The role of cysteine residues in the sulphate transporter, SHST1: Construction of a functional cysteine-less transporter

We investigated the role of cysteine residues in the sulphate transporter, SHST1, with the aim of generating a functional cysteine-less variant. SHST1 contains five cysteine residues and none was essential for function. However, replacement of C421 resulted in a reduction in transport activity. Sulphate transport by C205 mutants was dependent on the size of the residue at this position. Alanine at position 205 resulted in a complete loss of function whereas leucine resulted in a 3-fold increase in sulphate transport relative to wild type SHST1. C205 is located in a putative intracellular loop and our results suggest that this loop may be important for sulphate transport. By replacing C205 with leucine and the other four cysteine residues with alanine, we constructed a cysteine-less variant of SHST1 that has transport characteristics indistinguishable from wild type. This construct will be useful for further structure and function studies of SHST1.     

Tie2Cre-mediated inactivation of plexinD1 results in congenital heart, vascular and skeletal defects

PlexinD1 is a membrane-bound receptor that mediates signals derived from class 3 secreted semaphorins. Although semaphorin signaling in axon guidance in the nervous system has been extensively studied, functions outside the nervous system including important roles in vascular patterning have also been demonstrated. Inactivation of plexinD1 leads to neo-natal lethality, structural defects of the cardiac outflow tract, peripheral vascular abnormalities, and axial skeletal morphogenesis defects. PlexinD1 is expressed by vascular endothelial cells, but additional domains of expression have also been demonstrated including in lymphocytes, osteoblasts, neural crest and the central nervous system. Hence, the cell-type specific functions of plexinD1 have remained unclear. Here, we describe the results of tissue-specific gene inactivation of plexinD1 in Tie2 expressing precursors, which recapitulates the null phenotype with respect to congenital heart, vascular, and skeletal abnormalities resulting in neonatal lethality. Interestingly, these mutants also have myocardial defects not previously reported. In addition, we demonstrate functions for plexinD1 in post-natal retinal vasculogenesis and adult angiogenesis through the use of inducible cre-mediated deletion. These results demonstrate an important role for PlexinD1 in embryonic and adult vasculature.     

False positive rate in newborn screening for congenital adrenal hyperplasia (CAH)-ether extraction reveals two distinct reasons for elevated 17α-hydroxyprogesterone (17-OHP) values

Background

While the sensitivity of newborn screening for the salt wasting form of congenital adrenal hyperplasia (CAH) is good, the positive predictive value is poor due to the high false positive rate of the immunological assays for 17-OHP. Cross-reactivity with steroid sulfates is one of the main causes for false positive results. Several approaches have been described to improve CAH screening: adjusting cut-off levels to gestational age or birth weight, and second-tier molecular genetic analysis or second-tier liquid chromatography-tandem mass spectrometry (TMS).

Methods

17-OHP was extracted with diethyl ether from dried blood spots in order to separate 17-OHP from polar steroids (like steroid sulfates). The dried ether extracts of calibrators, controls, and patient samples were redissolved and measured with the 17-OHP test kit (Wallac).

Results

760 normal, 1049 false positive, and 232 samples of confirmed cases with CAH were analysed. Mean 17-OHP values were significantly lower after extraction: Normal samples: 17.5 nmol/L vs. 3.2 nmol/L; false positive samples: 97.0 nmol/L vs. 25.9 nmol/L; CAH: 275 nmol/L vs. 205 nmol/L. With a cut-off value of 11.9 nmol/L (mean + 3 SD of the normal values), 404 of the false positives turned out to be normal. Ether extraction revealed two distinct subgroups of initially false positives rather than a continuum with normal distribution of 17-OHP values.

Conclusion

Diethyl ether extraction provided evidence for two causes of false positive results in CAH screening. It reduced the rate of false positives by about 40% without loss of sensitivity.     

Screening for OST deficiencies in unsolved CDG-I patients

Congenital Disorders of Glycosylation (CDG) are a group of inherited disorders caused by deficiencies in glycosylation. Since 1980, 14 CDG type I (CDG-I) defects have been identified in the endoplasmic reticulum, all affecting the assembly of the oligosaccharide precursor. However, the number of unsolved CDG-I (CDG-Ix) patients displaying protein hypoglycosylation in combination with an apparently normal assembly of the oligosaccharide precursor is currently expanding.We hypothesized that the hypoglycosylation observed in some of these patients could be caused by a deficiency in the transfer of the oligosaccharide precursor onto protein, a reaction catalyzed by the oligosaccharyltransferase (OST) complex. For this purpose, the different subunits of the OST complex were screened in 27 CDG-Ix patients for whom structural analysis of the lipid-linked oligosaccharides revealed a normal level and intact structure of the oligosaccharide precursor. Among these 27 patients, one was identified with a homozygous missense mutation (c.1121G > A; p.G374D) in the ribophorin 2 (RPN2) subunit of the OST complex. The pathogenic nature of this mutation remains unproven due to the complexity of tackling a possible OST defect.     

