Idh1-mutated transgenic zebrafish lines: An in-vivo model for drug screening and functional analysis

Introduction The gene encoding isocitrate dehydrogenase 1 (IDH1) is frequently mutated in several tumor types including gliomas. The most prevalent mutation in gliomas is a missense mutation leading to a substitution of arginine with histidine at the residue 132 (R132H). Wild type IDH1 catalyzes oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) whereas mutant IDH1 converts α-KG into D2-hydroxyglutarate (D2HG). Unfortunately, there are few in vivo model systems for IDH-mutated tumors to study the effects of IDH1 mutations in tumor development. We have therefore created transgenic zebrafish lines that express various IDH1 mutants. Materials and methods IDH1 mutations (IDH1R132H, IDH1R132C and loss-of-function mutation IDH1G70D), IDH1wildtype or eGFP were cloned into constructs with several brain-specific promoters (Nestin, Gfap or Gata2). These constructs were injected into fertilized zebrafish eggs at the one-cell stage. Results In total more than ten transgenic zebrafish lines expressing various brain-specific IDH1 mutations were created. A significant increase in the level of D2HG was observed in all transgenic lines expressing IDH1R132C or IDH1R132H, but not in any of the lines expressing IDH1wildtype, IDH1G70D or eGFP. No differences in 5-hydroxymethyl cytosine and mature collagen IV levels were observed between wildtype and mutant IDH1 transgenic fish. To our surprise, we failed to identify any strong phenotype, despite increased levels of the oncome-tabolite D2HG. No tumors were observed, even when backcrossing with tp53-mutant fish which suggests that additional transforming events are required for tumor formation. Elevated D2HG levels could be lowered by treatment of the transgenic zebrafish with an inhibitor of mutant IDH1 activity. Conclusions We have generated a transgenic zebrafish model system for mutations in IDH1 that can be used for functional analysis and drug screening. Our model systems help understand the biology of IDH1 mutations and its role in tumor formation

Cytogenetic characterization of telomeres in the holocentric chromosomes of the lepidopteran Mamestra brassicae

Telomeres of the Mamestra brassica holocentric chromosomes were studied by Southern blotting, in-situ hybridization and Bal31 assay evidencing the presence of the telomeric (TTAGG)(n) repeat. Successively, molecular analysis of telomeres showed that TRAS1 transposable elements were present at the subtelomeric regions of autosomes but not in the NOR-bearing telomeres of the Z and W sex chromosomes. TRAS1 appeared to be transcriptionally active and non-methylated, as evaluated by RT-PCR and digestion with MspI and HpaII. Finally, dot-blotting experiments showed that the 2.8 +/- 0.5% of the M. brassicae genome consists of TRAS1

Metachromatic leukodystrophy genotypes in The Netherlands reveal novel pathogenic ARSA variants in non-Caucasian patients

Metachromatic leukodystrophy (MLD) is an autosomal recessively inherited sulfatide storage disease caused by deficient activity of the lysosomal enzyme arylsulfatase A (ASA). Genetic analysis of the ARSA gene is important in MLD diagnosis and screening of family members. In addition, more information on genotype prevalence will help interpreting MLD population differences between countries. In this study, we identified 31 different ARSA variants in the patient cohort (n = 67) of the Dutch expertise center for MLD. The most frequently found variant, c.1283C > T, p.(Pro428Leu), was present in 43 (64%) patients and resulted in a high prevalence of the juvenile MLD type (58%) in The Netherlands. Furthermore, we observed in five out of six patients with a non-Caucasian ethnic background previously unreported pathogenic ARSA variants. In total, we report ten novel variants including four missense, two nonsense, and two frameshift variants and one in-frame indel, which were all predicted to be disease causing in silico. In addit

Creatine and guanidinoacetate reference values in a French population

Creatine and guanidinoacetate are biomarkers of creatine metabolism. Their assays in body fluids may be used for detecting patients with primary creatine deficiency disorders (PCDD), a class of inherited diseases. Their laboratory values in blood and urine may vary with age, requiring that reference normal values are given within the age range. Despite the long known role of creatine for muscle physiology, muscle signs are not necessarily the major complaint expressed by PCDD patients. These disorders drastically affect brain function inducing, in patients, intellectual disability, autistic behavior and other neurological signs (delays in speech and language, epilepsy, ataxia, dystonia and choreoathetosis), being a common feature the drop in brain creatine content. For this reason, screening of PCDD patients has been repeatedly carried out in populations with neurological signs. This report is aimed at providing reference laboratory values and related age ranges found for a large scale population of patients with neurological signs (more than 6 thousand patients) previously serving as a background population for screening French patients with PCDD. These reference laboratory values and age ranges compare rather favorably with literature values for healthy populations. Some differences are also observed, and female participants are discriminated from male participants as regards to urine but not blood values including creatine on creatinine ratio and guanidinoacetate on creatinine ratio values. Such gender differences were previously observed in healthy populations; they might be explained by literature differential effects of testosterone and estrogen in adolescents and adults, and by estrogen effects in prepubertal age on SLC6A8 function. Finally, though they were acquired on a population with neurological signs, the present data might reasonably serve as reference laboratory values in any future medical study exploring abnormalities of creatine metabolism and transport