Pseudohypoparathyroidism Type Ib Associated with Novel Duplications in the GNAS Locus

ContextPseudohypoparathyroidism type 1b (PHP-Ib) is characterized by renal resistance to PTH (and, sometimes, a mild resistance to TSH) and absence of any features of Albright''s hereditary osteodystrophy. Patients with PHP-Ib suffer of defects in the methylation pattern of the complex GNAS locus. PHP-Ib can be either sporadic or inherited in an autosomal dominant pattern. Whereas familial PHP-Ib is well characterized at the molecular level, the genetic cause of sporadic PHP-Ib cases remains elusive, although some molecular mechanisms have been associated with this subtype.ObjectiveThe aim of the study was to investigate the molecular and imprinting defects in the GNAS locus in two unrelated patients with PHP-Ib.DesignWe have analyzed the GNAS locus by direct sequencing, Methylation-Specific Multiplex Ligation-dependent Probe Amplification, microsatellites, Quantitative Multiplex PCR of Short Fluorescent fragments and array-Comparative Genomic Hybridization studies in order to characterize two unrelated families with clinical features of PHP-Ib.ResultsWe identified two duplications in the GNAS region in two patients with PHP-Ib: one of them, comprising ∼320 kb, occurred ‘de novo’ in the patient, whereas the other one, of ∼179 kb in length, was inherited from the maternal allele. In both cases, no other known genetic cause was observed.ConclusionIn this article, we describe the to-our-knowledge biggest duplications reported so far in the GNAS region. Both are associated to PHP-Ib, one of them occurring ‘de novo’ and the other one being maternally inherited.     

Reconstructing the History of Mesoamerican Populations through the Study of the Mitochondrial DNA Control Region

The study of genetic information can reveal a reconstruction of human population’s history. We sequenced the entire mtDNA control region (positions 16.024 to 576 following Cambridge Reference Sequence, CRS) of 605 individuals from seven Mesoamerican indigenous groups and one Aridoamerican from the Greater Southwest previously defined, all of them in present Mexico. Samples were collected directly from the indigenous populations, the application of an individual survey made it possible to remove related or with other origins samples. Diversity indices and demographic estimates were calculated. Also AMOVAs were calculated according to different criteria. An MDS plot, based on FST distances, was also built. We carried out the construction of individual networks for the four Amerindian haplogroups detected. Finally, barrier software was applied to detect genetic boundaries among populations. The results suggest: a common origin of the indigenous groups; a small degree of European admixture; and inter-ethnic gene flow. The process of Mesoamerica’s human settlement took place quickly influenced by the region’s orography, which development of genetic and cultural differences facilitated. We find the existence of genetic structure is related to the region’s geography, rather than to cultural parameters, such as language. The human population gradually became fragmented, though they remained relatively isolated, and differentiated due to small population sizes and different survival strategies. Genetic differences were detected between Aridoamerica and Mesoamerica, which can be subdivided into “East”, “Center”, “West” and “Southeast”. The fragmentation process occurred mainly during the Mesoamerican Pre-Classic period, with the Otomí being one of the oldest groups. With an increased number of populations studied adding previously published data, there is no change in the conclusions, although significant genetic heterogeneity can be detected in Pima and Huichol groups. This result may be explained because populations historically assigned as belonging to the same group were, in fact, different indigenous populations.     

Validation of the isothermal Schistosoma haematobium Recombinase Polymerase Amplification (RPA) assay,coupled with simplified sample preparation,for diagnosing female genital schistosomiasis using cervicovaginal lavage and vaginal self-swab samples