Mislocalization of large ARF-GEFs as a potential mechanism for BFA resistance in COG-deficient cells

Defects in subunits of the conserved oligomeric Golgi (COG) complex represent a growing subset of congenital disorders of glycosylation (CDGs). In addition to altered protein glycosylation and vesicular trafficking, Cog-deficient patient fibroblasts exhibit a striking delay in the Golgi-disrupting effects of brefeldin A (BFA). Despite the diagnostic value of this BFA resistance, the molecular basis of this response is not known. To investigate potential mechanisms of resistance, we analyzed the localization of the large ARF-GEF, GBF1, in several Cog-deficient cell lines. Our results revealed mislocalization of GBF1 to non-Golgi compartments, in particular the ERGIC, within these cells. Biochemical analysis of GBF1 in control and BFA-treated fibroblasts demonstrated that the steady-state level and membrane recruitment is not substantially affected by COG deficiency, supporting a role for the COG complex in the localization but not membrane association of GBF1. We also showed that pretreatment of fibroblasts with bafilomycin resulted in a GBF1-independent BFA resistance that appears additive with the resistance associated with COG deficiency. These data provide new insight into the mechanism of BFA resistance in Cog-deficient cells by suggesting a role for impaired ARF-GEF localization.     

Identification of S-acyl glutathione conjugates of bile acids in human bile by means of LC/ESI-MS

Previous work from this laboratory has reported the biotransformation of bile acids (BA) into the thioester-linked glutathione (GSH) conjugates via the intermediary metabolites formed by BA:CoA ligase and shown that such GSH conjugates are excreted into the bile in healthy rats as well as rats dosed with lithocholic acid or ursodeoxycholic acid. To examine whether such novel BA-GSH conjugates are present in human bile, we determined the concentration of the GSH conjugates of the five BA that predominate in human bile. Bile was obtained from three infants (age 4, 10, and 13 months) and the BA-GSH conjugates quantified by means of liquid chromatography (LC)/electrospray ionization (ESI)-linear ion trap mass spectrometry (MS) in negative-ion scan mode, monitoring characteristic transitions of the analytes. By LC/ESI-MS, only primary BA were present in biliary BA, indicating that the dehydroxylating flora had not yet developed. GSH conjugates of chenodeoxycholic and lithocholic acid were present in concentrations ranging from 27 to 1120 pmol/ml, several orders of magnitude less than those of natural BA N-acylamidates. GSH conjugates were not present, however, in the ductal bile obtained from 10 adults (nine choledocholithiasis, one bile duct cancer). Our results indicate that BA-GSH conjugates are formed and excreted in human bile, at least in infants, although this novel mode of conjugation is a very minor pathway.     

Partial requirement of endothelin receptor B in spiral ganglion neurons for postnatal development of hearing

Impairments of endothelin receptor B (Ednrb/EDNRB) cause the development of Waardenburg-Shah syndrome with congenital hearing loss, hypopigmentation, and megacolon disease in mice and humans. Hearing loss in Waardenburg-Shah syndrome has been thought to be caused by an Ednrb-mediated congenital defect of melanocytes in the stria vascularis (SV) of inner ears. Here we show that Ednrb expressed in spiral ganglion neurons (SGNs) in inner ears is required for postnatal development of hearing in mice. Ednrb protein was expressed in SGNs from WT mice on postnatal day 19 (P19), whereas it was undetectable in SGNs from WT mice on P3. Correspondingly, Ednrb homozygously deleted mice (Ednrb(-/-) mice) with congenital hearing loss showed degeneration of SGNs on P19 but not on P3. The congenital hearing loss involving neurodegeneration of SGNs as well as megacolon disease in Ednrb(-/-) mice were markedly improved by introducing an Ednrb transgene under control of the dopamine β-hydroxylase promoter (Ednrb(-/-);DBH-Ednrb mice) on P19. Neither defects of melanocytes nor hypopigmentation in the SV and skin in Ednrb(-/-) mice was rescued in the Ednrb(-/-);DBH-Ednrb mice. Thus, the results of this study indicate a novel role of Ednrb expressed in SGNs distinct from that in melanocytes in the SV contributing partially to postnatal hearing development.     

Unroofed coronary sinus newly diagnosed in adult patients after corrected congenital heart disease

Patients with congenital heart disease corrected in early childhood may later in life present with cardiac symptoms caused by other associated congenital anomalies that were initially not diagnosed. Nowadays, several noninvasive imaging modalities are available for the visualisation of cardiac anatomy in great detail. We describe two patients with an unroofed coronary sinus, a rare congenital anomaly which could be diagnosed using a combination of modalities including echocardiography, cardiac CT and cardiac MRI.