Identification of salivary N-glycoproteins and measurement of glycosylation site occupancy by boronate glycoprotein enrichment and liquid chromatography/electrospray ionization tandem mass spectrometry

RATIONALE Diseases including cancer and congenital disorders of glycosylation have been associated with changes in the site-specific extent of protein glycosylation. Saliva can be non-invasively sampled and is rich in glycoproteins, giving it the potential to be a useful biofluid for the discovery and detection of disease biomarkers associated with changes in glycosylation. METHODS Saliva was collected from healthy individuals and glycoproteins were enriched using phenylboronic acid based glycoprotein enrichment resin. Proteins were deglycosylated with peptide-N-glycosidase F and digested with AspN or trypsin. Desalted peptides and deglycosylated peptides were separated by reversed-phase liquid chromatography and detected with on-line electrospray ionization quadrupole-time-of-flight mass spectrometry using a 5600 TripleTof instrument. Site-specific glycosylation occupancy was semi-quantitatively determined from the abundance of deglycosylated and nonglycosylated versions of each given peptide. RESULTS Glycoprotein enrichment identified 67 independent glycosylation sites from 24 unique proteins, a 3.9-fold increase in the number of glycosylation sites identified. Enrichment of glycoproteins rather than glycopeptides allowed detection of both deglycosylated and nonglycosylated versions of each peptide, and thereby robust measurement of site-specific occupancy at 21 asparagines. Healthy individuals showed limited biological variability in occupancy, with partially modified sites having characteristics consistent with inefficient glycosylation by oligosaccharyltransferase. Inclusion of negative controls without enzymatic deglycosylation controlled for spontaneous chemical deamidation, and identified asparagines previously incorrectly annotated as glycosylated. CONCLUSIONS We developed a sample preparation and mass spectrometry detection strategy for rapid and efficient measurement of site-specific glycosylation occupancy on diverse salivary glycoproteins suitable for biomarker discovery and detection of changes in glycosylation occupancy in human disease.

MRI reveals individual level deformities in the growing scoliotic spine that clinically are masked by the Cobb angle

Clinically, the Cobb angle method measures the overall scoliotic curve in the coronal plane but does not measure individual vertebra and disc wedging. The contributions of the vertebrae and discs in the growing scoliotic spine were measured using sequential MRI scans to investigate coronal plane deformity progression with growth. Sequential MRI data showed complex patterns of deformity progression. Changes to the wedging of individual vertebrae and discs may occur in patients who have no increase in overall Cobb angle measure; the Cobb method alone may be insufficient to capture the complex mechanisms of deformity progression.

A semiautomatic method to identify vertebral end plate lesions (Schmorl's nodes)

Background Context There are differences in definitions of end plate lesions (EPLs), often referred to as Schmorl’s nodes, that may, to some extent, account for the large range of reported prevalence (3.8 to 76%). Purpose To develop a technique to measure the size, prevalence and location of EPLs in a consistent manner. Study Design/Setting This study proposed a method using a detection algorithm which was applied to five adolescent females (average age 15.1 years, range 13.0 to 19.2 years) with idiopathic scoliosis (average major Cobb angle 60°, range 55 to 67°). Methods Existing low-dose, computed tomography scans were segmented semi-automatically to extract 3D morphology of each vertebral endplate. Any remaining attachments to the posterior elements of adjacent vertebrae or endplates were then manually sectioned. An automatic algorithm was used to determine the presence and position of EPLs. Results EPLs were identified in 15 of the 170 (8.8%) endplates analysed with an average depth of 3.1mm. 11/15 of the EPLs were seen in the lumbar spine. The algorithm was found to be most sensitive to changes in the minimum EPL gradient at the edges of the EPL. Conclusions This study describes an imaging analysis technique for consistent measurement of the prevalence, location and size of EPLs. The technique can be used to analyse large populations without observer errors in EPL definitions.

