding recombinant herpes simplex virus-1 or control rats. These data demonstrate the efficacy of the preproenkephalin A encoding vector and suggest that it should help in elucidating the role of Met-enkephalin-containing primary afferent fibers in pain transmission and/or control.

Allelotype of pediatric rhabdomyosarcoma.

An allelotype covering all autosomes was constructed for the embryonal form of childhood rhabdomyosarcoma (ERMS) in order to identify regions encompassing tumorsuppressor genes (TSG) involved in ERMS. Thusfar most studies were focussed on chromosome 11p15.5, which frequently shows loss of heterozygozity (LOH) in embryonal tumors like RMS and Wilms' tumor (WT). In this study we show that, besides LOH of chromosome 11p15.5 (72%), LOH of chromosome 16q was present in 54% of the tumors analysed. Delineation of these two regions shows that the smallest region of overlap (SRO) for chromosome 11 was between D11S988 and D11S922. This region, estimated to be 7 cM and 3-5 Mb, is also the location of the putative Wilms' tumor WT2 TSG. It contains several genes including IGF2 and potential tumorsuppressor genes like H19 and p57kip2, which might contribute to the carcinogenesis of RMS. Analysis of chromosome 16q LOH defined the SRO between D16S752 and D16S413. LOH of chromosome 16 is also found in other tumors, including WT. Our data suggest that genes involved in the development of RMS and WT may not only be similar for chromosome 11 but also for chromosome 16.

DNA-analysis is mandatory in case of an uncommon malignancy.

In a child a diagnosis of sweat gland carcinoma was made on basis of a surgical specimen presumed to be taken from an occipital lymph node. DNA analysis confirmed mixing of specimens in the referring hospital.

Accurate topological comparison of two recombinant human growth hormones by optical surface plasmon resonance.

A strategy for the comparison of two recombinant derived human growth hormones (r-hGH) has been developed using surface plasmon resonance (SPR). Statistical analysis was systematically used on the results obtained with several batches derived from two different Escherichia coli strains. Monoclonal antibodies (MAb) directed against four different domains in the tertiary structure of natural human growth hormone were used to compare the epitopic maps of the three (two recombinant and one natural) hGH by SPR analysis. Topological studies show the homogeneity of the epitopic maps of the three hGH. The kinetic parameters, association rate, and dissociation rate constants were also analyzed for the binding of each hGH batch to all MAbs. They were found to be homogeneous between the three hormones. Furthermore, the two r-hGH were compared by more classical approaches examining recognition of lactogenic or somatogenic receptors using, respectively, a bioassay of Nb2 cell proliferation and binding to rat liver microsomes. Specific bioactivities and IC50 values calculated in radioreceptor assays did not significantly differ between different r-hGH. The method was sensitive enough to show slight differences on koff value for one MAb (3C11) between (natural) hormone and two r-hGH. These differences are discussed in relation to previous observation made in the literature and the presence of isoforms in the natural product. The strategy developed here was very useful as a new tool to establish the equivalence of the two r-hGH.

Malignant astrocytoma-derived region of common amplification in chromosomal band 17p12 is frequently amplified in high-grade osteosarcomas.

Recently, we reported a new amplification event that involves marker D17S67 in 17p12 in three malignant astrocytomas of patients with a very short survival. The amplified region may contain an oncogene implicated in astrocytoma tumorigenesis. To determine the extent of the amplified regions, we constructed a yeast artificial chromosome contig spanning the D17S67 region and tested the amplification status of markers that map to the contig. We determined a commonly amplified region between markers D17S1311 and D17S1875 with a maximal length of 1,630 kb. By using marker 745R, from within the commonly amplified region, we screened 60 high-grade astrocytomas but could not detect additional tumors with the amplification event. This suggests that the incidence of the amplification event in high-grade astrocytoma is low (5%). It has recently been shown by comparative genomic hybridization that amplification of 17p11-p12 is a frequent event in high-grade osteosarcomas, occurring in 20-30% of cases. Since the commonly amplified region is within 17p12, we tested 745R in 20 osteosarcomas, including 6 lung metastases, and detected amplification in 9 cases (45%). Marker 745R was found to be amplified in 4 of the 6 lung metastases (66%). From this frequent involvement and the association with clinically aggressive astrocytomas we conclude that for both tumor types presence of the amplification event seems to correlate with aggressive clinical behaviour.

[Iodobenzylguanide-131 therapy as first choice in non-metastasized neuroblastoma]. / Jobenguaan-131-therapie als eerste keus bij het niet gemetastaseerde neuroblastoom.

Three children with a localized pelvic neuroblastoma are presented. Because of progression and clinical signs of compression of adjacent structures therapy was necessary. Targeted radiotherapy with 131I-MIBG was used as the first mode of therapy. This new strategy proved to be feasible, effective and non-toxic. Application of 131I-MIBG as first-line therapy should be considered more often in neuroblastoma patients.