BackgroundFemale genital schistosomiasis (FGS) is a neglected and disabling gynecological disease that can result from infection with the parasitic trematode Schistosoma haematobium. Accurate diagnosis of FGS is crucial for effective case management, surveillance and control. However, current methods for diagnosis and morbidity assessment can be inaccessible to those at need, labour intensive, costly and unreliable. Molecular techniques such as PCR can be used to reliably diagnose FGS via the detection of Schistosoma DNA using cervicovaginal lavage (CVL) samples as well as lesser-invasive vaginal self-swab (VSS) and cervical self-swab samples. PCR is, however, currently unsuited for use in most endemic settings. As such, in this study, we assessed the use of a rapid and portable S. haematobium recombinase polymerase amplification (Sh-RPA) isothermal molecular diagnostic assay, coupled with simplified sample preparation methodologies, to detect S. haematobium DNA using CVL and VSS samples provided by patients in Zambia.Methodology/Principal findingsVSS and CVL samples were screened for FGS using a previously developed Sh-RPA assay. DNA was isolated from VSS and CVL samples using the QIAamp Mini kit (n = 603 and 527, respectively). DNA was also isolated from CVL samples using two rapid and portable DNA extraction methods: 1) the SpeedXtract Nucleic Acid Kit (n = 223) and 2) the Extracta DNA Tissue Prep Kit (n = 136). Diagnostic performance of the Sh-RPA using VSS DNA extacts (QIAamp Mini kit) as well as CVL DNA extracts (QIAamp Mini kit, SpeedXtract Nucleic Acid Kit and Extracta DNA Tissue Prep Kit) was then compared to a real-time PCR reference test.Results suggest that optimal performance may be achieved when the Sh-RPA is used with PuVSS samples (sensitivity 93.3%; specificity 96.6%), however no comparisons between different DNA extraction methods using VSS samples could be carried out within this study. When using CVL samples, sensitivity of the Sh-RPA ranged between 71.4 and 85.7 across all three DNA extraction methods when compared to real-time PCR using CVL samples prepared using the QIAamp Mini kit. Interestingly, of these three DNA extraction methods, the rapid and portable SpeedXtract method had the greatest sensitivity and specificity (85.7% and 98.1%, respectively). Specificity of the Sh-RPA was >91% across all comparisons.Conclusions/SignificanceThese results supplement previous findings, highlighting that the use of genital self-swab sampling for diagnosing FGS should be explored further whilst also demonstrating that rapid and portable DNA isolation methods can be used to detect S. haematobium DNA within clinical samples using RPA. Although further development and assessment is needed, it was concluded that the Sh-RPA, coupled with simplified sample preparation, shows excellent promise as a rapid and sensitive diagnostic tool capable of diagnosing FGS at the point-of-care in resource-poor schistosomiasis-endemic settings.     

Genome-wide analysis of gene expression during Xenopus tropicalis tadpole tail regeneration

Background

During liver development, intrahepatic bile ducts are thought to arise by a unique asymmetric mode of cholangiocyte tubulogenesis characterized by a series of remodeling stages. Moreover, in liver diseases, cells lining the Canals of Hering can proliferate and generate new hepatic tissue. The aim of this study was to develop protocols for three-dimensional visualization of protein expression, hepatic portal structures and human hepatic cholangiocyte tubulogenesis.

Results

Protocols were developed to digitally visualize portal vessel branching and protein expression of hepatic cell lineage and extracellular matrix deposition markers in three dimensions. Samples from human prenatal livers ranging from 7 weeks + 2 days to 15½ weeks post conception as well as adult normal and acetaminophen intoxicated liver were used. The markers included cytokeratins (CK) 7 and 19, the epithelial cell adhesion molecule (EpCAM), hepatocyte paraffin 1 (HepPar1), sex determining region Y (SRY)-box 9 (SOX9), laminin, nestin, and aquaporin 1 (AQP1). Digital three-dimensional reconstructions using CK19 as a single marker protein disclosed a fine network of CK19 positive cells in the biliary tree in normal liver and in the extensive ductular reactions originating from intrahepatic bile ducts and branching into the parenchyma of the acetaminophen intoxicated liver. In the developing human liver, three-dimensional reconstructions using multiple marker proteins confirmed that the human intrahepatic biliary tree forms through several developmental stages involving an initial transition of primitive hepatocytes into cholangiocytes shaping the ductal plate followed by a process of maturation and remodeling where the intrahepatic biliary tree develops through an asymmetrical form of cholangiocyte tubulogenesis.