A mouse with an N-ethyl-N-nitrosourea (ENU) induced Trp589Arg Galnt3 mutation represents a model for hyperphosphataemic familial tumoural calcinosis

Mutations of UDP-N-acetyl-alpha-D-galactosamine polypeptide N-acetyl galactosaminyl transferase 3 (GALNT3) result in familial tumoural calcinosis (FTC) and the hyperostosis-hyperphosphataemia syndrome (HHS), which are autosomal recessive disorders characterised by soft-tissue calcification and hyperphosphataemia. To facilitate in vivo studies of these heritable disorders of phosphate homeostasis, we embarked on establishing a mouse model by assessing progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU), and identified a mutant mouse, TCAL, with autosomal recessive inheritance of ectopic calcification, which involved multiple tissues, and hyperphosphataemia; the phenotype was designated TCAL and the locus, Tcal. TCAL males were infertile with loss of Sertoli cells and spermatozoa, and increased testicular apoptosis. Genetic mapping localized Tcal to chromosome 2 (62.64-71.11 Mb) which contained the Galnt3. DNA sequence analysis identified a Galnt3 missense mutation (Trp589Arg) in TCAL mice. Transient transfection of wild-type and mutant Galnt3-enhanced green fluorescent protein (EGFP) constructs in COS-7 cells revealed endoplasmic reticulum retention of the Trp589Arg mutant and Western blot analysis of kidney homogenates demonstrated defective glycosylation of Galnt3 in Tcal/Tcal mice. Tcal/Tcal mice had normal plasma calcium and parathyroid hormone concentrations; decreased alkaline phosphatase activity and intact Fgf23 concentrations; and elevation of circulating 1,25-dihydroxyvitamin D. Quantitative reverse transcriptase-PCR (qRT-PCR) revealed that Tcal/Tcal mice had increased expression of Galnt3 and Fgf23 in bone, but that renal expression of Klotho, 25-hydroxyvitamin D-1α-hydroxylase (Cyp27b1), and the sodium-phosphate co-transporters type-IIa and -IIc was similar to that in wild-type mice. Thus, TCAL mice have the phenotypic features of FTC and HHS, and provide a model for these disorders of phosphate metabolism. © 2012 Esapa et al.

Sequential MRI reveals individual level deformities in the growing scoliotic spine that are clinically masked by the Cobb angle

Introduction Clinically, the Cobb angle method measures the overall scoliotic curve in the coronal plane but does not measure individual vertebra and disc wedging. The contributions of the vertebrae and discs in the growing scoliotic spine were measured to investigate coronal plane deformity progression with growth. Methods A 0.49mm isotropic 3D MRI technique was developed to investigate the level-by-level changes that occur in the growing spine of a group of Adolescent Idiopathic Scoliosis (AIS) patients, who received two to four sequential scans (spaced 3-12 months apart). The coronal plane wedge angles of each vertebra and disc in the major curve were measured to capture any changes that occurred during their adolescent growth phase. Results Seventeen patients had at least two scans. Mean patient age was 12.9 years (SD 1.5 years). Sixteen were classified as right-sided major thoracic Lenke Type 1 (one left sided). Mean standing Cobb angle at initial presentation was 31° (SD 12°). Six received two scans, nine three scans and two four scans, with 65% showing a Cobb angle progression of 5° or more between scans. Overall, there was no clear pattern of deformity progression of individual vertebrae and discs, nor between patients who progressed and those who didn’t. There were measurable changes in the wedging of the vertebrae and discs in all patients. In sequential scans, change in direction of wedging was also seen. In several patients there was reverse wedging in the discs that counteracted increased wedging of the vertebrae such that no change in overall Cobb angle was seen. Conclusion Sequential MRI data showed complex patterns of deformity progression. Changes to the wedging of individual vertebrae and discs may occur in patients who have no increase in Cobb angle measure; the Cobb method alone may be insufficient to capture the complex mechanisms of deformity progression.

Identifying vertebral end plate lesions (schmorl's nodes) using a semi-automatic method

