Background Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterized by hypotonia, ataxia, cognitive impairment, abnormal eye movements, respiratory control disturbances, and a distinctive mid-hindbrain malformation. JS demonstrates substantial phenotypic variability and genetic heterogeneity. This study provides a comprehensive view of the current genetic basis, phenotypic range and gene-phenotype associations in JS. Methods We sequenced 27 JS-associated genes in 440 affected individuals (375 families) from a cohort of 532 individuals (440 families) with JS, using molecular inversion probe-based targeted capture and next generation sequencing. Variant pathogenicity was defined using the Combined Annotation Dependent Depletion (CADD) algorithm with an optimized score cut-off. Results We identified presumed causal variants in 62% of pedigrees, including the first B9D2 mutations associated with JS. 253 different mutations in 23 genes highlight the extreme genetic heterogeneity of JS. Phenotypic analysis revealed that only 34% of individuals have a “pure JS” phenotype. Retinal disease is present in 30% of individuals, renal disease in 25%, coloboma in 17%, polydactyly in 15%, liver fibrosis in 14% and encephalocele in 8%. Loss of CEP290 function is associated with retinal dystrophy, while loss of TMEM67 function is associated with liver fibrosis and coloboma, but we observe no clear-cut distinction between JS-subtypes. Conclusion This work illustrates how combining advanced sequencing techniques with phenotypic data addresses extreme genetic heterogeneity to provide diagnostic and carrier testing, guide medical monitoring for progressive complications, facilitate interpretation of genome-wide sequencing results in individuals with a variety of phenotypes, and enable gene-specific treatments in the future.
The molybdenum blue reactlon for the determination of phosphate Is used as a model to Illustrate the extension of the use of hydrophoblc sorbents In flow Injection analyds for the preconcentratlon of an anlon and for on-cdumn detec#on, Le., optosenslng. Optosenslng provides for real-time monltorlng of the rate of slgnai change, dA /d T , so that the rate of color devebpment durlng the reductlon step of the analysis can be measured. The synerglstlc relationship between the rates of formation of the phosphate and sHlcate heteropdy complexes is examlned. A kinetic optosenslng method has been developed In which the difference In reduction rate for the heteropoly complexes allows simultaneous determlnatlon of phosphate, In the parts per bllllon range, and slllcate, in the parts per mllllon range. The system used an Inexpensive mlnlspec-20 spectrophotometer wtth a net path length through the sorbent of less than 2 mm. Partlal least-squares analyds was used to analyze the data, and predlctlon errors of approxlmately 10 % were obtained for both components. INTRODUCTIONEnhancement of the selectivity of instrumental methods by flow injection analysis (FIA) has been accomplished by various means, such as the use of solvent extraction ( 1 ) and gas diffusion techniques (2). These may be considered homogeneous techniques in that the chemistry occurs in the homogeneous media of the flowing stream itself. Additionally, sensitivity has been increased by on-line preconcentration using ion exchange (3). The latter technique may be viewed as heterogeneous since isolation of the analyte on a surface has occurred, creating a region of increased concentration within the flowing stream and may be considered similar to solute focusing (4). Only very recently, attention is being given to the concept of sorbent extraction in FIA, which has been used so far only for the preconcentration of metal complexes (5) using hydrophobic interactions on C-18. Further increases in sensitivity can be achieved by flow injection optosensing (6), which is based on the measurement of an analyte retained on the solid sorbent surface. Use of cation exchangers for this purpose has been described by Yoshimura and Waki (7) while Valcarcel and co-workers (8) used an anion exchanger in a detailed study of the iron(II1) thiocyanate complex. This paper deals, for the first time, with the use of sorbent extraction for the optosensing of anionic complexes and exploits in a novel way the differential kinetics of competing species.The present study of differential kinetics allows for the simultaneous determination of two analytes or the determination of one analyte in the presence of an interferent. Thus, while to date the use of sorbents has been driven by the "all or nothing" philosophy that focuses on steady-state chemistry, it is shown in this work how the horizon of applications for sorbent use in FIA can be greatly broadened with the incorporation of kinetic techniques.Previous work in this laboratory has focused on the enhancement of instrumental response at roo...
The compound 2,6-di-tert-butyl-4-nitrophenol (DBNP), a potentially powerful uncoupler of ATP-generating oxidative phosphorylation, has been physically and spectroscopically characterized using X-ray crystallography, differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), GC-MS spectrometry, Fourier-transformed IR (FTIR) spectrophotometry, UV-Vis spectrophotometry, and FT 'Hand I3C-NMR spectroscopy. However, DBNP is not commercially available; therefore, it had to be synthesized in the laboratory prior to toxicity studies. The DBNP was prepared from 2,6-di-tert-butylphenol (DBP) precursor in hexane through an electrophilic aromatic substitution process using NOz. A collective yield of 75% was obtained by using two empirically determined end points that prevented the coprecipitation of reaction by-products and resulted in the formation of DBNP in high purity. Excessive amounts of NO2 in reaction mixtures resulted in the decomposition of preformed DBNP. With a pK, value of 6.8 and a higher degree of lipophilicity, DBNP may prove to be a stronger uncoupler of oxidative phosphorylation than 2,4-dinitrophenol (pK, = 4.09) due to the expected enhancement of passive-diffusion kinetics across biological membranes at the physiological p H of 7.4. The present study is intended to provide analytical toxicologists, industrial hygiene monitors, and other professionals involved in chemical health and safety with a comprehensive source of basic information on the synthesis and analytical chemistry of DBNP. Keywojds-Synthesis 2,6-Di-tert-butyl-4-nitrophenol
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