Congenital X-linked neutropenia with myelodysplasia and somatic tetraploidy due to a germline mutation in SEPT6.

Septins play key roles in mammalian cell division and cytokinesis but have not previously been implicated in a germline human disorder. A male infant with severe neutropenia and progressive dysmyelopoiesis with tetraploid myeloid precursors was identified. No known genetic etiologies for neutropenia or bone marrow failure were found. However, next-generation sequencing of germline samples from the patient revealed a novel, de novo germline stop-loss mutation in the X-linked gene SEPT6 that resulted in reduced SEPT6 staining in bone marrow granulocyte precursors and megakaryocytes. Patient skin fibroblast-derived induced pluripotent stem cells (iPSCs) produced reduced myeloid colonies, particularly of the granulocyte lineage. CRISPR/Cas9 knock-in of the patient's mutation or complete knock-out of SEPT6 was not tolerated in non-patient-derived iPSCs or human myeloid cell lines, but SEPT6 knock-out was successful in an erythroid cell line and resulting clones revealed a propensity to multinucleation. In silico analysis predicts that the mutated protein hinders the dimerization of SEPT6 coiled-coils in both parallel and antiparallel arrangements, which could in turn impair filament formation. These data demonstrate a critical role for SEPT6 in chromosomal segregation in myeloid progenitors that can account for the unusual predisposition to aneuploidy and dysmyelopoiesis.

Prenatal diagnosis of chromosomal mosaicism in over 1600 cases using array comparative genomic hybridization as a first line test.

OBJECTIVE: The aim of this study was to assess the detection of chromosomal mosaicism in chorionic villus (CVS) and amniotic fluid (AF) samples using array comparative genomic hybridization (aCGH) and quantitative fluorescent polymerase chain reaction. METHODS: All patients undergoing invasive prenatal testing by aCGH at a specialist prenatal screening service were included in the study. A total of 1609 samples (953 CVS and 656 AF) underwent quantitative fluorescent polymerase chain reaction and targeted aCGH without concurrent conventional G-banded karyotyping. RESULTS: Chromosomal mosaicism was detected in 20 of the 1609 cases (1.24%); of which 17 were derived from 953 CVS (1.78%), and three from 656 AF (0.46%). Mosaicism was observed at a level as low as 9%. Four cases were likely confined placental mosaicism, 12 were likely true fetal mosaicism, and four cases were unable to be classified into either group. CONCLUSIONS: This study demonstrates that the use of aCGH as a first line test is able to identify chromosomal mosaicism down to 9%, which is lower than the level reliably detected using standard cytogenetic analysis. aCGH avoids the disadvantages of culturing, which include culture bias, artifact, and culture failure.

Simple inhibitors of histone deacetylase activity that combine features of short-chain fatty acid and hydroxamic acid inhibitors.

Butyric acid and trichostatin A (TSA) are anti-cancer compounds that cause the upregulation of genes involved in differentiation and cell cycle regulation by inhibiting histone deacetylase (HDAC) activity. In this study we have synthesized and evaluated compounds that combine the bioavailability of short-chain fatty acids, like butyric acid, with the bidentate binding ability of TSA. A series of analogs were made to examine the effects of chain length, simple aromatic cap groups, and substituted hydroxamates on the compounds' ability to inhibit rat-liver HDAC using a fluorometric assay. In keeping with previous structure-activity relationships, the most effective inhibitors consisted of longer chains and hydroxamic acid groups. It was found that 5-phenylvaleric hydroxamic acid and 4-benzoylbutyric hydroxamic acid were the most potent inhibitors with IC50's of 5 microM and 133 microM respectively.

A Two-test Protocol for the Precise Determination of the Maximal Lactate Steady State.

The purpose of this study was to determine the efficacy of a two-test method for precisely identifying the Maximal Lactate Steady State (MLSS). Eight male competitive cyclists performed two bouts on a cycle ergometer. Following a maximal oxygen consumption (V̇O2max) test (66.91 ± 5.29 mL·kg-1·min-1) we identified the lactate deflection point using the visual deflection (TVis), Log-Log (TLog), Dmax (TDmax), RER = 1.00 (TRER), ventilatory threshold (TVent), and the 1.0 mmol·L-1 increase above baseline (T+1) methods. The second incremental test (SIT) consisted of 6-7 stages (5 min each) starting 20-30 W below to 20-30 W above the predetermined deflection point, in 10 W increments. Comparison of the two tests yielded different threshold estimates (range 11-46W) for all methods (P = 0.001-0.019) except the TLog (P = 0.194) and TRER (P = 0.100). The SIT resulted in significantly (P = 0.007) more narrow range of thresholds (27.5 ± 11.01W) compared to the V̇O2max test (70 ± 42.51W). The TVis from the SIT was identified as the MLSS and was verified using three 45-minute steady-state exercise bouts at 95%, 100%, and 105% of MLSS intensity (average increment 12.8 W). Blood lactate and V̇O2 were recorded every 5 minutes and differed between the three intensities at every time point (P < 0.001). V̇O2 increased from the 5th to the 45th minute by 7.02 mL·kg-1·min-1 (100% MLSS), 3.63 mL·kg-1·min-1 (95% MLSS) and 7.5 mL·kg-1·min-1 (105% MLSS, to the 30th minute). These results indicate that the MLSS was identified correctly by the SIT, the single incremental test overestimated the MLSS intensity, and the TVis provides a very accurate determination of the lactate breakpoint. The use of a second submaximal test is required for a precise identification of MLSS.