INTRODUCTION There is a large range in the reported prevalence of end plate lesions (EPLs), sometimes referred to as Schmorl's nodes in the general population (3.8-76%). One possible reason for this large range is the differences in definitions used by authors. Previous research has suggested that EPLs may potentially be a primary disturbance of growth plates that leads to the onset of scoliosis. The aim of this study was to develop a technique to measure the size, prevalence and location of EPLs on Computed Tomography (CT) images of scoliosis patients in a consistent manner. METHODS A detection algorithm was developed and applied to measure EPLs for five adolescent females with idiopathic scoliosis (average age 15.1 years, average major Cobb 60°). In this algorithm, the EPL definition was based on the lesion depth, the distance from the edge of the vertebral body and the gradient of the lesion edge. Existing low-dose, CT scans of the patients' spines were segmented semi-automatically to extract 3D vertebral endplate morphology. Manual sectioning of any attachments between posterior elements of adjacent vertebrae and, if necessary, endplates was carried out before the automatic algorithm was used to determine the presence and position of EPLs. RESULTS EPLs were identified in 15 of the 170 (8.8%) endplates analysed with an average depth of 3.1mm. 73% of the EPLs were seen in the lumbar spines (11/15). A sensitivity study demonstrated that the algorithm was most sensitive to changes in the minimum gradient required at the lesion edge. CONCLUSION An imaging analysis technique for consistent measurement of the prevalence, location and size of EPLs on CT images has been developed. Although the technique was tested on scoliosis patients, it can be used to analyse other populations without observer errors in EPL definitions.

The vertebral venous system in healthy and scoliotic adolescent spines - a 3D MRI investigation

Introduction. The venous drainage system within vertebral bodies (VBs) has been well documented previously in cadaveric specimens. Advances in 3D imaging and image processing now allow for in vivo quantification of larger venous vessels, such as the basivertebral vein. Differences between healthy and scoliotic VB veins can therefore be investigated. Methods. 20 healthy adolescent controls and 21 AIS patients were recruited (with ethics approval) to undergo 3D MRI, using a 3 Tesla, T1-weighted 3D gradient echo sequence, resulting in 512 slices across the thoraco-lumbar spine, with a voxel size of 0.5x0.5x0.5mm. Using Amira Filament Editor, five transverse slices through the VB were examined simultaneously and the resulting observable vascular network traced. Each VB was assessed, and a vascular network recorded when observable. A local coordinate system was created in the centre of each VB and the vascular networks aligned to this. The length of the vascular network on the left and right sides (with a small central region) of the VB was calculated, and the spatial patterning of the networks assessed level-by-level within each subject. Results. An average of 6 (range 4-10) vascular networks, consistent with descriptions of the basivertebral vein, were identifiable within each subject, most commonly between T10-L1. Differences were seen in the left/right distribution of vessels in the control and AIS subjects. Healthy controls saw a percentage distribution of 29:18:53 across the left:centre:right regions respectively, whereas the AIS subjects had a slightly shifted distribution of 33:25:42. The control group showed consistent spatial patterning of the vascular networks across most levels, but this was not seen in the AIS group. Conclusion. Observation and quantification of the basivertebral vein in vivo is possible using 3D MRI. The AIS group lacked the spatial pattern repetition seen in the control group and minor differences were seen in the left/right distribution of vessels.

Sequential MRI of growing scoliotic spines reveals individual level deformities that are clinically masked by the cobb angle

INTRODUCTION. Clinically, the Cobb angle method measures the overall scoliotic curve in the coronal plane but does not measure individual vertebra and disc wedging. The contributions of the vertebrae and discs in the growing scoliotic spine were measured to investigate coronal plane deformity progression with growth. METHODS. A 0.49mm isotropic 3D MRI technique was developed to investigate the level-by-level changes that occur in the growing spine of a group of Adolescent Idiopathic Scoliosis (AIS) patients, who received two to four sequential scans (spaced 3-12 months apart). The coronal plane wedge angles of each vertebra and disc in the major curve were measured to capture any changes that occurred during their adolescent growth phase. RESULTS. Seventeen patients had at least two scans. Mean patient age was 12.9 years (SD 1.5 years). Sixteen were classified as right-sided major thoracic Lenke Type 1 (one left sided). Mean standing Cobb angle at initial presentation was 31° (SD 12°). Six received two scans, nine three scans and two four scans, with 65% showing a Cobb angle progression of 5° or more between scans. Overall, there was no clear pattern of deformity progression of individual vertebrae and discs, nor between patients who progressed and those who didn’t. There were measurable changes in the wedging of the vertebrae and discs in all patients. In sequential scans, change in direction of wedging was also seen. In several patients there was reverse wedging in the discs that counteracted increased wedging of the vertebrae such that no change in overall Cobb angle was seen. CONCLUSION. Sequential MRI data showed complex patterns of deformity progression. Changes to the wedging of individual vertebrae and discs may occur in patients who have no increase in Cobb angle measure; the Cobb method alone may be insufficient to capture the complex mechanisms of deformity progression.

