Neuron-Derived Neurotrophic Factor Is Mutated in Congenital Hypogonadotropic Hypogonadism

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disorder characterized by infertility and the absence of puberty. Defects in GnRH neuron migration or altered GnRH secretion and/or action lead to a severe gonadotropin-releasing hormone (GnRH) deficiency. Given the close developmental association of GnRH neurons with the olfactory primary axons, CHH is often associated with anosmia or hyposmia, in which case it is defined as Kallmann syndrome (KS). The genetics of CHH are heterogeneous, and >40 genes are involved either alone or in combination. Several CHH-related genes controlling GnRH ontogeny encode proteins containing fibronectin-3 (FN3) domains, which are important for brain and neural development. Therefore, we hypothesized that defects in other FN3-superfamily genes would underlie CHH. Next-generation sequencing was performed for 240 CHH unrelated probands and filtered for rare, protein-truncating variants (PTVs) in FN3-superfamily genes. Compared to gnomAD controls the CHH cohort was statistically enriched for PTVs in neuron-derived neurotrophic factor (NDNF) (p = 1.40 x 10(-6)). Three heterozygous PTVs (p.Lys62*, p.Tyr128Thrfs*55, and p.Trp469*, all absent from the gnomAD database) and an additional heterozygous missense mutation (p.Thr201Ser) were found in four KS probands. Notably, NDNF is expressed along the GnRH neuron migratory route in both mouse embryos and human fetuses and enhances GnRH neuron migration. Further, knock down of the zebrafish ortholog of NDNF resulted in altered GnRH migration. Finally, mice lacking Ndnf showed delayed GnRH neuron migration and altered olfactory axonal projections to the olfactory bulb; both results are consistent with a role of NDNF in GnRH neuron development. Altogether, our results highlight NDNF as a gene involved in the GnRH neuron migration implicated in KS.Peer reviewe

Application of ABTS Radical Cation for Selective On-Line Detection of Radical Scavengers in HPLC Eluates

The radical cation 2,2 ' -azinobis-(3 -ethylbenzothiazoline-6-sulfonate), (ABTS(.+)) was utilized in an on-line HPLC method for the detection of radical scavengers in complex matrixes. The HPLC-separated analytes react postcolumn with the preformed ABTS(.+), and the induced bleaching is detected as a negative peak by an absorbance detector at 734 nm, An optimized instrumental and experimental setup is presented. The method is suitable for both isocratic and gradient HPLC runs using mobile phases containing 100% organic solvent or,its solution in water, weak acids, or buffers (pH 3-7.4), The method is sensitive, selective, relatively simple, applicable to compounds of different chemical natures; uses common instruments and inexpensive reagents; and has a time-saving, non-laborious experimental protocol. It can also be used for quantitative analysis. The method was applied to several pure natural antioxidants and plant extracts. The minimum detectable concentration varied from 0.02 to 0.13 mug/mL, depending on the compound tested, The method can be applied to perform kinetic studies, which is illustrated by determination of Trolox equivalent antioxidant capacities (TEAC) of several known antioxidants in now injection mode.</p

Restricted-access material-based high-molecular-weight protein depletion coupled on-line with namo-liquid chromatography-mass spectrometry for proteomics applications

Proteomics samples often contain both abundant proteins and low-level proteins and peptides. Highly abundant proteins can mask and/or bind those of lower abundance and thereby hinder their analysis. In particular, we were concerned with samples containing large amounts of albumin (up to 4.0 mu M). In this study, a novel set-up for multidimensional nano-liquid chromatography-mass spectrometry (nanoLC-MS) with three columns coupled on-line was developed and characterised. A 1-mm-I.D. restricted-access-material (RAM) cartridge and a 100-mu m-I.D. reversed-phase trap column are coupled in forward-flush mode to remove albumin before on-line separation on a 50 mu m I.D. reversed-phase capillary analytical column. Volumes up to 100 mu L of a complex matrix (containing 0.4 or 4.0 mu M albumin) could be injected onto this system, enabling a 5000-fold volume reduction. Up to 99.7% of the albumin present in samples could be efficiently removed over the RAM cartridge. The total analysis time was about 40 min. Using Substance P as a model peptide, separations were efficient, with a peak width of 10s at half height. Moreover, separations were highly reproducible (relative standard deviation (RSD) on retention time similar to 3% over 1 week). The set-up proved to be robust and was used for about 750 analyses without exchanging one of the columns. Flexibility with respect to the stationary phase material in the sample preparation cartridge allows for other separation modes to be applied as well. (c) 2007 Elsevier B.V. All rights reserved

Improvement of recovery and repeatability in liquid chromatography-mass spectrometry analysis of peptides

Poor repeatability of peak areas is a problem frequently encountered in peptide analysis with nanoLiquid Chromatography coupled on-line with Mass Spectrometry (nanoLC-MS). As a result, quantitative analysis will be seriously hampered unless the observed variability can be corrected in some way. Currently, labeling techniques or addition of internal standards are often applied for this purpose. However, these procedures are elaborate and error-prone and may render complex samples even more complex. Moreover, whenever poor repeatability results from variable recovery, not just quantification, but also sensitivity is affected. We have studied the parameters influencing the repeatability of chromatographic peak areas for a model set of proteolytic peptides (i.e., a cytochrome c tryptic digest) in nanoLC-MS analysis. It is demonstrated that repeatability issues are mainly due to poor recovery of peptides from the sample vial. Problems are largely resolved by addition of an organic modifier to the sample vial to improve solubility of the peptides, but care needs to be taken not to lose peptides due to reduced affinity for reversed-phase materials. Good results are obtained when applying dimethylsulfoxide (DMSO) for this purpose. When applying DMSO, repeatability increases, and the limit of detection (LOD) decreases. For the most hydrophobic peptides, a gain in LOD of at least an order of magnitude is obtained. In an aqueous sample containing 0.1% formic acid (FA), it is possible to detect 100-200 fmol of peptide, whereas +/- 10 fmol can be detected in a sample containing 5% FA and 25% DMSO (10 mu L injections)