Conclusions

The developed protocols provide a novel and sophisticated three-dimensional visualization of vessels and protein expression in human liver during development and disease.     

1.15 A crystal structure of the X. tropicalis Spred1 EVH1 domain suggests a fourth distinct peptide-binding mechanism within the EVH1 family

The recently described Spred protein family has been implicated in the modulation of receptor tyrosine kinase signalling. We report the crystal structure of the Enabled/vasodilator-stimulated phosphoprotein homology-1 (EVH1) domain from Xenopus tropicalis Spred1, solved to 1.15 A resolution. This structure confirms that the Spred EVH1 adopts the pleckstrin-homology fold, with a similar secondary structure to Enabled. A translation of one of the peptide-binding groove beta-strands narrows this groove, whilst one end of the groove shows structural flexibility. We propose that Spred1 will bind peptides that are less proline-rich than other EVH1 domains, with conformational changes indicating an induced fit.     

Bacteriophage-Based Genetic System for Selection of Nonsplicing Inteins

A genetic selection system that detects splicing and nonsplicing activities of inteins was developed based on the ability to rescue a T4 phage strain with a conditionally inactive DNA polymerase. This phage defect can be complemented by expression of plasmid-encoded phage RB69 DNA polymerase. Insertion of an intein gene into the active site of the RB69 DNA polymerase gene renders polymerase activity and phage viability dependent on protein splicing. The effectiveness of the system was tested by screening for thermosensitive splicing mutants. Development of genetic systems with the potential of identifying protein splicing inhibitors is a first step towards controlling proliferation of pathogenic microbes harboring inteins in essential proteins.     

Structural insights into the catalytic mechanism of Trypanosoma cruzi trans-sialidase

Sialidases are a superfamily of sialic-acid-releasing enzymes that are of significant interest due to their implication as virulence factors in the pathogenesis of a number of diseases. However, extensive studies of viral and microbial sialidases have failed to provide a comprehensive picture of their mechanistic properties, in part because the structures of competent enzyme-substrate complexes and reaction intermediates have never been described. Here we report these structures for the Trypanosoma cruzi trans-sialidase (TcTS), showing that catalysis by sialidases occurs via a similar mechanism to that of other retaining glycosidases, but with some intriguing differences that may have evolved in response to the substrate structure.     

Structural analysis of P450 AmphL from Streptomyces nodosus provides insights into substrate selectivity of polyene macrolide antibiotic biosynthetic P450s

AmphL is a cytochrome P450 enzyme that catalyzes the C8 oxidation of 8-deoxyamphotericin B to the polyene macrolide antibiotic, amphotericin B. To understand this substrate selectivity, we solved the crystal structure of AmphL to a resolution of 2.0 Å in complex with amphotericin B and performed molecular dynamics (MD) simulations. A detailed comparison with the closely related P450, PimD, which catalyzes the epoxidation of 4,5-desepoxypimaricin to the macrolide antibiotic, pimaricin, reveals key catalytic structural features responsible for stereo- and regio-selective oxidation. Both P450s have a similar access channel that runs parallel to the active site I helix over the surface of the heme. Molecular dynamics simulations of substrate binding reveal PimD can “pull” substrates further into the P450 access channel owing to additional electrostatic interactions between the protein and the carboxyl group attached to the hemiketal ring of 4,5-desepoxypimaricin. This substrate interaction is absent in AmphL although the additional substrate -OH groups in 8-deoxyamphotericin B help to correctly position the substrate for C8 oxidation. Simulations of the oxy-complex indicates that these -OH groups may also participate in a proton relay network required for O₂ activation as has been suggested for two other macrolide P450s, PimD and P450eryF. These findings provide experimentally testable models that can potentially contribute to a new generation of novel macrolide antibiotics with enhanced antifungal and/or antiprotozoal efficacy.