Variations in vertebral venous vasculature

INTRODUCTION Cadaveric studies have previously documented typical patterns of venous drainage within vertebral bodies (VBs) [1,2,3], comprised primarily of the basivertebral vein, a planar tree like structure at the mid-height of the VB. These studies, however, are limited in the number of samples available, and so have not examined any potential differences in this anatomy in conditions such as scoliosis. MRI is able to create 3D images of soft tissue structures in the spine, including the basivertebral vein without the use of contrast. As a non-invasive imaging technique this opens up the possibility of examining the venous network in multiple VBs within the same subject, in healthy controls as well as in subjects with abnormal anatomy such as adolescent idiopathic scoliosis (AIS). CONCLUSIONS High resolution MRI scans allow in vivo quantification of the vertebral venous system at multiple levels on healthy and scoliotic populations for the first time. The length of the basivertebral vein was seen to have a significant bias to the right hand side of the VB in both healthy and AIS adolescents. The spatial pattern of this vein showed large variations in branching both within and across individuals.

Predicting surgical outcomes for paediatric spinal deformity patients using subject specific finite element models

INTRODUCTION Adolescent idiopathic scoliosis (AIS) is a spinal deformity, which may require surgical correction by attaching rods to the patient’s spine using screws inserted into the vertebrae. Complication rates for deformity correction surgery are unacceptably high. Determining an achievable correction without overloading the adjacent spinal tissues or implants requires an understanding of the mechanical interaction between these components. Our novel patient specific modelling software creates individualized finite element models (FEM) representing the thoracolumbar spine and ribcage of scoliosis patients. We have recently applied the model to investigate the influence of increasing magnitudes of surgically applied corrective force on predicted deformity correction...

Sequential MRI reveals individual level deformities in the growing scoliotic spine that are not seen clinically by the cobb angle

Clinically, the Cobb angle method measures the overall scoliotic curve in the coronal plane but does not measure individual vertebra and disc wedging. The contributions of the vertebrae and discs in the growing scoliotic spine were measured to investigate coronal plane deformity progression with growth. Sequential MRI data in this project showed complex patterns of deformity progression. Changes to the wedging of individual vertebrae and discs may occur in patients who have no increase in Cobb angle measure; the Cobb method alone may be insufficient to capture the complex mechanisms of deformity progression.

Short-rib polydactyly and jeune syndromes are caused by mutations in WDR60

Short-rib polydactyly syndromes (SRPS I-V) are a group of lethal congenital disorders characterized by shortening of the ribs and long bones, polydactyly, and a range of extraskeletal phenotypes. A number of other disorders in this grouping, including Jeune and Ellis-van Creveld syndromes, have an overlapping but generally milder phenotype. Collectively, these short-rib dysplasias (with or without polydactyly) share a common underlying defect in primary cilium function and form a subset of the ciliopathy disease spectrum. By using whole-exome capture and massive parallel sequencing of DNA from an affected Australian individual with SRPS type III, we detected two novel heterozygous mutations in WDR60, a relatively uncharacterized gene. These mutations segregated appropriately in the unaffected parents and another affected family member, confirming compound heterozygosity, and both were predicted to have a damaging effect on the protein. Analysis of an additional 54 skeletal ciliopathy exomes identified compound heterozygous mutations in WDR60 in a Spanish individual with Jeune syndrome of relatively mild presentation. Of note, these two families share one novel WDR60 missense mutation, although haplotype analysis suggested no shared ancestry. We further show that WDR60 localizes at the base of the primary cilium in wild-type human chondrocytes, and analysis of fibroblasts from affected individuals revealed a defect in ciliogenesis and aberrant accumulation of the GLI2 transcription factor at the centrosome or basal body in the absence of an obvious axoneme. These findings show that WDR60 mutations can cause skeletal ciliopathies and suggest a role for WDR60 in ciliogenesis.

