Preventive medicine of von Hippel-Lindau disease-associated pancreatic neuroendocrine tumors

Pancreatic neuroendocrine tumors (PanNETs) are rare in von Hippel-Lindau disease (VHL) but cause serious morbidity and mortality. Management guidelines for VHL-PanNETs continue to be based on limited evidence, and survival data to guide surgical management are lacking. We established the European-American-Asian-VHL-PanNET-Registry to assess data for risks for metastases, survival and long-term outcomes to provide best management recommendations. Of 2330 VHL patients, 273 had a total of 484 PanNETs. Median age at diagnosis of PanNET was 35 years (range 10-75). Fifty-five (20%) patients had metastatic PanNETs. Metastatic PanNETs were significantly larger (median size 5 vs 2\u2009cm; P\u20091.5\u2009cm in diameter were operated. Ten-year survival was significantly longer in operated vs non-operated patients, in particular for PanNETs <2.8\u2009cm vs 652.8\u2009cm (94% vs 85% by 10 years; P\u2009=\u20090.020; 80% vs 50% at 10 years; P\u2009=\u20090.030). This study demonstrates that patients with PanNET approaching the cut-off diameter of 2.8\u2009cm should be operated. Mutations in exon 3, especially of codons 161/167 are at enhanced risk for metastatic PanNETs. Survival is significantly longer in operated non-metastatic VHL-PanNETs

Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology.

Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease

Western blot.

<p>Lysates of knock-out homozygous <i>aldh7a1</i> zebrafish embryos (at 8 dpf) western blot analysis showing no aldh7a1 protein compared to wild-type.</p

Preventive medicine of von Hippel-Lindau disease-associated pancreatic neuroendocrine tumors

© 2018 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain. Pancreatic neuroendocrine tumors (PanNETs) are rare in von Hippel-Lindau disease (VHL) but cause serious morbidity and mortality. Management guidelines for VHL-PanNETs continue to be based on limited evidence, and survival data to guide surgical management are lacking. We established the European-American-Asian-VHL-PanNET-Registry to assess data for risks for metastases, survival and long-term outcomes to provide best management recommendations. Of 2330 VHL patients, 273 had a total of 484 PanNETs. Median age at diagnosis of PanNET was 35 years (range 10-75). Fifty-five (20%) patients had metastatic PanNETs. Metastatic PanNETs were significantly larger (median size 5 vs 2cm; P&lt;0.001) and tumor volume doubling time (TVDT) was faster (22 vs 126 months; P=0.001). All metastatic tumors were ≥2.8cm. Codons 161 and 167 were hotspots for VHL germline mutations with enhanced risk for metastatic PanNETs

Characterization of the first knock-out <i>aldh7a1</i> zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology

<div><p>Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in <i>ALDH7A1 (</i>PDE-<i>ALDH7A1</i>) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out <i>aldh7a1</i> zebrafish model using CRISPR-Cas9 technology. Zebrafish <i>aldh7a1</i> mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of <i>aldh7a1</i> knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out <i>aldh7a1</i> zebrafish has homozygous 5 base pair (bp) mutation in <i>ALDH7A1</i>. Knock-out <i>aldh7a1</i> embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out <i>aldh7a1</i> embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out <i>aldh7a1</i> embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out <i>aldh7a1</i> zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-<i>ALDH7A1</i> disease.</p></div

Daily survival of heterozygous 5 bp deletion male and female zebrafish embryos.

<p>Y axis to the left indicates total number of embryos in the tank. Y axis to the right indicates the number of dead embryos genotyped and presented as a histogram. X axis shows dpf.</p

EEG results of knock-out homozygous <i>aldh7a1</i> embryos and wild type embryos at 9 dpf.

<p>A) This shows spike discharges in a homozygous with no response to 100 μM diazepam, but almost normalization of EEG on 25 mM pyridoxine compared to wild type. B) This shows another homozygous with no response to phenobarbital, but almost normalization of EEG on pyridoxine (25 mM) compared to wild type. C) This shows normal EEG with spikes on pentylenetetrazole, and normalization of EEG on diazepam.</p

Locomotor behavior of zebrafish embryos using Viewpoint Videotrack for Zebrafish^TM.

<p>A) This represents cumulative plots of the position and velocity of a single knock-out homozygous and heterozygous 5 bp deletion <i>aldh7a1</i>embryo and wild type embryo at 10 dpf during one hour of behavioral recording. B) This represents average of total time spent moving (seconds), average of total distance travelled and average of velocity for homozygous (n = 20), heterozygous (n = 20) and wild type (n = 11) embryo at 10 dpf during one hour of behavioral recording. Embryos were placed in individual wells of a flat-bottom 96-well plate and acclimated to the recording chamber before tracking began. One hour of movement data were collected. Data shown are sums of 60-minute (average ± standard error). Error bars shows standard error of the mean (SEM). * is confidence level in comparison with wild type: * is p<0.05, *** is p<0.001. † is confidence level in comparison with heterozygous 10 dpf († - p<0.05, ††† - p<0.001).</p

QIAxcel system, Sanger sequencing and HRM analysis results.

<p>A) Gel image produced by QIAxcel system revealing wild type <i>aldh7a1</i> sequence, as well as the heterozygous and homozygous 5bp deletion. The DNA sequences shown in the lower panel reveal the complement reverse sequences. The broken line indicates the nucleotides that were deleted by CRISPR/Cas9 in the homozygous fish. B) The HRM melting plots are displayed. The heterozygous sequence is identified by the double peak. The homozygous sequence is identified by a peak at 76.5 degrees Celsius, and the wild type sequence is identified by a peak at 77.5 degrees Celsius.</p

Metabolite measurement of alpha-AASA, P6C and PA.

<p>3dpf zebrafish embryo lysates (n = 15) were analyzed by liquid chromatography tandem mass spectrometry for homozygous and heterozygous for 5 bp deletion <i>aldh7a1</i> embryos and wild-type embryos. These show elevated levels of alpha-AASA, P6C and PA compared to wild type.</p