A next-generation newborn screening pilot study: NGS on dried blood spots detects causal mutations in patients with inherited metabolic diseases.

The range of applications performed on dried blood spots (DBS) widely broadened during the past decades to now include next-generation sequencing (NGS). Previous publications provided a general overview of NGS capacities on DBS-extracted DNA but did not focus on the identification of specific disorders. We thus aimed to demonstrate that NGS was reliable for detecting pathogenic mutations on genomic material extracted from DBS. Assuming the future implementation of NGS technologies into newborn screening (NBS), we conducted a pilot study on fifteen patients with inherited metabolic disorders. Blood was collected from DBS. Whole-exome sequencing was performed, and sequences were analyzed with a specific focus on genes related to NBS. Results were compared to the known pathogenic mutations previously identified by Sanger sequencing. Causal mutations were readily characterized, and multiple polymorphisms have been identified. According to variant database prediction, an unexplained homozygote pathogenic mutation, unrelated to patient's disorder, was also found in one sample. While amount and quality of DBS-extracted DNA are adequate to identify causal mutations by NGS, bioinformatics analysis revealed critical drawbacks: coverage fluctuations between regions, difficulties in identifying insertions/deletions, and inconsistent reliability of database-referenced variants. Nevertheless, results of this study lead us to consider future perspectives regarding "next-generation" NBS.

Energy-rich phosphates and transintestinal transport in rat intestine incubated in vitro at different temperatures.

In the present work, the transported fluid and the tissue content of ATP, ADP and AMP has been evaluated in the jejunum rat intestine which was everted and incubated in vitro both at 28 degrees C and at 38 degrees C for 1 h. The energy-rich phosphates have been measured in the tissue at the beginning and at the end of the experiment as well as in vivo. These determinations have been made in the total intestine and in the scraped mucosa. ATP and ADP content are higher in vivo and lower but constant at 28 degrees C in vitro; on the contrary, at 38 degrees C in vitro, the initial and final content of these adenilic nucleotides are both lower than at 28 degrees C. Under all these conditions the AMP content does not vary appreciably. Wet weight to dry weight ratios ahve been reported for mucosal and submucosal tissues in unincubated and incubated intestines. In some experiments, fluid transport (measured as an actual serosal volume increase) was determined every 20 min during a 1-h incubation. At 28 degrees C, fluid transport is constant throughout the time of the experiment, but at 38 degrees C, there is a progressive decrease of the transported fluid. Fluid transport and ATP content of the intestine seem to be directly related. The transport activity which is lower at 38 degrees C than at 28 degrees C, seems to be due to a low availability of energy-rich phosphates.

Effect of ethyl acetate on the transport of sodium and glucose in the hamster small intestine in vitro.

The effect of ethyl acetate on Na+, water and glucose transport, as well as on glucose and electrolyte intracellular concentrations in everted and cannulated sacs of hamster jejunum, have been studied. Ethyl acetate, a substance that easily penetrates and delivers energy to the cell, strongly stimulates net glucose and Na+ transport. The explanation of the experimental results takes into account the possibility of the existence of an active extrusion of glucose at the level of the basolateral membrane of the enterocyte.

In vivo and in vitro sugar transport in frog intestine.

In the frog intestine, both in vitro and in vivo, experiments were carried out in order to increase knowledge of the mechanism of sugar exit across the basolateral membrane of the enterocyte. The frog intestine was chosen because it lacks crypt cells and, consequently, any external fluid circuit mechanism during sugar transport can be avoided. Therefore, the sugar concentration in the absorbate collected on the serosal side is likely to be similar to that present underneath the basolateral membrane of the enterocyte. Under this condition, cell and absorbate sugar concentrations are similar; yet there is a concomitant net transintestinal sugar transport. Moreover, in in vivo experiments a net transintestinal sugar transport takes place even against a concentration difference. These results suggest that sugar exit across the basolateral membrane is not simply due to a chemically facilitated diffusion.

Vaginal agenesis.

Vaginal agenesis is not rare. Many ingenious procedures have been devised for its correction; some are simple, and some are complicated. Not enough emphasis has been placed on the emotional aspects when patients with this problem are treated. Proper timing of the operation and selection of the best procedure are discussed. We feel that procedure of choice is the simplest, least dangerous one which will give good results in the individual patient. Rather than using the same routine procedure in each patient, we fitted the treatment to each patient. Our experience, particularly with the simple vulvovaginoplasty technique, is given. Postoperative support and encouragement are needed to have a successful outcome.