A novel ACVR1 mutation in the glycine/serine-rich domain found in the most benign case of a fibrodysplasia ossificans progressiva variant reported to date

Fibrodysplasia Ossificans Progressiva (FOP) is a rare, autosomal dominant condition, classically characterised by heterotopic ossification beginning in childhood and congenital great toe malformations; occurring in response to a c.617 G>A ACVR1 mutation in the functionally important glycine/serine-rich domain of exon 6. Here we describe a novel c.587 T>C mutation in the glycine/serine-rich domain of ACVR1, associated with delayed onset of heterotopic ossification and an exceptionally mild clinical course. Absence of great toe malformations, the presence of early ossification of the cervical spine facets joints, plus mild bilateral camptodactyly of the 5th fingers, together with a novel ACVR1 mutation, are consistent with the 'FOP-variant' syndrome. The c.587 T>C mutation replaces a conserved leucine with proline at residue 196. Modelling of the mutant protein reveals a steric clash with the kinase domain that will weaken interactions with FKBP12 and induce exposure of the glycine/serine-rich repeat. The mutant receptor is predicted to be hypersensitive to ligand stimulation rather than being constitutively active, consistent with the mild clinical phenotype. This case extends our understanding of the 'FOP-variant' syndrome.

Health-related quality of life of individuals with transfemoral amputation fitted with the Transcutaneous Bone Anchoring Prosthesis following the OGAAP

Background The benefits and safety transcutaneous bone anchored prosthesis relying on a screw fixation are well reported.[1-17] However, most of the studies on press-fit implants and joint replacement technology have focused on surgical techniques.[3, 18-23] One European centre using this technique has reported on health related quality of life (HRQOL) for a group of individuals with transfemoral amputation (TFA).[3] Data from other centres are needed to assess the effectiveness of the technique in different settings. Aim This study aimed at reporting HRQOL data at baseline and up to 2-year follow-up for a group of TFAs treated by Osseointegration Group of Australia who followed the Osseointegration Group of Australia Accelerated Protocol (OGAAP), in Sydney between 08/12/2011 and 09/04/2014. Method A total of 16 TFAs (7 females and 9 males, age 51 ± 12 y, height 1.73 ± 0.12 m, weight 83 ±18 kg) participated in this study. The cause of amputation was trauma or congenital limb deficiency for 11 (69%) and 5 (31%) participants, respectively. A total of 12 (75%) participants were prosthetic users while 4(25%) were wheelchair bound prior the surgery. The HRQOL were obtained from Questionnaire for Persons with Transfemoral Amputation (Q-TFA) using the four main scales (i.e., Prosthetic use, Mobility, Problem, Global) one year before and between 6.5 and 24 months after the Stage 1 of the surgeries for the baseline and follow-up, respectively. Results The lapse of time before and after Stage 1 was -6.19±3.54 and 10.83±3.58 months respectively. The raw score and percentage of improvement are presented in Figures 1 and 2, respectively. Discussion & Conclusion The average results demonstrated an improvement in each domain, particularly in the reduction of problems and an increase in global state. Furthermore, 56%, 75%, 94% and 69% of the participants reported an improvement in Prosthetic use, Mobility, Problem, Global scales, respectively. These results were comparable to previous studies relying of screwed fixation confirming that press-fit implantation is a viable alternative for bone-anchored prostheses.[1, 7, 8]

Autosomal dominant spondylocostal dysostosis is caused by mutation in TBX6

In humans, congenital spinal defects occur with an incidence of 0.5-1 per 1000 live births. One of the most severe syndromes with such defects is spondylocostal dysostosis (SCD). Over the past decade, the genetic basis of several forms of autosomal recessive SCD cases has been solved with the identification of four causative genes (DLL3, MESP2, LFNG and HES7). Autosomal dominant forms of SCD have also been reported, but to date no genetic etiology has been described for these. Here, we have used exome capture and next-generation sequencing to identify a stoploss mutation in TBX6 that segregates with disease in two generations of one family. We show that this mutation has a deleterious effect on the transcriptional activation activity of the TBX6 protein, likely due to haploinsufficiency. In mouse, Tbx6 is essential for the patterning of the vertebral precursor tissues, somites; thus, mutation of TBX6 is likely to be causative of SCD in this family. This is the first identification of the genetic cause of an autosomal dominant form of SCD, and also demonstrates the potential of exome sequencing to identify genetic causes of dominant diseases even in small families with few affected individuals.